**2. Biogenesis during trans-Golgi network vesicle trafficking**

Secreted proteins are translated at the rough endoplasmic reticulum and transit from the ER, through the ER-Golgi Intermediate Compartment (ERGIC) to the Golgi and subsequently the trans-Golgi network (TGN) (Shilo and Schejter 2011). Post-translational modifications such as glycosylation occur in the ER and Golgi. Membranes on the trans side of the Golgi apparatus form dynamic tubular reticular structures having a large surface area (De Matteis and Luini 2008). This network of saccules and tubes is continuously remodeled such that both the structure and size of the TGN varies depending on the secretory activity and the cell type (Trucco et al. 2004). Selective trafficking at the TGN will sort cargo into vesicles or carrier tubules (De Matteis and Luini 2008), and this sorting requires the genesis of carrier vesicles targeted to specific membranes within the cell. As noted above, these vesicles are distinguished by the combination of coat proteins on the cytosolic face of the membrane which determine the target membrane for that vesicle. In some pathways additional sorting occurs at the recycling endosome (reviewed in (De Matteis and Luini 2008; Santiago-Tirado and Bretscher 2011)).

The initiating event in vesicle biogenesis may be driven by the local membrane lipid composition where asymmetry in the types of lipid in the two faces of the membrane bilayer can induce bending (van Meer and Sprong 2004). Initiation may also involve membrane rafts, which are reported to be present in the TGN of all cells (Park and Loh 2008), and on membranes of secretory granules (Hosaka et al. 2004; Lang 2007; Guerriero et al. 2008). While membrane rafts are well characterized to play important roles in endocytosis at the plasmalemma, their role in vesicle formation at the TGN is not as well understood. Many vesicle coat proteins have been localized to lipid rafts on vesicles (Puri and Roche 2006). This has been suggested to be important for the formation of the coat protein complexes on the TGN for the initial creation of vesicle buds and selective trafficking (Simons and Sampaio 2011). For example, SNARE proteins are enriched in cholesterol-dependent rafts in beta-cells and PC-12 cells (Lang 2007). However, technical issues have called into question the validity of some methods for isolation of 'lipid rafts', leading to a more stringent definition and the term 'membrane rafts' (Lang 2007). Nonetheless, even with the more stringent approach, SNAREs such as VAMP2 and VAMP3 are enriched in membrane rafts of vesicles. Membrane rafts can contain different complements of proteins due to specific protein interactions. One model for the formation of vesicle buds is that membrane rafts on the TGN (with associated transmembrane cargo proteins) coalesce creating a lipid domain that is favorable to bending the membrane, and containing transmembrane proteins which can facilitate the decoration of the cytosolic face of the membrane with adaptor and other coat proteins (De Matteis and Luini 2008; Simons and Sampaio 2011). Testing the relevance of this model to selective sorting for secretion in living cells is important, and requires determining whether trafficking vesicles in different pathways (e.g., regulated secretion versus constitutive secretion pathways) contain different types of membrane rafts, or that

Phosphatidylinositol Bisphosphate Mediated Sorting of Secretory Granule Cargo 43

has not been well characterized in any cell type. Recently, PtdIns(3,4)P2 was found to be transiently synthesized at the plasmalemma as a second messenger of platelet-derived growth factor (PDGF) (Hogan et al. 2004). Importantly, our understanding of the roles of PtdInsPs derives primarily from studies of yeast and a few mammalian cell lines. It is unclear whether these generalizations carry over to well differentiated cells in tissues. For example, we find high levels of expression of PtdIns(3,4)P2 in parotid secretory granule

Discrete and dynamic localization of PtdIns-kinases (PI-kinases) and PI-phosphatases regulate the production of PtdInsPs. Of these enzyme families, PI4-kinases have the dominant roles in Golgi function and secretion, although PI3-kinases and PI5-kinases also have roles in secretion (Wang et al. 2003; Roth 2004; Balla and Balla 2006). Several PI4 kinases are localized to the Golgi, TGN, or endosomes, and form complexes with several coat proteins including GGA and FAPP1/2 (D'Angelo et al. 2008). Careful immunofluorescence co-localization studies with MDCK cells have found PI4KIIα in the TGN and PI4KIIIβ in the cis/medial Golgi (Weixel et al. 2005). This is consistent with PtdIns4P being the most abundant PtdInsP in the Golgi. PI4KIIIβ is recruited by the coat protein Arf1 to the Golgi, and in turn PI4KIIIβ recruits Rab11 and the PtdIns4P-binding protein FAPP1 (Godi et al. 2004). Knock-down of PI4KIIα by RNAi has little effect on intra-Golgi transit, but inhibits TGN export of vesicles (Wang et al. 2003). These studies are interpreted as showing that PI4-kinases produce PtdIns4P on the cytosolic leaflet of the TGN and vesicles, which anchors essential adaptor proteins (Santiago-Tirado and Bretscher 2011). Importantly, class I PI3-kinases are also present on Golgi membranes, and are essential for tumor necrosis factor (TNF) secretion by macrophages (Low et al. 2010). Similarly, PI3 kinase was localized to secretory granules of PC12 cells by both immunofluorescence and cell fractionation (Meunier et al. 2005). Transfection of a PtdIns3P-binding domain (FYVE), or a catalytically-inactive PI3-kinase, blocked regulated secretion, possibly by interfering with fusion of the granule at the plasmalemma (Meunier et al. 2005). In summary, certain PIkinases decorate the Golgi and TGN, interact with coat proteins, and produce PtdInsPs. These PtdInsPs are important for vesicle trafficking, however, the identities and roles of PIkinases have not been investigated in cells that are highly specialized for bulk exocrine

The presence of multiple different trafficking pathways for secretion has been described in many cell types (Dikeakos et al. 2007; De Matteis and Luini 2008; Park et al. 2008; Folsch et al. 2009; Perez et al. 2010; Stanley and Lacy 2010; Lacy and Stow 2011; Santiago-Tirado and Bretscher 2011). The best characterized of these pathways have a specific cargo protein that is a unique marker of that route, which is essential for molecular characterization (Lara-Lemus et al. 2006). For example, the cytolytic protein perforin of natural killer cells undergoes polarized secretion into the immune synapse, whereas the same cells secrete TNF in a non-polarized pattern (Reefman et al. 2010) thereby marking a different pathway. Also, some pituitary gonadotropes segregate luteinizing hormone into separate granules than follicle-stimulating hormone for different regulated secretion dynamics (Nicol et al. 2004). Alternatively, pathways can be marked by the use of different coat proteins. For example, the coat protein FAPP1 is essential for secretion by the basolateral pathway, whereas FAPP2

membranes.

secretion, such as the parotid.

