# EMERGING FUNCTIONS OF SEPTINS

EDITED BY: Manoj B. Menon and Matthias Gaestel PUBLISHED IN: Frontiers in Cell and Developmental Biology

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ISSN 1664-8714 ISBN 978-2-88945-287-3 DOI 10.3389/978-2-88945-287-3

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# **EMERGING FUNCTIONS OF SEPTINS**

Topic Editors:

**Manoj B. Menon**, Hannover Medical School, Germany **Matthias Gaestel**, Hannover Medical School, Germany

Confocal immunofluorescent image of fibroblasts co-stained for SEPT7 (red), tubulin (green) and actin (blue), depicting the septin, microtubule and the microfilament cytoskeletal networks respectively.

Image by Manoj B. Menon and Alexey Kotlyarov

Together with the microfilament, microtubule and intermediate-filament networks, septins constitute an integral part of the eukaryotic cytoskeleton. Historically identified as proteins critical for septum formation in the budding yeast *Saccharomyces cerevisiae*, septin family GTPases are expressed and participate in the process of cytokinesis in most eukaryotes except higher plants. More than a dozen septin genes in mammals, together with various splice variants displaying tissue-specific expression patterns and flexible hetero-polymeric higher-order assembly achieve an unfathomable complexity superior to the other cytoskeletal components. Even though the initial studies in the septin field was restricted to their evolutionarily conserved role in cell division, strong expression of septins in the non-dividing cells of the brain generated great interest in understanding their role in neuronal morphogenesis and other aspects of cellular function. On one hand, recent developments indicate complex non-canonical roles for septins in diverse processes ranging from neuronal development to immune response and calcium signaling. On the other hand several lines of data including those from knockout models question the universal role for septins in animal cell cytokinesis. Mammalian hematopoietic cells seem to proliferate and efficiently undergo cytokinesis in the absence of pivotal septin proteins in a context-dependent manner. The lack of septin-dependence of hematopoiesis also opens the possibility of safely targeting septin-dependent cytokinesis for solid-tumor therapy. Thus the septin field is perfectly poised with novel roles for septins being discovered and the basic understanding on septin assembly and its canonical functions constantly revisited.

The objective of this research topic was to provide an exclusive platform for discussing these rapid advances in the septin field. With a mixture of reviews and research articles encompassing diverse areas of septin research, ranging from the humble yeast model to human cancer, this ebook will be an interesting reading material for both experts as well as new comers to the septin field.

**Citation:** Menon, M. B., Gaestel, M., eds. (2017). Emerging Functions of Septins. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-287-3

# Table of Contents

#### **Chapter 1: Focus on yeast and fungal septins**

Molly McQuilken, Maximilian S. Jentzsch, Amitabh Verma, Shalin B. Mehta, Rudolf Oldenbourg and Amy S. Gladfelter

*40 Septins Focus Cellular Growth for Host Infection by Pathogenic Fungi* Michelle Momany and Nicholas J. Talbot

#### **Chapter 2: Septin functions in higher eukaryotes**

#### **Chapter 3: Septins in health and disease**

# Editorial: Emerging Functions of Septins

Manoj B. Menon\* and Matthias Gaestel\*

Institute for Cell Biochemistry, Hannover Medical School, Hannover, Germany

Keywords: septin, cytokinesis, cytoskeleton, SEPT, GTPase, signaling

**Editorial on the Research Topic**

#### **Emerging Functions of Septins**

Septins are a family of conserved cytoskeletal GTPases with unique heteropolymerization properties and diverse physiological functions (reviewed in Mostowy and Cossart, 2012). Originally discovered in the budding yeast Saccharomyces cerevisiae as factors crucial for the mother-bud separation, septin genes were later identified in almost all eukaryotes except higher plants. Initial studies on septins were limited to their functions in yeast, with a major focus on cell division. However, much information has been gathered about mammalian septins in the present century and septins are gaining importance as a distinct fourth component of the mammalian cytoskeleton. The presence of multiple septin genes with possibly redundant roles and the lack of potent and specific septin inhibitors have hindered functional characterization of the septin cytoskeleton. These difficulties have been effectively tackled by the widespread use of RNAi technologies and at least 10 out of the 13 mouse septin genes have been successfully knocked out, aiding in the identification of several general and isoform-specific physiological functions of septins. This Frontiers Research topic on the emerging functions of septins compiles contributions from the diverse field of septin research dealing with complex issues from yeast cell division to human cancer, from cellular morphogenesis to protein stability and from plant pathogens to human infections.

Historically, the name "septin" originated from their role in "septa" formation in the budding yeast. Septins participate in crucial cell cycle phases in S. cerevisiae and the mini-review from Glomb and Gronemeyer summarizes our current understanding on septin functions in this model organism, with a special focus on the subcellular organization and septin assembly. There is a long-standing view that septins could act as signaling scaffolds facilitating localized regulatory events. Perez et al. discuss septins as kinase scaffolds explaining the relevance of these septin associated kinases in the regulation of different cell cycle phases in the budding yeast. They describe the salient features of septin-dependent cell-cycle checkpoints, which determine the precise time of mitotic entry, exit, and cytokinesis. In addition to the role of septin scaffolds in coupling cell morphology to cell cycle decisions, the different signaling events regulating septin assembly are also discussed here. The complexity in the regulatory role for septins in yeast budding is further addressed by McQuilken et al. by monitoring septin reorganization at the bud neck using advanced polarized fluorescence microscopy. By the systematic analysis of multiple mutants lacking septin interacting proteins, they further establish a complex role for kinases in septin remodeling at the bud-neck. While the above mentioned articles focus on the model organism S. cerevisiae, Momany and Talbot present a different view on how septins are essential for fungal pathogenesis. Taking examples from human and plant pathogenic fungi they discuss the importance of septin-driven morphological changes as a prerequisite for focusing invasive force in an efficient and directed manner to facilitate pathogenesis.

#### Edited and reviewed by:

Ana Cuenda, Consejo Superior de Investigaciones Científicas (CSIC), Spain

#### \*Correspondence:

Manoj B. Menon<EMAIL_ADDRESS>Matthias Gaestel<EMAIL_ADDRESS>
#### Specialty section:

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

> Received: 14 July 2017 Accepted: 04 August 2017 Published: 24 August 2017

#### Citation:

Menon MB and Gaestel M (2017) Editorial: Emerging Functions of Septins. Front. Cell Dev. Biol. 5:73. doi: 10.3389/fcell.2017.00073

Septins associate with membranes and can facilitate selective diffusion of components, a property of septin family contributing to processes including neuronal morphogenesis, ciliogenesis, and sperm motility. In a thought-provoking mini review article, Palander et al. discuss the current understanding and possible functions of septins in the biogenesis of cilia and sperm flagella. The research article by Kaplan et al. addresses the functional redundancy of SEPT2 family members in neuronal morphogenesis, concluding that SEPT2, SEPT4, and not SEPT1 can compensate for the loss of SEPT5 in dendritic branching. In the context of membrane association of septins, the mini review by Song et al. deals with the evidences which indicates a role for septins in endo-lysosomal membrane traffic. They discuss the role of septins in vesicular trafficking and autophagy, as indicated by multiple septin-interactors with essential roles in these processes. In a completely different approach, Neubauer and Zieger summarize the current status of the mammalian septin interactome with important additional insights into their previous findings on clinically relevant septin interactors of human platelets and endothelial cells. Septins are described here as key mediators of platelet degranulation and, accordingly, the authors also deliberate on the clinical relevance of the SEPT5 gene locus in bleeding disorders.

Since septins are crucial player in multiple stages of cell division and septin gene translocations and fusions were detected in leukemia, septins have been implicated in cancer development (Cerveira et al., 2011). While a direct functional role for septin in tumorigenesis has not been established yet, there are several studies linking septins to diverse forms of human cancer, such as the use of SEPT9 gene methylation as a biomarker for colon cancer (Warren et al., 2011). In a very unique contribution on this subject, Poüs et al. look at the cancer related functions of septins through the prism of their subcellular localization. This alternate view of localization-dependent septin functions in cancer may ignite the interest of researchers from diverse fields to investigate septin functions in tumorigenesis. This article is complemented by a thought-provoking review article of Angelis and Spiliotis who provide a comprehensive view of septin mutations in human cancers. By an approach coupling large scale sequence data analysis and basic septin biochemistry, the authors convince the readers that despite the complexity associated with multiple septin genes and diverse cancers, there are recurring mutations on conserved amino acids across the septin family. Interesting observations on the possible effects of these mutations on septin oligomerization and function are described. Being the most investigated septin gene in the field of cancer, the authors also give due importance to SEPT9, and separately discuss the mutations of the unique N-terminal domain of SEPT9.

The immune-related functions of septins are also often linked to their membrane association and interplay with the cortical actin cytoskeleton. Despite normal development of hematopoietic lineages on pan-septin depletion in the Sept7 knockout mouse model (Menon et al., 2014), septins regulate lymphocyte migration (Tooley et al., 2009) and associate with macrophage phagosomes (Huang et al., 2008), indicating a clear participation in the immune response. A significant role for septins in host cell entry and invasiveness of intracellular bacterial pathogens in non-phagocytic cells was one of the more recent discoveries in the field (Mostowy et al., 2010). In their mini review Torraca and Mostowy discuss this role of septins in bacterial infection together with its interplay with the autophagy pathway. The diverse methods employed by bacterial pathogens to take advantage of the host cytoskeleton to gain entry into cells and the counter mechanisms in place are nicely described by the authors here. In the context of bacterial pathogenesis, it was recently shown that the intracellular longevity of Clostridium botulinum neurotoxin Bont/A arise from their association with septins (Vagin et al., 2014). Vagin and Beenhouwer discuss this concept in their mini review dealing with the role of septins as regulators of protein stability. They broadly look into the role played by septins in regulating the stability of pathogenic toxins as well as other signaling molecules, which could be relevant to cancer as well as immunity.

One of the latest discovered functions of septins concerns its involvement in the store operated calcium entry (SOCE). While an siRNA screen identified SEPT2 family members as mediators of calcium signaling in HeLa cells (Sharma et al., 2013), T cell calcium signaling was not affected in a conditional Sept7 knockout mouse model (Mujal et al., 2016). Studies in the Drosophila model also suggests an isoform specific role for septins in regulating SOCE (Deb et al., 2016). A perspective article by Deb and Hasan discuss the intricacies associated with these findings and their possible relevance to mammalian neuronal function.

Our understanding on the canonical functions of septins in cell division is constantly being revisited. In addition, the foot print is spreading far and wide as more and more physiologically relevant functions emerge for the septin family proteins. Collectively, the articles in this research topic including original research, perspectives and reviews on diverse aspects of septin research comprehensively summarizes the advances in the field. The contributions also provide ideas of promising directions for future investigations.

### AUTHOR CONTRIBUTIONS

Both authors equally contributed to the drafting and revision of the editorial and approved it for publication.

## FUNDING

Supported by the Deutsche Forschungsgemeinschaft and the LOM program of the Hannover Medical School.

# ACKNOWLEDGMENTS

We thank all contributing authors and reviewers for their support to the Research Topic.

# REFERENCES

**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 © 2017 Menon and Gaestel. 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) or licensor 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.

# Septin Organization and Functions in Budding Yeast

#### Oliver Glomb and Thomas Gronemeyer\*

*Department of Molecular Genetics and Cell Biology, Ulm University, Ulm, Germany*

The septins are a conserved family of GTP-binding proteins present in all eukaryotic cells except plants. They were originally discovered in the baker's yeast *Saccharomyces cerevisiae* that serves until today as an important model organism for septin research. In yeast, the septins assemble into a highly ordered array of filaments at the mother bud neck. The septins are regulators of spatial compartmentalization in yeast and act as key players in cytokinesis. This minireview summarizes the recent findings about structural features and cell biology of the yeast septins.

Keywords: septins, budding yeast, GTPase, crystal structure, cytokinesis

#### *Edited by:*

*Manoj B. Menon, Hannover Medical School, Germany*

#### *Reviewed by:*

*Ravi Manjithaya, Jawaharlal Nehru Centre for Advanced Scientific Research, India Markus Proft, Instituto de Biomedicina de Valencia (CSIC), Spain*

> *\*Correspondence: Thomas Gronemeyer<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 31 August 2016 Accepted: 19 October 2016 Published: 03 November 2016*

#### *Citation:*

*Glomb O and Gronemeyer T (2016) Septin Organization and Functions in Budding Yeast. Front. Cell Dev. Biol. 4:123. doi: 10.3389/fcell.2016.00123* The classical Hartwell screen revealed genes involved in cell cycle regulation of the budding yeast Saccharomyces cerevisiae (Hartwell, 1971). Beside many others, the screen showed mutations in the four genes CDC3, CDC10, CDC11, and CDC12 (**c**ell **d**ivision **c**ycle). These were later classified as members of the septins, a new family of cytoskeletal proteins in the budding yeast (Sanders and Field, 1994; Pringle, 2008). Another mitotic septin, Shs1 (**s**eventh **h**omolog of **s**eptin), was identified more than two decades after the initial screen (Mino et al., 1998). In meiosis, Cdc11 and Cdc12 are replaced by two other septins, Spr28 and Spr3, respectively (Ozsarac et al., 1995; De Virgilio et al., 1996). These two septins are expressed under an own promoter and the resulting meiosis-specific septin complexes exhibit distinct functionalities than their mitotic counterparts (Garcia et al., 2016). In this review we will focus on the mitotic septins.

