that CREB3L1 levels in tumors responsive to doxorubicin chemotherapy were significantly higher than in those resistant to this treatment (Denard et al., 2018). It has been postulated that CREB3L1 is a metastasis suppressor and that it may function in a similar way to p53 as a regulator of cell proliferation (Denard et al., 2011). In contrast, CREB3L1 is up-regulated in a metastatic subtype of triple-negative breast cancer cells that have activated both PERK signaling and the epithelial-to-mesenchymal transition program (Feng et al., 2017). In this tumor subtype CREB3L1 expression promotes invasion through the activation of extracellular matrix genes such as Col1a and FN1.

#### CREB3L2

CREB3L2 also known as BBF2 human homolog on chromosome 7 (BBF2H7) was identified as a novel human protein whose C-terminal region is fused to the FUS (fusion) genes in low-grade fibromyxoid sarcoma as a result of chromosomal translocation. Changes in CREB3L2 levels were first described in C6 glioma, HEK293 and MEF cells treated with thapsigargin (Kondo et al., 2007). Moreover, CREB3L2 mRNA levels were detected in different cell types and tissues (Kondo et al., 2007; Panagopoulos et al., 2007).

One of the findings that link CREB3L2 to the regulation of the secretory pathway is its participation in the differentiation of hepatic stellate cells (HSCs) to myofibroblast-like cells, a critical event in hepatic fibrosis. This process is characterized by enlargement of the ER and Golgi complex. Interestingly, another feature of this process is the up-regulation of Sec23A and Sec24D, components of the coat protein complex II (COPII), mediated by CREB3L2 (Tomoishi et al., 2017).

CREB3L2 mRNA is enriched in the developing notochord of Xenopus laevis embryos, where it also regulates genes of the secretory pathway (Tanegashima et al., 2009). Moreover, during early embryonic development of medaka fish, CREB3L2 is required for transcriptional regulation of a complete set of genes (Sec23a/24d/13/31a, Tango1, Sedlin, and KLHL12) essential for the enlargement of COPII vesicles to accommodate type II collagen for export from the ER (Ishikawa et al., 2017). In agreement with this, the well described zebrafish feelgood mutation, which disrupts head skeleton and notochord development through loss of secretory capacity, consists of a missense mutation in the DNA-binding domain of CREB3L2 and this results in decreased expression of sec23a and sec24d genes (Melville et al., 2011). An interesting fact about CREB3L2 is that, in developing cartilage, both the N- and C-terminal of the protein have important roles. After CREB3L2 cleavage, the N- terminus exerts its activity as transcription factor by promoting secretion of extracellular matrix proteins through induction of Sec23a expression (Saito et al., 2009; Hino et al., 2014). The C- terminal part of the protein, on the other hand, promotes the proliferation of chondrocytes and inhibits hypertrophic differentiation via regulating the Indian hedgehog (Ihh)/parathyroid hormonerelated protein signaling pathway (Saito et al., 2014). CREB3L2, as well as some cartilage matrix genes like Col2a1, are targets of Sox9 (Hino et al., 2014). The result of this transcriptional activation axis is the acceleration of cartilage matrix protein secretion during chondrocyte differentiation. In chondrocytes, CREB3L2 also functions as a target of the F-Box protein Fbxw7, a component of SKP1-CUL1-F-box protein type ubiquitin ligase which contributes to stem cell maintenance and cell differentiation. Fbxw7 targets the nuclear form of CREB3L2 for degradation in mesenchymal cells, thereby contributing to chondrogenesis (Yumimoto et al., 2013). Data obtained by Al-Maskari et al. (2018) showed that CREB3L2 expression increases during human B-cell transition to antibody secreting cells, which is a logic finding considering that this event implies great secretory overload (Shi et al., 2015) and that CREB3L2 has already been reported to up-regulate genes involved in the secretory pathway in similar cellular contexts.

The role of CREB3L2 in cancer is mainly represented by the specific translocation t(7;16)(q33;p11) that results in the creation of the chimeric gene FUS-CREB3L2, which is responsible for low-grade fibromyxoid sarcoma (LGFMS); a rare, slow-growing type of cancer that usually forms in the deep soft tissues of the legs or trunk (chest and abdomen) (Panagopoulos et al., 2007; Bartuma et al., 2010). In this context, the chimeric gene is believed to regulate CD24 (Moller et al., 2011). It was also described that, in malignant glioma, an FRS2/PAK1 activated RAS/MAPK signaling cascade up-regulates CREB3L2, which directly binds to the ATF5 promoter resulting in ATF5 transcription, an anti-apoptotic factor which plays a role in cell survival (Sheng et al., 2010).

#### CREB3L3

CREB3L3 (also known as CREB-H) was originally isolated as a transcription factor expressed in a liver-specific manner (Omori et al., 2001). CREB3L3 is also expressed in the stomach and small intestine. The roles for this transcription factor include triglyceride metabolism in the liver (Lee et al., 2011), reduction of cholesterol absorption (Kikuchi et al., 2016), glucose and lipid metabolism (Nakagawa et al., 2016b; Nakagawa and Shimano, 2018), acute phase response activation (Zhang et al., 2006) and hepcidin-mediated iron metabolism (Vecchi et al., 2009). CREB3L3 expression is regulated by a number of nuclear receptors, including PPARα (Danno et al., 2010), HNF4α (Luebke-Wheeler et al., 2008), GR (Lee et al., 2010), and ERRγ (Misra et al., 2014).

Surprisingly, CREB3L3 knock-out mice are viable, fertile, have a normal lifespan and do not display any gross physical or behavioral abnormalities. No anatomical or histological differences were found between liver and gastrointestinal tract of CREB3L3−/− mouse embryos respect to the controls. This information argues in favor of the notion that CREB3L3 is not essential for hepatogenesis and hepatocyte differentiation in the mouse. However, CREB3L3−/− mice showed a strong decrease in the transcript levels of acute phase genes compared to control animals when tunicamycin was administered to induce ER stress (Luebke-Wheeler et al., 2008). When intestinal CREB3L3 knock-out mice were compared to floxed mice, there were no

apparent differences in metabolic parameters. On the other hand, the liver CREB3L3 knock-out mice showed hyperlipidemia due to increased expression levels of genes related to cholesterol synthesis relative to floxed mice (Nakagawa et al., 2016a). Moreover, a whole genome expression analysis performed recently on liver samples from CREB3L3−/− mice subjected to ketogenic diet underscored the relevance of CREB3L3 in regulating apolipoprotein metabolism (Ruppert et al., 2019).

It has been demonstrated that CREB3L3 is required for the acute inflammatory response by regulating transcription of CRP and SAP genes which encode C-reactive protein and serum amyloid P-component proteins, respectively. Moreover, CREB3L3 and ATF6 interact with each other to synergistically activate expression of their target genes upon ER stress (Zhang et al., 2006). CREB3L3 has also been strongly linked to the regulation of cell proliferation. Interestingly, it is significantly underexpressed in hepatocellular carcinoma tissues and cells. Also, the loss of CREB3L3 function in hepatocellular carcinoma might contribute to the initiation and/or progression of cancer (Chin et al., 2005).

# CREB3L4

CREB3L4, also known as AIbZIP, CREB4 or TISP40, was first described in 2002 by two independent research groups. One of these groups was originally interested in identifying androgenregulated genes in human prostate cancer cells, and so CREB3L4 cDNA was isolated from LNCaP human prostate cancer cells treated with the synthetic androgen R1881. Because the cDNA analysis revealed that it contains a region with extensive similarity to the bZIP domain of CREB/ATF transcription factors, the protein was designated AIbZIP (Androgen-Induced bZIP protein; Qi et al., 2002).

The main function of CREB3L4 appears to be related to the tissue where it is mostly expressed; the prostate. This transcription factor has been shown to be involved in the proliferation of prostate cancer cells promoted by the Androgen Receptor (AR) and IRE1α (Kim et al., 2017). Interestingly, it was described that CREB3L4 can interact with CREB3L1 to inhibit its nuclear translocation in LNCaP cells. This results in p21 suppression and, consequently, increase in cell proliferation (Cui et al., 2016). Also, CREB3L4 has been linked to the process of cellular differentiation into adipocytes (Kim et al., 2014). Another group has reported its participation in male germ cell development, underlying that whereas CREB3L4 knockdown moderately impairs spermatogenesis, it is not sufficient to produce infertility in mice (Adham et al., 2005). Downstream target genes of CREB3L4 are tightly associated with prostate cell proliferation. In this context, the direct interaction between CREB3L4 and AR has been reported. Ben Aicha et al. (2007) demonstrated that a number of different genes with diverse functions are induced by CREB3L4: transcription factors, genes involved in protein processing, genes encoding channels and transporters, genes in charge of lipid and sugar metabolism and signal transduction genes, among others. These data imply once again the concept that the functions of CREB3 proteins, in this case CREB3L4, might not only be limited to the response to ER stress.

CREB3L4 is predominantly expressed in prostatic tissue and in breast cancer and prostate cancer cell lines. Interestingly, its expression is higher in cancerous prostate cells compared with non-cancerous prostate cells. Two independent research groups generated CREB3L4 knock-out mice, and both reported these mice to be healthy and fertile. Their findings using these murine models are slightly different between each other, but they both point to mild defects in spermatogenesis. More specifically, it was found that acetylated H2A and H4 histones are abnormally retained in epididymal sperm, implying that CREB3L4 might be regulating sperm head nuclei maturation in the mouse (Nagamori et al., 2006).

## CREB3 FAMILY IN THE CENTRAL NERVOUS SYSTEM

An RNA-sequencing transcriptome study performed with mouse brain cells (Zhang et al., 2014) indicated that CREB3L3 and CREB3L4 are minimally detected in different cell types of the CNS, while CREB3, CREB3L1 and CREB3L2 are co-expressed in most of them (**Figure 2**). In this section we review their role in physiological and pathological processes of the CNS.

#### Sampieri et al. CREB3 Family in Cellular Homeostasis

#### CREB3

CREB3 is the family member with the highest levels of expression in different cell types of the nervous system (Zhang et al., 2014, **Figure 2**). As we mentioned above, CREB3 was identified for its interaction with the transcriptional co-activator HCF (Freiman and Herr, 1997; Lu et al., 1997). During herpes virus infection, CREB3 and HCF together with the virion transactivator factor, VP16 induce expression of virion genes. Moreover, it has been postulated that, in neurons of the trigeminal ganglia, CREB3 is required for the establishment of herpes virus latency (Lu and Misra, 2000). Interestingly, CREB3 is also able to activate promoters of genes critical for herpes virus reactivation suggesting a complex role of the CREB3-HCF interaction in this process. The complexity of the CREB3-HCF interaction is harder to understand in dorsal root ganglion neurons where HCF and CREB3 poorly colocalized and exhibited Golgi and ER patterns, respectively (Kolb and Kristie, 2008). Another link between CREB3 and viral infection in the CNS is provided by the CREB3-Herp (homocysteine-induced ER protein) pathway in the Poliovirus (PV)-induced apoptosis. Herp is a CREB3 direct transcriptional target (Liang et al., 2006) involved in Ca2+ regulation in neurons. Down-regulation of CREB3 or Herp expression in IMR5 cells, a neuroblastoma cell line, increased PVinduced apoptosis (Mirabelli et al., 2016). Further analysis will be necessary to understand the participation of CREB3 and Herp in maintaining the balance of pro and antiapoptotic signals during the PV-induced neuropathogenesis of poliomyelitis.

Furthermore, CREB3 regulates intrinsic elongating form of axonal growth linked to injury-associated axonal ER responses (Ying et al., 2014). In dorsal root ganglion sensory neurons CREB3 localizes to the soma and the axonal ER. In response to nerve injury, CREB3 is synthesized in axons and transported to the nuclei of injured neurons via importin-dependent retrograde transport (Hasmatali et al., 2019). Interestingly, immunoprecipitation assays indicated that CREB3 interacts with importin, but CREB3-target genes involved in axon growth were not identified.

CREB3 knock-out mouse model (Penney et al., 2018) used to study the hypothalamic pituitary adrenal (HPA) axis and the glucocorticoid (GC) response linked CREB3 function in CNS to the secretory pathway. These animals have low levels of corticosterone and high levels of the glucocorticoid receptor (GR). Chromatin immunoprecipitation assays performed with mouse embryonic hippocampal cells indicated that CREB3 binds to the promoter region of genes that contain GC response elements (GRE). Interestingly, CREB3 also acts as a GR co-factor since it interacts with GR and enhances GR activity. Furthermore, in hippocampal cells, CREB3 binds to the promoter of genes encoding COPII components, regulating their expression.

 Up-Regulation of its expression was also demonstrated in reactive astrocytes proximal to a spinal cord injury (Nikaido et al., 2002). Studies performed in CREB3L1 knock-out mice indicated that, in astrocytes, CREB3L1 promotes glial scars formation, impedes axon growth and functional recovery after spinal injury (Sumida et al., 2018). Furthermore, CREB3L1 protein expression was detected in astrocytes and in neuronal primary cultures obtained from hippocampi of mice, but its mRNA up-regulation was detected only in astrocytes after treatment with kainic acid (KA). However, pyramidal neurons in the hippocampi of CREB3L1−/− mice were more susceptible to the toxicity induced by KA than those of wild-type mice (Chihara et al., 2009) suggesting a protective role of astrocytes against the KA-induced neuronal damage. One CREB3L1 target identified in astrocytes is the chondroitin 6-O-sulfate transferase 1 (C6ST1) gene, which encodes the major sulfotransferase of proteoglycan chondroitin sulfate (CSPG, Okuda et al., 2014). In vitro luciferase assays indicated that CREB3L1 binds to the first intron region of mouse C6ST1 gene. In contrast to the protective role that astrocytes play against KA-induced neuronal damage, astrocytes derived from wild-type CREB3L1 mice inhibited neurite outgrowth of cultured hippocampal neurons, whereas astrocytes from CREB3L1 knock-out mice did not. Therefore, CREB3L1 induction in reactive astrocytes in the injured brain may help to establish a non-permissive microenvironment for regenerating axons (Okuda et al., 2014). Another example of changes in the levels of CREB3L1 that modulate the function of the nervous system is given in the human retinal pigment epithelial cells, ARPE-19 (Miyagi et al., 2013). In these cells CREB3L1 transcriptionally regulates the vascular endothelial growth factor-A (VEGFA) which not only acts as a mediator of angiogenesis but also as a trophic and protective factor of retinal neurons (Foxton et al., 2013).