**3. Selective trafficking for secretion**

some pathways lack membrane rafts. Importantly, Guerriero et al. recently found that raftindependent and raft-associated proteins collect in distinct sites at the Golgi, and likely enter different vesicles (Guerriero et al. 2008).

#### **2.1 Phosphatidylinositol phosphates in biogenesis of secretory vesicles**

In all cell types, the earliest events that are strongly linked to the biogenesis of secretory vesicles is the binding of coat proteins to phosphatidylinositol phosphate lipids (PtdInsP) and PI-kinases in the TGN membrane. The phosphoinositides and small GTPases of the Arf and Rab families define the identity of the membrane and recruit additional coat proteins (Di Paolo and De Camilli 2006; De Matteis and Luini 2008; Santiago-Tirado and Bretscher 2011). PtdInsPs are recognized as being critical for selective trafficking of vesicles within cells (Di Paolo and De Camilli 2006; D'Angelo et al. 2008; Vicinanza et al. 2008; Graham and Burd 2011). Phosphatidylinositol comprises less than 10% of membrane phospholipids, and the individual phosphorylated forms total less than 1.5% of lipids (Di Paolo and De Camilli 2006; Roth 2004). Phosphatidylinositol can be phosphorylated on any combination of carbons 3, 4, or 5 of the inositol ring (Fig. 1). The most abundant, PtdIns4P, occurs at approximately 0.05% of membrane lipids, whereas the low abundance forms such as PtdIns(3,4)P2 or PtdIns(3,4,5)P3 are approximately 0.0001% each (Cullen 2011). Subcellular pools of the 7 different PtdInsPs have diverse regulatory roles in cytoskeleton remodeling, second messenger signaling, endosomal trafficking, membrane trafficking, osmotic stress, nuclear signaling, and other aspects of cell physiology (Godi et al. 2004; Balla and Balla 2006; Di Paolo and De Camilli 2006). Cellular effects can be mediated by signaling through production of second messengers (diacylglycerol, and inositol trisphosphate). However, many cellular effects are mediated by the localized anchoring of cytosolic proteins having specific PtdInsP-binding domains (e.g., PH, FYVE, PX, ENTH-domains) (De Matteis et al. 2005; Balla and Balla 2006). For example, Arf1 directly binds and recruits PI4-Kinases to the TGN. This produces PtdIns4P which aids in recruitment of coat proteins such as AP1, GGAs, VAMPs, and FAPP1/2 most of which interact with PtdIns4P as well as other proteins in the coat matrix (Balla and Balla 2006). This interaction provides identity to the membrane (reviewed in (Santiago-Tirado and Bretscher 2011)). PI3K-C2α is also present on the TGN. Each type of PtdInsP is localized to specific membranes within the cell. The most abundant phosphoinositides, PtdIns4P, and PtdIns(4,5)P2, are predominantly localized to the Golgi and plasma membrane, respectively, whereas PtdIns3P and PtdIns(3,5)P2 are predominantly localized to early and late endosomes (Di Paolo and De Camilli 2006; Santiago-Tirado and Bretscher 2011). PtdIns(3,4)P2 is rare in resting cells, but is present in the plasmalemma and multivesicular and early endosomes (Roth 2004; Di Paolo and De Camilli 2006). PtdIns(3,4)P2 is not noted as being present in the Golgi or TGN (De Matteis et al. 2005), however, this

Fig. 1. Structure of Phosphatidylinositol (3,4)bisphosphate. PtdIns(3,4)P2 is an exceedingly rare membrane lipid that is highly localized to specific subcellular membranes within the cell.

some pathways lack membrane rafts. Importantly, Guerriero et al. recently found that raftindependent and raft-associated proteins collect in distinct sites at the Golgi, and likely enter