The mitotic yeast septin proteins can be recombinantly expressed and purified from E. coli (Bertin et al., 2008; Renz et al., 2013). Analysis of mutated or truncated recombinant septin preparations uncovered the organization of the individual subunits: They assemble into hetero-oligomers, called rods, with the order Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 with Shs1 sometimes replacing the terminal subunit Cdc11 (Bertin et al., 2008; Garcia et al., 2011). These rods appear in vitro as short filaments of 32 nm length under high salt conditions (Kaplan et al., 2015). Septin rods can be induced to form long septin filaments in vitro by lowering the salt concentration of a buffer that otherwise keeps the rods stable in solution (Bertin et al., 2008; Renz et al., 2013). These filaments are about 1.5 µM long and exhibit a thickness within the range observed for microtubuli (Kaplan et al., 2015). Hexameric rods lacking the terminal subunit Cdc11 fail to form filaments suggesting that filament formation occurs end-over-end via the terminal subunit Cdc11 (Bertin et al., 2010). This observation was further fostered by applying single molecule localization microscopy to rods with their central and terminal subunits labeled with fluorescent dyes (Kaplan et al., 2015). Another study using septin preparations labeled with suitable FRET donor and acceptor dyes confirmed the end-over-end assembly via Cdc11 and showed that this interaction has a high affinity with a K<sup>D</sup> in the nano-molar range (Booth et al., 2015). Alternative modes for filament formation via other subunit contacts could be excluded by evaluation of different combinations of subunits labeled with the FRET compatible dyes (Booth et al., 2015).

Septin octamers containing Shs1 instead of Cdc11 as terminal subunit do not form linear filaments in vitro but rather curved bundles that assemble into closed rings (Garcia et al., 2011) suggesting that Shs1 is necessary to form also curved structures in vivo. Recombinant septin rods exclusively capped with Shs1 do not assemble end-over-end into filaments whereas Shs1-Cdc11 contacts are formed in vitro (Booth et al., 2015).

Crystal structures of the human subunits SEPT2 and SEPT7 provided invaluable insight into the architecture of subunit contacts and filament formation (Sirajuddin et al., 2007, 2009; Zent et al., 2011). Structurally, septins are GTP-binding proteins sharing similarities with the small GTPases of the Ras family (Leipe et al., 2002). A central conserved G-domain is flanked by variable N- and C-terminal extensions. The G-domain core resembles the conserved Ras structure with six β-strands and five α-helices. Structural elements from Ras like the switch I and switch II motifs and the P-loop (Schweins and Wittinghofer, 1994) classify the septins as bona fide GTPases. Besides these canonical structural elements, the septin G-domain harbors a septin unique element at its C-terminus that forms a distinct α-helix, α6 following the domain nomenclature from Ras, which points away from the G-domain in a 90◦ angle. Other septin specific features are the presence of a short N-terminal helix, α0, and a distinct loop with two antiparallel strands β7 and β8.

The crystal structure of a septin filament consisting of SEPT7, SEPT6, and SEPT2 revealed the nature of the binding interface of the septin subunits within the filament (Sirajuddin et al., 2007): Interactions between two adjacent G-domains (called G-interface) alternate with interactions of two adjacent N- and C-termini of two subunits (called NC-interface). The filament subunit is a hexamer with the order SEPT7-SEPT6- SEPT2-SEPT2-SEPT6-SEPT7. Here SEPT7 forms a G-interace with SEPT6, SEPT6, and SEPT2 form a NC-interface, SEPT2 and SEPT2 a G-interface and so on. The α6 helix is the prominent element of the NC-interface. C-terminal extensions like predicted coiled-coils do not seem to contribute to the NCinterface (Sirajuddin et al., 2007). The G-interface is composed of a complex network of interactions between different amino acid residues of the respective opposite subunit. Especially the interacting Val, His, and Trp residues located in the β7 and β8 sheets are highly conserved among human and fungal septins. Based on the high sequence homology between fungal and human septins one would assume that the architecture of the G-interface is shaped similarly in yeast septins. However, in the recently solved crytsal structure of Cdc11 (Brausemann et al., 2016, **Figure 1**), the terminal subunit of the S. cerevisiae septin rod, the loop containing the β7 and β8 sheets is not structured and the base of the loop points away from the presumed G-interface in an approximetaly 90◦ angle (**Figure 1**). This, together with the finding that Cdc11 dimers are maintained via the C-terminal coiled-coil extensions of the Cdc11 monomers and not via a NC- or G-interface, suggests that at least some yeast septins have interaction interfaces that might differ from the "classic" NC- and G-interfaces from the human septins.

Cdc11 exhibits one more unique feature: The crystal structure is from the apo form and does consequently not contain a nucleotide (Brausemann et al., 2016). All yeast and human septins harbor an absolute conserved lysine residue in the

canonical P-loop sequence GXXXXGKS/T. The P-loop is usually in contact with the β-phosphate of the bound GTP or GDP. Instead of the conserved lysine, Cdc11 has an arginine in this position of the P-loop whose side chain would collide with the β-phosphate of any bound nucleotide (**Figure 1**). Is Cdc11 not capable of binding a nucleotide? This assumption is fostered by the finding that cells expressing cdc11 alleles that contain mutants in the P-loop show wild type like phenotype at 30◦C (Casamayor and Snyder, 2003). Furthermore, only Cdc10 and Cdc12 have been shown to bind and hydrolyze GTP whereas GTPase activity could not be shown for Cdc3 and Cdc11 (Versele and Thorner, 2004).

vers. 1.10.2. from PDB structure 5AR1.

In the living yeast cell, the septin filaments assemble at the bud neck in an organized array, the so-called septin ring. This ring undergoes different cell cycle dependent architectural transitions (**Figure 2**). In early G1-phase, the septins are recruited to and accumulate at the presumptive bud site in a patch-like structure. Shortly before bud emergence, the septins assemble in a Cdc42 dependent manner into a ring marking the future site of bud growth and cytokinesis (Gladfelter et al., 2002; Caviston et al., 2003; Iwase et al., 2006). This initial recruitment of the septins to the future bud site depends besides Cdc42 on its effectors Gic1 and Gic2, and the action of the cyclin-dependent kinases Cdc28 and Pho85 (Tang and Reed, 2002; Iwase et al., 2006; Egelhofer et al., 2008; Okada et al., 2013). Evaluation of the mechanisms underlying the annealing of recombinant septin rods and formation of filaments on lipid bilayers suggests that septin filaments are formed in vivo at the plasma membrane from small complexes that diffuse in two dimensions (Bridges et al., 2014). Initial recruitment to the plasma membrane is supposed to be mediated by phospholipids. In an in vitro lipid

monolayer model PIP2 enhanced the filament assembly rate of septin hetero-octamers (Bertin et al., 2010).

After bud formation, the septin ring expands into a stable hourglass-shaped collar that is present at the bud neck until the onset of mitosis (Vrabioiu and Mitchison, 2006; Oh and Bi, 2011; Ong et al., 2014). Before cytokinesis, the septin collar splits into two distinct rings, one located at the mother and one at the daughter site of the bud neck. The contractile actomyosin ring (AMR), which powers the ingression of the cleavage furrow and septum formation, is assembled between the two septin rings (Wloka and Bi, 2012). Myo1, one essential constituent of the AMR, is recruited to the site of cytokinesis via the septin interacting protein Bni5 (Fang et al., 2010; Schneider et al., 2013). Bni5 in turn associates with the Cterminal extensions of the septin subunits Cdc11 and Shs1 (Finnigan et al., 2015).

After completion of cell separation, the old septin rings are disassembled and septin subunits are partially replaced and recycled for the next round of the cell cycle (McMurray and Thorner, 2008).

Taken together, the septins act mainly as scaffold for other proteins that are recruited to the bud neck or the site of cytokinesis, respectively. Additionally, the septins function as a diffusion barrier for proteins and organelles at the cortex of the bud neck (Luedeke et al., 2005; Shcheprova et al., 2008; Caudron and Barral, 2009; Orlando et al., 2011).

The transitions that the septins undergo throughout the cell cycle are supposed to be regulated by posttranslational modifications: Transition of the septin ring into a stable septin collar after bud emergence is associated with the phosphorylation and acetylation of certain subunits (Mitchell et al., 2011; Hernández-Rodríguez and Momany, 2012). For example, the p21-activated kinase Cla4 phosphorylates Cdc10 after bud emergence and a deletion of CLA4 strongly affects the timely formation of septin structures (Dobbelaere et al., 2003; Kadota et al., 2004; Versele and Thorner, 2004). The splitting of the septin collar at the onset of cytokinesis is supposed to be initiated by a collective switch in the orientation of the septin filaments from parallel to perpendicular to the growth axis of the cell (DeMay et al., 2011). The switch is accompanied by at least two different modifications. First, the bud neck kinase Gin4 phosphorylates Shs1 at residues different from those being modified in G1-phase (Mortensen et al., 2002). Gin4 is recruited together with the septins to the presumptive bud site, co-localizes with the septins at the bud neck for the complete cell cycle and disappears from the bud neck after splitting of the septin collar (Longtine et al., 1998; Mortensen et al., 2002; Au Yong et al., 2016). Second, the small ubiquitin-like modifier (SUMO) Smt3 is covalently attached to Cdc3, Cdc11, and Shs1 at the mother site of the bud (Johnson and Blobel, 1999). Phosphorylation events play apparently an important role in septin structure transitions and septin organization and several more kinases have been identified to interact with the septins: Gin4 (Dobbelaere et al., 2003), Kcc4 (Okuzaki and Nojima, 2001; Kozubowski et al., 2005), Ste20 (Ptacek et al., 2005), Hsl1 (Finnigan et al., 2016), Elm1 (Kang et al., 2016), and Kin2, the ortholog of animal MARK/PAR-1 kinase (Yuan et al., 2016), were all identified as septin interactors.

However, for most of these regulatory proteins the exact timing of the interaction with the septins remains unknown and one can speculate that much more interactions exist but are not identified so far. Recently, Renz et al., performed a systematic screen for septin interaction partners throughout the cell cycle by combining cell cycle synchronization at stages with a distinct septin structure (**Figure 2**) with quantitative mass spectrometry to timely resolve the septin interactome (Renz et al., 2016). Yeast cells were synchronized in G1 phase with alpha factor, in S phase with hydroxyurea and in late anaphase with the help of a tempertaure sensitive cdc15 allele. This strategy allowed to link interaction partners to specific transition states of septin structures. Distinct sets of regulatory proteins were found to interact with the septins at certain stages of the cell cycle: Gin4 and the anillin-like protein Bud4, a known septin interactor (Kang et al., 2013), were identified both in S-phase and anaphase. The kinases Hsl1, Mck1, and Prk1 are specifically associated with the septins in S-phase cells. Ste20 and yeast SUMO (Smt3) were only identified as specific septin interactors in cells with split septin rings. An activity of Mck1 and Prk1 on the septins has not been reported and remains to be confirmed by supplementary studies. This screen strengthened also evidence that septins play a role in endocytosis: Syp1 is a negative regulator of the WASP-Arp23 complex and involved in endocytic site formation, but was also classified as septin organizing protein (Qiu et al., 2008; Merlini et al., 2015). It interacts with the septins in anaphase (Renz et al., 2016). In S-phase, the septins interact with Sla2, an adaptor protein that links actin to clathrin and endocytosis

#### REFERENCES

(Skruzny et al., 2015). In alpha facor arrested cells, Vps1 interacts with the septins (Renz et al., 2016). Vps1 is a dynamin related protein that functions at several stages of membrane trafficking including endocytic scission (Smaczynska-de Rooij et al., 2010, 2015).

The septins influence the composition of the ER at the bud neck and thereby render diffusion of ER membrane proteins from mother to bud. Septins, septin dependent kinases, the membrane protein Bud6 and sphingolipids were shown to be required for the ER diffusion barrier (Luedeke et al., 2005; Clay et al., 2014). Interestingly, the published septin interactome contains a hit for Erp1 (Renz et al., 2016), a member of the p24 complex that was reported to be involved in ER retention and ER-Golgi transport (Copic et al., 2009). Another constituent of COPII vesicles that interacts with p24 family members, Sfb3/Lpt1 (Manzano-Lopez et al., 2015), was additionally identified in the MS screen. These findings support the previsously published model of a septin dependent ER diffusion barrier.

Recapitulatory, the past few years have witnessed a considerable increase of published results concerning yeast septins, the majority of these in budding yeast. Efforts were made in all areas of septin biology, from structural investigations using EM, light microscopy and X-ray diffraction to reconstruction of septin assembly in complex in vitro systems and to elucidation of septin related processes in the living cell. All these studies together establish and/or approve the budding yeast as a prime model organism for septin research. While structural information of several of the human septin subunits is now avaliable (the PDB provides crystal structures for SEPT2, SEPT7, SEPT9, SEPT3 and the septin trimer 2/6/7), only one structure from a yeast septin was solved (Cdc11). Structure determination, the evaluation of the role of posttranslational modifications of the septins and the uncovering of so far unknown septin related processes in the living cell will represent the challenges for septin biologists for the next decade.

#### AUTHOR CONTRIBUTIONS

TG and OG wrote the manuscript. OG prepared the figures.

### FUNDING

OG was supported by Deutsche Forschungsgemeinschaft grant JO 187/8-1.

and function. Mol. Cell. Biol. 23, 2762–2777. doi: 10.1128/MCB.23.8.2762-27 77.2003

like protein Vps1 during endocytosis in yeast. J. Cell Sci. 123, 3496–3506. doi: 10.1242/jcs.070508

**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 © 2016 Glomb and Gronemeyer. 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) or licensor 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.

