InteractiveFly: GeneBrief

Transport and Golgi organization 1: Biological Overview | References

Exit of secretory cargo from the endoplasmic reticulum (ER) takes place at specialized domains called ER exit sites (ERESs). In mammals, loss of TANGO1 and other MIA/cTAGE (melanoma inhibitory activity/cutaneous T cell lymphoma-associated antigen) family proteins prevents ER exit of large cargoes such as collagen. This study shows that Drosophila melanogaster Tango1, the only MIA/cTAGE family member in fruit flies, is a critical organizer of the ERES-Golgi interface. Tango1 rings hold COPII (coat protein II; see Sec23) carriers and Golgi in close proximity at their center. Loss of Tango1, present at ERESs in all tissues, reduces ERES size and causes ERES-Golgi uncoupling, which impairs secretion of not only collagen, but also all other cargoes examined. Further supporting an organizing role of Tango1, its overexpression creates more and larger ERESs. These results suggest that spatial coordination of ERES, carrier, and Golgi elements through Tango1's multiple interactions increases secretory capacity in Drosophila and allows secretion of large cargo (Liu, 2017).

Secreted proteins reach the extracellular space through a controlled series of membrane traffic events ensuring fusion of cargo-containing secretory vesicles with the plasma membrane. After translocation into the ER, secretory cargo is collected at specialized cup-shaped regions of the ER and then loaded into membrane vesicles that transfer the cargo to the Golgi compartment. These specialized regions of the ER are known as ER exit sites (ERESs) or transitional ER, the latter emphasizing their dynamic relation with the Golgi. At the ERES, vesicles budding from the ER in the direction of the Golgi are generated by the coat protein II (COPII) complex, a set of proteins highly conserved in all eukaryotes. Structural studies have shown that budding of COPII vesicles from ERES is mediated by the assembly of a vesicle-enclosing cage of 60-90 nm in diameter, yet many secreted proteins exceed the dimensions of this cage and are efficiently secreted by cells, raising the question of how this happens. Examples of large secreted proteins include collagens, the main component of extracellular matrices in all animals, for which trimers assemble in the ER into long semirigid rods (Liu, 2017).

TANGO1, a protein belonging to the MIA/cTAGE family (melanoma inhibitory activity/cutaneous T cell lymphoma-associated antigen, has been shown to be involved in the transport of collagens from the ERES to Golgi. Tango1 was discovered in a screening for genes affecting secretion in Drosophila melanogaster S2 cells and confirmed in a second similar screening. It was later found that human TANGO1 was required for the secretion of collagen but not other secreted proteins. This was supported by a TANGO1 knockout mutant mouse which indeed showed defects in the deposition of multiple types of collagens (Wilson, 2011). TANGO1 is a transmembrane protein localized specifically at ERES. The luminal portion of TANGO1 contains an SH3-like domain at its N terminus that is capable of binding collagen at the ER lumen (Saito, 2009) through the chaperone Hsp49 (Ishikawa, 2016). The cytoplasmic portion contains a region with two presumed coiled coils and a Pro-rich region at its C terminus through which TANGO1 may interact with the COPII coat (Saito, 2009). It has been proposed that TANGO1 collects collagen at ERESs as a specific receptor while at the same time ensuring that a large enough vesicle is formed to package that cargo. Activities of TANGO1 in both retarding COPII coat assembly and recruiting ER-Golgi intermediate compartment (ERGIC) membranes to nascent vesicles have been proposed as mechanisms by which TANGO1 can mediate formation of such megacarrier vesicles (Liu, 2017).

Apart from TANGO1, the human genome contains additional TANGO1-like proteins of the MIA/cTAGE family. These include a short splice variant of TANGO1 (TANGO1S) and eight other members of the MIA/cTAGE family of proteins. Common to all these TANGO1-elike proteins is the presence of transmembrane, coiled-coil and Pro-rich regions highly similar to the cytoplasmic portion of TANGO1. In contrast to full-length TANGO1, however, they lack the SH3-like domain and extended intraluminal region. Nonetheless, a function in secretion has been shown for some of these proteins. TANGO1S, lacking the signal peptide and luminal domain of the full protein but preserving its transmembrane domain, is involved in collagen secretion (Maeda, 2016). Also involved in collagen secretion is cTAGE5. Finally, TALI, a chimeric protein resulting from fusion of MIA2 and cTAGE5 gene products, is required for the secretion of ApoB-containing large lipoparticles (Liu, 2017).