Increased CREB3L1 expression was also shown in the hypothalamus, specifically in the supraoptic and paraventricular nuclei of dehydrated and salt-loaded rats. The CREB3L1 upregulation occurs in arginine vasopressin (AVP) neurons, where CREB3L1 activates AVP gene transcription in vivo (Greenwood et al., 2014, 2015). In the mouse pituitary cell line AtT20, CREB3L1 expression is up-regulated by cAMP in vitro, and orphan nuclear receptor Nr4a1 is the transcription factor controlling the expression of CREB3L1. Furthermore, the ability to activate CREB3L1 by Nr4a1 is related to the level of methylation of the CpG island within the CREB3L1 proximal promoter (Greenwood et al., 2017).

Moreover, a direct link between CREB3L1 levels and changes in neurons was shown in Drosophila class IV dendritic arborization (C4da) neurons where nuclear polyglutamine (polyQ) toxicity reduced CREB3L1/CREBA levels. Additionally, polyQ led to down-regulation of genes involved in the secretory pathway and to the loss of Golgi outposts (GOPs). Furthermore, C4da neurons exhibited defective terminal dendrite elongation and decreased supply of plasma membrane (Chung et al., 2017). In C4da neurons, CREB-binding protein directly regulates CrebA transcription in cooperation with the Drosophila transcription factor Cut (Chung et al., 2017). Consistent with the data obtained in Drosophila, rat hippocampal primary neurons exhibited reduced number of dendrites spines and GOPs due to polyQ toxicity. Also, CREB3L1/CrebA overexpression restored the loss of GOPs and the down-regulation of COPII-related genes induced by polyQ. However, no significant changes exist in

the branching and elongation of terminal dendrites for the overexpression of CREB3L1 alone.

#### CREBL2

Expression of CREB3L2 in vivo was analyzed by immunohistochemistry in a mouse model of permanent focal brain ischemia where CREB3L2 was detected in the region closer to the infarction region in the striatum, especially in neurons (labeled with MAP2). In contrast to CREB3L1, no expression of CREB3L2 was detected in astrocytes in this model. The role of CREB3L2 in the brain was studied using SK-N-SH cells, a human neuroblastoma cell line. Overexpression and siRNA transfection assays indicate that CREB3L2 reduces and increases the sensitivity to ER stress-induced cell death, respectively (Kondo et al., 2007). Although CREB3L2-depleted cells die more than control cells by the action of thapsigargin, the levels of typical ER-stress markers such as GRP78/BiP, XBP1, CHOP, and PDI were not modified. CREB3L2-target genes that protect against cell death induced by thapsigargin were not explored in SK-N-SH cells. Participation of CREB3L2 in cell survival was also reported in mouse malignant glioma GL261 cells (Sheng et al., 2010), where CREB3L2 was identified as one of the 12 genes required for expression of ATF5 (activating transcription factor 5), an anti-apoptotic factor which plays a role in cell survival (Monaco et al., 2007). Sheng et al. (2010) also showed that expression of CREB3L2 in human malignant glioma was higher than in normal brain and that individuals with ATF5-positive glioblastomas had shorter survival times than those with ATF5-negative glioblastomas. Moreover, expression of CREB3L2, ATF5 and its target, the oncogene MCL1 (myeloid cell leukemia sequence 1, Le Gouill et al., 2004) decreased after serum-induced GS9-6 differentiation indicating that these proteins are enriched in undifferentiated cells. ATF5 is required for terminal differentiation and survival of olfactory

TABLE 1 | CREB3 family members in the central nervous system, list of their functions, upstream regulators and downstream target genes.

N/D, not defined.

sensory neurons according to homozygous Atf5 knock-out mice (Wang et al., 2012).

CREB3L2 levels also increase during oligodendrocytes (OL) maturation where Chd7 (chromodomain-helicase-DNA-binding protein 7) and Sox10 activates a transcriptional program for OL differentiation (He et al., 2016). CREB3L2 and Osterix are Chd7 targets and their down-regulation in OL precursor cells inhibited expression of genes related to myelination (He et al., 2016). Moreover, in the dorsal-root ganglion neurons-like F11 cell line, CREB3L2 was identified as a direct target gene of the transcription factors nuclear factor IL-3 (NFIL3) and the tree isoforms of the CCAAT-enhancer-binding proteins (C/EBPα, C/EBPβ, and C/EBPδ; Macgillavry et al., 2011). It has been postulated that these transcription factors co-regulate CREB3L2 expression during forskolin-induced neurite outgrowth model. NFIL3 and C/EBPs integrate a complex transcriptional regulatory network that fine-tunes the expression of neuronal outgrowthrelated genes.

# OUTLOOK

CREB3 transcription factors are widely expressed in different tissues and they regulate a broad range of developmental, physiological and pathological processes playing fundamental roles in cellular homeostasis. Most of them show a tissuespecific preferential expression, however, CREB3, CREB3L1, and CREB3L2 co-express in different cells of the CNS (**Figure 2**) where they participate in essential processes (**Table 1** and **Figure 3**). For example, CREB3L1 and CREB3L2 are involved in neurite outgrowth (Macgillavry et al., 2011; Okuda et al., 2014) while CREB3 and CREB3L1 modulate (directly or indirectly) axonal growth after an injury (Ying et al., 2014; Sumida et al., 2018). CREB3 regulates pathogenic mechanisms of herpes and polio virus (Lu and Misra, 2000; Liang et al., 2006), and in non-CNS cells a CREB3-ARF4 signaling pathway mediates the susceptibility to pathogens (Reiling et al., 2013). As we mentioned above, CREB3 transcription factors can also modulate cell survival by activating expression of anti-apoptotic factors (Sheng et al., 2010). Also, CREB3 and CREB3L1 contribute to neuroendocrine regulation of the hypothalamic/pituitary/adrenal axis modulating the GR activity and the AVP gene transcription (Greenwood et al., 2014; Penney et al., 2018). Most of these processes require the adaptation of the secretory pathway which is regulated by CREB3 family members in multiple cell types (Fox and Andrew, 2015). In line with this, CREB3 regulates expression of genes encoding COPII components and formation of Golgi outposts in hippocampal cells (Chung et al., 2017; Penney et al., 2018).

CREB3 transcription factors emerge as signaling hubs for the regulation of Golgi homeostasis, integrating stimuli from multiple sources to control secretion, protein post-translational modification and trafficking, impacting on membrane expansion and composition. Their contribution to the CNS is crucial since neurons are specially sensitive to Golgi stress and Golgi fragmentation, events tightly connected to neurodegenerative diseases (Gonatas et al., 2006; Machamer, 2015; Lopez et al., 2017). Although some upstream regulators and downstream target genes of CREB3 transcription factors have been identified

(**Table 1**), many questions remain open: What are the specific stimuli that trigger the activation of these factors and how are they sensed? How are CREB3 family genes regulated in each cell type of the CNS? What are the consequences of lacking one or more CREB3 transcription factors in a cell? Can they replace each other? How do they participate in neuronal development? Comprehensive understanding of how CREB3 transcription factors function promises not only to explain fundamental biological questions, but also to provide new options for therapeutic intervention.

#### AUTHOR CONTRIBUTIONS

CA, LS, and PD wrote the manuscript. CA contributed with funding support.

#### REFERENCES

#### FUNDING

LS and PD are both CONICET doctoral fellows. CA is an investigator of CONICET and a Professor at Universidad Nacional de Córdoba. Work in the Álvarez lab is supported by the grants PICT2016-0042-Prestamo BID from the Ministry of Science and Technology of Argentina, SECyT-UNC Tipo A (2016 and 2018) from Universidad Nacional de Córdoba.

#### ACKNOWLEDGMENTS

The authors thank Susana Genti-Raimondi (Universidad Nacional de Córdoba, Córdoba, Argentina) and Hector A. Saka (Universidad Nacional de Córdoba, Córdoba, Argentina) for their comments after reading the manuscript, special thanks to HAS for his contribution with figure design.

concentration increase and apoptosis induced by poliovirus. J. Gen. Virol. 97, 2194–2200. doi: 10.1099/jgv.0.000544

regulator of secretion. Front. Mol. Neurosci. 11:352. doi: 10.3389/fnmol.2018. 00352

with high-grade metastatic breast cancers with poor prognosis and is prevalent in triple negative breast cancers. Breast Cancer Res. 18:12.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Sampieri, Di Giusto and Alvarez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# New Insights Into the Golgi Stacking Proteins

Erpan Ahat<sup>1</sup> , Jie Li<sup>1</sup> and Yanzhuang Wang1,2 \*

<sup>1</sup> Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, United States, <sup>2</sup> Department of Neurology, University of Michigan School of Medicine, Ann Arbor, MI, United States

The Golgi stacking proteins, GRASP55 and GRASP65, are best known for their roles in Golgi structure formation. These peripheral Golgi proteins form trans-oligomers that hold the flat cisternal membranes into stacks. Depletion of both GRASP proteins in cells disrupts the Golgi stack structure, increases protein trafficking, but impairs accurate glycosylation, and sorting. Golgi unstacking by GRASPs depletion also reduces cell adhesion and migration in an integrin-dependent manner. In addition to Golgi structure formation and regulation of cellular activities, GRASPs, in particular GRASP55, have recently drawn attention in their roles in autophagy, and unconventional secretion. In autophagy, GRASP55 senses the energy level by O-GlcNAcylation, which regulates GRASP55 translocation from the Golgi to the autophagosome-lysosome interface, where it interacts with LC3 and LAMP2 to facilitate autophagosome-lysosome fusion. This newly discovered function of GRASP55 in autophagy may help explain its role in the stress-induced, autophagosome-dependent unconventional secretion. In this review, we summarize the emerging functions of the GRASP proteins, focusing on their roles in cell adhesion and migration, autophagy, unconventional secretion, as well as on novel GRASP-interacting proteins.

#### Edited by:

Daniel Ungar, University of York, United Kingdom

#### Reviewed by:

Antonino Colanzi, Istituto di Biochimica delle Proteine (IBP), Italy Paul Anthony Gleeson, The University of Melbourne, Australia Adam Graham Grieve, University of Oxford, United Kingdom

> \*Correspondence: Yanzhuang Wang<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Traffic, a section of the journal Frontiers in Cell and Developmental Biology

Received: 18 April 2019 Accepted: 03 July 2019 Published: 16 July 2019

#### Citation:

Ahat E, Li J and Wang Y (2019) New Insights Into the Golgi Stacking Proteins. Front. Cell Dev. Biol. 7:131. doi: 10.3389/fcell.2019.00131 Keywords: Golgi, stacking, GRASP65, GRASP55, O-GlcNAcylation, autophagy, unconventional secretion

# INTRODUCTION

The Golgi apparatus is an essential membrane-bound organelle in the cell that functions as a "post station" in the secretory pathway (Klute et al., 2011). In mammalian cells, Golgi membranes are organized as stacks of multiple flat cisternae, which are further linked into a ribbon-like structure located in the perinuclear region (Klumperman, 2011). The Golgi functions as a protein modification and sorting center in the secretory pathway, with different modification enzymes residing in different subcompartments, including cis-Golgi network (CGN), cis-, medial-, and trans-cisternae, and trans-Golgi network (TGN) (Goldfischer, 1982). The Golgi receives newly synthesized proteins and lipids from the endoplasmic reticulum (ER), sequentially modifies, and dispatches them to distinct destinations by protein sorting at the TGN (Marsh and Howell, 2002; Brandizzi and Barlowe, 2013).

The highly ordered Golgi stack structure, which is the functional unit of the Golgi, is believed to facilitate sequential protein modification, and processing in mammals. Using in vitro assays mimicking Golgi disassembly and reassembly that occur during the cell cycle, two Golgi peripheral membrane proteins, GRASP65 and GRASP55 (Golgi ReAssembly and Stacking Protein), were identified as Golgi stacking factors (Barr et al., 1997; Shorter et al., 1999; Wang et al., 2003). Both GRASPs were further characterized and confirmed to control Golgi stacking and ribbon linking in vivo (Wang et al., 2003; Puthenveedu et al., 2006; Feinstein and Linstedt, 2008; Xiang and Wang, 2010). GRASP65 is mainly targeted to cis-Golgi, whereas GRASP55 localizes

to medial- and trans-cisternae. GRASP65 and GRASP55 have similar domain structures. The conserved GRASP domain at the N-terminus contains a membrane anchor and can form dimers and trans-oligomers. The more divergent serine proline-rich (SPR) domain at the C-terminus contains multiple phosphorylation sites, whose phosphorylation inhibits GRASP oligomerization in mitosis (Wang et al., 2005; Vielemeyer et al., 2009; Tang et al., 2012) and perhaps also in stress and pathological conditions (**Figures 1**, **2A**; Joshi et al., 2014, 2015; Joshi and Wang, 2015). In coordination with GRASPs, GRASPs interacting proteins, including the GRASP65 binding partner GM130, and GRASP55 binding protein Golgin-45, may also be involved in Golgi structure formation (Lee et al., 2014).

Since depletion or inhibition of GRASP55 and GRASP65 impairs Golgi structure formation, these proteins have been recently used as tools to disrupt the Golgi structure and thereby determine the functional consequence of Golgi structural disruption. In addition, a number of novel GRASP-interacting proteins have been identified and GRASPs have been linked to autophagy, unconventional secretion and other cellular activities, such as cell adhesion, migration, and growth. In this review, we attempt to summarize these new discoveries on GRASP functions and discuss the potential links between these new findings.