# Septin-Associated Protein Kinases in the Yeast *Saccharomyces cerevisiae*

Adam M. Perez, Gregory C. Finnigan † , Françoise M. Roelants and Jeremy Thorner\*

*Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA*

Septins are a family of eukaryotic GTP-binding proteins that associate into linear rods, which, in turn, polymerize end-on-end into filaments, and further assemble into other, more elaborate super-structures at discrete subcellular locations. Hence, septin-based ensembles are considered elements of the cytoskeleton. One function of these structures that has been well-documented in studies conducted in budding yeast *Saccharomyces cerevisiae* is to serve as a scaffold that recruits regulatory proteins, which dictate the spatial and temporal control of certain aspects of the cell division cycle. In particular, septin-associated protein kinases couple cell cycle progression with cellular morphogenesis. Thus, septin-containing structures serve as signaling platforms that integrate a multitude of signals and coordinate key downstream networks required for cell cycle passage. This review summarizes what we currently understand about how the action of septin-associated protein kinases and their substrates control information flow to drive the cell cycle into and out of mitosis, to regulate bud growth, and especially to direct timely and efficient execution of cytokinesis and cell abscission. Thus, septin structures represent a regulatory node at the intersection of many signaling pathways. In addition, and importantly, the activities of certain septin-associated protein kinases also regulate the state of organization of the septins themselves, creating a complex feedback loop.

Keywords: cell cycle, cell signaling, cytoskeletal element, morphology, protein phosphorylation

# INTRODUCTION

Septins are a conserved family of GTPases (Pan et al., 2007; Peterson and Petty, 2010; Nishihama et al., 2011) that serve multiple biological functions in diverse cell types (Weirich et al., 2008; Hall and Russell, 2012; Hernandez-Rodriguez and Momany, 2012; Mostowy and Cossart, 2012; Fung et al., 2014). All eukaryotes examined to date (except higher plants) encode multiple septin genes, ranging from just two in the nematode Caenorhabditis elegans (John et al., 2007), to five in Drosophila melangaster (O'Neill and Clark, 2016), to seven in Saccharomyces cerevisiae (Garcia et al., 2016), to 13 in humans (Peterson and Petty, 2010; Hall and Russell, 2012; Fung et al., 2014).

Budding yeast (S. cerevisiae) has served as a path-finding model eukaryote in which to explore the structure, function, and regulation of septins and septin-associated proteins. The products of the yeast septin genes assemble into linear, apolar hetero-oligomeric rods that are the fundamental building block of all septin-based structures (Bertin et al., 2008; Bertin and Nogales, 2012), as has now also been shown for other organisms. These rods can self-associate end-to-end to form filaments and can, depending in their subunit composition, also interact in other modes to form

#### *Edited by:*

*Manoj B. Menon, Hannover Medical School, Germany*

#### *Reviewed by:*

*Andrew Truman, University of North Carolina at Charlotte, USA Francesc Posas, Pompeu Fabra University, Spain*

> *\*Correspondence: Jeremy Thorner<EMAIL_ADDRESS>
#### *† Present Address:*

*Gregory C. Finnigan, Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS, USA*

#### *Specialty section:*

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

*Received: 03 September 2016 Accepted: 14 October 2016 Published: 01 November 2016*

#### *Citation:*

*Perez AM, Finnigan GC, Roelants FM and Thorner J (2016) Septin-Associated Protein Kinases in the Yeast Saccharomyces cerevisiae. Front. Cell Dev. Biol. 4:119. doi: 10.3389/fcell.2016.00119* more elaborate super-structures, such as spirals, rings, braids, and gauze-like lattices (Rodal et al., 2005; Garcia et al., 2011; Oh and Bi, 2011; Bertin et al., 2012; Ong et al., 2014). Other factors nucleate the assembly of septin structures at discrete subcellular locations (Chen et al., 2011; Bi and Park, 2012) where they serve as both scaffolds (Shulewitz et al., 1999; Sakchaisri et al., 2004; Wloka et al., 2011; Bridges and Gladfelter, 2015) and diffusion barriers (Takizawa et al., 2000; Caudron and Barral, 2009) and thereby dictate, via direct and indirect interactions, the subcellular distribution of numerous other proteins (Gladfelter et al., 2001; McMurray and Thorner, 2009; Finnigan et al., 2016a). In particular, as discussed here, septin-based structures recruit, and thereby localize (and, in some cases, regulate the activity of) a multiplicity of protein kinases that integrate multiple inputs into signaling pathways and ultimately initiate ensuing biological responses (**Figure 1**).

Septins were first discovered in S. cerevisiae as cell division cycle (cdc) mutants whose compromised function resulted in abnormal growth patterns and the inability of cells to execute cytokinesis (Hartwell, 1971; Hartwell et al., 1974). Electron microscopy revealed a prominent set of filaments that encircle the yeast bud neck (Byers and Goetsch, 1976) and, later, antibody staining demonstrated that septins were constituents of those filaments (Haarer and Pringle, 1987; Kim et al., 1991). The advent of GFP and other genetically encoding fluorescent protein tags to follow protein dynamics in live cells in real time revealed further that septin organization undergoes dramatic changes during cell cycle progression. Septins first accumulate as a patch at the incipient site of bud emergence that rapidly resolves into a filamentous ring (Kozubowski et al., 2005; Okada et al., 2013), which then expands, concomitant with bud growth, into an hourglass-shaped tube or collar composed of at least 30–40 gyres of circumferential filaments at the bud neck (Byers and Goetsch, 1976; McMurray et al., 2011; Finnigan et al., 2015b; Patasi et al., 2015). At the onset of cytokinesis, the collar splits into two fllamentous bands of roughly equal size with a prominent gap in between (Dobbelaere et al., 2003; Dobbelaere and Barral, 2004) wherein factors needed for actomyosin contractile ring assembly and new plasma membrane (PM) and cell wall (CW) synthesis accumulate (Bi et al., 1998; Nishihama et al., 2009). After cytokinesis and cell separation, each daughter cell disassembles the half of the collar it inherited (Johnson and Blobel, 1999; Tang and Reed, 2002) before constructing a new septin-based site for the next bud to emerge. An N-terminal F-BAR domain—and C-terminal Muniscin/Mu homology domain (MHD)-containing protein Syp1 localizes prominently at the bud neck and has been implicated in the processes required for disassembly of the septin ring (Qiu et al., 2008). Syp1 is a highly phosphorylated protein (Albuquerque et al., 2008; Soulard et al., 2010; Swaney et al., 2013). In this regard, it is interesting that eight of the phospho-sites detected in Syp1 fit the -T/S-P- consensus for the protein kinase Cdk1/Cdc28 that is the major cell cycle driver (Verma et al., 1997; Mok et al., 2010) and five of them fit the consensus sequence (-R-x-x-S/T-) determined for the bud neck-localized protein kinase Gin4 (Mok et al., 2010; Roelants et al., 2015). However, Syp1 also localizes prominently to cortical puncta and functions as an endocytic adaptor that is involved in cargo selection and negative regulation of Las17 (yeast WASp)- Arp2/3 complex activity (Boettner et al., 2009; Reider et al., 2009) during the early stages of endocytic patch formation (Stimpson et al., 2009). Although eukaryotic proteins with such apparently disparate functions certainly exist, it is nonetheless a little hard to reconcile mechanistically these two rather distinct roles attributed to Syp1.

In S. cerevisiae, five (CDC3, CDC10, CDC11, CDC12, and SHS1) of its seven septin genes are expressed in mitoticallydividing haploid and diploid cells (Versele et al., 2004; Versele and Thorner, 2004, 2005), whereas the remaining two septin genes (SPR3 and SPR28) are expressed only in diploid cells undergoing meiosis and sporulation (Garcia et al., 2016). The mitotic septins assemble into two types of hetero-octameric rods that differ only in their terminal subunit: Cdc11-Cdc12- Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 (Bertin et al., 2008) and Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Shs1 (Garcia et al., 2011). In vitro the former can associate end-to-end into paired filaments in low-salt solution and into sheets of paired filaments on the surface of a lipid monolayer containing phosphatidylinositol-4,5-bisphosphate (Bertin et al., 2010). Mutagenesis studies have shown that the residues needed for these characteristic in vitro behaviors are also essential for viability in vivo (Bertin et al., 2008; McMurray et al., 2011; Finnigan et al., 2015b). The Shs1-containing hetero-octamers associate laterally in a staggered fashion, rather than end-on-end, forming curved bundles, rings, and bird's nest-like structures in vitro (Garcia et al., 2011). Although cells lacking Shs1 are viable, the septin structures formed in its absence are aberrant (Garcia et al., 2011), most likely because, as observed in vitro (Booth et al., 2015), Cdc11-capped rods and Shs1-capped rods are able to form heterotypic end-on-end junctions, and likely also do so in vivo (Finnigan et al., 2015a,b). The dynamic interplay between these two types of hetero-octamers may facilitate the massive reorganizations of septin architecture that occur over the course of the cell cycle (Vrabioiu and Mitchison, 2006; Ong et al., 2014).

Diverse protein kinases are associated with septin structures at various points throughout progression through the cell division cycle. However, it is still not completely clear how many of these enzymes contribute directly to installing posttranslational modifications on septins and/or septin-associated proteins that drive the observed dynamic changes in septin structure during cell cycle progression and how many of these enzymes are recruited to septin structures as "readers" of the status of septin assembly to phosphorylate other substrates and thereby drive subsequent downstream events. Here, we highlight key regulatory pathways that use the septin cytoskeleton as a signaling platform to direct other orchestrated events required for successful passage through the cell cycle.

# A MORPHOGENESIS CHECKPOINT

Eukaryotes have evolved quality control mechanisms, collectively dubbed checkpoints (Hartwell and Weinert, 1989; Paulovich et al., 1997; Ibrahim, 2015), by which to ensure that the events required for successful cell division are only executed

at the proper time and in the proper order. Virtually all recognized checkpoint mechanisms involve regulation by reversible protein phosphorylation mediated by protein kinases and phosphoprotein phosphatases (Domingo-Sananes et al., 2011; Rhind and Russell, 2012). In yeast, a checkpoint delays the decision to pass from G2 to M phase if there is some incompleteness (or abnormality) in cell morphogenesis that needs time to be finished (or repaired). It was initially thought that this control was exerted in response to defects in actin cytoskeleton assembly and/or function (McMillan et al., 1998). However, it was demonstrated shortly thereafter that it is the state of septin collar assembly that is monitored by this checkpoint (Shulewitz et al., 1999), a conclusion that was amply confirmed subsequently (Lew, 2003; Howell and Lew, 2012). In essence, when the septin collar is properly formed, it recruits and thereby serves as a congregation point for information exchange among the regulatory factors required to release the cell from cell cycle blockade (**Figure 2A**, upper). This arrangement provides a feedback circuit by which the state of septin organization is temporally and spatially integrated with other processes necessary for cell cycle progression. Because chromosome segregation and cytokinesis cannot proceed productively if the bud neck is occluded, this checkpoint delays initiation of the G2-M transition until the septin collar has been erected properly.

In brief, the cyclin B (Clb2)-bound form of protein kinase Cdk1 (Cdc28), which is the primary driver of mitosis, is held in check by inhibitory phosphorylation on Tyr19 in its P-loop, a modification installed by protein kinase Swe1 (the S. cerevisiae ortholog of mammalian Wee1) (Booher et al., 1993). This phosphorylation is reversed, in part, by the action of phosphoprotein phosphatase Mih1 (yeast ortholog of mammalian Cdc25) (Russell et al., 1989). However, to lift Swe1 mediated inhibition of Cdk1 completely, Swe1 is also degraded in a highly regulated manner, as follows. In conjunction with the joint actions of the protein-arginine N-methyltransferase Hsl7 (Cid et al., 2001; Sayegh and Clarke, 2008) and the protein kinase Hsl1 (Ma et al., 1996; Barral et al., 1999; Shulewitz et al., 1999) (closest mammalian orthologs are the AMPK-related protein kinase family members, BRSK1, and BRSK2), Swe1 is exported from the nucleus (Keaton et al., 2008), captured at the bud neck, and marked there for timely cyclosome/anaphasepromoting complex (APC) protein-ubiquitin ligase-dependent ubiquitinylation (Simpson-Lavy and Brandeis, 2010; Lianga et al., 2013) and proteasome-mediated destruction via phosphorylation by protein kinase Cla4 (yeast ortholog of mammalian PAK1) and especially by protein kinase Cdc5 (yeast ortholog of mammalian Plk1) (Sakchaisri et al., 2004). Compared to Hsl1, two other septin-associated protein kinases, Gin4 and Kcc4 (which are apparent paralogs, but less related to Hsl1) have a supporting, but less significant role, in these events (Ma et al., 1996; Barral et al., 1999; Kusch et al., 2002). It should be appreciated that because Clb2-Cdc28 phosphorylation of Swe1 creates phosphosites that are docking motifs for binding the Polo boxes of Cdc5, thus priming Swe1 for Cdc5-mediated phosphorylation (Asano et al., 2005; Harvey et al., 2005), the Hsl7- and Hsl1-dependent down-regulation of the nuclear pool of Swe1 partially alleviates Clb2-Cdc28 inhibition. This initially modest increase in Clb2- Cdc28 activity then is able to initiate a self-reinforcing burst of autocatalytic activation of Cdk1 by targeting more and more of the population of Swe1 molecules for even more efficient Cdc5 mediated phosphorylation, thereby unleashing more and more Clb2-Cdk1 activity. Thus, once initiated, these processes lead to rapid hyper-phosphorylation and nearly complete destruction of Swe1 (Shulewitz et al., 1999; Sreenivasan and Kellogg, 1999).