In all cell types, the earliest events that are strongly linked to the biogenesis of secretory vesicles is the binding of coat proteins to phosphatidylinositol phosphate lipids (PtdInsP) and PI-kinases in the TGN membrane. The phosphoinositides and small GTPases of the Arf and Rab families define the identity of the membrane and recruit additional coat proteins (Di Paolo and De Camilli 2006; De Matteis and Luini 2008; Santiago-Tirado and Bretscher 2011). PtdInsPs are recognized as being critical for selective trafficking of vesicles within cells (Di Paolo and De Camilli 2006; D'Angelo et al. 2008; Vicinanza et al. 2008; Graham and Burd 2011). Phosphatidylinositol comprises less than 10% of membrane phospholipids, and the individual phosphorylated forms total less than 1.5% of lipids (Di Paolo and De Camilli 2006; Roth 2004). Phosphatidylinositol can be phosphorylated on any combination of carbons 3, 4, or 5 of the inositol ring (Fig. 1). The most abundant, PtdIns4P, occurs at approximately 0.05% of membrane lipids, whereas the low abundance forms such as PtdIns(3,4)P2 or PtdIns(3,4,5)P3 are approximately 0.0001% each (Cullen 2011). Subcellular pools of the 7 different PtdInsPs have diverse regulatory roles in cytoskeleton remodeling, second messenger signaling, endosomal trafficking, membrane trafficking, osmotic stress, nuclear signaling, and other aspects of cell physiology (Godi et al. 2004; Balla and Balla 2006; Di Paolo and De Camilli 2006). Cellular effects can be mediated by signaling through production of second messengers (diacylglycerol, and inositol trisphosphate). However, many cellular effects are mediated by the localized anchoring of cytosolic proteins having specific PtdInsP-binding domains (e.g., PH, FYVE, PX, ENTH-domains) (De Matteis et al. 2005; Balla and Balla 2006). For example, Arf1 directly binds and recruits PI4-Kinases to the TGN. This produces PtdIns4P which aids in recruitment of coat proteins such as AP1, GGAs, VAMPs, and FAPP1/2 most of which interact with PtdIns4P as well as other proteins in the coat matrix (Balla and Balla 2006). This interaction provides identity to the membrane (reviewed in (Santiago-Tirado and Bretscher 2011)). PI3K-C2α is also present on the TGN. Each type of PtdInsP is localized to specific membranes within the cell. The most abundant phosphoinositides, PtdIns4P, and PtdIns(4,5)P2, are predominantly localized to the Golgi and plasma membrane, respectively, whereas PtdIns3P and PtdIns(3,5)P2 are predominantly localized to early and late endosomes (Di Paolo and De Camilli 2006; Santiago-Tirado and Bretscher 2011). PtdIns(3,4)P2 is rare in resting cells, but is present in the plasmalemma and multivesicular and early endosomes (Roth 2004; Di Paolo and De Camilli 2006). PtdIns(3,4)P2 is not noted as being present in the Golgi or TGN (De Matteis et al. 2005), however, this

Fig. 1. Structure of Phosphatidylinositol (3,4)bisphosphate. PtdIns(3,4)P2 is an exceedingly rare membrane lipid that is highly localized to specific subcellular membranes within the

**2.1 Phosphatidylinositol phosphates in biogenesis of secretory vesicles**

different vesicles (Guerriero et al. 2008).

cell.

has not been well characterized in any cell type. Recently, PtdIns(3,4)P2 was found to be transiently synthesized at the plasmalemma as a second messenger of platelet-derived growth factor (PDGF) (Hogan et al. 2004). Importantly, our understanding of the roles of PtdInsPs derives primarily from studies of yeast and a few mammalian cell lines. It is unclear whether these generalizations carry over to well differentiated cells in tissues. For example, we find high levels of expression of PtdIns(3,4)P2 in parotid secretory granule membranes.

Discrete and dynamic localization of PtdIns-kinases (PI-kinases) and PI-phosphatases regulate the production of PtdInsPs. Of these enzyme families, PI4-kinases have the dominant roles in Golgi function and secretion, although PI3-kinases and PI5-kinases also have roles in secretion (Wang et al. 2003; Roth 2004; Balla and Balla 2006). Several PI4 kinases are localized to the Golgi, TGN, or endosomes, and form complexes with several coat proteins including GGA and FAPP1/2 (D'Angelo et al. 2008). Careful immunofluorescence co-localization studies with MDCK cells have found PI4KIIα in the TGN and PI4KIIIβ in the cis/medial Golgi (Weixel et al. 2005). This is consistent with PtdIns4P being the most abundant PtdInsP in the Golgi. PI4KIIIβ is recruited by the coat protein Arf1 to the Golgi, and in turn PI4KIIIβ recruits Rab11 and the PtdIns4P-binding protein FAPP1 (Godi et al. 2004). Knock-down of PI4KIIα by RNAi has little effect on intra-Golgi transit, but inhibits TGN export of vesicles (Wang et al. 2003). These studies are interpreted as showing that PI4-kinases produce PtdIns4P on the cytosolic leaflet of the TGN and vesicles, which anchors essential adaptor proteins (Santiago-Tirado and Bretscher 2011). Importantly, class I PI3-kinases are also present on Golgi membranes, and are essential for tumor necrosis factor (TNF) secretion by macrophages (Low et al. 2010). Similarly, PI3 kinase was localized to secretory granules of PC12 cells by both immunofluorescence and cell fractionation (Meunier et al. 2005). Transfection of a PtdIns3P-binding domain (FYVE), or a catalytically-inactive PI3-kinase, blocked regulated secretion, possibly by interfering with fusion of the granule at the plasmalemma (Meunier et al. 2005). In summary, certain PIkinases decorate the Golgi and TGN, interact with coat proteins, and produce PtdInsPs. These PtdInsPs are important for vesicle trafficking, however, the identities and roles of PIkinases have not been investigated in cells that are highly specialized for bulk exocrine secretion, such as the parotid.

#### **3. Selective trafficking for secretion**

The presence of multiple different trafficking pathways for secretion has been described in many cell types (Dikeakos et al. 2007; De Matteis and Luini 2008; Park et al. 2008; Folsch et al. 2009; Perez et al. 2010; Stanley and Lacy 2010; Lacy and Stow 2011; Santiago-Tirado and Bretscher 2011). The best characterized of these pathways have a specific cargo protein that is a unique marker of that route, which is essential for molecular characterization (Lara-Lemus et al. 2006). For example, the cytolytic protein perforin of natural killer cells undergoes polarized secretion into the immune synapse, whereas the same cells secrete TNF in a non-polarized pattern (Reefman et al. 2010) thereby marking a different pathway. Also, some pituitary gonadotropes segregate luteinizing hormone into separate granules than follicle-stimulating hormone for different regulated secretion dynamics (Nicol et al. 2004). Alternatively, pathways can be marked by the use of different coat proteins. For example, the coat protein FAPP1 is essential for secretion by the basolateral pathway, whereas FAPP2