Besides TANGO1 and TANGO1-like proteins, loss of several factors potentially involved in general secretion have been shown to affect preferentially collagen secretion in mammalian cells. These include the TRAPP tethering complex component Sedlin, ubiquitination of Sec31 by the ubiquitin ligase KLHL12, Syntaxin 18, and the SNARE regulator Sly1. Notably, mutations in the Sec23A subunit of COPII led to craniofacial development defects attributable to aberrant collagen secretion. These studies suggest that secretion of collagen or large cargo, though using the same basic transport machinery as other cargoes, could be especially sensitive to impairments in that machinery (Liu, 2017).

The fruit fly Drosophila, in which Tango1 was first found, provides a very distinct advantage for studying the early secretory pathway in the form of limited gene redundancy compared with mammals. For instance, single Sar1 and Sec23 homologues are found in Drosophila. Similarly, only one Tango1 protein exists in Drosophila, in contrast to the presence of multiple TANGO1-like proteins with possible overlapping functions in humans. In addition, most proteins shown to play an essential role in secretory pathway function and organization have homologues encoded in the Drosophila genome as well, including Rab small GTPases, COPI and COPII coat components, SNAREs, Golgi matrix proteins, and Golgins. One of the main differences in secretory pathway organization between mammalian and Drosophila cells is that in mammals, ERES-derived vesicles fuse to form an ERGIC, where cargo transits en route to a single juxtanuclear Golgi ribbon. In flies, however, Golgi elements remain dispersed throughout the cytoplasm in close proximity to ERESs, forming ERES-Golgi units. Because this mode of organization is characteristic not just of flies, but probably of all nonmammalian animals and also plants, it is certain that ERES-ERGIC-Golgi secretory pathway organization in mammals is an elaboration on an ancestral, more simple theme represented by functionally independent ERES-Golgi units (Liu, 2017).

Besides its advantages for secretory pathway studies, the fruit fly Drosophila has strongly emerged in recent years as a convenient model to study the biology of collagen and the extracellular matrix. Compared with the 28 types of collagen found in mammals, Drosophila possesses a reduced complement of collagens, consisting of basement membrane Collagen IV and Multiplexin. Expression of Multiplexin, related to Collagens XV and XVIII, is restricted to the heart and central nervous system and is dispensable for viability. Collagen IV, in contrast, is abundantly present in all fly tissues. In Drosophila, as in all animals, Collagen IV is the main component of basement membranes, polymers of extracellular matrix proteins that underlie epithelia and surround organs and provide structural support to tissues. Drosophila Collagen IV is a heterotrimer composed of α chains encoded by Collagen at 25C (Cg25C; α1 chain) and viking (Vkg; α2 chain). The length of the Drosophila Collagen IV trimer is 450 nm, with a predicted molecular mass of 542.4 kD and increased flexibility caused by imperfections of the triple helix (Liu, 2017).

Having shown previously that Drosophila Tango1 is required for secretion of Collagen IV by fat body cells, their main source in the Drosophila larva (Pastor-Pareja, 2011), this study set out to characterize the expression of Tango1, loss-of-function phenotype, and specificity toward Collagen IV. In the course of this study, it was found that Tango1 is required to maintain the size and integrity of ERES-Golgi units, its loss of function impairing not only Collagen IV secretion, but also general secretion (Liu, 2017).

In this study, imaging of ERESs through super-resolution microscopy revealed close proximity of COPII carriers and cis-Golgi elements in the center of Tango1 rings (see also Raote, 2017). When the effects of Tango1 loss are examined, this study found that ERESs were reduced in size and frequently uncoupled from Golgi, indicating a requirement of Tango1 in the normal organization of ERES-Golgi units. Moreover, supporting an important role of Tango1 in the morphogenesis of Drosophila ERESs, overexpression of Tango1 created more and larger ERESs (Liu, 2017).