# GRASP55 AND GRASP65 AS TOOLS TO PROBE THE BIOLOGICAL SIGNIFICANCE OF GOLGI STRUCTURE FORMATION

The role of GRASPs in Golgi stack formation and the impact of GRASP depletion or inhibition on Golgi functions have been explored using a number of experimental approaches. Inhibition of GRASP65 by microinjecting inhibitory antibodies, knocking down (KD) GRASPs by siRNA, or knocking out (KO) GRASPs by CRISPR/Cas9, all significantly impair Golgi stack formation (Wang et al., 2003; Tang et al., 2010; Bekier et al., 2017). Research from the Rothman and Wang labs demonstrated that depletion of GRASPs, which results in Golgi fragmentation, increases the trafficking of selected cargo molecules, including vesicular stomatitis virus G-protein (VSVG), α5 integrin, and CD8 (Wang et al., 2008; Xiang et al., 2013; Lee et al., 2014). A plausible explanation for this result is that Golgi unstacking increases the membrane surface for vesicle formation and thus accelerates protein trafficking (Wang et al., 2008; Zhang and Wang, 2015; Bekier et al., 2017; Ahat et al., 2019). Need to mention, in contrast to the results described above, D'Angelo et al. (2009) reported that GRASP55/65 bind the C-terminal hydrophobic tail of specific transmembrane proteins such as CD8, and this interaction is required for CD8 trafficking through the Golgi stack. These controversial results could be caused by the different constructs used in the studies; the first two labs used full length CD8 (or a fusion protein with full length CD8), and while the last one used a VSVG-CD8α (C-terminal tail) chimera. Other factors, such as the knockdown efficiency, may also be involved. Considering that there is currently no other way to disrupt Golgi stacking other than manipulating GRASPs, this subject requires further investigation.

Glycomic analysis by mass spectrometry showed that GRASP depletion, especially GRASP55 single depletion or GRASP55/65 double-depletion, results in a reduction in the overall glycan abundance, complexity, and glycoprotein composition at the plasma membrane. Interestingly, GRASP depletion-mediated Golgi unstacking also causes mis-sorting of lysosome enzymes such as cathepsin D (Xiang et al., 2013; Zhang et al., 2015). Therefore, it has been proposed that cisternal stacking impedes the intra-Golgi trafficking speed by reducing the accessibility of coat proteins to Golgi membranes, which ensures accurate glycosylation, and sorting (Zhang and Wang, 2015, 2016; Huang and Wang, 2017). Recently, GRASP55 and GRASP65 single knockout mice have been reported, with only limited defects in Golgi structure and function (Veenendaal et al., 2014; Chiritoiu et al., 2019). One possibility is that the knockout effect of one GRASP may be compensated by the redundancy of the other GRASP protein. It has been demonstrated that when one GRASP is depleted in cells, the level of the other GRASP protein may increase to compensate for the knockout effect (Bekier et al., 2017). GRASP55 and GRASP65 double knockout mice have not been reported so far.

# EFFECTS OF GOLGI DESTRUCTION ON CELL ATTACHMENT, MIGRATION, AND GROWTH

The effect of Golgi unstacking induced by GRASP depletion on cellular activities such as cell attachment, migration, and growth have recently been investigated. GRASP KD or KO in HeLa cells reduces cells adhesion to fibronectin-coated dishes (Ahat et al., 2019). GRASP depletion also reduces cell migration in HeLa and MDA-MB-231 cells. While the effect was significant when a single GRASP was depleted, it was more robust when both GRASPs were removed. Because cell attachment and migration are mediated by cell adhesion molecules, in particular integrins, the level of a variety of integrins in GRASP KO cells was assessed. Among the 10 integrin subunits tested, which can form 8 different heterodimers, α5β1 integrins, the major and well-characterized integrin complex in HeLa, and MDA-MB-231 cells that utilizes fibronectin as its ligand (Mierke et al., 2011), exhibited the most robust reduction upon GRASP depletion. Further analysis showed that GRASP depletion reduces α5β1 integrin levels not only in the cell, but also at the cell surface, providing a reasonable explanation how GRASP depletion reduces cell attachment, and migration (Ahat et al., 2019). Consistently, exogenous expression of α5β1 integrins rescues the attachment and migration defects in GRASP-depleted cells. The effects of GRASP depletion on α5β1 integrins are specific for Golgi unstacking, as disruption of the Golgi ribbon by knocking down Golgin-84 or by nocodazole treatment, or destroying the Golgi structure by brefeldin A treatment, does not reduce integrin levels in cells (Xiang et al., 2013).

There are three possibilities to reduce the α5β1 integrin protein levels in cells by GRASP depletion: decreased synthesis,

accelerated degradation, or both. As Golgi structural defects caused by GRASP-depletion may impair protein glycosylation, which plays an important role in protein stability (Shental-Bechor and Levy, 2008), it was initially speculated that Golgi unstacking may impair α5β1 glycosylation and thus reduces their stability. However, the results demonstrated that the reduction of α5β1 integrin is due to decreased protein synthesis rather than increased degradation (Ahat et al., 2019). Interestingly, GRASPdepletion significantly increases total protein synthesis and accelerates cell proliferation (Ahat et al., 2019), consistent with the previous report that GRASP65 depletion accelerates cell cycle progression (Tang et al., 2010). How GRASP depletion selectively decreases the synthesis of α5β1 integrin while increasing overall protein synthesis remains a mystery. One possibility is that the overall protein synthesis is increased due to accelerated cell proliferation and enhanced protein trafficking. In addition, it has been proposed that the Golgi functions as a signal hub, as many signaling molecules are docked on the Golgi membranes and respond to different cellular stresses (Mayinger, 2011; Makhoul et al., 2018). So it is also possible that α5β1 integrin synthesis is regulated by signaling pathways on the Golgi in response to Golgi structural changes caused by GRASP depletion.

# NEW DISCOVERIES ON GRASP55 AND GRASP65 INTERACTING PROTEINS

Recently, some new discoveries have been made on the known interacting partners of GRASPs. Golgin-45 is one of the earliest identified GRASP55-binding proteins, which is involved in vesicle tethering and Golgi structure regulation (Short et al., 2001; Lee et al., 2014). The structural basis of GRASP55 interaction with Golgin-45 has recently been revealed. The last C-terminal residues of Golgin-45, QGELIAL, insert into the canonical PDZpeptide binding pocket in the PDZ1 domain of GRASP55, while the upstream residues of the C-terminal sequence of Golgin-45, TRYENITFNCCNHC, interacts with both PDZ domains by inserting into the cleft between them. Furthermore, the C-terminus of Golgin-45 also binds the PDZ2 domains of the two neighboring GRASP55 molecules, which enhances GRASP55 oligomerization. This is thought to play an important role in Golgi stacking. The third interaction site between Golgin-45 and GRASP55 is a unique zinc finger-like structure formed between Cys393/Cys<sup>396</sup> of Golgin-45 and His<sup>18</sup> (β1)/Cys<sup>103</sup> (β2) of GRASP55 (Zhao et al., 2017). Similar to the Golgin-45 and GRASP55 interaction, GM130 interacts with GRASP65 via its C-terminal KITVI sequence that binds PDZ1, and via the IPFFY sequence that interacts with both PDZ domains by inserting into the hydrophobic cleft between them. But unlike the Golgin-45 and GRASP55 interaction, GRASP65 undergoes conformational change on PDZ domain upon GM130 interaction but does not form a zinc-finger structure on GM130-GRASP65 interaction (Hu et al., 2015; Zhao et al., 2017).

In the past several years, a number of new GRASP binding proteins have been identified (**Figure 1** and **Table 1**). Two interacting proteins have recently been discovered for GRASP65, the actin elongation factor Mena (mammalian enabled homolog), and the Hsc70 (heat shock cognate 71 kDa protein)

co-chaperone DjA1 (DnaJ homolog subfamily A member 1). Mena is recruited to the Golgi membranes by GRASP65 to facilitate actin polymerization and GRASP65 oligomerization, and thus functions as a bridging protein in Golgi ribbon linking (Tang et al., 2016). DjA1 binds to GRASP65 and promotes GRASP65 oligomerization in a Hsc70-independent manner (Li et al., 2019b).

Most recently, several novel GRASP55 binding partners have been identified that are related to the newly discovered function of GRASP55 in autophagy. GRASP55 not only facilitates autophagosome-lysosome fusion via the interactions with LC3 on autophagosomes and LAMP2 on lysosomes (Zhang and Wang, 2018a,b; Zhang et al., 2018), but also directly binds Beclin-1 (BECN1) and UVRAG to facilitate the assembly

and membrane association of the phosphatidylinositol 3-kinase (PtdIns3K, or PI3K) complex (Zhang et al., 2019), and therefore plays an important role in autophagosome maturation during both glucose depletion and amino acid starvation. Moreover, GRASP55 binds cystic fibrosis transmembrane conductance regulator (CFTR) and transforming growth factor beta 1 (TGFβ1) to facilitate their unconventional secretion (Gee et al., 2011; Nüchel et al., 2018). These new findings reveal novel roles for GRASPs in cellular activities outside of the Golgi. While some of these findings have been recently reviewed elsewhere (Li et al., 2019a), several key advances are discussed below in detail.

### GRASP55 REGULATES AUTOPHAGOSOME-LYSOSOME FUSION

How the Golgi copes with different stresses and whether there is a Golgi stress sensor have not been systematically studied. In an effort to explore how the Golgi responds to energy deprivation, a number of Golgi proteins were examined for O-GlcNAcylation, a cytosolic glycosylation that serves as an energy sensor to regulate cellular pathways (Zhang et al., 2018). In this study, Zhang et al. discovered that GRASP55, but not other Golgi matrix proteins examined, including GRASP65, GM130 and Golgin-45, is O-GlcNAcylated under growth condition. Upon glucose starvation, GRASP55 is de-O-GlcNAcylated and forms puncta outside of the Golgi area. Since glucose starvation induces autophagy, the function of GRASP55 in autophagy was then tested. Indeed, depletion of GRASP55, but not GRASP65, increased the number of autophagosomes but decreased the autophagic flux, indicating a defect in autophagosome-lysosome fusion.

How does a Golgi protein like GRASP55 regulate autophagy? The study by Zhang et al. (2018) revealed that GRASP55 de-O-GlcNAcylation upon glucose deprivation allows some of the GRASP55 molecules to colocalize with autophagosomes. GRASP55 targeting to autophagosomes is regulated by O-GlcNAcylation, as it is enhanced by mutating the O-GlcNAcylation sites on GRASP55 as well as by glucose starvation which reduces GRASP55 O-GlcNAcylation. Further biochemical studies demonstrate that GRASP55 interacts with LC3-II on autophagosomes, and this interaction is enhanced by GRASP55 de-O-GlcNAcylation. In addition, GRASP55 also interacts with LAMP2 on lysosomes. These together suggest a possibility that GRASP55 may function as a bridging protein to facilitate LC3-LAMP2 interaction as well as autophagosomelysosome fusion. Indeed, this possibility was subsequently confirmed using in vivo and in vitro approaches (**Figure 2B**; Zhang et al., 2018). In cells, GRASP55 depletion reduces LC3 and LAMP2 colocalization as well as autophagosome-lysosome fusion. In vitro, GRASP55 facilitates autophagosome-lysosome fusion in an in vitro fusion assay. Furthermore, the addition of recombinant GRASP55 enhances LC3 and LAMP2 coimmunoprecipitation from cell lysates (Zhang et al., 2018). Thus, like in Golgi stacking, GRASP55 oligomers serve as membrane tethers to facilitate autophagosome-lysosome fusion.

The role of GRASP55 in autophagosome-lysosome fusion is not limited to glucose starvation, but also in amino acid starvation. Upon amino acid starvation, GRASP55 not only physically interacts with LC3 and LAMP2, but also regulates the formation of the PI3K UVRAG complex that is known to facilitate autophagosome-lysosome fusion (Zhang et al., 2019). Here, GRASP55 directly interacts with Beclin-1, induces the UVRAG PI3K complex formation and increases its membrane association. These reports identified GRASP55 as a specific energy and nutrient sensor on the Golgi to regulate autophagy.

Since GRASP55 still appears on autophagosomes in the presence of the protein synthesis inhibitor cycloheximide, it is speculated that GRASP55 is targeted to autophagosomes from an existing pool upon autophagy induction (Zhang et al., 2018). One remaining interesting question concerns how GRASP55 is targeted to autophagosomes. GRASP55 is unlikely translocated to the autophagosomes with the entire Golgi as cargo for autophagic degradation, since no other Golgi markers are found in the newly formed autophagosomes, and GRASP55 is localized on the outer membrane of autophagosomes instead of the lumen (Zhang et al., 2018, 2019). One possibility is through vesicular transport, similar to the transmembrane protein Atg9 that is normally enriched in the Golgi and is translocated to autophagosomes upon autophagy induction. However, unlike Atg9, GRASP55 does not have a transmembrane domain, and how the translocation occurs selectively on de-O-GlcNAcylated GRASP55 remains unknown. Alternatively, it is possible that a small pool of GRASP55 constantly shuttles between the Golgi, cytosol, and autophagosomes; and the equilibrium is regulated by GRASP55 O-GlcNAcylation. Nevertheless, how GRASP55 is targeted to autophagosomes under stress conditions requires further investigation.

While the discovery of GRASP55 as a membrane tether in autophagosome-lysosome fusion is exciting, it is unknown how GRASP55 interplays with other known tethering proteins, in particular Rab7 and the HOPS complex, as well as the STX17- SNAP29-VAMP7/8 SNARE complex that mediates the fusion (Jager et al., 2004; Itakura et al., 2012; Jiang et al., 2014). It is possible that these proteins function sequentially during autophagosome-lysosome fusion. Alternatively, GRASP55 may function as an independent mechanism in autophagosomelysosome fusion.

#### GRASP55/65 AND UNCONVENTIONAL SECRETION

It was long believed that only proteins with canonical ER signal peptides at the N-terminus can be secreted out of the cell. Since the discovery of the Golgi-independent unconventional secretion of the cytokine interleukin 1 beta (IL-1β) (Muesch et al., 1990), more proteins without ER signal sequences (leaderless proteins) and some integral membrane proteins have been reported to be transported or secreted in a Golgi-independent manner (Kinseth et al., 2007; Schotman et al., 2008; Gee et al., 2011). Collectively, the non-canonical Golgi-independent secretion is referred to as unconventional protein secretion (UPS). Interestingly, although

UPS itself is Golgi-independent, it requires two important Golgi proteins, GRASP55 and GRASP65, in mammalian cells and their homologs in other model organisms. Whether unconventional secretion is a by-product of loss of Golgi functions, or viceversa, has not been ruled out due to the lack of molecular tools to manipulate Golgi structure, and function without affecting GRASPs. In a previous review (Rabouille, 2017), unconventional secretion is generally classified into four categories, type I is direct translocation of cargo molecules across the plasma membrane via pore formation, type II is unconventional secretion through ATPbinding cassette (ABC) transporters, type III is vesicle-mediated secretion of cytosolic proteins, and type IV is the Golgi bypassing transportation of integral membrane proteins. GRASPs and their homologs have been reported to play a role in type III and type IV UPS, which is the focus of this review in terms of unconventional secretion.