Most importantly, Hsl1 is only active in aiding the targeting of Swe1 for destruction when Hsl1 is associated with a correctly

kinases phosphorylate targets that help promote cytokinesis. Gin4 remains septin collar-associated from G1 through cytokinesis.

assembled septin collar at the bud neck (McMillan et al., 1999, 2002; Shulewitz et al., 1999; Finnigan et al., 2016b). Disruption of the septin collar using mutant septin alleles results in failure to recruit Hsl1 and Hsl7 to the bud neck, thereby stabilizing Swe1, which is then able to impose a pronounced cell cycle delay that is manifest by the formation of markedly elongated buds (Shulewitz et al., 1999; Longtine et al., 2000), a phenotype that is not observed if cells lack Swe1 (Barral et al., 1999; McMillan et al., 1999; McMurray et al., 2011).

Directly fusing Swe1 to a septin subunit does not fully bypass the requirement for Hsl1 and Hsl7 in Swe1 degradation (King et al., 2012), indicating that simple tethering of Swe1 at the septin collar for subsequent phosphorylation by Cla4 and Cdc5 is not the sole function of the Hsl1 and Hsl7 enzymes. Binding of Hsl1 to septins occurs via uniquely evolved septin-binding sequences (residues 611–950) downstream of its N-terminal kinase domain, which, to operate efficiently, must act in concert with a C-terminal phosphatidylserine-binding element (KA-1 domain) (Finnigan et al., 2016a,b). Thus, Hsl1 recruitment to the bud neck likely serves as a dual "sensor," reporting that both septin collar assembly and the plasma membrane lipid composition have achieved the proper state to initiate the destruction of Swe1 and thereby license passage from G2 to M phase. Hsl1 catalytic activity is dispensable for its recruitment to the septins (Finnigan et al., 2016b), but may be required, directly or indirectly, in recruiting Hsl7 to the bud neck (Kang et al., 2016). Hsl7 is a substrate of Hsl1 (Cid et al., 2001), and Hsl1 is also extensively autophosphorylated (Barral et al., 1999), but the functional consequences of these modifications remain to be determined.

Hsl1 (and the checkpoint) may have additional roles besides sensing the state of septin organization and plasma membrane composition. A recent study has suggested that some of these same components, in conjunction with an additional bud necklocalized protein kinase, Elm1, have a role in monitoring bud size/shape prior to licensing entry into mitosis (Kang et al., 2016). Elm1 is able to phosphorylate the activation loop in Kcc4 and Gin4 in vitro (Asano et al., 2006), and presumably serves as their upstream activating kinase in vivo (Koehler and Myers, 1997; Bouquin et al., 2000). In contrast, under the same conditions, Elm1 does not directly phosphorylate Hsl1 in vitro (Asano et al., 2006), but there are claims that Elm1-mediated phosphorylation is required for full Hsl1 activity in vivo (Szkotnicki et al., 2008). It has been suggested that Elm1 interacts with Hsl7, thereby impeding engagement of Hsl7 with Hsl1; in this way, Swe1 recruitment to the bud neck and its degradation are delayed to allow time for sufficient bud growth (Kang et al., 2016). It has been reported that localization of septins, Hsl7 and Elm1 all depend upon local membrane curvature (Bridges et al., 2016; Kang et al., 2016); it is possible, therefore, that these factors serve as sensors of cell geometry, thereby integrating this input into the timing of the decision of when to allow passage through G2-M. Although Gin4 and Kcc4 have been implicated in septin collar assembly (Longtine et al., 1998) and in the Swe1 degradation pathway (Barral et al., 1999), more recent evidence indicates that these enzymes likely exert their effects more indirectly by modulating the plasma membrane lipid distribution (Roelants et al., 2015) (see further below). Indeed, like Hsl1, Gin4 and Kcc4 also possess phosphatidylserine-binding C-terminal KA-1 domains that are required for their function in vivo (Moravcevic et al., 2010).

### MITOTIC EXIT

In addition to the role of the septin collar in integrating signals that determine when to initiate entry into mitosis, growing evidence implicates septin structures at the bud neck in coordinating certain aspects of the signaling network that regulates exit from mitosis. Mitotic exit is facilitated, in large part, by a protein kinase signaling cascade, dubbed the Mitotic Exit Network (MEN) (Bardin and Amon, 2001; McCollum and Gould, 2001), that gauges successful spindle elongation by monitoring the location and status of the spindle pole bodies (SPBs) (Smeets and Segal, 2002; Hotz and Barral, 2014; Falk et al., 2016). Once anaphase has been achieved, signals emanating from the SPB in the daughter cell initiates a pathway that serves to reverse the actions of Cdk1 by unleashing the phosphoprotein phosphatase Cdc14 (Mocciaro and Schiebel, 2010; Bremmer et al., 2012). Components of the MEN regulatory circuitry in yeast have features that suggest that it may be the antecedant of the Hippo tumor-suppressor pathway in animal cells (Avruch et al., 2012; Harvey and Hariharan, 2012; Rock et al., 2013).

The core components of MEN (**Figure 2A**, lower) are the small Ras-related GTPase Tem1, which, in its GTP-bound state, binds to and activates the protein kinase Cdc15, which, in turn, is the upstream activator of two paralogous protein kinases Dbf2 and Dbf20 that, to be activated by Cdc15 and functional, need to associate with an essential regulatory subunit Mob1. The primary role of activated Dbf2/Dbf20-Mob1 complexes is to phosphorylate Cdc14, which both releases it from its inhibitory anchor in the nucleolus (Azzam et al., 2004) and prevents its nuclear import (Mohl et al., 2009), allowing its activity to spread throughout the cell to inactivate Clb-bound Cdk1 and reverse the Cdk1-dependent phosphorylation of many Cdk1 substrates (Visintin and Amon, 2000; Meitinger et al., 2012).

Whether Tem1 is in its inactive GDP-bound state or in its activated GTP-bound state is controlled by events at the SPB, completely independent of the septin collar. In brief, when no SPB has been segregated into the daughter bud, the Tem1 GTPase-activating protein (GAP) complex, Bfa1-Bub2, is activated by the protein kinase Kin4 located at the cortex of the mother cell and, hence, Tem1 remains in its inactive state (D'Aquino et al., 2005; Pereira and Schiebel, 2005). This regulatory scheme is referred to as the spindle positioning/orientation checkpoint (SPOC) (Caydasi and Pereira, 2012; Ibrahim, 2015). However, there is a clear connection to events at the septin collar. First, it was observed that septin-defective mutants exhibited aberrations in MEN signaling outputs (Castillon et al., 2003). Second, Kin4 (and its paralog Frk1) are, like Gin4, Kcc4, and Hsl1, members of the branch of yeast AMPK-like protein kinases (Rubenstein and Schmidt, 2007); and, indeed, it has been shown that, as for Gin4 and Kcc4, Elm1 is the upstream protein kinase responsible for phosphorylation of T209 in the activation loop of Kin4 and, further, that this modification is essential for Kin4 catalytic activity (Caydasi et al., 2010; Moore et al., 2010). Moreover, localization of Elm1 to the bud neck is critical for its phosphorylation and activation of Kin4 (Caydasi et al., 2010; Moore et al., 2010).

The action of phosphoprotein phosphatase PP2A bound to one of its two, yeast, B-type regulatory subunits, Rts1, was also implicated as a positive factor required for maintaining Kin4 at the mother cell cortex and at mother-cell SPBs (Chan and Amon, 2009). However, it was previously shown that Rts1-bound PP2A is required for maintenance of septin ring organization during cytokinesis (Dobbelaere et al., 2003). Thus, the role of Rts1-PP2A is likely indirect and simply to preserve the structure of the septin collar and, thus, the localization of active Elm1 there, supporting maintenance of adequate levels of activated Kin4 in the mother cell.

The daughter cell-specific protein Lte1, despite its sequence resemblance to other guanine nucleotide exchange factors (GEFs), does not appear to function as the GEF for Tem1. Rather, it appears to promote formation of GTP-bound Tem1 by binding to and inactivating Kin4 and excluding the kinase from the daughter SPB, thereby preventing activation of the Bfa1-Bub2 GAP (Bertazzi et al., 2011; Falk et al., 2011).

All of the MEN components, including the protein kinases Cdc5 (Sakchaisri et al., 2004), Cdc15 (Lee et al., 2001), and Dbf2/Dbf20 (Frenz et al., 2000), localize first to the SPB and then to the bud neck (**Figure 2**). At the SPB, Cdc5 functions in MEN by acting in concert with Tem1 to promote SPB recruitment and activation of Cdc15 (Rock et al., 2013). Cdc5 function depends on its cellular location, as revealed by experiments in which Cdc5 recruitment was artificially directed primarily to the SPB or primarily to the bud neck; at the SPB, Cdc5 was necessary for efficient mitotic exit and, at the bud neck, Cdc5 clearly promoted Swe1 degradation (Sakchaisri et al., 2004). The two C-terminal polo boxes of Cdc5 are required for stable association of this protein kinase at each of these two subcellular locations (Song et al., 2000; Lowery et al., 2005), but the protein target (s) at each location that carry the phospho-epitopes to which the polo boxes bind have not been well defined. Given the role that members of the polo family of protein kinases, including Cdc5, play in driving multiple cell cycle events subsequent to initial substrate phosphorylation by cyclin B-bound Cdk1 (Barr et al., 2004; Archambault and Glover, 2009), and the role that the septin collar plays in regulating the sub-cellular distribution of Cdc5, the coordination between septin dynamics and Cdc5 localization, as well as between septin dynamics and Elm1 localization (Thomas et al., 2003), provide regulatory inputs that contribute to ensuring that Swe1 degradation (and mitotic entry) always precedes mitotic exit. Indeed, the septin collar is the passageway through which any and all components segregated between a mother and daughter cell must pass and, hence, is a cellular structure ideally situated to monitor such cell cycle events.

#### CYTOKINESIS

In addition to its role in tethering of factors involved in controlling the spatial and temporal aspects of the cell cycle, the septin collar also has functions in regulating membrane dynamics. At the cortex of a mother cell or its bud, where the ER is in close apposition to the PM, there are periodic intimate protein-and lipid-mediated connections between the two, referred to as PM-ER junctions (Manford et al., 2012; Gatta et al., 2015). However, at the bud neck, the septin filaments tightly coat the cytosolic surface of the PM at this location (Byers and Goetsch, 1976; Bertin and Nogales, 2012), preventing sterically the formation of such ER-PM contact sites. Although extensions of the ER can be seen to pass through the bud neck, at the location of the septin collar per se, the ER surface appears denuded of ribosomes and, further, that this band of smooth ER acts as a barrier to diffusion of ER membrane proteins (but not ER lumenal proteins) (Luedeke et al., 2005). On the one hand, it has been reported that establishment of this specialized ER domain and its function in restricting diffusion depends on localized recruitment of the actin—and formin-associated protein Bud6 by the septin Shs1 (Luedeke et al., 2005), whereas another more recent study claims that a bridged interaction between Shs1 and the integral ER membrane protein Scs2 is responsible for erecting this ER diffusion barrier (Chao et al., 2014). The evidence in support of both claims is not compelling for several reasons. First, more recent analysis indicates that very little of the total cellular pool of Bni6 is located at the bud neck [see entry for Bud6/Aip3/Ylr319c at the Yeast Protein Localization Plus Database (YPL+) at the Univ. of Graz, Austria; http://yplp.uni-graz.at/index.php], and Scs2 is a demonstrated component of a major class of the ER-PM junctions. Second, because shs11 cells are viable and do not display severe growth and morphology defects under most conditions (Iwase et al., 2007; Finnigan et al., 2015b), this specialized, purportedly Shs1-mediated, domain in the transbud neck ER doesn't seem to be very important for viability or efficient cell cycle progression. Finally, affixing the ER to the septin collar at the bud neck, and thereby risking impeding the passage of chromosomes and other cellular organelles, would not seem to make much sense physiologically for efficient cell division.

In any event, to complete each cell cycle, the spindle midbody remnant must be removed, the plasma membranes resealed, and the CW septa deposited between them, to create two, separate and independent yeast cells. In S. cerevisiae, cytokinesis and cell separation proceed via the concerted action of actomyosin ring (AMR) formation and contraction (Bi et al., 1998) with concomitant synthesis of the chitinaceous primary septum (Nishihama et al., 2009). To permit these events to happen following anaphase, the septin collar is split into two gasketlike bands via a mechanism that is still completely unknown (McMurray and Thorner, 2009; Bi and Park, 2012; Wloka and Bi, 2012; Juanes and Piatti, 2016). However, after septin collar assembly and release from the morphogenesis checkpoint, the elevation of Clb2-Cdc28 activity commences the process of promoting AMR formation early in M phase and, once localized to the septin collar, Cdc5 generates a local pool of activated (GTPbound) Rho1 at the bud neck via phosphorylation and activation of one of the primary Rho1 GEFs (Tus1) (Yoshida et al., 2006). Activated Rho1, in turn, is known to stimulate formindependent assembly of the cytokinetic AMR and type II myosin contractility (Yoshida et al., 2006; Ramkumar and Baum, 2016). In addition, activated Rho1 also binds directly to and stimulates protein kinase Pkc1 (Kamada et al., 1996), which controls the transcription of genes needed for CW synthesis (Jung and Levin, 1999) and other events needed for cell cycle completion (Darieva et al., 2012), as well as two enzymes required for CW production (the 1,3-β-D-glucan synthase Fks1 and its paralog Gsc2/Fks2) (Mazur and Baginsky, 1996). In this way, the septins provide a platform by which the cell cycle machinery is linked to both the cytoskeletal machinery and the CW synthesis machinery that each contribute to the successful execution of yeast cytokinesis. Difficult to reconcile with these findings (and the evidence discussed earlier for its role in septin ring disassembly), however, is the claim that F-BAR and MHD protein Syp1 is phosphorylated in a Rho1- and Pkc1-dependent manner to promote septin collar assembly (Merlini et al., 2015).