Overall, the results are consistent with a model in which the spatial organization of the ERES-Golgi interface provided by Tango1's multiple interactions helps build enlarged COPII carriers that canalize traffic in the center of ERESs. The proximity of ERESs and Golgi in Drosophila leads to an additional proposal that direct ERES-Golgi contact might be the way in which large cargo normally transfers from the ER to the Golgi in flies. Direct contact between ER and Golgi has been suggested as a mode of ER-to-Golgi transport in the yeast Saccharomyces cerevisiae and in plants, where ERESs and Golgi are, like in Drosophila, closely juxtaposed and possibly attached physically through a matrix. ERES-ERGIC contact also has been suggested as a transport mechanism in mammals. Careful electron tomography analysis and in vivo imaging could be used in the future to investigate in more detail the dynamics of cargo transfer among ERESs, COPII carriers, and Golgi at the center of Tango1 rings. Given the necessity to secrete not only Collagen IV or lipoprotein particles but also giant cuticular proteins like the 2,500-mol-wt protein Dumpy, ER-to-Golgi carriers in Drosophila must be necessarily large. Taking into account this and the narrow space in which Drosophila ERES-Golgi transport takes place, it is possible that such large carriers start fusion with the Golgi before having separated from the ERES, effectively creating intermittent tubular connections (Liu, 2017).

These experiments, importantly, revealed a wider role in secretion for Tango1, its knockdown causing intracellular retention of the multiple cargoes. Thus, large carriers or tubular connections built with the assistance of Tango1 may mediate not only the transport of large cargo, but also a significant portion of the total flow of general cargo. This is in contrast to the specific roles in secretion of collagens (TANGO1, TANGO1S, and cTAGE5) or lipoprotein particles (TANGO1 and TALI) proposed for mammalian members of the MIA/cTAGE family. Apart from Collagen IV (Pastor-Pareja, 2011), the ECM proteins Perlecan, Tiggrin, SPARC, and Laminin were previously observed to accumulate intracellularly in the absence of Drosophila Tango1, raising the possibility that these defects were secondary to Collagen IV retention or, alternatively, that Tango1 were required for secretion of large ECM proteins in general. The current results, however, show that small non-ECM cargoes like plain GFP were inefficiently secreted in the absence of Tango1 as well. Further supporting a general role of Drosophila Tango1 in secretion, Tango1 is expressed in all tissues of the larva, inconsistent with a relation with specific cargoes. The highest expression of Tango1 was found in the salivary gland, a dedicated secretory organ where genes encoding secretory pathway components are highly expressed as a group, including COPII and COPI genes. It would seem, therefore, that Tango1 expression correlates with secretory activity, but not with Collagen IV secretion because Collagen IV is not expressed in the salivary gland (Pastor-Pareja, 2011; Liu, 2017 and references therein).

Supporting both an organizing function of Tango1 at the ERES-Golgi interface and a wider role in secretion, the cytoplasmic part of Tango1 could rescue Tango1 loss in the fat body. The result of this rescue experiment additionally posits the question of what is the role of the intraluminal part of the Drosophila protein, through which mammalian TANGO1 is thought to interact with cargo. The intraluminal SH3-like domain of Tango1 is conserved among Drosophila and mammals, a sure sign of a biological role, and it is possible that this domain in Drosophila still has a role in binding cargoes, either directly or through several adaptors. Nonetheless, the results clearly show that Tango1 loss impairs general secretion and that the cytoplasmic part of the protein is by itself capable of enhancing Collagen IV secretion independent of the intraluminal part. Although it is conceivable that Drosophila Tango1 and mammalian TANGO1 have diverged in their function, the possibility that MIA/cTAGE5 family members are partially redundant in facilitating general secretion beyond any roles they may have as specific cargo adaptors is worth considering in light of these findings (Liu, 2017).