#### GRASPs and Unconventional Trafficking of Integral Membrane Proteins CFTR

The Phe508 deletion from CFTR results in the inhibition of its trafficking to the plasma membrane and ER-associated degradation. It has been shown that GRASP55 and GRASP65 are required for the unconventional secretion of both WT and DeltaF508 CFTR when ER-to-Golgi trafficking is inhibited by expressing a dominant negative Sar1 or Arf1, or by inhibiting vesicle fusion via overexpression of syntaxin 5 (Gee et al., 2011). GRASP55 is phosphorylated at Ser441 during certain ER stresses, monomerized, and located near the ER to regulate unconventional secretion of CFTR, which requires CFTR-GRASP55 interaction (**Figure 2C**; Kim et al., 2016; Gee et al., 2018). Similar to GRASP55, GRASP65 overexpression rescued the secretion defect of mutant CFTR, although GRASP65 was not extensively tested in the same studies.

One contradiction in these reports is that overexpression of the GRASP55-G2A mutant inhibits CFTR secretion, whereas expression of N-terminally tagged GRASP55, which has been shown to abolish its membrane targeting similar to the GRASP-G2A mutant, increases CFTR secretion (Gee et al., 2011; Heinrich et al., 2014; Kim et al., 2016). Considering that GRASP55 regulates CFTR secretion by direct binding, GRASP55 may serve as a cytosolic chaperone to regulate CFTR sequestration by autophagosomes or by multivesicular bodies (MVBs) (Noh et al., 2018). Here, GRASP55 phosphorylation and re-localization in response to ER stress is necessary for CFTR secretion. A recent report showed that GRASP55 regulates the IRE1 ER unfolded protein response (UPR) pathway (Chiritoiu et al., 2019). It is unclear why the medial/trans-Golgi protein GRASP55, but not the cis-Golgi protein GRASP65, is the main player in regulating this activity as GRASP65 is localized on the Golgi compartments closer to the ER.

#### Integrin

dGRASP, the Drosophila homolog of GRASP, regulates the Golgi bypassing, non-canonical trafficking of the alpha subunit of integrin during Drosophila wing development (Schotman et al., 2008). Unlike normal situations in which dGRASP is localized on the Golgi, during wing development, dGRASP localizes near the plasma membrane to regulate non-canonical secretion of alpha-integrin. It was proposed that dGRASP regulates integrin secretion via facilitating the fusion of integrin-containing vesicles with the plasma membrane (Schotman et al., 2008). Alternatively, GRASP depletion may cause mis-sorting of an unknown, plasma membrane destined vesicle fusion protein (i.e., SNARE), which subsequently affects the non-canonical secretion of integrin. Unlike CFTR, it is unclear if dGRASP binding is required for integrin secretion. Considering that GRASP55 translocates to the ER in CFTR secretion while dGRASP translocates to near the plasma membrane area in integrin secretion, GRASP may regulate the secretion of these two transmembrane cargoes through different mechanisms.

## GRASP55 and Endosome/ Autophagosome-Dependent Unconventional Secretion of Cytosolic Proteins

#### IL-1β

Interleukin 1 beta, the major cytosolic regulator of inflammation, was one of the first identified cargoes of UPS (Muesch et al., 1990). IL-1β and another cytokine, TGFβ1, are secreted via a non-canonical secretory pathway distinct from the conventional ER-Golgi pathway (Nüchel et al., 2018). IL-1β is exported either through vesicle mediated secretion or via Gasdermin-Dmediated pore formation at the plasma membrane (Kayagaki et al., 2015). More recently, it was revealed that IL-1β is first restrained in the intermembrane space of autophagosomes and then secreted by the fusion of autophagosomes with the plasma membrane (**Figure 2D**). This fusion process is mediated by SNAREs including Sec22b on autophagosomes, syntaxin 3 and syntaxin 4 on the plasma membrane, and SNAP-23 and SNAP-29 from the cytosol (Dupont et al., 2011; Kimura et al., 2017). Most recently, a question was raised on whether autophagy is indeed involved in IL-1β secretion (Chiritoiu et al., 2019).

Interestingly, depletion of GRASP55 or GRASP65 reduces IL-1β secretion (Zhang et al., 2015; **Figure 2D**). GRASP55 may regulate the secretion of IL-1β through its role in autophagy or by modulating the IREα/XBP-1 UPR pathway (Dupont et al., 2011; Zhang and Wang, 2018a; Chiritoiu et al., 2019; van Ziel et al., 2019). Considering the function of GRASP55 in autophagosomelysosome fusion, it is reasonable to speculate that GRASP55 may also be involved in autophagosome-plasma membrane fusion in this scenario.

#### AcbA/Acb1

GRASP65 and GRASP55 were originally identified in mammalian cells to regulate Golgi stacking and ribbon linking. Kinseth et al. (2007) provided the first evidence that GRASP regulates unconventional secretion in Dictyostelium. This unexpected finding came from the observation that knockout of GrpA, the GRASP homolog in Dictyostelium, results in a secretion defect of AcbA, a protein that is processed in the extracellular environment to produce the spore differentiation

factor-2 (SDF 2) (Kinseth et al., 2007; Levi and Glick, 2007). Later, it was reported that Acb1, the budding yeast homolog of ACBP, is also unconventionally secreted, which depends on Grh1 (the GRASP homolog in the budding yeast), core Atg genes, ESCRT machinery and SNAREs, but is independent of COPII coated vesicles (**Figure 2D**; Duran et al., 2010; Manjithaya et al., 2010).

Although GRASP is required for Acb1 secretion, it is not clear at which step GRASP is involved. It seems that the vesicles carrying Acb1 are distinct from autophagosomes because GFP-Grh1 colocalizes with neither the autophagosome marker Atg8 nor the phagophore marker Ape1 (Bruns et al., 2011). Instead, a novel membrane-bound compartment called CUPS (compartment for UPS) is required for Acb1 secretion. However, the formation and identity of this membrane structure are largely unknown (Ye, 2018).

#### OUTLOOK

It is clear that GRASP65 and GRASP55 have both Golgidependent and Golgi-independent functions. On the Golgi, GRASP trans-oligomers are the primary machineries for Golgi stack formation. The recent finding of reduced cell adhesion and migration under GRASP depletion strengthens the essential role of Golgi stacking in protein trafficking, modification, and signaling.

Under certain stress conditions or at certain stages of development, GRASPs also function outside of the Golgi, likely as membrane tethers as within the Golgi stacks. The findings are interesting, but several outstanding questions remain. For example, how is GRASP55 targeted to different locations outside of the Golgi? Do GRASPs function as tethers in the regulation of unconventional secretion? The recent findings of GRASP55 in autophagy may help address these questions. Furthermore, the role of GRASPs in unconventional secretion has been confirmed in a variety of systems, but not all types of unconventional secretion require GRASPs. For example, unconventional secretion of a misfolding-associated protein secretion (MAPS) cargo GFP1-10 and a cilia transmembrane protein Peripherin/rds is reported to be GRASP-independent (Tian et al., 2014; Lee et al., 2016). This indicates a heterogeneity of unconventional secretion and the specificity of GRASPs in the regulation of unconventional

#### REFERENCES

secretion of certain substrates. Future studies by comparing different cargo molecules may help understand the exact roles of GRASPs in unconventional secretion.

It is clear that GRASP proteins play important roles in unconventional secretion, but the underlying mechanism remains largely unknown. Both unconventional secretion of certain cargoes and autophagy are augmented under stress conditions. The newly uncovered roles of GRASP55 as an energy sensor in the Golgi and a membrane tether in autophagy indicate that it may serve as a stress sensor and an effector in stress response; and these roles may be linked to unconventional secretion of certain cargo molecules. GRASP55 may coordinate Golgi-dependent and Golgi-independent trafficking pathways in the cell under different conditions. In addition to autophagy, the emerging role of GRASP55 in the regulation of ER stress and UPR indicates that GRASP55 may affect unconventional secretion through UPR (Chiritoiu et al., 2019; van Ziel et al., 2019). Systematic studies on Golgi response to different stress stimuli and identification of novel GRASP interacting proteins under normal and stress conditions may shed light on the mechanism of GRASP55 and GRASP65 in Golgi-dependent and Golgi-independent functions.

# AUTHOR CONTRIBUTIONS

All authors contributed to the design, conception and manuscript preparation. All authors approved the publication of this study.

# FUNDING

This work was supported by the National Institutes of Health (Grants GM112786, GM105920, and GM130331), M-Cubed, and the Fastforward Protein Folding Disease Initiative of the University of Michigan to YW.

# ACKNOWLEDGMENTS

We thank Wang Lab members for their stimulating discussions. The figures are created using BioRender.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Ahat, Li and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Multifaceted Rho GTPase Signaling at the Endomembranes

#### Santosh Phuyal\* and Hesso Farhan\*

Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway

The Rho family of small GTPases orchestrates fundamental biological processes such as cell cycle progression, cell migration, and actin cytoskeleton dynamics, and their aberrant signaling is linked to numerous human diseases and disorders. Traditionally, active Rho GTPase proteins were proposed to reside and function predominantly at the plasma membrane. While this view still holds true, it is emerging that active pool of multiple Rho GTPases are in part localized to endomembranes such as endosomes and the Golgi. In this review, we will focus on the intracellular pools and discuss how their local activation contributes to the shaping of various cellular processes. Our main focus will be on Rho signaling from the endosomes, Golgi, mitochondria and nucleus and how they regulate multiple cellular events such as receptor trafficking, cell proliferation and differentiation, cell migration and polarity.

#### Edited by:

Jaakko Saraste, University of Bergen, Norway

#### Reviewed by:

Stéphanie Miserey-Lenkei, Centre National de la Recherche Scientifique (CNRS), France Heike Folsch, Northwestern University, United States

#### \*Correspondence:

Santosh Phuyal<EMAIL_ADDRESS>Hesso Farhan<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Traffic, a section of the journal Frontiers in Cell and Developmental Biology Received: 19 April 2019 Accepted: 28 June 2019

Published: 16 July 2019

#### Citation:

Phuyal S and Farhan H (2019) Multifaceted Rho GTPase Signaling at the Endomembranes. Front. Cell Dev. Biol. 7:127. doi: 10.3389/fcell.2019.00127 Keywords: Rac1, Cdc42, RhoA, RhoB, RhoD, Golgi, endocytosis, membrane trafficking

#### THE RHO GTPase FAMILY

The founding member of the Rho GTPase, termed Rho for Ras homolog, was identified in 1985 (Madaule and Axel, 1985). Shortly thereafter, two back-to-back papers elegantly demonstrated the functional importance of Rho and Rac in actin cytoskeleton assembly (Ridley and Hall, 1992; Ridley et al., 1992) driving the expansion of Rho GTPase biology. Since then, the Rho family of small GTPases has grown to include 20 separate proteins divided into seven subfamilies: Rho, Rac, Cdc42, Rnd, RhoD, RhoBTB, and RhoH (Hodge and Ridley, 2016). The majority of Rho family members undergo conformational switching between GTP-bound active and GDP-bound inactive states. This GTP/GDP cycling is tightly regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs function as Rho GTPases activator by catalyzing the exchange of GDP for GTP, whereas GAPs facilitate hydrolysis of GTP leading to their inactivation. An additional layer of complexity is provided by guanine-nucleotide dissociation inhibitors (GDIs) that bind to the inactive pool of Rho GTPases and sequester them in cytosol (Garcia-Mata et al., 2011). Additional factors contributing to the complexity of Rho GTPase signaling is the crosstalk between its family members, distinct subcellular distribution of their GEFs and GAPs, and the post-transcriptional modifications such as phosphorylation, ubiquitination, and palmitoylation that regulate stability and spatial distribution of Rho GTPases.

The switch I and switch II domains of Rho GTPases undergo conformational change upon GTP-binding. Once active, Rho GTPases associate with membranes and selectively interact with downstream effectors and other scaffolding proteins to mediate a myriad of biological processes including reorganization of the actin cytoskeleton, regulation of membrane trafficking, cell motility and polarity. A large body of literature deals with signaling initiated by Rho GTPases and their effectors at or around the plasma membrane although intracellular pool of Rho GTPases and their

regulators are increasingly apparent. It is becoming clear that coordination of functionally active Rho GTPases, their GEFs and GAPs, as well as their effectors extend beyond the plasma membrane to multiple intracellular organelles. Understanding how cells decode these spatio-temporal signals to generate biologic outputs is a major area of investigation.

Here, we review emerging knowledge on biological processes and signaling events mediated by intracellular pools of Rho GTPases, with special emphasis on signaling patterns that are triggered or initiated by endomembrane localized Rho GTPases. In particular, we summarize recent studies that provide direct evidence for endosomal, Golgi, mitochondrial and nuclear pools of Rho GTPase signaling.

## RHO GTPase SIGNALING FROM ENDOSOMES

The endosomal system consists of pleomorphic membranous carriers that processes and transports a range of cargoes including active signaling receptors. The ability of endocytic organelles in generating compartmentalized signaling patterns by directional shuttling and/or retaining of signaling molecules into specific locations within the cell is well documented and has been extensively reviewed elsewhere (Palfy et al., 2012). In this review, we will summarize Rho GTPase signaling originating directly from the endosomal system and elaborate on how Rho family members utilize endosomes for signal propagation along the endocytic pathway (**Figure 1**).

From the Rho GTPase family, RhoB was among the first ones to be predominantly detected in the endocytic compartments (Adamson et al., 1992; Gampel and Mellor, 2002). Today, the list of endosome localized Rho GTPases has grown to include RhoD, Rac1, Cdc42, TCL, and TC10, although the underlying mechanism of their recruitment to endosomes remains to be clarified in the majority of cases. In the case of RhoB, the type of prenylation was suggested to be the major determinant of its precise localization. The geranylgeranylated form of RhoB was localized to late endosomes, while the farnesylated form was detected predominantly at the plasma membrane (Wherlock et al., 2004). Whether the kind of lipid modification is a deciding factor for all other endosome localized Rho GTPases remains to be addressed. Furthermore, the identity, localization and regulation of the prenylation factors responsible for differential RhoB prenylation is also poorly characterized. Additionally, it is also unclear how GEFs/GAPs for Rho GTPases are targeted to the endosomes. Nevertheless, the recruitment of Rho GTPases to endosomes generates spatially restricted signals that, as we will discuss in the following sections, has consequences for numerous cellular processes.