Phosphatidylinositol Bisphosphate Mediated Sorting of Secretory Granule Cargo 45

(Saito et al. 2011). Within the lumen, phogrin can bind to carboxypeptidase E (CPE), and contributes to sorting of the complex (Saito et al. 2011). Hence, information about the identity of the vesicle encoded by the coat proteins, is related through phogrin to determine the luminal cargo. Phogrin also has PI-phosphatase activity which may be important for

Other sorting receptors have been reported in neural and endocrine cells. CPE itself binds the granule membrane. The C-terminus of CPE can span the membrane, although only 5 amino acid residues are cytosolic (Dhanvantari et al. 2002). Recycling of CPE from the plasmalemma requires ARF6 apparently due to direct binding to the coat protein. CPE acts as a sorting receptor for proopiomelanocortin (POMC) and proBDNF trafficking into the regulated secretion pathway (reviewed in (Park and Loh 2008)). Secretogranin III (SgIII) can also serve as a sorting receptor. Despite the absence of a transmembrane domain, SgIII binds to granule membranes or to cholesterol-rich liposomes, and anchors chromograninA (CgA) to the membrane (Hosaka et al. 2004). SgIII also interacts with CPE, POMC, and adrenomedullin (Hosaka et al. 2005; Han et al. 2008). It is unclear how SgIII is targeted to specific granules, but this may occur due to selective interactions with membrane rafts, or

Dikeakos et al. (Dikeakos et al. 2007; Dikeakos and Reudelhuber 2007) have shown that a hydrophobic patch in short amphipathic alpha helices is sufficient to sort cargo proteins. Helical domains are implicated in sorting of somatostatin, CPE, prohormone convertase enzymes (PC1/3, PC2), and chromogranin A (CgA). The proposed mechanism for this sorting is that the hydrophobic patch of the helix embeds into the membrane of the forming granule (Dikeakos and Reudelhuber 2007). One of the sorting-competent helical domains (Hels13-5) bound liposomes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) with a Kd=9.7 μM (Kitamura et al. 1999). This Hels13-5 peptide integrated into the non-polar layer to a variable extent depending on the pH of the liposome. No cholesterol was necessary for this interaction. In addition, the helical domain of PC1/3 was shown to directly interact with CHAPS detergent micelles (Dikeakos et al. 2009). Characterization of both natural and artificial helices which confer sorting of a cargo protein at the TGN is an important step forward, however, at this point it is unclear how the identity of the vesicle (determined by the cytosolic coat proteins) directs selective sorting of

For many years, aggregation of secreted proteins has been seen as critical to sorting into the regulated secretory pathway in neuroendocrine cells (Gorr et al. 2005). Dense-core secretory granules of the regulated pathway contain large aggregates comprising chromogranins secretogranins and other secreted proteins, and which are not present in the constitutive secretion pathway. Aggregation of granins and many hormones can be demonstrated *in vitro* in a Ca++ and pH-dependent fashion. The pH of the TGN is approximately 6.2. In AtT20 cells the pH decreases further as the granule matures, reaching pH 5.5 in mature secretory granules (Wu et al. 2001). The acidic pH and high Ca++ present in the regulated secretory pathway is essential for aggregation. This relatively non-specific interaction may allow trafficking of large aggregates of proteins even where only a few specific interactions with transmembrane sorting receptors are present. Nonetheless, it must be recognized that aggregation is a fairly ill-defined concept, and it will be necessary to characterize these interactions in order to determine how some cargo is excluded from the aggregate to be

regulating glucose-stimulated insulin secretion (Caromile et al. 2010).

due to interaction with CPE and, indirectly, phogrin.

such cargo into the correct pathway.

sorted into the constitutive pathway.

directs vesicles to the apical pathway (Godi et al. 2004; Vieira et al. 2005). Importantly, mutation of proteins involved with sorting between trafficking pathways can cause disease. This is also seen with non-genetic diseases, such as pancreatitis during which inappropriate basolateral (endocrine) secretion of cargo proteins occurs. Hence, pancreatic amylase or lipase in the serum are standard clinical markers of this disease. Given the presence of multiple different pathways for secretion, the key issue is to understand the molecular interactions of proteins destined for each pathway, which cause sorting to the correct immature granule or tubule as it forms, or which cause retention of the protein in the granule as it matures.

The best characterized model for specific sorting of soluble cargo proteins involves a transmembrane sorting receptor protein (Fig. 2). The receptor protein is present in the TGN membrane and is able to interact with coat proteins on the cytosolic side of the vesicle bud, and can also bind luminal cargo proteins. The transmembrane receptor is localized to the vesicle bud by the appropriate coat proteins, and in turn, selects the correct cargo. Hence, a single protein serves to coordinate the identity of the vesicle with the luminal contents. This model is exemplified by mannose-6-phosphate sorting receptors (MPRs), which are type I transmembrane receptors present in the TGN (Ghosh et al. 2003). Both the cation-dependent (MPR300) and the cation-independent (MPR46) MPRs deliver lysosomal enzymes from the TGN to endosomes for subsequent transfer to lysosomes. The cytosolic tails of MPRs have specific binding sites for multiple adaptor proteins, including AP1, AP2, GGAs and PACs1 (reviewed in (Ghosh et al. 2003)). In addition, the portion of MPR in the lumen of the vesicle binds to mannose 6-phosphate tags on cargo proteins. The mannose 6-phosphate is a specific posttranslational modification on the N-glycans of over 60 of acid hydrolases which need to be transported to lysosomes. Failure of this sorting causes lysosomal sorting disease, Mucolipidosis type II alpha/beta (I-cell disease) (Ghosh et al. 2003). A similar sorting mechanism has been described for stabilin-1 which binds GGAs and serves as a sorting receptor for a chitinase-like enzyme.

Fig. 2. Transmembrane sorting receptor model. The coat proteins localize a transmembrane sorting receptor, which collects the appropriate luminal cargo.