Recently, suppression of mammalian TFG expression has been shown to result in smaller ERESs that remain functional for the export of many secretory cargoes, but not collagen. TFG, a protein first characterized in the roundworm Caenorhabditis elegans, has been proposed to act in mammals by forming oligomeric assemblies that physically join ERESs and ERGIC (Johnson, 2015). Human and C. elegans TFG have no clear homologue in Drosophila. Conversely, C. elegans has no Tango1 homologue. This is despite the fact that C. elegans possess all four major basement membrane components, numerous collagens, and multiple other large ECM proteins. In this evolutionary context, work on Drosophila Tango1 shows that alternative mechanisms acting in ERES organization may exist in animal cells to increase capacity of ER-to-Golgi transport in terms of both cargo size and the amount of cargo to be secreted. Furthermore, because small COPII vesicles have seldom been observed in animal cells, it is possible that animals have largely abandoned these in favor of larger COPII-dependent carriers built with help from proteins like TFG and Tango1. Such proteins might have initially evolved to enable secretion of metazoan ECM and other large cargoes, creating in the process a mode of transport that increased efficiency of general ER export as well (Liu, 2017).

Tango1 coordinates the formation of ER/Golgi docking sites to mediate secretory granule formation

Regulated secretion is a conserved process occurring across diverse cells and tissues. Current models suggest that the conserved cargo receptor Tango1 mediates the packaging of collagen into large coat protein complex II (COPII) vesicles that move from the endoplasmic reticulum (ER) to the Golgi apparatus. However, how Tango1 regulates the formation of COPII carriers and influences the secretion of other cargo remains unknown. Through high-resolution imaging of Tango1, COPII, Golgi and secretory cargo (mucins) in Drosophila larval salivary glands, this study found that Tango1 forms ring-like structures that mediate the formation of COPII rings, rather than vesicles. These COPII rings act as docking sites for the cis-Golgi. Moreover, nascent secretory mucins were observed emerging from the Golgi side of these Tango1/COPII/Golgi complexes, suggesting that these structures represent functional docking sites/fusion points between the ER exit sites and the Golgi. Loss of Tango1 disrupted the formation of COPII rings, the association of COPII with the cis-Golgi, mucin O-glycosylation and secretory granule biosynthesis. Additionally, this study identified a Tango1 self-association domain that is essential for formation of this structure. These results provide evidence that Tango1 organizes an interaction site where secretory cargo is efficiently transferred from the ER to Golgi and then to secretory vesicles. These findings may explain how the loss of Tango1 can influence Golgi/ER morphology and affect the secretion of diverse proteins across many tissues (Reynolds, 2019).

Secretion of proteins is a highly conserved event occurring in all eukaryotic species and across many tissues. This process begins in the ER where proteins destined to be secreted are synthesized and transported to the cis-region of the Golgi apparatus in a COPII-dependent process. Appropriately modified and folded proteins are packaged into secretory vesicles emanating from the trans-Golgi network that then await appropriate signals before fusing with the plasma membrane to release their contents. However, the mechanisms whereby bulky cargo is efficiently packaged into small vesicular transport vehicles and moved between compartments of the secretory apparatus are largely unknown (Reynolds, 2019).

Recently studies have identified an essential cargo receptor (Tango1 or MIA Src homology 3 (SH3) domain ER export factor 3; MIA3) responsible for the efficient packaging and secretion of high-molecular-weight collagen. Tango1, a type I transmembrane protein located at the ER exit sites (ERES) was first identified in a screen for genes that affect general secretion and Golgi morphology in Drosophila cells (Bard, 2006). Subsequent studies demonstrated a role for Tango1 in collagen secretion, whereby it is thought to mediate the formation of large COPII megacarriers capable of transporting the large procollagen rods from the ER to the Golgi apparatus (Saito, 2009; Wilson, 2011). Current models suggest that the luminal SH3 domain of Tango1 binds to the procollagen chaperone HSP47 (Ishikawa, 2016), directing procollagen to sites of COPII vesicle formation. Tango1 is thought to modulate the size of the COPII vesicles by recruiting factors that limit Sar1GTPase activity, thus allowing the vesicles to grow in size to accommodate this large cargo (Reynolds, 2019).