It can be expected that endosome-specific Rho GTPases represent a functionally distinct subset from those existing at the plasma membrane. Indeed, the farnesylated pool (plasma membrane localized) of RhoB appeared functionally distinct from the geranylgeranylated (endosome localized) pool in that treatment of cells with farnesyl-transferase inhibitors, which abolishes the farnesylated pool of RhoB, resulted in an increased recycling of endocytosed epidermal growth factor (EGF) receptor (Wherlock et al., 2004). In agreement with this, EGF-triggered signaling was prolonged in cells treated with farnesyl-transferase inhibitors. Endosomal RhoB was also shown to recruit the Ser/Thr kinase PRK1 to this compartment, resulting in its activation. Active PRK1 on endosomes then regulated the trafficking of EGFR in a way leading to prolonged signaling and preventing its degradation (Mellor et al., 1998; Gampel et al., 1999). Interestingly, PRK1 has been reported to phosphorylate the intermediate filament proteins vimentin and neurofilament as well as interact with α-actinin (Mukai, 2003). Whether RhoB-PRK1 regulated kinetics of EGF signaling shows actindependency remains to be explored in greater details. The fact that RhoB promotes endosome recycling appears to be true in several cell types. While RhoB controls EGFR recycling in epithelial cells, it also controls recycling of the integrin LFA-1 in T-lymphocytes, which regulates their migration (Samuelsson et al., 2017). These studies imply that RhoB targeted to endosomal compartment is an autonomous signaling entity. Supporting this view, cytoplasmic endosomes harboring RhoB were found to pick up inactive Src kinase and stimulate its activity en route to the plasma membrane (Sandilands et al., 2004). This type of translocation and activation of Src required the presence of RhoB containing endosomes since inactive pool of Src kinase accumulated around the perinuclear region in RhoB−/<sup>−</sup> cells growing on fibronectin (Sandilands et al., 2004). The exact molecular mechanism underlying RhoB mediated endosomal motility, however, remains elusive. Proteins central to actin dynamics such as Scar1, Dia1, and mDia2 are also associated with RhoB-positive endosomes (Sandilands et al., 2004; Fernandez-Borja et al., 2005; Wallar et al., 2007) suggesting that RhoBpositive cytoplasmic entities could regulate endosomal trafficking in a manner dependent on rearrangement of actin cytoskeleton. It is currently unclear which GEFs activate RhoB on endosomes and which GAPs control this activity. In addition, it is unknown whether endocytic Rab GTPases contribute to the regulation of the endosomal RhoB pool. A possible candidate is the Rhospecific GAP, DLC3 which has been localized to Rab8-positive membrane tubules, reminiscent of the endocytic recycling compartment (Braun et al., 2015). Depletion of DLC3 impairs transferrin receptor endocytosis and this effect was neutralized by simultaneous depletion of RhoA and RhoB.

Several Rho family members regulate some aspects of actin cytoskeleton dynamics, and accordingly, multiple studies have demonstrated the importance of endosomal pool of Rho GTPases for actin-based endocytic vesicle movement. For example, a role for RhoD in controlling the endocytic vesicle movement has been documented. It was shown that cells possessing active RhoD display reduced velocity of early endosome movement, thereby slowing down the membrane trafficking events (Murphy et al., 1996). This effect of RhoD likely involves actin-based mechanisms since RhoD-mediated recruitment of its effector hDia2C to early endosomes aligns them along actin filaments (Gasman et al., 2003). RhoD has also been reported to bind to the Rab5 effector Rabankyrin-5 on early endosomes (Nehru et al., 2013). Thereby, Rab5 and RhoD may cooperate to regulate internalization of EGFR. It remains to be tested if RhoB and

FIGURE 1 | Schematic illustration depicting Rho GTPase signaling from endosomes. A number of Rho family members residing at endosomes generate localized signaling output to regulate a wide array of biological functions.

RhoD might control bidirectional endosomal movement by directly regulating each other's activity. Such a crosstalk between Rho GTPases has already been demonstrated for RhoB and Rac1 (Marcos-Ramiro et al., 2016). In cytokine pre-treated vascular and microvascular endothelial cells, endosomal RhoB retains Rac1 in endosomes and negatively affects its activity to prevent endothelial barrier reformation (Marcos-Ramiro et al., 2016).

While RhoD and Rab5 cooperate to regulate endocytic trafficking, Rac1 required Rab5 for its endosomal recruitment (Palamidessi et al., 2008). Rac1 was detected by several groups on endosomes in different eukaryotic organisms (Strehle et al., 2006; Palamidessi et al., 2008; Menard et al., 2014). It was shown that formation of Rab5-positive early endosomes was a pre-requisite for endosomal recruitment of Rac1 and its GEF Tiam1 leading to Rac1 activation. This active, endosomal pool of Rac1 was then delivered to specific plasma membrane domains turning them into regions of localized actin cytoskeleton remodeling. The process of endocytic Rac1 delivery required another small GTPase, Arf6 (**Figure 1**). The Rab5-Rac1-Arf6 signaling circuit was crucial not only for localized actin dynamics and the morphology of cancer cells, but also for directed cell migration (Palamidessi et al., 2008). An analogous mechanism was found to account for concentration of Cdc42 at the leading edge of astrocytes (Osmani et al., 2010). In migrating astrocytes, Cdc42 and its GEF β-PIX were shown to co-localize on endosome-like structures. The localization of Cdc42 on endosome-like vesicles required Rab5, and the directed delivery of Cdc42 to the leading edge depended on Arf6, as was observed for Rac1. Hence, the concept of coupling of multiple small GTPases might be more universal than currently anticipated. While these studies further provide evidence for the intriguing possibility that endosomes serve as a hub for Rho GTPase activation and spatiotemporal signal generation, they also raise the question about how this mechanism is controlled. The kinase LRRK2 is unusual in that it harbors a GTPase (ROC) domain. LRRK2 was found to localize to endosomes and to play an important role in negatively controlling Rac1 activity on this site (Schreij et al., 2015). Loss of LRRK2 resulted in hyperactive Rac1 and loss of dendritic spines in neuronal dendrites. It is unclear whether LRRK2 regulates Rac1 through phosphorylation, or through a scaffolding effect. There is evidence for phosphorylation-dependent regulation of Rho GTPases (Kwon et al., 2000; Schoentaube et al., 2009; Schwarz et al., 2012). LRRK2 also possesses GTPase activity (Liu and West, 2017) and it remains to be tested whether this activity is involved in Rac1 regulation. Notably, LRRK2 is mutated in familial Parkinson's disease and was very recently shown to be activated by the Rab29 on endosomes as well as the trans-Golgi network (Purlyte et al., 2018). This provides new avenues for

future investigations of crosstalk of Rac1 or Cdc42 with Rab29 and linking this to the pathogenesis of Parkinson's disease.

Rac1 and Cdc42 signaling from endosomes extends beyond Rab5-positive endosomal entities. For instance, Rab11 positive recycling endosomes were shown to traffic Rac1 to immunological synapses (Bouchet et al., 2016). The interaction of Rac1 with Rab11 on recycling endosomes was facilitated by a Rab11 effector FIP3. This Rab11-FIP3-Rac1 tripartite complex controlled T-cell spreading, cortical rigidity and immunological synapse symmetry in a manner dependent on Rac1 (Bouchet et al., 2016). The effects of FIP3 on T-cell spreading and synapse symmetry were Rac1 dependent, because this effect was abolished in the presence of Rac1 inhibitor, NSC23766. We note here that this inhibitor disrupts the interaction of Rac1 with its GEF Tiam/Trio. Thus, it is possible that FIP3 recruits Tiam/Trio to endosomes to mediate localized Rac1 activation. Alternatively, it is possible that Rac1 is recruited to recycling endosomes in its active form. In some cases, endocytosis of certain cell surface receptors may initiate endosomal Rac1 activation. Indeed, c-Met internalization and its trafficking to perinuclear endosomes in response to HGF-stimulation was essential for Rac1 activation (Menard et al., 2014). Notably, optimal Rac1 activation required interaction of c-Met with Rac1 specific GEF Vav2 in the perinuclear endosomes to initiate Rac1-driven cell migration (Menard et al., 2014). Late endosomal Rac1 together with Cdc42 also responds to growth factor independent signals. Using fluorescence resonance energy transfer (FRET)-based biosensors, it was shown that constitutively active Rac1 and Cdc42 promoted actin-nucleation events to drive sorting of cargo into intraluminal vesicles (Kajimoto et al., 2018). Interestingly, this process was initiated by sphingosine 1-phosphate signaling to multivesicular endosomes localized Rac1 and Cdc42, and also required activity from GEFs PLEKHG2 and P-Rex1 (Kajimoto et al., 2018).

Finally, the Rho GTPase family members TCL and TC10 have both been described to reside in early endosomal compartments to control endocytic pathway (de Toledo et al., 2003; Kawase et al., 2006). Apart from that, TCL and TC10 have been relatively underexplored and a clear picture of signaling originating from endosomal pools of these two Rho GTPases is lacking. The activity of TC10 on exocytic vesicles was reported using a FRETbased probe. It was shown that TC10 activity drops sharply just before fusion of the vesicle with the plasma membrane (Kawase et al., 2006). This result indicates that GTP hydrolysis by TC10 is a critical step for vesicle exocytosis. This role of TC10 appears to be applicable to several types of vesicles such as delivery of EGFR to the cell surface (Kawase et al., 2006) as well as translocation of the glucose transporter GLUT4 to the cell surface upon insulin stimulation (Chiang et al., 2001). Because TC10 was also shown to localize to subdomains of the plasma membrane (Liu and West, 2017) it remains to be determined which pool of TC10 regulates exocytic vesicle trafficking.

Taken together, these studies establish that Rho GTPases and their GEFs/GAPs assemble into signaling entities at early/late endosomes (**Figure 1**). However, it is unclear whether active Rho GTPases at the early/late endosomal compartments stem from plasma membrane internalized active pool. So far, the requirement of GEFs for optimal endosomal Rho GTPase signaling output points toward active recruitment of Rho GTPases to the endosomes. However, such a demonstration by selective manipulation of local pools of GEFs in contrast to depletion of total cellular pools of GEFs is currently missing.

# RHO GTPase SIGNALING FROM THE GOLGI

Over two decades ago, Cdc42 was already noticed to localize to the Golgi (Erickson et al., 1996). Since then a number of other Rho GTPase family members, their GEFs and GAPs, and interaction partners of Rho GTPases have been detected at the Golgi apparatus (**Figure 2**). At the Golgi, Cdc42 interacts with components of the COPI coat, which was originally shown to be important for cellular transformation (Wu et al., 2000). A major function of coatomer is to mediate formation of COPI vesicles for retrograde transport from the Golgi back to the ER (Letourneur et al., 1994). Accordingly, Cdc42 was shown to regulate membrane deformation by coatomer components (Park et al., 2015). However, rather than affecting COPI transport back to the ER, Cdc42 appeared to promote intra-Golgi anterograde transport. Thus, active Cdc42 might introduce a bias toward anterograde versus retrograde COPI trafficking (Park et al., 2015). It should be mentioned that these results were primarily obtained with a fast cycling mutant of Cdc42 (Cdc42-F28L) and that it remains to be shown whether Cdc42 can introduce this traffic bias under physiologic conditions. Notably, the fact that several tumors exhibit secretion of ER chaperones, might be interpreted as a failure of COPI-based retention. Future work should test the possibility whether higher Cdc42 activity at the Golgi is involved in this phenomenon.

An important question is whether Cdc42 is active at the Golgi, which is best answered by live cell imaging. The first detection of active Cdc42 at the Golgi was made possible by FRET-based reporters (Nalbant et al., 2004). However, it remained unclear whether this pool is functionally relevant for canonical Cdc42 related processes such as regulation of the cytoskeleton, cell polarity or cell migration. An early indication that this pool is functionally relevant for these processes was the finding that coatomer-bound (i.e., Golgi localized) Cdc42 stimulates actin assembly, but inhibits recruitment of the molecular motor dynein to the nascent vesicles (Chen et al., 2005). This observation suggests a model where Cdc42 disassociates from the coatomer once the vesicle formation is completed, allowing dynein to bind and transport vesicles. In addition, the Golgi pool of Cdc42 was reported to mediate dynein and microtubule-dependent Golgi positioning in directionally migrating cell (Hehnly et al., 2010). Later, the Golgi matrix protein GM130 was proposed to act as a factor that selectively regulates the Golgi pool of Cdc42 without affecting the plasma membrane (Baschieri et al., 2014). GM130 binds to RasGRF, which was previously shown to inhibit Cdc42 activity (Calvo et al., 2011). Using GM130 to specifically modulate Cdc42 activity at the Golgi, it was demonstrated that this pool acts as a reservoir that supplies the leading edge plasma membrane of directionally migrating cells with active Cdc42 (Baschieri et al., 2014). This was in agreement with previous reports showing that

intra-Golgi trafficking and actin dynamics at the Golgi. The figure highlights function of some of the Rho GTPases at the Golgi.

cytoplasmic vesicles carrying Cdc42 are delivered to the leading edge (Osmani et al., 2010).

Another interesting question is whether the Golgi harbors GAPs and GEFs that would mediate Cdc42 activity at this organelle. A GAP for Cdc42, ARHGAP10, was reported to be recruited to the Golgi by active Arf1 (Dubois et al., 2005). It was revealed that active Arf1 via its interaction with ARHGAP10 controls Cdc42 activity at the Golgi, and in this way regulates Golgi structure and actin cytoskeleton dynamics at the Golgi (Dubois et al., 2005). Earlier report suggested that the GEF Tuba might play a role in modulating Cdc42 activity at the Golgi (Kodani et al., 2009). However, four independent groups have failed to localize Tuba to the Golgi, but rather found it to be confined to cytoplasmic vesicles or the cell surface (Salazar et al., 2003; Kovacs et al., 2006; Baschieri et al., 2014; Bruurs et al., 2018). Very recently, the Golgi matrix protein GCC88 was found to interact with the long form of intersectin-1, a Cdc42 GEF (Makhoul et al., 2019). Intersectin-1 was convincingly localized to the Golgi providing us with an excellent candidate that might mediate local activation of Cdc42. Further supporting a role for intersectin-1 at the Golgi is an earlier report showing that a small molecule (ZCL278) that inhibited Cdc42-intersectin1 interaction disrupted Golgi structure (Friesland et al., 2013). It will be interesting to see whether this drug could be used to test some of the aforementioned effects of Golgi-based Cdc42 functions such as COPI transport or cell transformation.