Various mechanisms have been suggested for sorting of secreted proteins into the regulated secretory pathway, as opposed to the constitutive secretory pathway or trafficking to intracellular targets (Dikeakos and Reudelhuber 2007; Park et al. 2008). Some proteins are secreted by the model discussed above. Phogrin (Ptprn2) is a type I transmembrane receptor present in the TGN and secretory granules of endocrine and neuroendocrine cells. Phogrin contains tyrosine and leucine motifs in the C-terminal (cytosolic) tail, which are important for localization to secretory granules, likely by interaction with coat proteins such as AP1

directs vesicles to the apical pathway (Godi et al. 2004; Vieira et al. 2005). Importantly, mutation of proteins involved with sorting between trafficking pathways can cause disease. This is also seen with non-genetic diseases, such as pancreatitis during which inappropriate basolateral (endocrine) secretion of cargo proteins occurs. Hence, pancreatic amylase or lipase in the serum are standard clinical markers of this disease. Given the presence of multiple different pathways for secretion, the key issue is to understand the molecular interactions of proteins destined for each pathway, which cause sorting to the correct immature granule or tubule as it forms, or which cause retention of the protein in the

The best characterized model for specific sorting of soluble cargo proteins involves a transmembrane sorting receptor protein (Fig. 2). The receptor protein is present in the TGN membrane and is able to interact with coat proteins on the cytosolic side of the vesicle bud, and can also bind luminal cargo proteins. The transmembrane receptor is localized to the vesicle bud by the appropriate coat proteins, and in turn, selects the correct cargo. Hence, a single protein serves to coordinate the identity of the vesicle with the luminal contents. This model is exemplified by mannose-6-phosphate sorting receptors (MPRs), which are type I transmembrane receptors present in the TGN (Ghosh et al. 2003). Both the cation-dependent (MPR300) and the cation-independent (MPR46) MPRs deliver lysosomal enzymes from the TGN to endosomes for subsequent transfer to lysosomes. The cytosolic tails of MPRs have specific binding sites for multiple adaptor proteins, including AP1, AP2, GGAs and PACs1 (reviewed in (Ghosh et al. 2003)). In addition, the portion of MPR in the lumen of the vesicle binds to mannose 6-phosphate tags on cargo proteins. The mannose 6-phosphate is a specific posttranslational modification on the N-glycans of over 60 of acid hydrolases which need to be transported to lysosomes. Failure of this sorting causes lysosomal sorting disease, Mucolipidosis type II alpha/beta (I-cell disease) (Ghosh et al. 2003). A similar sorting mechanism has been described for stabilin-1 which binds GGAs and serves as a sorting

Fig. 2. Transmembrane sorting receptor model. The coat proteins localize a transmembrane

Various mechanisms have been suggested for sorting of secreted proteins into the regulated secretory pathway, as opposed to the constitutive secretory pathway or trafficking to intracellular targets (Dikeakos and Reudelhuber 2007; Park et al. 2008). Some proteins are secreted by the model discussed above. Phogrin (Ptprn2) is a type I transmembrane receptor present in the TGN and secretory granules of endocrine and neuroendocrine cells. Phogrin contains tyrosine and leucine motifs in the C-terminal (cytosolic) tail, which are important for localization to secretory granules, likely by interaction with coat proteins such as AP1

sorting receptor, which collects the appropriate luminal cargo.

granule as it matures.

receptor for a chitinase-like enzyme.

(Saito et al. 2011). Within the lumen, phogrin can bind to carboxypeptidase E (CPE), and contributes to sorting of the complex (Saito et al. 2011). Hence, information about the identity of the vesicle encoded by the coat proteins, is related through phogrin to determine the luminal cargo. Phogrin also has PI-phosphatase activity which may be important for regulating glucose-stimulated insulin secretion (Caromile et al. 2010).

Other sorting receptors have been reported in neural and endocrine cells. CPE itself binds the granule membrane. The C-terminus of CPE can span the membrane, although only 5 amino acid residues are cytosolic (Dhanvantari et al. 2002). Recycling of CPE from the plasmalemma requires ARF6 apparently due to direct binding to the coat protein. CPE acts as a sorting receptor for proopiomelanocortin (POMC) and proBDNF trafficking into the regulated secretion pathway (reviewed in (Park and Loh 2008)). Secretogranin III (SgIII) can also serve as a sorting receptor. Despite the absence of a transmembrane domain, SgIII binds to granule membranes or to cholesterol-rich liposomes, and anchors chromograninA (CgA) to the membrane (Hosaka et al. 2004). SgIII also interacts with CPE, POMC, and adrenomedullin (Hosaka et al. 2005; Han et al. 2008). It is unclear how SgIII is targeted to specific granules, but this may occur due to selective interactions with membrane rafts, or due to interaction with CPE and, indirectly, phogrin.

Dikeakos et al. (Dikeakos et al. 2007; Dikeakos and Reudelhuber 2007) have shown that a hydrophobic patch in short amphipathic alpha helices is sufficient to sort cargo proteins. Helical domains are implicated in sorting of somatostatin, CPE, prohormone convertase enzymes (PC1/3, PC2), and chromogranin A (CgA). The proposed mechanism for this sorting is that the hydrophobic patch of the helix embeds into the membrane of the forming granule (Dikeakos and Reudelhuber 2007). One of the sorting-competent helical domains (Hels13-5) bound liposomes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) with a Kd=9.7 μM (Kitamura et al. 1999). This Hels13-5 peptide integrated into the non-polar layer to a variable extent depending on the pH of the liposome. No cholesterol was necessary for this interaction. In addition, the helical domain of PC1/3 was shown to directly interact with CHAPS detergent micelles (Dikeakos et al. 2009). Characterization of both natural and artificial helices which confer sorting of a cargo protein at the TGN is an important step forward, however, at this point it is unclear how the identity of the vesicle (determined by the cytosolic coat proteins) directs selective sorting of such cargo into the correct pathway.