The "split" septin collar appears to have two main roles. For certain proteins needed for AMR assembly, e.g., Bni5 (Lee et al., 2002), the septins continue to serve their scaffold function by mediating direct physical association with these factors, thereby anchoring and concentrating them at the bud neck (Patasi et al., 2015; Finnigan et al., 2015a, 2016a). For other proteins, e.g., Sec3 involved in localized deposition of secretory vesicles (Dobbelaere and Barral, 2004) and Chs2 involved in septum construction (Foltman et al., 2016), the two septin bands of the split collar seems to act like corrals and barriers to diffusion, thereby physically trapping these factors between them and thus confining them at this location indirectly (Dobbelaere and Barral, 2004; McMurray et al., 2011; Finnigan et al., 2016a).

In addition to Cdc5, the other two protein kinases of the MEN cascade, Cdc15 and Dbf2/Dbf20-Mob1 complexes, are recruited to the split septin rings during late anaphase, shortly after Cdk1 inactivation (Frenz et al., 2000; Song et al., 2000; Xu et al., 2000; Luca et al., 2001; **Figure 2B**). At this location, these enzymes recruit and phosphorylate many factors directly involved in the coordination of AMR contraction and primary septum formation. In particular, at the bud neck, Dbf2- Mob1 phosphorylates another F-BAR protein, Hof1 (Meitinger et al., 2011), which also contains a C-terminal SH3 domain important for its function (Oh et al., 2013). Hof1 associates with septin structures throughout G1-S phase and while the AMR is assembled between the rings of the split septin collar primarily via direct interaction with Cdc10 (and Cdc12) (Vallen et al., 2000; Oh et al., 2013; Finnigan et al., 2016a). Association of Hof1 with the septins is mediated by a coiled-coil element in Hof1, and phosphorylation at a single Ser within this region by bud neck-localized Dbf2-Mob1 (an event that requires priming by prior phosphorylation of Hof1 by Cdk1 and Cdc5) promotes disassociation of Hof1 from the septin rings (Meitinger et al., 2013). This displacement then allows Hof1 to associate with the AMR and initiate contraction, but the mechanism by which it does so has not been elucidated yet.

In addition to its binding to the AMR, Hof1 also forms a complex with Inn1 and Cyk3 at the septin ring in late anaphase (Sanchez-Diaz et al., 2008; Nishihama et al., 2009). Recruitment of these three components to the bud neck requires MEN signaling activity (Meitinger et al., 2010), and thus presumably their phosphorylation, and all are required for efficient cytokinesis because they contribute to coordinating AMR contraction with primary septum formation (Meitinger et al., 2012; Wloka and Bi, 2012; Juanes and Piatti, 2016). As mentioned above, formation of the primary septum requires the localized activity of chitin synthase Chs2 between the bands of the split septin collar. After Chs2 has been sequestered at this position, Dbf2-Mob1 translocates to the split rings and directly phosphorylates and activates Chs2 (Oh et al., 2012). The dynamic relocation of the protein kinases of the MEN cascade to the split septin collar provides an elegant solution to help ensure that cell division only occurs after successful chromosome segregation. However, the mechanisms that promote recruitment of these kinases to the septins are unknown. Moreover, the SPOC protein kinase Kin4 also localizes to the septin rings late in anaphase, yet its function at the bud neck is not understood.

Finally, cytokinesis cannot be completed unless and until two intact and separable PMs have been generated. In this regard, there is evidence that two types of septin-associated protein kinases spatially and temporally modulate the PM lipid bilayer assymmetry at the bud neck. At sites of polarized growth, the protein kinase Fpk1 (and its paralog Fpk2) phosphorylate and stimulate two PM-localized flippases (Dnf1 and Dnf2), which translocate phosphatidylethanolamine (PtdEth) and phosphatidylserine from the outer to the inner leaflet of the PM (Nakano et al., 2008; Roelants et al., 2010). However, at the bud neck, septin-anchored protein kinase Gin4 phosphorylates and inhibits Fpk1 (and Fpk2) function (Roelants et al., 2015). Thus, flipping of aminoglycerophospholipids is prevented in a highly localized manner. Preventing flipping of aminoglycerophospholipids in this way contributes to enhancing the efficiency of cell division because alterations that compromise flippase function suppress the inviabiity phenotype of a variety of mutations (hof1, inn1, cyk3, and myo1, the type II myosin of the AMR) known to cause defects in cytokinesis (Roelants et al., 2015). Moreover, Gin4 is released from the septin collar just before it splits and the AMR contracts, whereas the bulk of Fpk1 remains (Roelants et al., 2015), presumably fine-tuning the timing of transbilayer lipid flipping necessary for the completion of PM scission and cell separation. The mechanisms responsible for the cell cycle-coupled ejection of Gin4 and the recruitment of Fpk1 are not known, although Gin4 is phosphorylated and activated in a cell cycle-dependent manner at the G2-M transition, apparently by Clb2-Cdc28 (Altman and Kellogg, 1997) and, conversely, the septins are required for the mitosis-specific activation of Gin4 (Carroll et al., 1998). In any event, by the spatio-temporal control that Gin4 exerts on Fpk1 activity, it is clear that the septins at the bud neck are critical for protein kinase-mediated regulation of localized PM remodeling.

# REGULATION OF SEPTIN ORGANIZATION

The actions of several septin-associated protein kinases also seem to regulate septin organization. The Cdc42-stimulated protein kinase Cla4 (yeast ortholog of mammalian PAKs) directly phosphorylates septins Cdc3 and Cdc10 both in vitro and in vivo, and Cla4 is necessary for both the formation of the septin collar and the regulation of septin dynamics at specific points in the cell cycle (Dobbelaere et al., 2003; Schmidt et al., 2003; Versele and Thorner, 2004).

Septin subunit Shs1 is the most extensively phosphorylated septin and is a substrate of Gin4 (Mortensen et al., 2002), as well as of the G1 cyclin-dependent protein kinases Cdc28/Cdk1 and Pho85 (yeast ortholog of mammalian Cdk5) (Egelhofer et al., 2008). The latter marks are removed at the end of mitosis via bud neck recruitment of Rts1-bound PP2A, an event thought to aid in initiating splitting of the septin collar (Dobbelaere et al., 2003), and which is coincident with Gin4 phosphorylation of Shs1 on a distinct subset of residues (Mortensen et al., 2002; Asano et al., 2006). These data suggest that cell cycle-dependent phosphorylation states of Shs1 are key in regulating septin dynamics. However, arguing against this hypothesis, strains expressing Shs1 alleles with most of the residues phosphorylated by Cdks mutated to either to Ala or phosphomimetic Asp displayed no discernible septindefective phenotype (Finnigan et al., 2015b). Furthermore, most of these phosphorylation sites are within a poorly conserved segment of Shs1, and deletion of this entire region does not lead to any loss of Shs1 function in vivo (Finnigan et al., 2015b). Perhaps phosphorylation of Shs1 by Cdks and Gin4 is redundant with additional mechanisms that regulate septin assembly, but the precise consequence of these phosphorylation events on septin structure and/or function remains unclear. In marked contrast, Shs1 is phosphorylated at a single site by the protein kinase Rad53 in response to DNA damage and replication stress (Smolka et al., 2006, 2007) and a phosphomimetic mutation of this single residue displays a prominent growth defect (Finnigan et al., 2015b), suggesting that this direct phosphorylation might represent a checkpoint whereby cell cycle progression can be delayed (via perturbation of the septin collar) to permit time for repair of the DNA damage.

The purported role that Gin4 has in proper septin collar assembly at the bud neck has very low penetrance (Longtine et al., 1998) and appears to represent only a kinetic delay (McMurray and Thorner, 2009). Given the close apposition of septin structures with the PM in the cell (Byers and Goetsch, 1976; Bertin et al., 2012) and the effect of lipids on the state of septin assembly in vitro (Bertin et al., 2010; Bridges et al., 2014), the phenotype exhibited by gin41 mutants might be explained by the lack of proper control of Fpk1 function and the ensuing effects on local PM lipid composition. A consequence of Gin4-mediated inhibition of flippase function (via inhibition of the flippase-activating protein kinase Fpk1) is a pronounced reduction in the inner leaflet concentration of PtdEth in the PM (Roelants et al., 2015). A low inner leaflet PtdEth level leads to activation of Cdc42 by suppressing the function of Cdc42-specific GAPs (Saito et al., 2007; Das et al., 2012), and activated Cdc42 plays a significant role in localized tethering of the factors needed for initial recruitment of septins to the site of incipient bud emergence (Iwase et al., 2006; Okada et al., 2013).

# OUTLOOK AND PROSPECTUS

Depending on their assembly state, septin-based structures provide dynamic platforms from which the action of a significant number of protein kinases can be directed both spatially and temporally. Moreover, as observed for other protein kinase-scaffold interactions (Ferrell, 2000; Good et al., 2011; Langeberg and Scott, 2015), signaling emanating from septin-associated kinases can be channeled to particular colocalized targets conferring specificity and can be insulated from improper substrates to ensure fidelity. Moreover, where necessary, co-recruitment of multiple protein kinases permits signal propagation in the appropriate sequence and enables cross-talk to elicit coincident and combinatorial outputs.

Moreover, these septin structures serve as sensors that transmit upstream cues, such as cell cycle timing and membrane curvature, to their associated kinases. Certain of these kinases also regulate septin structure and organization, establishing an extremely complex feedback system which is yet to be fully understood. As highlighted through the course of the above discussion, there are still many mechanistic aspects of the control of septin-associated protein kinases that remain to be delineated. Hence, this area of cell biology and biochemistry remains a fertile area for exploring the role of cellular structures in regulating signaling enzymes, and vice versa.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

AP, GF, and FR prepared the draft and JT edited and submitted the final version of the manuscript.

#### ACKNOWLEDGMENTS

This work was supported by a postdoctoral fellowship from the Adolph C. and Mary Sprague Miller Institute for Basic Research in Science (to GF), and National Institutes of Health R01 Research Grants GM101314 (to JT and colleague Eva Nogales) and GM21841 (to JT).

promoting cleavage furrow and septum formation in S. cerevisiae. J. Cell Biol. 185, 995–1012. doi: 10.1083/jcb.200903125

signaling during polarized cell growth via Rga GTPase-activating proteins. Dev. Cell 13, 743–751. doi: 10.1016/j.devcel.2007.09.014

**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 © 2016 Perez, Finnigan, Roelants and Thorner. 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) or licensor 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.

# Analysis of Septin Reorganization at Cytokinesis Using Polarized Fluorescence Microscopy

Molly McQuilken1, 2, Maximilian S. Jentzsch<sup>2</sup> , Amitabh Verma<sup>3</sup> , Shalin B. Mehta<sup>3</sup> , Rudolf Oldenbourg3, 4 and Amy S. Gladfelter 1, 3 \*

*<sup>1</sup> Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, <sup>2</sup> Department of Biological Sciences, Dartmouth College, Hanover, NH, USA, <sup>3</sup> Marine Biological Laboratory, Bell Center for Regenerative Medicine, Woods Hole, MA, USA, <sup>4</sup> Department of Physics, Brown University, Providence, RI, USA*

Septins are conserved filament-forming proteins that act in diverse cellular processes. They closely associate with membranes and, in some systems, components of the cytoskeleton. It is not well understood how filaments assemble into higher-order structures *in vivo* or how they are remodeled throughout the cell cycle. In the budding yeast *S. cerevisiae*, septins are found through most of the cell cycle in an hourglass organization at the mother-bud neck until cytokinesis when the collar splits into two rings that disassemble prior to the next cell cycle. Experiments using polarized fluorescence microscopy have suggested that septins are arranged in ordered, paired filaments in the hourglass and undergo a coordinated 90◦ reorientation during splitting at cytokinesis. This apparent reorganization could be due to two orthogonal populations of filaments disassembling and reassembling or being preferentially retained at cytokinesis. In support of this idea, we report a decrease in septin concentration at the mother-bud neck during cytokinesis consistent with other reports and the timing of the decrease depends on known septin regulators including the Gin4 kinase. We took a candidate-based approach to examine what factors control reorientation during splitting and used polarized fluorescence microscopy to screen mutant yeast strains deficient in septin interacting proteins. Using this method, we have linked known septin regulators to different aspects of the assembly, stability, and reorganization of septin assemblies. The data support that ring splitting requires Gin4 activity and an anillin-like protein Bud4, and normal accumulation of septins at the ring requires phosphorylation of Shs1. We found distinct regulatory requirements for septin organization in the hourglass compared to split rings. We propose that septin subpopulations can vary in their localization and assembly/disassembly behavior in a cell-cycle dependent manner at cytokinesis.