In addition to Cdc42, active RhoA have also been localized to Golgi (Quassollo et al., 2015). Using FRET-based biosensors, active RhoA was detected at Golgi outposts (GOPs) in neuronal dendrites (Quassollo et al., 2015). Reportedly, the biogenesis of GOPs occurs from dendrite-localized ER exit sites (Horton et al., 2005). However, it remained unclear whether GOPs could emanate through fission from the somatic Golgi. RhoA at the Golgi was activated downstream of lysophosphatidic acid initiating the activation of a cascade involving ROCK, LIMK1 and PKD1, thereby resulting in tubulation of the somatic Golgi and elongation of these tubules into dendrites (Quassollo et al., 2015). RhoA was also involved in activating dynaminmediated fission events in dendrites leading to separation of the Golgi tubules and the formation of GOPs. Whether this is a main pathway for GOP formation has to be tested in the future. Because GOPs are important for dendrite formation and branching, it is conceivable that they play an important role in synaptic integration and plasticity. Thus, potential roles

of Golgi-based signaling of RhoA in these processes merits future investigations.

A very recent example of an active role of RhoA at the Golgi is the identification of a cascade triggered by protease activated receptors at the cell surface that activate a RhoA GEF called GEF-H1. GEF-H1 mediates RhoA activation at the trans-Golgi, which in turn activates PKD, a Ser/Thr kinase of great importance for the biogenesis of post-Golgi carriers. Thereby, RhoA was shown to regulate cargo delivery for localized exocytosis at focal adhesions (Eisler et al., 2018).

More recently, Golgi localization of RhoD and its role in maintaining Golgi homeostasis was reported (Blom et al., 2015). Endogenous RhoD colocalized with the Golgi resident proteins, whereas ectopic expression of constitutively active and inactive RhoD alone or with its binding partner WHAMM (WASP homolog associated with actin, membranes, and microtubules) led to Golgi fragmentation (Blom et al., 2015). Using temperature sensitive VSV-G to monitor trafficking, it was found that RhoD deregulates anterograde vesicular transport from the endoplasmic reticulum (ER) to the plasma membrane. In cells where RhoD activity was perturbed, VSV-G was scattered in vesicular structures positive for GM130 that were sensitive to endoglycosidase-H cleavage, indicating that the VSV-G containing Golgi derived vesicles were not fully functional (Blom et al., 2015). This suggests a role for RhoD in the secretory pathway.

The recently identified RhoBTB-1,-2, and -3 are much larger than classical small GTPases, possess additional domains and are not regulated by the conventional GTPase cycle (Aspenstrom et al., 2007; Ji and Rivero, 2016). In fact, RhoBTB3 – the only RhoBTB family member that was reported to localize to Golgi – functions as an ATPase (Espinosa et al., 2009). RhoBTB3 is anchored at the trans-side of the Golgi, where it functions as an effector for Rab9, a GTPase localized on late endosomes that traffics to the trans-Golgi. Arrival of these Rab9-positive carriers to the Golgi, induces activation of the ATPase activity of RhoBTB3, which is required to remove the coating off these vesicles, thereby preparing them to fuse with the Golgi (Espinosa et al., 2009). Corroborating this, it was further demonstrated that Golgi-residing RhoBTB3 is important for maintaining Golgi architecture since its depletion resulted in Golgi fragmentation (Lu and Pfeffer, 2013). At the Golgi RhoBTB3 is part of a Cul3-RING-E3 ubiquitin ligase complex, which binds Cyclin E and targets its proteasomal degradation (Lu and Pfeffer, 2013). This finding highlights that a Golgilocalized signaling molecule plays a role in the cell cycle by regulating G1-S-phase entry, a phase where Golgi has not been involved previously.

A major function of Rho GTPases is to regulate the cytoskeleton as well as molecular motors. The actin-nucleating Arp2/3 complex was shown to localize to the Golgi and to play a role in its polarization during cell migration (Magdalena et al., 2003). The dynamics of Arp2/3 at the Golgi were later shown to be regulated by the Golgi-pool of Cdc42 (Dubois et al., 2005). Cdc42 was also shown to regulate actin-assembling formin family members FMNL -2 and -3 at the Golgi and to thereby regulate the architecture of this organelle (Kage et al., 2017). Further evidence linking Cdc42 and Golgi architecture via regulation of actin dynamics was provided by identifying the Cdc42 exchange factor ITSN-1 as an interaction partner for the Golgi matrix protein GCC88 (Makhoul et al., 2019). Importantly, this interaction was suggested to play a role in Golgi dispersal that is observed in neurodegeneration.

## RHO GTPase SIGNALING FROM MITOCHONDRIA

The first ever description of mitochondria localized Rho GTPase was provided by Boivin and Beliveau (1995). The authors subjected outer cortical region of kidney from Sprague–Dawley male rats to subcellular fractionation and probed for RhoA, Cdc42 and Rac1 in different subcellular fractions. While all three Rho GTPases were detected in plasma membrane and cytosolic fractions, only Rac1 was detected in mitochondriaenriched fractions (Boivin and Beliveau, 1995). Corroborating this, Velaithan et al. (2011) showed direct physical interaction between mitochondrial Rac1 and Bcl-2 in human cancer cell lines and clinical biopsies from B-cell lymphoma patients. A year later, Rac1 was also detected in mitochondria in alveolar macrophages isolated from asbestosis patients (Osborn-Heaford et al., 2012). This study further revealed that the C-terminal cysteine (Cys-189) residue of Rac1 is required for its mitochondrial import. Subsequent studies have since included neuronal cells in the repertoire of cell types exhibiting mitochondrial Rac1 (Natsvlishvili et al., 2015; Pan et al., 2018).

In addition to describing localization of Rac1, these studies also provide compelling evidence for Rac1 signaling on mitochondria. The direct interplay of Rac1 and Bcl-2 at mitochondria maintained a mild pro-oxidant intracellular milieu through increased intracellular superoxide levels, successively promoting the death inhibitory activity of Bcl-2 (Velaithan et al., 2011; **Figure 3**). Interestingly, inhibition of Rac1-Bcl-2 interaction in lymphoma cells restored death signaling in response to common chemotherapeutic agents (Velaithan et al., 2011). Elevated mitochondrial Rac1 activity in alveolar macrophages via its interaction with cytochrome c, increased oxidative stress and contributed to the development of pulmonary fibrosis (Osborn-Heaford et al., 2012). Furthermore, increased Rac1 signaling was proposed to play important roles in the regulation of neuroplasticity and prevention of apoptosis and autophagy via its association with sigma-1 receptor, inositol 1,4,5-trisphosphate receptor and Bcl-2 at the mitochondrial membrane (Natsvlishvili et al., 2015). In contrast to these findings, interference of Rac1-Bcl-2 complex either by inhibition or siRNA mediated depletion of Rac1 relieved mitochondrial oxidative stress and promoted neuronal survival in a focal cerebral ischemia in vivo in a diabetic rat model and a hyperglycemia-exposed PC-12 cell in vitro model (Pan et al., 2018). Hence, Rac1 signaling at the mitochondria could produce cell-type specific outcomes under different conditions.

Generally, mitochondria have largely been overlooked as sites for Rho GTPase signaling. Whether members of Rho GTPase

family other than Rac1 also localize to mitochondria and might have been missed because of their rapid shuttling in and out of the mitochondria remains a subject for future studies.

# RHO GTPASE SIGNALING FROM NUCLEUS

Because the outer nuclear membrane is part of the ER and it also is a membrane enclosed organelle, we will briefly review evidence for Rho GTPase signaling in and from this location. The C-terminal polybasic region of Rac1 harbors functional nuclear localization signals (K-K-R-K and K-R-K-R) that promote its nuclear translocation (Lanning et al., 2004; Navarro-Lerida et al., 2015). Reportedly, Rac1 also contains two internal nuclear export motifs (Navarro-Lerida et al., 2015). It was revealed that interaction of Rac1 with nucleophosmin-1 mediates its efficient nuclear export (Navarro-Lerida et al., 2015; **Figure 3**). Consequently, wealth of data describe nucleocytoplasmic shuttling of Rac1 and postulate functional implications of nuclear Rac1 signaling (Kraynov et al., 2000; Michaelson et al., 2008; Menard et al., 2014; Navarro-Lerida et al., 2015; Woroniuk et al., 2018). Such a nuclear localization signal is also present in the C-terminal polybasic region of several other Rho GTPase family members (Williams, 2003). Whether other Rho GTPases also shuttle in and out of the nucleus in a manner similar to Rac1 remains to be investigated. Rac1 cycling in and out of the nucleus seemingly depends on the cell cycle, with increased nuclear Rac1 during the late G2 phase (Michaelson et al., 2008). In addition to Rac1, its GEF Tiam2 is also present at the outer nuclear membrane (Woroniuk et al., 2018), providing the basis for Rac1 signaling at the nuclear envelope (**Figure 3**). The question next arises is what functional consequences does Rac1 signaling in nucleus or on its envelope might have. Given its pivotal role in actin dynamics, Rac1 signaling in the nucleus could lead to changes in nuclear shape and position in actin-dependent fashion. This could have consequences for cancer cell invasion since structural changes in shape and size, and deformity of the nucleus are decisive factors for invasion through tight gaps in the extracellular matrix (Friedl et al., 2011). An additional effect of Rac1 signaling was altered nuclear membrane fluidity and order (Navarro-Lerida et al., 2015), which might further be an important factor regulating the ability of the nucleus to deform during invasion. The actin mesh around the nucleus might also play a role in positioning of this organelle, because depletion of Rac1 GEF Tiam2 resulted in a failure to position the nuclei with cellular axis of migration (Woroniuk et al., 2018). Moreover, aggressive tumors display higher nuclear Rac1, a phenomenon that results in increased invasiveness in vitro by potentiating cytoplasmic RhoA signaling (Navarro-Lerida et al., 2015). Alternatively, Rac1 may directly interact with nuclear proteins to induce actin-independent changes in cells. On one hand, active Rac1 directly interacts with STAT3 and regulates its activity by promoting its phosphorylation (Simon et al., 2000). On the other hand, Rac1 interaction with nucleophosmin-1 attenuates Rac1 signaling and inhibits cell spreading (Zoughlami et al., 2013). Moreover, increased Rac1 shuttling into the nucleus accelerates cell division (Michaelson et al., 2008). Finally, a fraction of active monomeric Rac1 segregated in the nucleus from dimeric and inactive Rac1 in the cytoplasm upon induction of DNA damage (Hinde et al., 2014).

Despite lacking a canonical nuclear localization signal, RhoA has been reported to translocate to the nucleus (Baldassare et al., 1997; Dubash et al., 2011; **Figure 3**). When quiescent fibroblasts were stimulated with α-thrombin, RhoA was found to translocate to the nucleus and stimulate enzymatic activity of PLD leading to the production of phosphatidic acid and diacylglycerol (Baldassare et al., 1997). The precise biological consequences of these lipid species in the nucleus remains to be fully understood. Later, Garcia-Mata and co-workers also demonstrated that a pool of RhoA is present in the nucleus with a subset of its GEFs (Net1, Ect2) and GAPs (DLC1, p190 RhoGAP). Notably, RhoA and Net1 activity selectively increased in the nucleus upon DNA damage implicating nuclear Net1/RhoA activity in DNA damage signaling (Dubash et al., 2011).

Another Rho GTPase with potential nuclear effects, but no clear nuclear localization signal is RhoB, which was shown to localize to the nuclear envelope, and to interact with and regulate the transcription factor DB1 (Lebowitz and Prendergast, 1998; Gerald et al., 2013; **Figure 3**). The RhoB-DB1 interaction was important for sprouting and proliferation in primary human blood endothelial cells (angiogenesis) in favor of lymphatic endothelial cells (lymphangiogenesis). Accordingly, RhoB knockout mice exhibited reduced angiogenesis but enhanced lymphangiogenesis in response to wounding (Gerald et al., 2013). Neither the stimulus that triggers RhoB translocation, nor the temporal dynamics of this subcellular pool were determined. Elucidating these details will be important to gain a full understanding of spatiotemporal RhoB signaling.

A recent study has described a biologic role for nuclear Cdc42 (Liu et al., 2018). When cultured under stiff mechanical environment, Cdc42 translocated from cytoplasm to the nucleus in tumor repopulating cells (i.e., cancer cells with stem-like properties) (Liu et al., 2018). The nuclear translocation of Cdc42 elevated expression of Tet2, an epigenetic modifier involved in chromatin methylation. Tet2 expression leads to increased expression of p21 and p27, which induce a G1 phase arrest and thus dormancy. The fact that stiffnessmediated dormancy was observed in vivo suggests that nuclear Cdc42 activity might also be relevant in cancer (Liu et al., 2018). Future work is needed to test how essential Cdc42 is for this process and whether identifying drugs that inhibit nuclear translocation of Cdc42 might be useful for cancer therapeutics.

#### REFERENCES

# CONCLUDING REMARKS

It is becoming increasingly apparent that signals emanating from distinct subcellular pools of Rho GTPases is spatially and temporally regulated to generate diverse physiological outcomes. Owing to the recent advancements in Rho GTPase biosensors and microscopy techniques, our knowledge of the complexity of Rho GTPase signaling in time and space has significantly advanced. Nevertheless, many new and interesting questions are yet to be addressed. For example, our understanding of any potential Rho GTPase signaling originating at the ER, the largest organelle in the cell, remains primitive. ER extends throughout the cytoplasm and forms membrane contact sites with endosomes, Golgi, mitochondria and plasma membrane. Membrane contact sites are known to play well-defined roles in bidirectional transport of molecules as well as signal transmission. Since Rho GTPases are able to signal from endosomes, Golgi, mitochondria and plasma membrane, it is tempting to speculate that they may participate in the biogenesis of membrane contact sites, or use them as passages to change subcellular localization. Such speculations are supported by the observation in yeast that the Rho-like GTPase Gem1 (the homolog of mammalian Miro1) is localized to the ER-mitochondrial contact sites (Kornmann et al., 2011). The molecular machinery that recruits Rho GTPases and their GEFs/GAPs to distinct endomembranes has yet to be thoroughly characterized. Finally, we need to incorporate the diverse Rho GTPase signaling units together to better understand their crosstalk and biological outcome as a whole. This makes it clear that using drugs or tools that globally activate or inhibit Rho GTPases is unlikely to be of great use. Future efforts should focus on identifying tools and methods to specifically modulate subcellular pools.