For many years, aggregation of secreted proteins has been seen as critical to sorting into the regulated secretory pathway in neuroendocrine cells (Gorr et al. 2005). Dense-core secretory granules of the regulated pathway contain large aggregates comprising chromogranins secretogranins and other secreted proteins, and which are not present in the constitutive secretion pathway. Aggregation of granins and many hormones can be demonstrated *in vitro* in a Ca++ and pH-dependent fashion. The pH of the TGN is approximately 6.2. In AtT20 cells the pH decreases further as the granule matures, reaching pH 5.5 in mature secretory granules (Wu et al. 2001). The acidic pH and high Ca++ present in the regulated secretory pathway is essential for aggregation. This relatively non-specific interaction may allow trafficking of large aggregates of proteins even where only a few specific interactions with transmembrane sorting receptors are present. Nonetheless, it must be recognized that aggregation is a fairly ill-defined concept, and it will be necessary to characterize these interactions in order to determine how some cargo is excluded from the aggregate to be sorted into the constitutive pathway.

Phosphatidylinositol Bisphosphate Mediated Sorting of Secretory Granule Cargo 47

the molecular interactions of PSP in the secretory granule in an attempt to understand how

We analyzed rat parotid granule membranes by mass spectrometry with the goal of identifying integral membrane proteins that may be candidate sorting receptors in the parotid gland. Parotid gland homogenates were fractionated on sucrose gradients to isolate the secretory granules, which were osmotically lysed. Membranes were washed and further enriched by an additional sucrose gradient. Sucrose gradient-purified granule membranes were electrophoresed on polyacrylamide gels, and trypsinized peptides identified by MS/MS as described (Uriarte et al. 2008). Numerous integral or membranebound proteins were identified, including several involved in vesicle trafficking and cytoskeletal proteins, as expected. However, potential sorting receptors such as SgIII (Han et al. 2008) or carboxypeptidase E (Dhanvantari et al. 2002) were not identified in parotid membranes by this method. Nonetheless, one salivary cargo protein, PSP, was identified. Other abundant soluble cargo proteins such as amylase and acidic Proline-Rich Protein (PRP) were not detected. To confirm the binding of PSP to granule membranes, western blot analysis was done with independent preparations of purified and extensively washed granule membranes. This confirmed that PSP is selectively bound, whereas amylase and PRP are absent from purified membranes (Fig. 3) (Venkatesh et al. 2011). While this approach failed to identify candidate sorting receptor proteins, it did demonstrate that PSP is a good marker for interactions with the membrane. In other cell types, putative sorting proteins such as secretogranin III (Hosaka et al. 2004), carboxypeptidase E (Dhanvantari et al. 2002), PC1/3, and PC2 (Jutras et al. 2000) are also associated with

Fig. 3. Western blots of purified secretory granule membranes, probed with either antiamylase or anti-PSP. Lanes contain either intact granules (G), purified granule membranes (M), or soluble cargo protein lysate (L). Equal proportions (0.5%) of each fraction was analyzed. The star indicates PSP in the purified membrane fraction. Mw: molecular size

Given the existing models for sorting receptors, we tested whether PSP was bound to a sorting receptor protein in the secretory granule membrane. Numerous experiments were done attempting to crosslink PSP to a membrane protein; however, PSP never crosslinked

**4.1 Parotid secretory protein (PSP) binds to secretory granule membranes**

sorting may be controlled (Venkatesh et al. 2011).

isolated secretory granule membranes.

standards.


**7. Clinical significance**

Sleep apnea worsens the symptoms of CKD such as daytime fatigue, sleepiness, and impaired neurocognitive function. Hypoxemia during sleep is associated with nocturnal hypertension, left ventricular hypertrophy, impaired sympathovagal balance, and increased risk of cardiovascular complications including death (68-69). Sleep apnea may exacerbate the infectious complications common in ESRD patients because sleep disruption and deprivation degrade immune function (26). Severe sleep apnea is an independent predictor of graft loss among female kidney transplant patients (27).


*3.7. Multivariate Analysis*

Multivariate analysis is a tool to find patterns and relationships between several variables simultaneously. To understand the relationship among 130 eggplant genotypes with various morpho-physiological traits, principal component, biplot, and heatmap analysis were done which revealed different clusters of genotypes that performed better in differ-

ent aspects. The genotypes by traits biplot were constructed from a two-way matrix of 10 morpho-physiological traits and 130 eggplant genotypes using the relative value of the trait (Figure 4). Again, biplot analysis showed the trait profiles of the genotypes, especially, those genotypes positioned far away from the origin and the results indicated a correlation between traits with genotypes. Again, traits on opposite sides of the origin are negatively correlated and traits near each other are positively correlated. Moreover, traits at 90◦ to each other are not correlated, concerning the origin. The principal component (PC) analysis identified a total of 10 principal components (PCs) for the morpho-physiological traits. Among them, the first two PC explained 82.26% of the entire morpho-physiological variations (Figure 4). This biplot revealed superior genotypes with higher levels of expression of favorable trait combinations. The total outcome proposed that TF, FD, SLA, FW, PH, and YPP could help to detect superior genotypes in elite germplasm.

**Figure 4.** Genotypes by traits (G × T) biplot based on 130 germplasm and 10 quantitative traits of eggplant. DFF =Days to first flowering (day), FD = Fruit Diameter (cm), FL = Fruit Length (cm), FW = Fruit Weight (g), YPP =Yield Per Plant (kg), NDVI = Normalized Difference Vegetation Index, PH = Plant height (cm), SLA = Single Leaf Area (cm2), SPAD = Soil Plant Analyses Development, TF = Total Number of Fruits.