Keywords: septins, cytokinesis, polarized light microscopy, Shs1

# INTRODUCTION

Septins are a conserved family of cytoskeletal GTP-binding proteins that function in many cellular processes including cytokinesis, cell polarity, and membrane remodeling in many eukaryotic cell types (Gilden and Krummel, 2010; Mostowy and Cossart, 2012; Khan et al., 2015). To contribute to these diverse processes, septins polymerize into filaments and then assemble into higher-order structures associated with the cell cortex (Frazier et al., 1998; Bertin et al., 2008; Spiliotis and Gladfelter, 2012; Bridges et al., 2014). In the budding yeast Saccharomyces

#### Edited by:

*Manoj B. Menon, Hannover Medical School, Germany*

#### Reviewed by:

*Gregory Charles Finnigan, Kansas State University, USA Qi Cao, University of Maryland, USA Anne Paoletti, Curie Institute, Poland*

> \*Correspondence: *Amy S. Gladfelter<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Signaling, a section of the journal Frontiers in Cell and Developmental Biology* Received: *13 January 2017*

Accepted: *05 April 2017* Published: *03 May 2017*

#### Citation:

*McQuilken M, Jentzsch MS, Verma A, Mehta SB, Oldenbourg R and Gladfelter AS (2017) Analysis of Septin Reorganization at Cytokinesis Using Polarized Fluorescence Microscopy. Front. Cell Dev. Biol. 5:42. doi: 10.3389/fcell.2017.00042* cerevisiae, septins assemble into a ring at the new bud site and this ring transitions into an hourglass which splits into two rings at cytokinesis (Haarer and Pringle, 1987; Ford and Pringle, 1991; Kim et al., 1991). The formation of higher-order septin structures is required for proper septin function, and for decades the underlying architecture of these higher-order structures remained uncertain (Byers and Goetsch, 1976; Rodal et al., 2005; Bertin et al., 2012). Recent platinum-replica transmission electron microscopy studies provided clear ultrastructural details of septin higher-order structures in yeast over the course of the cell cycle (Ong et al., 2014). In this approach, predominantly paired filaments parallel to the mother-bud axis were detectable in the hourglass early in the cell cycle, whereas at cytokinesis two orthogonal arrays (parallel and circumferential to axis) of paired septin filaments could be seen and finally after splitting all filaments appeared to be circumferential. The exact ultrastructure in a mature hourglass prior to cytokinesis is not clear and it is possible that the circumferential filaments assemble prior to the onset of cytokinesis as the intensity of septins continues to increase through time in G2/M (Chen et al., 2011). It is not yet clear what the function is of the process of splitting the hourglass into two rings. There is evidence that the split structure forms a corral for the cytokinetic apparatus to trap it locally however it is not clear that the corral is needed for efficient cytokinesis (Dobbelaere and Barral, 2004; Wloka et al., 2011).

The budding yeast S. cerevisiae, where septins were first discovered, contains 5 mitotic septins: three septins that assembly into the core of the heteromeric rod (Cdc12, Cdc10, Cdc3) while either Cdc11 or Shs1 occupies the terminal position (Hartwell, 1971; Bertin et al., 2008; Garcia et al., 2011). In vivo it is not yet definitive the proportion of rods that have Shs1 or Cdc11 at the terminal positions or if there are rods that are polar and have Cdc11 on one end and Shs1 on the other end. The molecular role of Shs1 is unclear, since it is clearly necessary for higher order structure organization but it is not required for cells to undergo cytokinesis except in sensitized backgrounds (Hartwell, 1971; Mino et al., 1998; Garcia et al., 2011; Ong et al., 2014; Booth et al., 2015). Previous studies have assessed the role of C-terminal extension (CTE) in Shs1 localization and function in both S. cerevisiae and Ashbya gossypii (Versele and Thorner, 2004; Meseroll et al., 2012, 2013; Finnigan et al., 2015). In A. gossypii, the CTE is required to build structures of a specific size and the CTE is phosphorylated on multiple sites in vivo. The phosphorylation is not essential for ring scaling but it does contribute to protein turnover in higher-order assemblies and phosphomimetic alleles are lethal (Meseroll et al., 2012). Similarly, the CTE of ScShs1 is highly phosphorylated and the sites are not essential for viability in a sensitized background (Egelhofer et al., 2008; Garcia et al., 2011). However, whether or not the sites contribute to reorganization or splitting of the ring at cytokinesis has not been investigated. A hypothesis we test in this paper is if regulation of Shs1 is required for septin reorganization at cytokinesis.

Insights into septin organization through the cell cycle in live cells have come from using polarized fluorescence microscopy. This microscopy technique provides organizational information in addition to localization of cellular structures through fluorescence imaging. The approach takes advantage of the existence of the dipole moment of GFP, which allows GFP to be preferentially excited by and emit linearly polarized light parallel to the dipole moment (Inoué et al., 2002). If the movement of GFP is restricted relative to its septin fusion partner, then the dipole moment of GFP will act as a readout of underlying septin organization (Vrabioiu and Mitchison, 2006, 2007). Previously, polarized fluorescence microscopy was used by our group and others to assess the organization of septin higher order structures over the course of the cell cycle and found septins to be highly anisotropic (ordered). It was shown that septins rapidly and dramatically reorganize their ensemble organization by 90◦ over the course of septin ring splitting (Vrabioiu and Mitchison, 2006, 2007; DeMay et al., 2011a,b).

We set out to understand the root of this striking rearrangement of the highly-ordered septin cortex during cytokinesis. The molecular basis for a large-scale rearrangement of a network of cytoskeletal filaments between two, highly anisotropic yet orthogonal states is difficult to envision. Several models for this transition have been proposed previously: (1) a concerted rotation of the entire higher-order assembly; (2) the selective departure of a majority population of filaments that then "reveals" a second, orthogonal population; and (3) a triggered disassembly and reassembly of filaments into a new ensemble that is perpendicular to the starting state (Vrabioiu and Mitchison, 2006; DeMay et al., 2011a). Work from the Barral lab showed that the collar becomes more "fluid" at cytokinesis based on recovery from photobleaching which could be consistent with either a rotation or disassembly/reassembly model (Dobbelaere and Barral, 2004). Analysis of septin filaments in vitro have shown them to be highly flexible, that they associate with the membrane through avidity and that longer filaments are more stably bound (Bridges et al., 2014, 2016). These properties could enable a regulated fragmentation and departure of a population of filaments, key components of models 2 and 3. Strong support for the third model came from EM ultrastructural and photoactivation studies from Bi and Svitkina, making it the most well substantiated at this point, although it is still possible that circumferential rings assemble in mature hourglasses and these could be retained at cytokinesis (Ong et al., 2014). The key question is: what promotes such a swift rearrangement of molecular order within such a small window of time and space?

Our goal in this study was to begin to identify the molecular requirements of septin reorganization at cytokinesis. We assessed the organization of septins in a panel of mutant yeast strains using polarized fluorescence microscopy. Our results suggest phosphorylation-based modifications of septins, Gin4 kinase activity and the anillin-like protein Bud4 are important for maintaining a subpopulation of septins at the bud neck before, during and after cytokinesis.

# MATERIALS AND METHODS

#### Yeast Strains and Strain Construction

We generated S. cerevisiae Cdc12-conGFP (AGY169; DHD5 yeast with AGB467: pRS416-ScCDC12-conGFP3) based on rationale from previous work (Vrabioiu and Mitchison, 2006, 2007; DeMay et al., 2011a,b). pRS416-ScCDC12-conGFP3 was generated by removing nucleotides encoding 3 amino acids of the N-terminal alpha-helix of GFP and 4 amino acids of the Cterminal region of Cdc12. AGB455 (pScCDC12) was generated by amplifying ScCDC12 from S. cerevisiae genomic DNA using the oligonucleotides AGO1181 and AGO1182. The PCR product was ligated into the pRS416 plasmid (AGB441) and verified by test digestions using PspXI and SlaI and sequencing (Dartmouth College Core Facility, Hanover, NH). Next, the plasmid AGB459 (pScCdc12-GFP) was generated by first amplifying the GFP insert from the plasmid AGB005 using the oligonucleotides AGO1187 and AGO1188 and ligating the product into the digested AGB455 plasmid. Again this plasmid was verified with test digestions and sequencing (Dartmouth College Core Facility, Hanover, NH). Constraining of GFP to Cdc12 was completed using PCR to amplify the entire AGB459 using the oligonucleotides AGO1203 and AGO1204 which contain homology over the CDC12-GFP linker and this amplification removed nucleotides encoding 4 amino acids from the N-terminus of GFP and 3 amino acids from the C-terminus of Cdc12. AGB467 (pRS416-ScCDC12 conGFP) was verified by sequencing (Dartmouth College Core Facility, Hanover, NH). Once verified, AGB467 was transformed into wild-type lab strain (AGY000), and the following yeast deletion strains: bnr1∆, bni1∆, bud4∆, cla4∆, gin4∆, rts1∆, and shs1∆ (kindly provided by Erfei Bi, AGY028-AGY035 to generate AGY066-AGY073). Doug Kellogg generously supplied us with shs1 phosphomutant strains (Egelhofer et al., 2008) and the plasmid AGB467 was transformed into these strains as well to generate AGY317-AGY320. To generate strains to measure septin intensity over the cell cycle the plasmid AGB553 (E1915 YIplac128-GFP-ScCDC3:LEU a gift from Erfei Bi) was integrated into the same strains as the AGB467 plasmid generating the strains AGY131-AGY137, and AGY323-AGY327. All strain, plasmid, and oligonucleotide information is present in **Tables 1**–**3**.

# Yeast Culture and Preparation

For imaging, S. cerevisiae cells were grown overnight in appropriate media with proper selection for the specific plasmids, collected by centrifugation, and resuspended in 2x low fluorescence minimal media (LFMM). Cells were mounted on gel pads of 1.4% agarose and LFMM on glass depression slide, covered with a coverslip (no. 1.5), sealed with Valap, and imaged.

#### Time-Lapse Imaging and Intensity Analysis

Time-lapse recordings for estimating septin intensity of mutant strains through the cell cycle were acquired with a Nikon Eclipse Ti-E inverted wide-field microscope equipped with a 60x (1.4 N.A.) plan-apochromat oil objective and a Andor Zyla 4.2 sCMOS camera. A Chroma DAPI/FITC/TRITC/Cy5 quad filter set was used for fluorescent imaging of GFP. The fluorescent light source was a Spectra LED lamphead and images were acquired with 12% laser power, 100 ms exposure, and 90 s time intervals.

Using FIJI, individual cells were cropped from the timelapse recordings and the mean background-subtracted septin intensity at the mother-bud neck were determined for every time point for a given cell using MATLAB. These septin intensity values were saved and plotted in an Excel file. For every cell, a time zero (**t0**) was manually determined. This **t0** represented the maximum septin intensity value close (not more than 4– 5 time points) to the visible ring split. In most cases the quantitatively determined **t0** was not more than two time points (3 min) away from the visible split. This quantitative approach was taken to account for different visible split times depending on how a given image was automatically contrasted by FIJI. **t0** therefore represented the quantitative onset of ring splitting. Since the primary focus of this study was the septin concentration change during ring splitting, all following analyses were limited to 24 min before and after **t0**. Percent change over the entire 24 min was determined and reported in **Table 4**. Since the majority of the strains analyzed showed biphasic septin disassembly rates over the course of ring splitting, the disassembly rate was determined for both phases of disassembly (phase 1 and phase 2). Phase 1 and phase 2 were distinguished by the different slopes of the biphasic intensity curves. If a strain did not show biphasic disassembly, then it is reported as monophasic and the intensity from t = 0 to t = 24 min after splitting; Phase 2 is the rate of disassembly from the last time point of Phase 1 to t = 24. After disassembly rates were determined, the fold change compared to wild-type was calculated.

### Polarization Microscopy

All polarization fluorescence images (except the shs1 phosphomutant strains) were acquired using the MF-PolScope (Abrahamsson et al., 2015). The MF-PolScope was set up on an inverted Olympus IX-83 microscope body, equipped with an Olympus 60x (1.3 N.A.) silicon-oil immersion objective, and the LC-compensator for polarization-controlled excitation light, with a green 515/30 nm emission filter (Semrock) placed before an EMCCD camera (Andor iXon-888). The fluorescence light source used was a blue LED module of an X-cite XLED1 (Lumen Dynamics). Images were acquired through Micro-manager (www.micro-manager.org) using the OpenPolScope software (www.openpolscope.org).

Polarized fluorescence measurements of the shs1 phosphomutant strains and bud4∆ splitting events were acquired with the Zeiss Axio Imager.M1 wide-field microscope with five linear polarized filters (Chroma 21033a) in a filter wheel (Ludl Electronic Products, cat. no. 99A075) and UV blocker 420 nm LP emission filter 32 nm (Chroma, cat. no. E420LPv2-32). The fluorescent light source used was an EXFO X-Cite 120 lamp. Images were acquired through Micro-manager (www.micro-manager.org) using the OpenPolScope software (www.openpolscope.org).