### AUTHOR CONTRIBUTIONS

SP and HF researched the relevant literature, conceived, and prepared the manuscript.

#### FUNDING

This research in the Farhan lab was supported by funding from the Norwegian Research Council, the Norwegian Cancer Society, and the Anders Jahre Foundation.

following injury by regulating VEZF1-mediated transcription. Nat. Commun. 4:2824. doi: 10.1038/ncomms3824

in Golgi polarity in scratch wound models. Mol. Biol. Cell 14, 670–684. doi: 10.1091/mbc.e02-06-0345

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Phuyal and Farhan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Organelle Inheritance Control of Mitotic Entry and Progression: Implications for Tissue Homeostasis and Disease

Fabiola Mascanzoni, Inmaculada Ayala and Antonino Colanzi\*

Institute of Biochemistry and Cell Biology, National Research Council, Naples, Italy

The Golgi complex (GC), in addition to its well-known role in membrane traffic, is also actively involved in the regulation of mitotic entry and progression. In particular, during the G2 phase of the cell cycle, the Golgi ribbon is unlinked into isolated stacks. Importantly, this ribbon cleavage is required for G2/M transition, indicating that a "Golgi mitotic checkpoint" controls the correct segregation of this organelle. Then, during mitosis, the isolated Golgi stacks are disassembled, and this process is required for spindle formation. Moreover, recent evidence indicates that also proper mitotic segregation of other organelles, such as mitochondria, endosomes, and peroxisomes, is required for correct mitotic progression and/or spindle formation. Collectively, these observations imply that in addition to the control of chromosomes segregation, which is required to preserve the genetic information, the cells actively monitor the disassembly and redistribution of subcellular organelles in mitosis. Here, we provide an overview of the major structural reorganization of the GC and other organelles during G2/M transition and of their regulatory mechanisms, focusing on novel findings that have shed light on the basic processes that link organelle inheritance to mitotic progression and spindle formation, and discussing their implications for tissue homeostasis and diseases.

Keywords: cell cycle, mitosis, golgi complex, organelles, mitotic spindle

# INTRODUCTION

Entry into mitosis requires major cell reorganization to allow the proper inheritance of the genetic material between the daughter cells (Champion et al., 2017). The cells follow a specific and coordinated series of events to complete a successful division cycle. Mitosis is triggered by a complex regulatory circuit that controls the activation of the Cyclin-dependent kinase 1 (CDK1)/CyclinB complex, which is the master regulator of the mitotic onset (Nigg, 2001). Entry into mitosis and its proper completion are under the surveillance of checkpoints, which arrest the progression of the cell cycle if DNA damage or spindle failures are detected (Nigg, 2001). The irreversible commitment to mitotic entry is associated with a rapid and profound reorganization of cell shape. The cells become progressively round, centrosomes separate, the microtubules (MTs) organize the spindle apparatus, chromatin is condensed, and the nuclear envelope is disassembled. The latter event allows the spindle to capture and segregate the chromosomes (Champion et al., 2017).

#### Edited by:

Jaakko Saraste, University of Bergen, Norway

#### Reviewed by:

Nobuhiro Nakamura, Kyoto Sangyo University, Japan Catherine Jackson, UMR7592 Institut Jacques Monod (IJM), France

> \*Correspondence: Antonino Colanzi<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Traffic, a section of the journal Frontiers in Cell and Developmental Biology

> Received: 18 April 2019 Accepted: 04 July 2019 Published: 23 July 2019

#### Citation:

Mascanzoni F, Ayala I and Colanzi A (2019) Organelle Inheritance Control of Mitotic Entry and Progression: Implications for Tissue Homeostasis and Disease. Front. Cell Dev. Biol. 7:133. doi: 10.3389/fcell.2019.00133

Interestingly, it is now evident that also the proper reorganization and segregation of cellular organelles during mitosis is indispensable to ensure a correct cell division. Several studies have demonstrated that perturbations of the redistribution of specific organelles result in profound alterations of the cell division process (Jongsma et al., 2015), with potential consequences on tissue development and homeostasis. For example, epithelia are specialized animal tissues that form protective barriers lining the organs and the body. Each epithelium is characterized by a specific structural organization and cell composition. In response to constant turnover and environmental insults, epithelia maintain homeostasis through a tight balance of cell duplication, differentiation, and death (Tai et al., 2019). Moreover, cell divisions must also follow a predetermined orientation to preserve correct tissue architecture. Thus, errors in the control of the number and orientation of cell divisions can have detrimental consequences, compromising tissue development and/or function, and potentially leading to tumor progression (Ragkousi and Gibson, 2014).

Here, we review new findings related to the mechanisms underlying the mitotic segregation of subcellular organelles, and discuss how they impact on the proper mitotic progression, with an emphasis on those involved in the coordination of Golgi complex (GC) inheritance with the cell cycle.

#### PREPARATION OF CELL ENTRY INTO MITOSIS

The primary event of cell division is the segregation of sister chromatids between the daughter cells. This essential task is achieved by the spindle apparatus, which is a very complex system that is organized by the centrosomes and composed of MTs and hundreds of regulatory proteins (Heald and Khodjakov, 2015). The recruitment of the γ-Tubulin Ring Complex (γ-TuRC) to the pericentrosomal region stabilizes MT minus ends and creates anchor points associated to the microtubule-organizing center (MTOC). The γ-TuRC act as a template for the polymerization of α/β tubulin heterodimers into MTs, which then undergo rapid growth and catastrophe phases (Petry and Vale, 2015; Petry, 2016). The centrosomes organize three types of MT fibers: kinetochore, polar, and astral MTs (**Figure 1**). The kinetochore MTs are directed toward the kinetochores and are responsible for the traction forces required to separate the sister chromatids. The polar MTs form antiparallel fibers that exert sliding forces mediated by kinesins, and thus, accomplish both separation and elongation of the spindle poles in mitosis. The astral MTs associate with specific domains of the cell cortex (**Figure 1**; Petry, 2016).

The assembly of the spindle is regulated by phosphorylation events. The kinase Aurora A associates with the centrosomes and is a crucial regulator of mitotic entry and centrosome maturation, which consists of the recruitment of proteins involved in MT nucleation and anchoring (Marumoto et al., 2002). Aurora B is a fundamental player in chromosome segregation because it controls the chromosome-microtubule attachment and sister chromatid cohesion (Hindriksen et al., 2017). Pololike kinase 1 (PLK1) is involved in spindle maintenance by regulating MT dynamics through the phosphorylation of Microtubule-associated proteins (MAPs) and kinesin motors (**Figure 1**; Bruinsma et al., 2012). The concerted lengthening and shortening of the kinetochore, and of the polar spindle MTs, control the proper alignment of chromosomes at the spindle midzone (Heald and Khodjakov, 2015; Petry, 2016). The correct attachment of the chromosomes to the mitotic spindle is monitored by a spindle assembly checkpoint (SAC), which is activated in the case of incorrectly attached kinetochores, resulting in a delay of anaphase onset. The impairment of this checkpoint can lead to aneuploidy, aging and cancer progression (Lara-Gonzalez et al., 2012).

An additional crucial event during cell division is the orientation of the spindle apparatus, as it determines the proper orientation of the cell division axis (Lu and Johnston, 2013). After its formation, the spindle apparatus is subjected to a rotation process. The astral MTs associate with specific cues at the cell cortex, from where they are subjected to pulling forces (**Figure 1**). Thus, complex signaling pathways coordinate MTmediated forces that are originated at the cell cortex and are applied on the spindle MTs (Lu and Johnston, 2013).

A critical preparatory step for spindle formation is the progressive cell reshaping from a flat to a spherical geometry that occurs during the G2/M transition (Champion et al., 2017). The rounding process becomes detectable during late prophase, but the preparatory steps begin during G2, when the focal adhesions (FA) undergo selective disassembly. This process is essential to achieve the progressive retraction of the cell margins and the formation of the actomyosin cortex, which are necessary for the cell rounding in metaphase (Cadart et al., 2014; Champion et al., 2017). Cell rounding is necessary to create a symmetric cell organization that allows the kinetochore MTs to capture the chromosomes. In fact, a reduced rounding can cause the scattering of chromosomes, which decreases the probability of being captured by the kinetochore MTs. Besides, an asymmetric cell geometry can induce spindle deformations, resulting in the splitting of the spindle poles and formation of acentriolar spindles, thus increasing the likelihood of multipolar cell division and aneuploidy. Therefore, alterations of mitotic rounding can affect spindle morphology, chromosome segregation, and timely mitotic progression (Champion et al., 2017). In addition, the geometry of the cell during mitosis also influences the orientation of the spindle and, as a consequence, the direction of the cell division axis, and thus tissue morphogenesis and differentiation (Morin and Bellaiche, 2011). Collectively, these findings emphasize that correct cell geometry and the controlled redistribution of cell components are crucial for the daughter cells fate, and errors in the process can lead to the development of diseases (Lancaster et al., 2013).

Entry into mitosis is also accompanied by an extensive reorganization of subcellular organelles, which undergo stereotyped reorganization of the structure and localization (Champion et al., 2017). For instance, during G2 the centrosomes move from the perinuclear area to the center

of the nucleus in order to be disengaged and separated (Sutterlin and Colanzi, 2010). The reorganization of organelle morphology can range from the full disassembly of the GC to the subtle modifications of the endosomes (Lowe and Barr, 2007; Jongsma et al., 2015). In the next paragraphs, we will summarize the current knowledge about the mitotic fate of the intracellular organelles, with a focus on the GC, and referring to several recent reviews for more mechanistic details.

# THE GOLGI COMPLEX

# Structural Reorganization of the Golgi Complex During Mitosis

The GC has a pivotal role in the secretory pathway, as it is involved in the modification and sorting of cargoes (Wei and Seemann, 2010). In mammalian cells, the GC is characterized by a ribbon structure, which is composed of several polarized stacks of cisternae that are laterally connected by tubules (Lowe, 2011). The structure of the GC is maintained by the Golgi Reassembly Stacking Protein of 55 and 65 kDa (GRASP55 and GRASP65) (Zhang and Wang, 2015) and by the members of the golgin family (Gillingham and Munro, 2016). All these proteins act as membrane tethers and concur in the stacking of the cisternae and in directing the formation of the membranous tubules connecting the stacks (Xiang and Wang, 2010; Witkos and Lowe, 2015). The perinuclear position of the GC is maintained through the association with MTs (Maia et al., 2013).

During late G2, the Golgi ribbon is divided into individual stacks (unlinking) (**Figures 2A,B**). The pro-ribbon role of the GRASPs is inhibited by phosphorylation. The basic regulatory elements have been identified, and they include the kinase PKD that leads to a RAF1/MEK1/ERK1-mediated phosphorylation of GRASP55 (Valente and Colanzi, 2015). In addition, a major inducer of ribbon unlinking is phosphorylation of GRASP65 by JNK2 and PLK1 (Acharya et al., 1998; Colanzi et al., 2000; Feinstein and Linstedt, 2007; Sengupta and Linstedt, 2010; Tang and Wang, 2013; Rabouille and Linstedt, 2016). Moreover, Golgi unlinking requires the fissioninducing protein BARS to cleave the tubules connecting the stacks (Hidalgo Carcedo et al., 2004; Colanzi et al., 2007). For more mechanistic details the reader is referred to several reviews (Lowe and Barr, 2007; Corda et al., 2012; Tang and Wang, 2013; Ayala and Colanzi, 2017; Wei and Seemann, 2017). During prophase (**Figure 2C**), the activation of CDK1 leads to the phosphorylation of additional sites on golgins and GRASPs, resulting in complete inhibition of the membrane tethering processes. Furthermore, CDK1 also induces the phosphorylation of adaptor proteins involved in membrane fusion, such as p47/VCIP135 and p37, causing inhibition of membrane fusion events (Uchiyama et al., 2003; Kaneko et al., 2010; Tang and Wang, 2013; Totsukawa et al., 2013). An additional contribution to the inhibition of the membrane fusion machineries is provided by the

centrosome; GC, Golgi complex; NU, nucleus. Adapted with permission from Ayala and Colanzi (2017).

HACE1-mediated monoubiquitination of the SNARE protein syntaxin 5 (Huang et al., 2016). As a result, the Golgi membranes are consumed by an extensive vesiculation during metaphase, when they become dispersed into vesicular/tubular clusters, with some intermediate compartment (IC) and Golgi proteins redistributed into the Endoplasmic Reticulum (ER) (**Figure 2D**; Saraste and Marie, 2018). Then, during telophase and cytokinesis, the dispersed Golgi proteins and membranes are gradually reassembled into a GC in each of the daughter cells (**Figure 2E**; Shorter and Warren, 2002; Altan-Bonnet et al., 2004; Colanzi and Corda, 2007).

Importantly, the disassembly of the GC is also a requirement for mitotic entry. Indeed, blocking the unlinking step induces a potent G2 block of the cell cycle, pointing out that a mitotic "Golgi checkpoint" oversees the correct premitotic cleavage of the GC (Sutterlin et al., 2002; Hidalgo Carcedo et al., 2004). Although the existence of a mechanism that controls the correct partitioning of the organelle could be surprising, recent findings are starting to reveal the general framework, and the evidence suggests that a physical/functional connection of the GC with the centrosomes and the MT network is at the basis of this novel checkpoint.