**10. Extubation technique**

aerosols and contamination [46], Front neck kit should be available immediately in case needed however it is better keep nearby the room not inside to avoid

A lot of COVID 19 patients need long time ventilation that many cases will undergo tracheostomy, currently there is debate about the proper time case selection tracheostomy technique. The major indication for tracheostomy is to facilitate mechanical ventilation for a long period, while reducing complications from a trans-laryngeal endotracheal tube and weaning from ventilation. Airway obstruction, laryngeal oedema might be an emerging feature of COVID-19 which indicate

When elective tracheostomy is done, an inflated tracheostomy tube cuff via which pressure support ventilation can be delivered affords a closed system for

Tracheostomy in COVID 19 patients is like intubation a high risk for aerosol generating same consideration will apply for intuatbion however based on recent reports for tracheostomised COVID 19 patients it is recommended to use of enhanced PPE, with PAPRs(powered air-purifying respirators), eye protection, fluid-repellent disposable surgical gown, and gloves If a PAPR is not available, we advise the use of a fit-tested filtering face piece 3 (FFP3) or N95 mask with an additional fluid shield,

Surgical tracheostomies were generally favored over percutaneous tracheostomies during the SARS outbreak, however, percutaneous techniques have subsequently advanced and no data are available to establish superiority of one approach over the other from the standpoint of infectious transmission or safety. Single-use bronchoscopes with a sealed ventilator circuit are preferable when doing percuta-

Good sedation should ensure during the procedure to avoid cough and muscle

Like intubation, extubation is an aerosol generating procedure (AGPs) and need

i. Strategies for supporting respiration after extubation, such as noninvasive ventilation (NIV) and high-flow nasal oxygen (HFNC), are relatively contraindicated because of their ability to aerosolize SARS-CoV-2

iv. Personal protective equipment (PPE) with airborne precautions is required during extubation and for personnel entering the room for a variable period

ii. Extubation should ideally take place in a negative pressure room, if

iii. All non-essential staff should exit the room before extubation.

of time after extubation, dependent on room ventilation.

minimizing number of people during the procedure is necessary.

Specific considerations during extubation include [49]:

contaminate it.

**8. Tracheostomy**

urgent tracheostomy [47, 48].

neous tracheostomies.

relaxant might be used.

available.

**14**

special attention in COVID 19 patients.

**9. Extubation**

controlled weaning of respiratory support.

*Special Considerations in Human Airway Management*

Many techniques have been described to minimize exposure to aerosols during extubation although no clear evidence behind them [50].


**3. Discussion**

The preceding description of prominent, relative extensive reflections and reflection sequences has emphasized their interpretation as mafic sills. The basis for these interpretations ranges from unequivocal (e.g., Siljan reflections—drilled) to likely (Arizona—outcrop) to speculative (central U.S. seismic character). There are a number of caveats that must be considered in evaluating the interpretations of these and other reported images of deep intrusions. For example, the seismic sections shown in this paper are all 2D. Without 3D control, apparently subhorizontal reflections could just as well be along-strike images of features that are actually dipping at right angles to the line of the section. However, most of the examples here are based on surveys that did include 3D control, either in the form of a local grid of surveys (e.g., Siljan [54], North Texas [65], Great Britain [81]) or as formal three D seismic arrays (e.g., the Winagami reflections sequence [61], layered reflections in eastern New Mexico [69]).

Another consideration is that igneous intrusions may be manifest with different seismic characteristics than that exemplified by the Siljan sequence—i.e., narrow bands of strong reflectivity separated by larger bands of non-reflectivity. For example, Figure 14 (Upper left) shows a sample of BIRPS marine deep seismic reflection profiling data from northwest Britain [88] that indicates a lower crust that is highly layered and strongly reflective, a pattern commonly referred to as Layered Lower Crust (LLC). However, the layering in this case is more of a lamination, without the distinctive separation between reflective units that has been used to associate many of the sill examples in this paper with Siljan. This laminated appearance is characteristic of a number of seismic profiles in western Europe, particularly in areas affected by post-Variscan extension [89]. Based on the strong amplitudes of reflections making up this lamination, Warner [88] argues that they are most likely due to igneous intrusions or the juxtaposition of contrasting metamorphic compositions by pervasive shearing, although this remains a matter of debate [18]. Meissner et al. [90] argue from seismic anisotropy that the LLC develops by ductile processes (extension?) within warm, low-viscosity felsic lower crust with intercalations of mafic intrusions. Numerical modeling by Gerya and Berg [91] illustrates how crustal rheology can control the geometry of mafic intrusion, with a warm lower crust resulting in the lateral spread of magma with coeval viscous deformation. Thus, the different presentations of sills like Siljan/Wollaston vs. the LLC may be due to a contrast in crustal rheology—i.e., hot and ductile for the LLC vs. cold and brittle for the Siljan and its analogous reflectors—modulated by the stress field at the time of emplacement. However, the seismic data from the extended Norwegian continental margin [4,92] suggest that this maybe an over-simplification, as they show relatively continuous, distinct lower crustal sill reflectors (e.g., Figure 14; upper right) that resemble Siljan more than the LLC around Britain.

**Figure 14.** (**Left**) laminated reflections in the lower crust of NW Britain from the BIRPS marine reflection profiling [88]. (**Right**) distinct reflections interpreted as sills in the extended lower crust of the Norwegian continental margin [4]. T is interpreted as a reflection from the top of a high velocity body (HVB; mafic underplating?).

A number of mafic sills in both outcrop and seismic sections from basins exhibit a characteristic "saucer" shape which has fueled recent discussion about intrusion mechanics in sedimentary sequences [4,27,29,93,94]. This distinctive saucer shape has also been reported for intrabasement reflections on a several deep seismic profiles [48,95,96]. Figure 15 shows one particular distinctive example, the Surrency Bright Spot (SBS) encountered during a COCORP survey of the inferred suture zone between Laurasia and Gondwanaland buried beneath the coastal plain sedimentary rocks of southeastern Georgia. The strong amplitude of this spatially limited reflection was originally interpreted to indicate fluid involvement [95] though magma was ruled out due to its location on a long inactive passive continental margin. The saucer shape of the SBS (Figure 15) is now recognized as identical to that documented not only in oil industry 3D seismic data for various sedimentary basins but in notable outcrops of mafic intrusions [27]. The presence of saucer-shaped reflectors deep in the continental basement provides new context to evaluate mechanical models proposed to explain this geometry within sedimentary strata [4,38,93]. Thus the SBS (Surrency Bright Saucer?) is most likely a mafic intrusion, perhaps emplaced during the rifting of the Atlantic margin.