Once acquired, all polarization images were analyzed using the OpenPolScope software. Analysis involved internal background corrections of every cell, performed by selecting a ROI in the cytoplasm, and a ROI in the septin hourglass structure and then processed for anisotropy. The anisotropy formula calculates <sup>I</sup>max−Imin Imax+Imin in each pixel, based on the bleach corrected fluorescence intensity values. Circular statistics were used to calculate the ensemble orientation and anisotropy for each

#### TABLE 1 | Yeast strains used in this study.

#### TABLE 2 | Plasmids used in this study.

given septin structure. To do this, a 6 × 6 pixel ROI was selected in the center of the septin structure so as to exclude edges of the septin structure with isotropic measures from GFP β-barrel orientation along the curve of the septin ring. The pixel-by-pixel orientation, anisotropy, and intensity for each septin structure was then exported, and used to calculate the

#### TABLE 3 | Oligonucleotides used in this study.

#### TABLE 4 | Average percent disassembly, rate change, rate fold change for each phase of disassembly observed.

*Rate fold change is normalized to the relevant wild-type background, Phase 1 was identified visually by the first rate of disassembly in the slopes of the biphasic intensity curves. If a strain did not show biphasic disassembly, then it is reported as monophasic and the intensity from t* = *0 to t* = *24 min after splitting; Phase 2 is the rate of disassembly from the last time point of Phase 1 to t* = *24.*

\**bud4*∆ *decreased in intensity faster than wild-type yeast; therefore, disassembly rate was determined only for the first 7.5 min.*

ensemble orientation weighted by anisotropy and intensity of each ring, and of the entire population of cells. In addition we also calculated the population variance (PopVar = 1 − ( Vector Average Anisotropy∗Intensity Scalar Anisotropy∗Intensity )). A detailed description of how to use the OpenPolScope software for polarization imaging and analysis is present in Current Protocols in Cell Biology (McQuilken et al., 2015).

#### RESULTS

### Septin Concentration at the Bud Neck Decreases at Ring Splitting for Multiple Septin Subunits

Previous data from our lab and others have shown that there is a 90◦ reorientation of the organization within the septin hourglass structure over the course of septin ring splitting at cytokinesis in S. cerevisiae cells (Vrabioiu and Mitchison, 2006; DeMay et al., 2011a,b). We had hypothesized in our work that one explanation of the apparent reorganization is that there are two populations of septins in the hourglass and this could be seen by Ong et al. (2014) at least at the onset of cytokinesis. Upon ring splitting, one of these sub-populations is thought to leave the bud neck and another population assembles and/or is retained perpendicular to the original population. What drives the disassembly and reassembly process of septins within such a small region of the cortex in a short window of the cell cycle?

We hypothesized that whether a given septin filament is released or stays localized at the neck could be explained by the composition of septin heteromeric rods, specifically the terminal subunit, which is occupied by either Cdc11 and/or Shs1. If Cdc11 or Shs1 specified which complexes were retained or exited from the neck, we predicted that Cdc11 and Shs1 would display different rates of change in concentration at splitting. To assess this, we monitored the abundance of 3 different septins at the mother-bud neck over the course of septin ring splitting. We used widefield fluorescence microscopy to image GFP-Cdc3, Shs1- GFP (in two different strain backgrounds), and Cdc11-GFP every 90 s to ensure sufficient time resolution for assessing intensity differences and kinetics (see Materials and Methods). Using two different Shs1-GFP strains we observed the accumulation of Shs1 in the septin hourglass is only ∼50% of the level of either Cdc3 or Cdc11 (**Figure 1A**). Interestingly, we also observed that the decrease of all three septins over the course of ring splitting occurred in a biphasic manner. The first phase occurred within the first ∼10 min after ring splitting. This first phase of septins leaving the bud neck is consistent with the timing of the isotropic "transition" period observed previously (DeMay et al., 2011a). Time-lapse imaging indicated that all septin proteins decrease in abundance with comparable rates during ring splitting and decrease by ∼50% in concentration over the first phase of disassembly (phase 1), regardless of their starting abundance

(**Figure 1A**, **Table 4**, and consistent with what was seen for Cdc3 in Dobbelaere et al., 2003; Wloka et al., 2011). When we measure the difference in disassembly rates over the first phase of disassembly between the different septins there was little difference in the rates (**Table 4**). Additionally, when Cdc11 and Shs1 are tagged in the same cell with different fluorophores, the decrease in abundance at the neck is nearly simultaneous for each protein, further indicating that these two subunits behave similarly with regard to septin dynamics in this part of the cell cycle (discrepancy between times of −0.3 ± 0.3 min, N = 40 cells, difference not significant from 0 with p > 0.41). These data indicate that the difference between the septin populations

FIGURE 1 | The formins, Bni1 and Bnr1, are not required for the reorganization of the septin ring. (A) Septin intensity of Cdc11-GFP (red), GFP-Cdc3 (blue), Shs1-GFP (green), and Sep7-GFP (purple) over the course of septin ring splitting (time = 0). Error bars represent standard error for each time point (*N* = 100 cells). (B) Septin intensity of GFP-Cdc3 in wild-type (black), *bni1*∆ (purple), and *bnr1*∆ (red) over the course of septin ring splitting (time = 0). Error bars represent standard error for each time point (*N* = 100 cells). (C) Representative polarization images of wild-type, *bni1*∆ and *bnr1*∆ cells expressing ScCdc12-conGFP in the hourglass and split structures. The blue lines represent the calculated dipole orientation and their length is scaled according to anisotropy. Scale bar 0.5 µm. (D) Polar plots of net dipole orientations for individual hourglass and split septin rings scaled by anisotropy (hourglass *N* > 45 cells, split rings *N* > 8 cells). (E) Scatter plot of net dipole orientations for individual hourglass and split septin rings in three different focal planes. Color scale indicates visible phenotypes of septin rings [broken ring (tan), improper ring size (orange), (hourglass *N* > 30 cells, split rings *N* > 8 cells)]. (F) Difference in net orientation measures between top and bottom focal planes for hourglass and split structures. Error bars denote standard error. (G) Population variance in hourglass and split ring structures.

that stay associated with the cortex and that are released into the cytoplasm is not simply the presence or absence of Cdc11 or Shs1.

# Septin Reorganization Does Not Require the Formins

We next used time-lapse imaging and polarization microscopy to assess the contribution of septin interacting proteins on the organization and behavior of septins in the splitting transition. We first tested the hypothesis that a subset of septins could be specified to disassemble or be anchored via connections with F-actin assemblies. The yeast formin Bnr1 is regulated by Shs1 indicating these two factors are functionally interacting at this window of the cell cycle (Buttery et al., 2012). We assessed how the two formins, Bni1 and Bnr1, influence septin intensity changes and reorganization over septin ring splitting (**Figure 1B**). In strains lacking formins we observed a decrease in the initial intensity of septins in the hourglass, suggesting the actin cytoskeleton contributes partially to the accumulation of septins at the mother bud neck prior to ring splitting. This defect was most severe in bni1∆, where septin intensity at the hourglass was reduced by ∼65% relative to the hourglass in wild-type cells. However, the rate at which the septin sub-population exited the bud-neck during ring splitting was again observed as a biphasic disassembly, was not substantially affected in any of the strains (0.7-fold faster for phase 1 and 0.5-fold slower for phase 2 in bni1∆ and 0.6-fold faster in phase 1 and no fold change difference for phase 2 bnr1∆, **Table 4**) consistent with previous reports (Feng et al., 2015).

We next asked if the formins were required for the characteristic 90◦ reorganization. To address this, we used polarization microscopy to measure the organization of the septin hourglass and split rings in wild-type, bni1∆, and bnr1∆ strains containing Cdc12-conGFP (see Materials and Methods, **Figures 1C–E**). Specifically, we used multifocus (MF-PolScope) microscopy to simultaneously capture different Z-positions along with polarized fluorescence information so as to determine if orientation was comparable at all positions in the ring at the same time (Abrahamsson et al., 2015). When the orientation is consistent between Z-slices of the top, middle, and bottom of septin assemblies, we have interpreted this to mean that the prevailing population of septins are "paired filaments" (DeMay et al., 2011a,b).

As previously published from our lab, the septin hourglass of wild-type cells expressing Cdc12-conGFP exhibit GFP dipoles with a net orientation perpendicular to the mother-bud axis, while septin rings exhibit a net orientation parallel to the mother-bud axis (DeMay et al., 2011a,b). The net orientation of GFP dipoles in bni1∆ and bnr1∆ are comparable to wild-type on average however the anisotropy is lower for both mutants suggesting a slightly less well-ordered structure (**Figure 1D**, **Table 5**). We categorized each orientation data point by the state of the ring (broken, aberrant size) but could not see any correlation between grossly misorganized rings and orientations substantially deviating from 90◦ (**Figure 1D**). There seems to TABLE 5 | Average anisotropy for all strains.

*HG* = *Hourglass and S* = *Split.*

be little difference between the net orientation in the top, middle, and bottom focal planes in the two formin mutants suggesting pairing or symmetric organization relative to the plasma membrane (**Figures 1E,F**). In addition, we also estimated the population variance, which is a measure of the difference in average dipole orientation between cells with the same type of structure, and found little difference in each strain over the entire population compared to wild-type cells (**Figure 1G**). These results suggest that although Bni1 and Bnr1 are required for the proper accumulation and organization of septins in the septin hourglass, they are not required for proper timing of disassembly and reorientation of septins at ring splitting (**Figures 1B–E**).

## Septins in Cells Lacking bud4 Have Highly Misorganized Rings after Splitting

We next examined other septin interacting proteins beginning with Bud4, an anillin-like protein. Bud4 has been shown to associate with septins and mutants do not have two split septin rings making them a key candidate in regulation of septin orientation in this process (Wloka et al., 2011). We looked at septin intensity through time over ring splitting in the bud4∆ mutant. We observed septins leave the bud neck in a monophasic manner (2-fold faster than wild-type in phase 1, and 3.5-fold faster than wild-type in phase 2). This rapid disassembly from the bud neck is consistent with previous data that Bud4 is required for stability of split septin rings during and after cytokinesis (**Figure 2A**, **Table 4**, Wloka et al., 2011; Eluère et al., 2012; Kang et al., 2013). The ring disassembled asymmetrically with one side much faster than the other but the analysis here was averaged across the whole ring to be consistent with other measurements in the study. Similar to bnr1∆ mutants, bud4∆ mutants showed a reduced septin accumulation within the hourglass with ∼70% the intensity observed in wild-type hourglasses.

Using polarization microscopy, we observed little difference in terms of septin hourglass orientation, a slight decrease

(D) Polar plots of net dipole orientations for individual hourglass and split septin rings scaled by anisotropy (hourglass *N* > 50 cells, *N* = 18 cells, respectively). (E) Difference in net orientation measures between top and bottom focal planes for hourglass and split structures. Error bars denote standard error. (F) Population

in anisotropy, and comparable population variance across all focal planes compared to wild-type (**Figures 2B–F**, **Table 5**). Any variability in GFP dipole orientation observed in the bud4∆ mutant could not be explained by visible septin defects (**Figure 2C**). However, when we analyzed the orientation of split rings in the bud4∆ we observed a severe decrease in anisotropy, across all focal planes suggesting that Bud4 is also required for proper organization of septin split ring structures (**Figures 2C–F**, **Table 5**). This is again consistent with previous data that Bud4 is required for the stability of the septin split rings (Wloka et al., 2011; Eluère et al., 2012; Kang et al., 2013).

variance in hourglass and split ring structures.

### Gin4, but Not Cla4 or Rts1, Contributes to Rate of Disassembly at Splitting

The isotropic transition between the two orthogonal organization states is quite transient, lasting just a few minutes, indicating that the cue to direct disassembly and/or reassembly could be a posttranslational modification. Recipients of the modification could be a subpopulation of septins, a septin regulator and/or modifiers of the local membrane composition.  Both gin4∆ and cla4∆ cells showed a decrease in septin accumulation in the hourglass, both exhibiting ∼50% reduction in intensity relative to the wild-type hourglass (**Figure 3A**, green and gold lines). In addition, we observed that septins do not leave the bud neck in a biphasic manner in the gin4∆ mutant (**Figure 3A**). Instead, septins leave the bud neck at a constant rate that is slower in both phases compared to wild-type cells (∼75% the rate of wild-type in phase 1, and ∼30% slower than the rate wild-type in phase 2, **Table 4**). In contrast, in cla4∆ cells septins still left the bud neck in a biphasic manner similar to the rate seen in wildtype cells (0.1-fold faster than wild-type in phase 1, and 0.4-fold faster than wild-type in phase 2, **Table 4**). Notably, Rts1 had little effect on septin accumulation in the hourglass, and no effect on disassembly rate relative to wild-type cells (**Figure 3A**, **Table 4**). Thus, Gin4, and to a lesser extent Cla4, are required for the sharp decrease in septin abundance at the neck that accompanies ring splitting.