## Role of the Centrosome in the Localization and Structure of the Golgi Complex in Interphase

It is already known that the structure and localization of the GC can be modulated by the centrosome through multiple mechanisms (Sutterlin and Colanzi, 2010; Rios, 2014). The centrosome is composed of two centrioles enclosed by pericentriolar material (PCM), which consists of a thick shell of multiprotein complexes. In most cell types, the centrosome represents the major MTOC, and is involved in the formation of radial MT fibers (Sanchez and Feldman, 2017; Muroyama and Lechler, 2017). The template for MT nucleation is the γ-TuRC, which is recruited to the centrosome. The centrosome is positioned at the cell center, close to the nucleus. Following a polarization stimulus, it is reoriented in the direction of the leading edge of the cell (Pouthas et al., 2008).

The centrosomal MTs form radial fibers that guide the positioning of the Golgi membranes toward the cell center thanks to dynein, which is a minus end-directed motor complex (Rios, 2014) that is recruited at the GC by Golgin160 (Yadav et al., 2012). Also, the actin cytoskeleton contributes to the maintenance of the ribbon, as it forms tracks for actin-based motors (Valderrama et al., 1998). Besides, a subset of MTs is nucleated at the GC (Chabin-Brion et al., 2001). Nucleation of MTs from the GC is driven by a multiprotein complex that is organized by the scaffold protein AKAP-450, which is recruited at the cis-Golgi by GM130 (Zhu and Kaverina, 2013). The newly nucleated MTs are then stabilized and anchored at the trans-Golgi network (TGN) through the MT binding proteins CLIP-associated proteins 1 and 2 (CLASP1/2), due to their ability to stabilize MT plus ends by suppressing MT catastrophes (Efimov et al., 2007; Wu et al., 2016). The simultaneous knockdown of both CLASPs reduces MT nucleation at the GC and causes mitotic defects, such as multipolar spindle formation, and consequent cytokinesis failure (**Table 1**; Mimori-Kiyosue et al., 2005; Lansbergen et al., 2006; Pereira et al., 2006).

The GC-based MT nucleation is crucial not only for the structural integrity of the GC, but also for the formation of asymmetric MTs that are essential for the orientation of the GC toward the leading edge during migration (Vinogradova et al., 2009; Kaverina and Straube, 2011; Wu and Akhmanova, 2017), and for the polarized delivery of cargoes (Miller et al., 2009; Sanders and Kaverina, 2015). Yet, the significance of the GC-centrosome proximity and of the ribbon organization are not completely understood. In particular, the knockdown of the golgin GMAP210 or Golgin160 induces the unlinking into separated stacks, which are still able to transport cargoes to the cell surface but that become unable to direct the secretion toward specific domains of the plasma membrane (PM) at the leading edge. As a result, the directional persistence of cell migration is reduced (Yadav et al., 2009). In agreement with these observations, depletion of GM130 using siRNA in UO2S or HeLa cells results in ribbon unlinking and reduced efficiency of cell migration (Kodani and Sutterlin, 2008). Additionally, experiments based on the expression of various N-terminal fragments of AKAP-450 led to the conclusion that the proximity of the GC to the centrosome, but not the presence of an intact ribbon, is the crucial factor for optimal directional cell migration (Hurtado et al., 2011). However, experiments based on RPE1 cells in which GM130 was knocked out led to the conclusion that a close association of the GC with the centrosome is not required for cell migration or protein transport (Tormanen et al., 2019). Therefore, more investigations are needed to better understand the functional consequences of perturbations of the GC structure, or of its proximity to the centrosome.

# Physical and Functional Relationships Between the Golgi Complex and the Centrosome During Mitosis

The structural reorganization of the GC during the cell cycle appears to be coordinated with those of the centrosome (Sutterlin and Colanzi, 2010). The centrosomes are duplicated during S-phase; then, during G2, they are pulled apart, in coincidence with the severing of the Golgi ribbon (**Figure 2B**; Persico et al., 2010). Thus, the GC is segregated into two groups of stacks, each of which is localized in proximity to a separated centrosome (**Figure 2C**). Furthermore, during this phase, the membranes of the IC remain closely associated with the centrosomes and become detached from the bulk of the GC, suggesting that the IC maintains its identity during mitosis and provides an intermediate station for Golgi dispersal (Marie et al., 2012; Saraste and Marie, 2018). In concomitance with these events, the composition and size of the PCM material are profoundly modified, involving the recruitment of other components, like Cep192/SPD-2, PCNT/PLP, and Cep215/Cnn, in a process that is defined as "centrosome maturation"(Joukov et al., 2014). The "mature" centrosomes reach their final position and orientation in metaphase, when they direct MT nucleation for the formation of the spindle, which is fundamental for correct segregation of the chromosomes into the daughter cells (Meraldi and Nigg, 2002). Defects in assembly and duplication of the centrosomes, and the consequent problems in MT nucleation, are the primary cause of the formation of aberrant spindles. In support of a functional GC-centrosome relationship, the G2-specific Golgi ribbon unlinking acts as a controller of the centrosomal recruitment of Aurora A (Persico et al., 2010), which is a major regulator of G2/M transition, centrosome maturation, and spindle formation (**Figures 2B,C**; Marumoto et al., 2002). In particular, Barretta et al. (2016) demonstrated that upon unlinking, Src is activated at the GC, then Src interacts with Aurora A and phosphorylates the residue Y148, increasing the kinase activity of Aurora A, which then is recruited to the centrosomes to induce their maturation (**Figure 2a**). Aurora A is a pivotal switch of spindle formation, as its inhibition or ablation causes formation of multipolar and/or fragmented spindles (Marumoto et al., 2002; Hoar et al., 2007; Malumbres and Perez de Castro, 2014). An additional line of evidence of the correlation between mitotic Golgi disassembly and centrosomebased functions, has been revealed thanks to an assay designed to prevent the disassembly of the Golgi stacks during mitosis through the controlled formation of 3,3<sup>0</sup> -diaminobenzidine (DAB) polymers in the Golgi lumen (Guizzunti and Seemann, 2016). Cells containing DAB polymers in the Golgi stacks entered into mitosis normally, but they arrested in metaphase with intact Golgi clusters associated with monopolar spindles, which caused SAC activation. Artificial disassembly of the GC relieved this block, suggesting that the disassembly of the Golgi stacks is required for progression through mitosis (Guizzunti and Seemann, 2016; Wei and Seemann, 2017).

In addition, several reports have shown a direct role of Golgi matrix proteins in assisting spindle formation. For example, the N-terminal domain of GM130 includes a nuclear localization signal (NLS) that has been shown to be essential for proper spindle assembly (Wei et al., 2015). During interphase, the NLS is masked by the interaction with the Golgi matrix protein p115. CDK1-mediated phosphorylation of GM130 dissociates p115 from GM130, and this triggers a crucial pathway of mitotic disassembly of the Golgi stacks (Nakamura et al., 1997; Nakamura, 2010; Wei et al., 2015), as it unmasks the NLS, which

TABLE 1 | Golgi located proteins involved in spindle formation.

becomes able to bind and sequester at the Golgi the nuclear pore component importin-α. Of note, before mitosis onset, importinα is bound to the spindle assembly factor TPX2 (**Figure 2b**), keeping this protein inactive. Accordingly, as a consequence of the binding of importin-α to GM130, TPX2 becomes free to interact with Aurora A, resulting in the increase of its kinase activity and local stimulation of MT nucleation required for the assembly of the spindle. Furthermore, once the spindle fibers are formed, they become "stabilized" by GM130 (**Figure 2c**), which directly binds and bundles MTs, thus linking Golgi membranes to the spindle (**Figures 2C,D**; Wei et al., 2015; Wei and Seemann, 2017). Probably correlated to this function, depletion of GM130 causes the formation of over duplicated centrosomes and multipolar spindles during mitosis (**Table 1**), resulting in metaphase arrest and cell death (Kodani and Sutterlin, 2008).

To further support the functional connection of the GC with the spindle, several reports have shown evidence of Golgi-associated proteins that influence spindle formation and mitotic progression. In this regard, the GM130 interactor p115 becomes associated with the mitotic spindle throughout mitosis. A specific armadillo-like fold of the N-terminus of p115 was responsible for its interaction with γ-tubulin and centrosomal targeting. Strikingly, p115 depletion causes spindle abnormalities, chromosome defects, and cytokinesis failure (**Table 1**; Radulescu et al., 2011). Also, the other GM130 interactor, GRASP65, has been endowed with mitosis-specific roles. Indeed, GRASP65 depleted cells show multiple disorganized and non-functional spindle asters (**Table 1**), indicating that GRASP65 regulates MT dynamics during entry into mitosis (Sutterlin et al., 2005). The list of Golgi-associated proteins with roles in spindle formation is not limited to the GM130-based protein complex. For instance, depletion of the Golgi-associated phosphoinositide phosphatase SAC1 causes perturbations of Golgi architecture and spindle abnormalities (**Table 1**; Liu et al., 2008). In addition, tankyrase-1 is an ADP-ribosyltransferase that is associated with the Golgi in interphase, and relocates to the spindle poles during mitosis. Its depletion causes mitotic arrest with abnormal chromosome segregation, bipolar spindle formation, and failure of telomere separation (**Table 1**; Chang et al., 2005). Also, the tankyrase-1 substrate Miki translocates from the GC to the centrosomes during the late G2/M phase. Depletion of Miki induces a pseudometaphase state that leads to the formation of multinucleated cells (**Table 1**; Ozaki et al., 2012). Another Golgiassociated protein implicated in cell cycle control is the Rad50 interacting protein RINT-1, whose depletion causes partial Golgi fragmentation, centrosome amplification during interphase, and increased formation of multiple spindle poles that culminate in frequent chromosome missegregation (**Table 1**; Lin et al., 2007).

More in general, the spindle recruits and directs the inheritance of Golgi matrix proteins that are involved in the formation of the Golgi ribbon, while a minimal set of proteins and membranes sufficient to reassemble functional Golgi stacks are inherited independently of the spindle (Wei and Seemann, 2009). It could be speculated that the Golgi matrix proteins recruited by the spindle are not simple passengers, but acquire different mitosis-specific functions. In support of this possibility, during mitosis, the small GTPase Arf1 becomes inactive and dissociates from the Golgi membranes (Altan-Bonnet et al., 2003), and this correlates with the dispersal of several peripheral Golgi proteins. If Arf1 is artificially kept active, Golgi membranes do not fragment, and the peripheral proteins remain associated with the GC throughout mitosis. These cells enter mitosis, but exhibit gross defects in chromosome segregation and cytokinetic furrow formation, resulting in multinucleation (Altan-Bonnet et al., 2003).

Thus, there is a substantial amount of evidence to conclude that an active functional interplay between the GC and the centrosome is crucial for spindle formation and, hence, for accurate segregation of the genetic material.

#### STRUCTURAL REORGANIZATION OF OTHER ORGANELLES DURING MITOSIS

#### Endoplasmic Reticulum

The ER is a large continuous membranous organelle that is responsible for the synthesis of the majority of the integral membrane proteins and lipids. It comprises three different

domains: the smooth ER (SER), the rough ER (RER), and the nuclear envelope. The ER constitutes a vast network of cisternae and tubules spread across the cytosol, and establishes contacts with several subcellular compartments (Phillips and Voeltz, 2016).

The ER undergoes marked structural modifications during mitosis. Specifically, the cisternae are transformed into mixed populations of tubules, the extent of which varies among cell lines (Puhka et al., 2007, 2012). In addition, during late prophase, the nuclear envelope is disassembled and its membranes reabsorbed into the bulk of the ER to expose the chromatin to the spindle apparatus. In prometaphase, the ER is split into two large pools of membranes that maintain continuity throughout mitosis (Lu et al., 2009; Schwarz and Blower, 2016). During anaphase and telophase, after chromosomal segregation, the nuclear envelope reassembles, and this marks the beginning of the reorganization of the ER compartment, although the underlying molecular mechanisms are poorly understood (Schwarz and Blower, 2016).

The ER is also a major hub for intracellular organization and signaling (Jongsma et al., 2015; Schwarz and Blower, 2016). Indeed, throughout interphase it forms multiple contact sites (CS) with the PM, endosomes, GC, and cytoskeleton (Phillips and Voeltz, 2016). Membrane CSs have crucial functions in interorganelle signaling and lipid transfer. Considering its role as a major organizing compartment, it is likely that the inheritance of the ER is regulated by yet unknown control mechanisms. Recently, it has been shown that the ER–PM CSs undergo significant changes in morphology and function during mitosis (Yu et al., 2019). One of the major functions of CSs is to control Ca2<sup>+</sup> signaling through the store-operated Ca2<sup>+</sup> entry (SOCE), which depends on the integrity of ER–PM CS to allow contact between the proteins STIM1 and Orai1 (Yu et al., 2019). During mitosis, the density of these specific CSs is decreased. Also, the average distance between the PM and the closest ER in mitosis is increased. Therefore, the down-regulation of ER–PM junctions in mitosis induces SOCE inhibition by preventing the interaction of STIM1 with Orai1. This inhibition could affect not only Ca2<sup>+</sup> signaling but also lipid metabolism and membrane structure (Yu et al., 2019).

#### Endosomes

The membranous endocytic system mediates the traffic of lipids, proteins, and other molecules among various intracellular locations. These features have an essential impact on signal transduction and nutrient acquisition. Even if the various cellular functions of the endolysosomal system are extensively investigated, the mechanisms of endosome inheritance are marginally known. The current view is that endosomes and lysosomes remain intact during mitosis, and that during cytokinesis these organelles accumulate in the proximity of the MTOC (Bergeland et al., 2001; Jongsma et al., 2015). Despite this limited knowledge, an interesting aspect is that during prophase, MT-dependent motors induce the clustering of Rab11 positive endosomes around the centrosomes (**Figures 2C,c**). This localization is important to prevent their transport to the PM and to bring MT-nucleating proteins to the centrosome (Hehnly and Doxsey, 2014). For this reason, Rab11 relocation to the centrosome is necessary for the formation of a functional and properly oriented mitotic spindle. The clustered Rab11 endosomes are then segregated by the mitotic spindles between the daughter cells (**Figures 2D,E**; Hehnly and Doxsey, 2014). Moreover, in line with the proposed role of membrane reservoir in mitotic cells, during anaphase, the recycling endosomes are transported toward the cleavage furrow (**Figure 2E**; Bergeland et al., 2001), where they are believed to provide the membranes required to complete the cytokinesis (Simon and Prekeris, 2008).

## Peroxisomes