Many recent studies have suggested that Tango1 is important for the secretion of additional molecules other than collagen. In mammalian cells, Tango1 affects the export of bulky lipid particles such as pre-chylomicrons/very low-density lipoproteins (Santos, 2016). In Drosophila, loss of Tango1 results in defects in the secretion of mucins, laminins, perlecan, and other extracellular matrix proteins (Lerner, 2013; Liu, 2017; Petley-Ragan, 2016; Rios-Barrera, 2017; Zhang, 2014; Ke, 2018). Additional genetic studies suggest that Tango1 influences general secretion rather than the specific secretion of certain proteins. These studies also identified many additional Golgi proteins that interact with Tango1, either directly or indirectly, such as Grasp65 and GM130, and suggest that there may exist more direct contacts between the ER and Golgi that are mediated by Tango1 (Liu, 2017). Work by Rios-Barrera (2017) suggests that although Tango1 is important for the secretion of bulky cargo, it has an additional role in ER-Golgi morphology. However, high-resolution visualization of Tango1 dynamics and COPII vesicle formation relative to endogenous cargo biosynthesis and packaging has been challenging given the small size of these structures and the resolution limits of light microscopy. Thus, the exact roles Tango1 plays in the packaging and secretion of diverse cargos, as well as ER-Golgi morphology, remain unclear (Reynolds, 2019).

This study used the Drosophila larval salivary gland (SG) to image the relationship between Tango1 and the synthesis and packaging of secretory cargo (mucins) in real time, taking advantage of the increased spatial resolution unique to this gland. The SG undergoes hormonally regulated secretory granule formation that results in secretory granules of 3-8 microns in diameter (~10-100x larger than those seen in mammalian systems) that are filled with highly O-glycosylated mucin proteins (19-23). Drosophila mucins are similar in structure to mammalian mucins (having serine/threonine-rich O-glycosylated regions) but are typically smaller in size. Fly lines expressing GFP-tagged versions of one secretory mucin (Sgs3-GFP) have allowed high-resolution, real-time imaging of secretory granule formation and secretion. The genetic tractability of Drosophila has also allowed the identification of factors that control secretory vesicle formation, morphology, and extrusion of bulky cargo, such as mucins. Through real-time imaging using this system, this study found that Tango1 undergoes regulated self-association and dynamic shape changes during hormonally induced secretion to form ring structures that mediate the formation of COPII rings rather than vesicles. These Tango1-COPII rings act as docking sites for the cis-Golgi. Moreover, nascent secretory mucins were imaged emerging from the Golgi side of these Tango1-COPII-Golgi complexes, suggesting that these structures represent functional docking sites/fusion points between the ER exit sites and the Golgi. Taken together, these data suggest that Tango1 acts as a scaffold for the formation of functional ER-Golgi junctions that allow the efficient synthesis, intraorganellar transport, and packaging of diverse secretory cargo (Reynolds, 2019).

Taking advantage of the high spatial resolution of secretory structures in the Drosophila larval SG, this study has demonstrated that Tango1 coordinates the formation of ER-Golgi interaction sites to mediate secretory vesicle formation. High resolution imaging of Tango1 relative to COPII, Golgi, and secretory cargo demonstrates that Tango1 self-associates to form rings that appear to orchestrate the formation of COPII rings. Moreover, cis-Golgi markers localize within these rings, forming a distinct Tango1-COPII-Golgi structure. In support of this Tango1-COPII-Golgi structure being a functional site of interaction between the ER and Golgi, secretory granules were found emanating from the trans-Golgi face of this structure. These results support a model where Tango1 mediates a functional interaction point between the ERES and Golgi to allow efficient transfer of cargo and the subsequent formation of secretory granules (Reynolds, 2019).

This unique structure and the formation of secretory vesicles are dependent on Tango1. This study found that loss of Tango1 resulted in the loss of the COPII rings, loss of the organized association of the cis-Golgi with COPII, and loss of secretory vesicles. Additionally, Tango1 overexpression in Drosophila cells was sufficient to drive COPII ring formation and Golgi association. Previous studies have demonstrated direct binding of Tango1 and COPII components, which likely orchestrates the overlapping ring formation. Likewise, COPII components are known to interact with various cis-Golgi proteins, which likely drives their association in this structure. The formation of this entire structure depends on Tango1 self-association via the CCD1 domain in the cytoplasmic region. This is similar to the domain responsible for Tango1 self-association in mammals, suggesting conserved aspects of Tango1 action between these species (Reynolds, 2019).