We next used polarization microscopy to measure the organization of the septin hourglass and split rings in cla4∆, gin4∆, and rts1∆ mutant strains (**Figures 3B–D**). In cla4∆ and rts1∆ cells the spread of orientations for the GFPdipole in the hourglass increased (thus the population variance increased), but the net orientation remained ∼90◦ with some decrease in anisotropy (**Figures 3C,D,F**, **Table 5**). In both cla4∆ and rts1∆ strains the split structure was not appreciably different from wildtype (**Figures 3B–D**). Unlike cla4∆ and rts1∆, organization within the septin hourglass in the gin4∆ mutant was highly disorganized, while the split structure maintained

pSc*CDC12*-conGFP, and *rts1*∆ containing Sc*CDC12*-conGFP in the hourglass and split structure. The blue lines represent the calculated dipole orientation and their length is scaled according to anisotropy. Scale bar 0.5 µm. (C) Scatter plot of net dipole orientations for individual hourglass and split septin rings in three different focal planes. Color scale indicates visible phenotypes of septin rings [broken ring (tan), improper ring size (orange), (hourglass *N* > 30 cells, split rings *N* > 9 cells)]. (D) Polar plots of net dipole orientations for individual hourglass and split septin rings scaled by anisotropy (hourglass *N* > 30 cells, split rings *N* > 9 cells). (E) Difference in net orientation measures between top and bottom focal planes for hourglass and split structures. Error bars denote standard error. (F) Population variance in hourglass and split ring structures.

its ordered, net orientation of ∼0/180◦ (**Figures 3B–D**). This data indicates that Gin4 is required for ordered septin hourglass organization and efficient disassembly but not the split ring organization. As with bni1∆, bnr1∆, and bud4∆, little difference between the net orientation in the top, middle, and bottom focal planes was observed, and the increase in variability in GFP-dipole orientations within the population could not be correlated with visible septin higher-order structure defects because morphologically aberrant and normal appearing rings had a similar spread of orientations (**Figures 3C,E**). These data are consistent with a well-appreciated role for Gin4 in establishing the organization of septins but also indicates it is involved in executing the switch-like disassembly process. However, the septins that are recruited/retained after ring splitting do not require Gin4 for normal orientation or order.

## Shs1 Contributes to Organization of Hourglass, Split Rings and Timing of Splitting

It is possible that phosphorylation on a subset of septin rod complexes could generate the two distinct pools of septins at cytokinesis that are either in a state of disassembly or stay assembled at the membrane. As Shs1 has the most evidence of phosphorylation, we examined septin organization in cells lacking Shs1 (Mortensen et al., 2002; Egelhofer et al., 2008).

We first looked at septin intensity over ring splitting in a shs1∆ mutant strain. We observed a defect in the accumulation of GFP-Cdc3 to the septin hourglass (∼70% reduction compared to wild-type intensity), and similar to what we observed in gin4∆ mutant cells, a monophasic disassembly rate of septins from the bud neck slower than either observed phase in wild-type cells (0.6-fold slower than wild-type phase 1, and 0.3-fold slower than wild-type phase 2, **Table 4**, **Figure 4A**) When observed by polarization microscopy, there was a dramatic increase in the disorganization of both the hourglass and split ring structures in the shs1∆ mutant strain, consistent with what has been seen for shs1∆ mutants by cryo-EM tomography and platinum replica EM (**Figures 4B–D**, Bertin et al., 2012; Ong et al., 2014). Septin disorganization was reflected in a wide spread of GFPdipole orientations and thus an increased variance within the population, and a decrease in anisotropy (**Figures 4D,F**, **Table 5**). As with the previous mutant strains we analyzed, the difference in spread of angles was not associated with visible defects in septin structures, and no obvious difference was observed in GFP-dipole net orientation between the top, middle, and bottom focal planes (**Figures 4C,E**).

It is clear that complete loss of Shs1 leads to gross abnormalities in the construction and organization of septin structures throughout the cell cycle making it difficult to disentangle assembly defects from a role of Shs1 in the splitting and reorganization process. We hypothesized the highly phosphorylated C-terminus of Shs1 could be contributing to septin organization. We therefore, examined cells carrying shs1∆C, where the C-terminus of Shs1 is eliminated. shs1∆C cells showed a similar decrease in septin accumulation in the hourglass as the shs1∆ null strains, but interestingly still showed biphasic disassembly of septins over the course of splitting (**Figure 4A**, **Table 4**). When septin organization was assessed using polarization microscopy, both hourglasses and split rings in shs1∆C were disorganized, similar to shs1∆ (**Figure 4**, **Table 5**). Taken together these data suggest the C-terminus of Shs1 is not important for the biphasic exit of septins from the mother-but neck but is necessary for proper organization of septin higher order structures throughout the cell cycle. What aspect of the Shs1 C-terminus makes it important for septin organization but not septin disassembly?

We next analyzed the organization of septin higher-order structures in mutants of Shs1 that have altered phosphorylation sites (Egelhofer et al., 2008). Several shs1 phosphorylation defective mutants were evaluated that had been previously generated based on results from SILAC mass spectrometry experiments in the Kellogg lab. These were ps1 (Pho85 sites), ps2

FIGURE 4 | The septin Shs1 is required for the proper organization of all septin higher order structures. (A) Septin intensity of GFP-Cdc3 in wild-type (black) and *shs1*∆ (blue) over the course of septin ring splitting (time = 0). Error bars represent standard error for each time point (*N* = 100 cells). (B) Representative polarization images of wild-type containing pSc*CDC12*-conGFP, *shs1*∆ containing pSc*CDC12*-conGFP, and *shs1*∆*C* containing pSc*CDC12*-conGFP in the hourglass and split structure. The blue lines represent the calculated dipole orientation and their length is scaled according to anisotropy. Scale bar 0.5 µm. (C) Scatter plot of net dipole orientations for individual hourglass and split septin rings in three different focal planes. Color scale indicates visible phenotypes of septin rings [broken ring (tan), improper ring size (orange), (hourglass *N* > 55 cells, split rings *N* > 10 cells)]. (D) Polar plots of net dipole orientations for individual hourglass and split septin rings scaled by anisotropy (hourglass *N* > 55 cells, split rings *N* > 10 cells). (E) Difference in net orientation measures between top and bottom focal planes for hourglass and split structures. Error bars denote standard error. (F) Population variance in hourglass and split ring structures.

(CDK sites), and ps4 (Pho85/CDK sites) (**Figure 5A**; Egelhofer et al., 2008). Septin disassembly rates were notably faster in the wild-type strain background of these mutant alleles, which could be due to background mutations such as in BUD4, which is consistent with the observed asymmetric splitting also observed in certain bud4∆ strains (Voth et al., 2005). The shs1 ps mutants showed strikingly distinct assembly and disassembly defects amongst each other. shs1-ps1 and shs1 ps4 had substantially diminished levels of septins in assembled hourglasses, ∼50 and 80% reduction in intensity relative to wildtype, respectively (**Figure 5B**). In contrast, shs1-ps2 cells had similar septin levels at the hourglass compared to wild-type as gauged by GFP-Cdc3 intensity. Interestingly, none of the shs1 ps mutants had a different rate of disassembly from wild-type (**Table 4**). This is potentially a result of the strain background interfering with any differences in rates relative to one another; however, these data still suggest that phosphorylation may be able to tune the affinity of septins for membrane at the neck or septin-septin interactions for the assembly of the hourglass.

All the shs1 phosphorylation-deficient mutants were comparably highly disorganized and showed a wide distribution of orientations and cell-to-cell variability in both hourglass and asymmetric split ring structures (**Figures 5C–F**, **Table 5**). The impact of these mutations is comparable to a complete null mutant of SHS1. These data attest to the key importance of Shs1 and likely its regulation by phosphorylation in modulating the abundance and organization of septin higher-order structures but do not support a major role of these modifications in the splitting process.

#### DISCUSSION

What could specify the bifurcation of behavior within a population of septins such that disassembly and reassembly/retention processes basically coexist in time and space? Our goal was to identify features of septins themselves and regulators that might be responsible for this transition in filament organization. One hypothesis we tested was that the septin Shs1 might specify which septins are prone to departure versus reassembly/retention. The difference in abundance of the two terminal septins within the hourglass is consistent with evidence that Shs1 has the ability to cap septin rods, but not polymerize with other Shs1 containing rods (Finnigan et al., 2015). However, we did not see a substantial difference in the rate

FIGURE 5 | Shs1 phosphorylation modulates septin abundance, splitting dynamics, and organization. (A) Schematic of Shs1 protein domains with mutated phosphorylation sites highlighted for each shs1-phosphomutant (adapted from Egelhofer et al., 2008). (B) Septin intensity of GFP-Cdc3 in W303 background (black), *shs1-ps1* (blue), *shs1-ps2* (purple), and *shs1-ps4* (green) over the course of septin ring splitting (time = 0). Error bars represent standard error for each time point (*N* = 100 cells). (C) Representative polarization images of W303 background containing Sc*CDC12*-conGFP, and *shs1-*phosphomutants containing Sc*CDC12*-conGFP in the hourglass and split structure. The blue lines represent the calculated dipole orientation and their length is scaled according to anisotropy. Scale bar 0.5 µm. (D) Scatter plot of net dipole orientations for individual hourglass and split septin rings in three different focal planes. Color scale indicates visible phenotypes of septin rings [broken ring (tan), improper ring size (orange), (hourglass *N* > 26 cells, split rings *N* > 14 cells)]. (E) Polar plots of net dipole orientations for individual hourglass and split septin rings scaled by anisotropy (hourglass *N* > 26 cells, split rings *N* > 14 cells]. (F) Population variance in hourglass and split ring structures.

at which Cdc11 and Shs1 depart the neck indicating that simply different terminal subunits do not specify the subpopulations at splitting. These results do not lead to a simple explanation for specifying the different fates of the hourglass and split ring septin filaments based on differential behavior of Cdc11 and Shs1 during the transition.

We hoped that through careful analysis of splitting kinetics and organization states that we would be able to dissect the molecular control of the transition. We identified several categories of defects in both the kinetics of splitting and septin organization by combining timelapse imaging with polarized fluorescence analysis (**Table 6**): (1) Decreased abundance in the hourglass (bni1∆, bnr1∆, bud4∆, gin4∆, cla4∆, rts1∆, shs1∆, shs1∆C, and shs1-ps1 and -ps4); (2) Increased rate of disassembly (bud4∆); (3) Monophasic rate of disassembly (gin4∆, shs1∆); (4) Misorganized hourglass but organized split rings (gin4∆); (5) Organized hourglass but misorganized split rings (bud4∆); (6) Misorganized hourglass and split rings (shs1∆, shs1∆C shs1-ps1,2 and 4); and (7) correctly oriented but less ordered (low anisotropy) assemblies (bni1∆, bnr1∆, cla4∆, rts1∆). Of note, many regulators (Class1), including those influencing Factin have an appreciable impact of the abundance of septins in the hourglass structure. It is unclear whether this is due to some altered membrane composition in these mutants or direct impacts on septins that may lower their affinity for the membrane.

In this study, we had aimed to identify mutants that would have specific defects in reorientation. However, the only cases where we see definite defects in reorientation is in scenarios where the ring fails to split or the split rings are unstable such as what happens in cells lacking Bud4. A clear challenge that emerged from this study is that perturbing many of the factors that we suspect might contribute to the process leads to defects in the starting higher-order structure of the septin hourglass. In many cases these defects were not detectable until analysis with polarized light. It is difficult to disentangle the degree to which defects arise due to poor construction as opposed to an inability to respond appropriately to cell cycle triggers of the transition. Future work will need to expand this screen to the vast array of septin regulators and

Frontiers in Cell and Developmental Biology | www.frontiersin.org May 2017 | Volume 5 | Article 42 |

interacting proteins that are known to exist with the hope of still identifying a regulator that might be responsible for this transition without contributing to the organization per se. It is likely that this next phase of screening will require identifying partial loss-of-function or separation-of-function alleles because it is probable that whatever responds to or executes the signal for reorientation also contributes to overall septin organization.

Despite the constellation of phenotypes, there are a few key aspects of the data that point to potential mechanisms relevant to the splitting transition. In particular, consistent with previous observations, Bud4 is required for a proper split ring state (Wloka et al., 2011; Eluère et al., 2012; Kang et al., 2013). We were interested to note that the reorientation did not occur in the absence of splitting. This suggests that the reorientation is concomitant with and potentially requires the splitting process. One possible reason for this is that the reorientation also involves reacting to changes in local membrane curvature and composition that are happening at cytokinesis and also contribute to the reassembly process. On a molecular level, the data indicate that either Bud4 is required for receiving the cell cycle cue of splitting and/or is an essential guide or anchor for the reassembly process that creates the split ring structures.

Another clue emerges from the similar behavior of gin4∆ and shs1∆ which both lose the biphasic disassembly observed in wild-type cells, and thus are slow in going through the transition. This suggests that Gin4 is targeting either Shs1 or is impacting a feature of the transition that Shs1 is required for as well. In the absence of Gin4 activity or Shs1, septins remain associated with the membrane longer. This could be due to Gin4 phosphorylating a subpopulation of septins or other substrates that influence septin affinity for the membrane. We assessed the role of septin phosphorylation (which may or may not be due to Gin4) and see that non-phosphorylatable mutants altered septin levels in the hourglass and monophasic disassembly. Unfortunately, uncertainty about the background of these mutants with regard to the BUD4 locus makes it challenging to interpret the rate of disassembly in these strains because the disassembly was more rapid than expected in controls. Analysis of an shs1 mutant that lacks the entire C-terminus was illuminating in that it also has a major defect in the recruitment of septins to the hourglass however it shows a biphasic disassembly that is not slowed compared to wildtype, unlike the shs1 null. This mutant still retains a phosphorylation site at T6 that is missing in the PS alleles, pointing to this residue as a potentially important site for regulating contact with the membrane for both assembly of the hourglass and release of septins at splitting. Overall, the data point to Gin4 and Shs1 as modulators of the rate of change in septin dynamics and orientation at late stages of the cell cycle. It is likely that this process involves phosphorylation-dependent changes in membrane composition and/or septin-membrane binding that change the affinity of septins for the membrane and possibly change the preference of septins to bind membranes of specific curvatures.