**Figure 15.** (**Left**) Unmigrated COCORP true amplitude section showing the Surrency Bright Spot [95]. (**Upper Right**) Blow up of migrated image of the Surrency Bright Spot [95]). (**Lower Right**) depth contour of the Surrency Bright Spot from the COCORP 3D seismic survey [97]. The "saucer" shape of this basement reflector is comparable to those associated with mafic intrusions imaged by 3D oil industry data and in outcrop [27].

Moho reflections on many deep seismic surveys also exhibit strong amplitudes (e.g., Figure 2; [98]) and/or a layered character [99]. These characteristics have been interpreted to represent "underplating", which may be another aspect of accreting/injecting mafic sills at the base of the crust [47,100].

Not every extensive prominent reflection or band of reflections is necessarily a sill. Setting aside the obvious example of sedimentary units (e.g., the finely layered, upper few seconds of the seismic sections shown in Figures 6 and 8–10). Other processes can produce distinct reflection bands traceable over large distance. Figure 16 (right) show seismic data from west central Sweden not too distant from Siljan [101]. Lacking any other constraints, it would be tempting to interpret these as sills, perhaps related to the Siljan reflections. However, the tectonic setting, outcropping geology, and—most definitively—drill holes make clear that these reflections are actually thrust faults, part of a crustal scale nappe complex emplaced during the Caledonian orogeny [101]. Regional detachment faults have also appeared on crustal reflections surveys as strong, subhorizontal reflections of regional extent. The southern Appalachian detachment traced on COCORP surveys in Tennessee and Georgia [102] and the Main Himalayan Thrust imaged by the INDEPTH surveys in southern Tibet [103] are prime examples.

**Figure 16.** High amplitude, subhorizontal, and west dipping layered reflections from deep seismic surveys in west-central Sweden. Drilling and outcrop correlations identify these as thrust structures rather than intrusion [101].

Implicit in our discussion of strong reflections is the presumption that the magmas involved—whether hot or frozen—represent emplacement from below. An alternative to consider is that the reflections arise from in situ melting. This was originally one interpretation of the seismic bright spots imaged by INDEPTH in Tibet [48,104]. A fluid magma at depth, whether a planar intrusion or an in-situ melt, should give rise to a prominent reflection (e.g., Table 1) regardless of its composition. It is unlikely that reflection data, or any geophysical observations, could distinguish between these two possibilities However, if a reflector under consideration is a frozen product of in situ melt, it is unlikely to have a significant reflection contrast with its surrounding country rock (of the same composition) unless substantial fractional crystallization is involved. On the other hand, a saucer-like reflection geometry in a strong indication that mechanical intrusion is involved. Of course, intrusions may well represent crustal as well as mantle melts, though the silicic composition of the former is less likely to generate a strong reflection unless it is still molten.

The seismic character of a reflector on a seismic section depends heavily on both details of acquisition and processing. This is particularly true for reflection amplitudes. For example, the upwardly convex geometry of saucer shaped intrusions implies some degree of amplification of the associated reflection due to focusing, which in turn is ameliorated by seismic migration [105]. Therefore, caution is warranted when basing interpretation on superficial similarities in appearance between reflections on seismic sections collected and processed by di fferent groups and individuals.


**4.2 Anomalous coronary artery from the opposite sinus (ACAOS)**

Anomalous origin of either the RCA to the left coronary sinus or the LCA to the right coronary sinus, the proximal anomalous coronary artery (CA) may run anterior to the pulmonary trunk (prepulmonic), posterior to the aorta (retroaortic), septal (subpulmonic), or between the pulmonary artery and the aorta (interarterial). Only those with an interarterial (aorta-pulmonary) course can increase risks of myocardial ischemia, arrhythmia, syncope, and sudden cardiac death considering life threatening and clinical guidelines recommend surgical correction [17].

Numerous mechanisms of ischaemia particularly during exercise have been suggested: (1) the compression of the anomalous vessel coursing between the aorta and the pulmonary artery during increased cardiac output and expansion of the great vessels; (2) the acute angle takeoff of the anomalous vessel with further stretch during exercise, possibility accounting for a flap-like closure of the coronary ostium; (3) spasm or kinking of the anomalous vessels; and (4) the course within the aortic wall ("intramural") of the proximal segment of the anomalous vessel [13, 17]. The intramural aortic course can explain the imaging feature (angiography and echo) of CA intussusceptions into the aoric wall: the proximal segment of the anomalous vessel (segmental hypoplasia) is narrowed, and the asymmetrical lateral compression of the anomalous vessel with a silt-like or ovoid rather than circular lumen, particularly during systole and stress.

In **Figure 2** (A and B) we present anomalous Coronary Artery from the Opposite Sinus (ACAOS).

#### **4.3 Coronary artery fistulas**

Coronary artery fistulas are defined as abnormal communications between a coronary artery and a cardiac chamber or major vessel, such as to the vena cava, right or left ventricle, pulmonary vein or pulmonary artery [18, 19]. Coronary artery fistulas are rare findings, identified in 10 (0.05%) of 18,272 diagnostic cardiac catheterizations [20].

#### **4.4 Myocardial bridging**

The three major coronary arteries generally course along the epicardial surface of the heart. On occasion, however, short coronary artery segments descend into the myocardium for a variable distance. This abnormality, termed myocardial bridging occurs in 5–12% patients and usually is confined to LAD [21].