Interestingly, the COPII structures present in this system exist as rings rather than spherical vesicular structures. Whether the COPII ring represents a structure unique to Drosophila or whether these structures might also be present in mammals awaits further investigation. However, evidence exists for diverse COPII structures in different systems, including tubules and protruding saccules from the ER membrane. The flexibility of the COPII coat may allow unique adaptations, depending on the biological context. Indeed, one recent study in mammalian cells suggests that procollagen transport occurs via a 'short-loop pathway' from the ER to the Golgi in the absence of large COPII vesicular carriers. This study offers support for the possibility that similar COPII-dependent ER-Golgi interaction sites may exist in mammals (Reynolds, 2019).

Previous work in Drosophila also supports a model where Tango1 mediates a connection to the Golgi. In this study, the authors demonstrated that overexpression of the Tango1 cytoplasmic domain can increase the size and density of ERES and increase the number of Golgi units, strongly suggesting a role for Tango1 in organizing both ERES and the Golgi (Ishikawa, 2016). Indeed, the authors propose that large COPII carriers may begin fusion with the Golgi before separating from the ERES. The current imaging clearly demonstrates that Tango1, COPII, and the Golgi lie in close proximity and that their spatial separation likely precludes the formation of a separate, large COPII vesicular carrier. Moreover, the finding that mucin cargo emerges from the trans-Golgi face of this structure strongly supports a model where Tango1 serves to organize ordered ERES/Golgi interactions sites through which cargo passes. The results and model would also explain previous Drosophila studies where loss of Tango1 affects Golgi structure as well as the secretion of diverse proteins. Tango1 was originally discovered in an RNAi screen in Drosophila cells for genes that affect both secretion and Golgi structure (Tango = transport and Golgi organization). Likewise, more recent studies have suggested that Tango1 plays a role in the interaction of the Golgi and ER that is independent of its role in trafficking bulky cargo proteins. Many of these studies also present evidence that loss of Tango1 affects constitutive secretion of all proteins, including small reporter proteins. If Tango1 functions to mediate docking sites between ERES and Golgi, then the loss of Tango1 would be expected to result in changes in Golgi structure. Indeed, evidence was seen of Golgi structural changes when Tango1 was deleted from this system. Likewise, if the rate of constitutive secretion also benefits from these contact sites, one would expect the loss of Tango1 to affect this as well. These results and model are therefore consistent with previous studies that suggest a role for Tango1 in Golgi structure and constitutive secretion (Reynolds, 2019).

Studies investigating the role of Tango1 in other systems have proposed diverse models for how Tango1 coordinates the secretion of specific cargo. In mammals, it is proposed that the SH3 region of Tango1 interacts with the HSP47 chaperone, which then binds collagen to mediate its entry into the nascent COPII vesicle. However, this model does not explain how diverse cargo and constitutive secretion can be affected by the loss of Tango1. Additionally, this model necessitates a second packaging event for bulky cargo on the trans-side of the Golgi that must take place. The data presented in this study suggest that tissues under a high secretory burden may use Tango1 to reduce the number of independent packaging steps required for bulky, highly glycosylated cargo (such as mucins) by forming direct connection points between the ER and Golgi. This may ensure the efficient production and packaging of large amounts of cargo into secretory vesicles over a short period of time (Reynolds, 2019).

The size of the secretory structures present in this genetically tractable system and its amenability to real-time imaging during the secretory process have led to key insights with regard to secretory granule biogenesis and secretion. Using this system, it was previously shown that clathrin and AP-1, which localize to the trans-Golgi network, are required for proper secretory granule formation. Subsequently, the activity of the phosphatidylinositol kinase PI4KII was shown to be essential for secretory granules to reach mature size, likely because of influences on homotypic fusion events. Previous work has identified a role for O-glycosylation in secretory granule morphology during granule maturation. Real-time imaging has outlined the steps involved in secretory granule fusion with the apical plasma membrane and identified factors required for proper secretion of the mucinous contents. The current results are consistent with these prior studies and shed light on how cargo moves efficiently from the ER to the Golgi through a unique secretory structure whose organization depends on Tango1. This structure may explain how Tango1 has diverse effects on both regulated and constitutive secretion across many cell and tissue types. Moving forward, this tractable genetic imaging system will be amenable to identifying additional factors responsible for the highly organized and incredibly robust secretory program of the SG. Moreover, understanding the mechanisms by which biological systems maximize secretory capacity and efficiency may provide insights into novel strategies to restore defective secretion in disease states (Reynolds, 2019).

Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements

Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix. Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum (ER) to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site (ERES) protein Tango1. This study reports an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, distribution of GFP-tagged Collagen IV was examined in live animals, and 88 gene hits were found for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically: PH4alphaEFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in the hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and RabX3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast, led to uncoupling of ERES and Golgi. Additionally a characterization of a screening hit, trabuco (tbc), is presented, encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, the success is discussed of this screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells (Ke, 2018).

Dual function for Tango1 in secretion of bulky cargo and in ER-Golgi morphology

Tango1 enables ER-to-Golgi trafficking of large proteins. Loss of Tango1, in addition to disrupting protein secretion and ER/Golgi morphology, causes ER stress and defects in cell shape. The previously observed dependence of smaller cargos on Tango1 is a secondary effect. If large cargos like Dumpy, which this study identifies as a Tango1 cargo, are removed from the cell, nonbulky proteins reenter the secretory pathway. Removal of blocking cargo also restores cell morphology and attenuates the ER-stress response. Thus, failures in the secretion of nonbulky proteins, ER stress, and defective cell morphology are secondary consequences of bulky cargo retention. By contrast, ER/Golgi defects in Tango1-depleted cells persist in the absence of bulky cargo, showing that they are due to a secretion-independent function of Tango1. Therefore, maintenance of ER/Golgi architecture and bulky cargo transport are the primary functions for Tango1 (Rios-Barrera, 2017).

The endoplasmic reticulum (ER) serves as a major factory for protein and lipid synthesis. Proteins and lipoproteins produced in the ER are packed into COPII-coated vesicles, which bud off at ER exit sites (ERES) and then move toward the Golgi complex where they are sorted to their final destinations. Regular COPII vesicles are 60-90 nm in size, which is sufficient to contain most membrane and secreted molecules. The loading of larger cargo requires specialized machinery that allows the formation of bigger vesicles to accommodate these bulky molecules. Tango1 (Transport and Golgi organization 1), a member of the MIA/cTAGE (melanoma inhibitory activity/cutaneous T cell lymphoma-associated antigen) family, is a key component in the loading of such large molecules into COPII-coated vesicles. Molecules like collagens and ApoB (apolipoprotein B)-containing chylomicrons are 250-450 nm long and rely on Tango1 for their transport out of the ER, by physically interacting with Tango1 or Tango1 mediators at the ERES (Rios-Barrera, 2017).

Tango1 is an ER transmembrane protein that orchestrates the loading of its cargo into vesicles by interacting with it in the ER lumen. The interaction of Tango1 with its cargo then promotes the recruitment of Sec23 and Sec24 coatomers on the cytoplasmic side, while it slows the binding of the outer layer coat proteins Sec13 and Sec31 to the budding vesicle. This delays the budding of the COPII carrier. Tango1 also recruits additional membrane material to the ERES from the Golgi intermediate compartment (ERGIC) pool, thereby allowing vesicles to grow larger. It also interacts directly with Sec16, which is proposed to enhance cargo secretion. A shorter isoform of mammalian Tango1 lacks the cargo recognition domain but nevertheless facilitates the formation of megacarrier vesicles (Rios-Barrera, 2017).

Apart from bulky proteins, some heterologous, smaller proteins like secreted horseradish peroxidase (ssHRP, 44 kDa) and secreted GFP (27 kDa) also depend on Tango1 for their secretion . Unlike for collagen or ApoB, there is no evidence for a direct interaction between Tango1 and ssHRP or secreted GFP. It is not clear why Tango1 would regulate the secretion of these molecules, but it has been proposed that in the absence of Tango1, the accumulation of nonbulky proteins at the ER might be due to abnormally accumulated Tango1 cargo blocking the ER; however, this has not been tested experimentally (Rios-Barrera, 2017).

Drosophila Tango1 is the only member of the MIA/cTAGE family found in the fruit fly, which simplifies functional studies. Like vertebrate Tango1, the Drosophila protein participates in the secretion of collagen. And as in vertebrates, ssHRP, secreted GFP, and other nonbulky molecules like Hedgehog-GFP also accumulate in the absence of Tango1. These results have led to the proposal that Tango1 participates in general secretion. However, most of the evidence for these conclusions comes from overexpression and heterologous systems that might not reflect the physiological situation (Rios-Barrera, 2017).

This study describes a tango1 mutant allele that was identified in a mutagenesis screen for genes affecting the structure and shape of terminal cells of the Drosophila tracheal system. Tracheal terminal cells form highly ramified structures with branches of more than 100 μm in length that transport oxygen through subcellular tubes formed by the apical plasma membrane. Their growth relies heavily on membrane and protein trafficking, making them a very suitable model to study subcellular transport. Terminal cells were used to study the function of Tango1, and loss of Tango1 was found to affect general protein secretion indirectly, and it also leads to defects in cell morphology and in the structure of the ER and Golgi. The defects in ER and Golgi organization of cells lacking Tango1 persist even in the absence of Tango1 cargo (Rios-Barrera, 2017).

These studies led to an explanation of why, in the absence of Tango1, nonbulky proteins accumulate in the ER despite not being direct Tango1 cargos. These cargos are retained in the ER as a consequence of nonsecreted bulky proteins interfering with their transport. However, the effect of loss of Tango1 on ER/Golgi morphology can be uncoupled from its role in bulky cargo secretion (Rios-Barrera, 2017).

This study has described a role of Tango1, which was initially identified through its function in tracheal terminal cells and other tissues in Drosophila embryos, larvae, and pupae. Due to their complex shapes and great size, terminal cells are a well-suited system to study polarized membrane and protein trafficking, with the easily scorable changes in branch number and maturation status providing a useful quantitative readout that serves as a proxy for functional membrane and protein trafficking machinery. Moreover, this analyses are conducted in the physiological context of different tissues in the intact organism (Rios-Barrera, 2017).

The loss-of-function allele tango1^2L3443 has a stop codon eight amino acids downstream of the PRD domain and eliminates the 89 C-terminal amino acids of the full-length protein. It is unlikely that the mutation leads to a complete loss of function. First, terminal cells expressing an RNAi construct against tango1 show stronger defects, with fewer branches per cell than homozygous tango1^2L3443 cells. Second, the mutant protein appears not to be destabilized nor degraded, but instead is present at apparently normal levels, albeit at inappropriate sites. Predictions of the deleted fragment of the protein suggest it is disorganized and that it contains an arginine-rich domain that has no known interaction partners and that is not present in human Tango1. In homozygous mutant terminal cells, the mutant tango1^2L3443 protein fails to localize at ERES. In mammalian Tango1, the Sec16-interacting region within the PRD domain is necessary for the localization of Tango1 to the ERES and for its interaction with Sec23 and Sec16, but since this domain is fully present in tango1^2L3443, the results mean that either the missing 89 C-terminal amino acids contain additional essential localization signals, or that the PRD domain is structurally affected by the truncation of the protein. The latter is considered less likely, as a truncation of eight amino acids downstream of the PRD domain is unlikely to destabilize the polyproline motifs, especially as the overall stability of the protein does not seem to be affected. Furthermore, this region shows a high density of phosphoserines (Ser-1345, Ser-1348, Ser-1390, and Ser-1392), suggesting it might serve as a docking site for adapter proteins or other interactors (Rios-Barrera, 2017).

Terminal cells lacking Tango1 have fewer branches than control cells and are often not properly filled with air. This loss-of-function phenotype is not due to a direct requirement for Tango1, as it is suppressed by the simultaneous removal of Dumpy (Dpy), an extracellular protein involved in epidermal-cuticle attachment, aposition of wing surfaces and trachea development. It also cannot be explained by the individual loss of crb, Piopio (Pio) a zona pellucida (ZP) domain protein that mediates the adhesion of the apical epithelial surface and the overlying apical extracellular matrix, or dpy, since knocking down any of these genes has no effect on cell morphology. Instead, it is proposed that the cell morphological defect is due at least in part to the activation of the ER stress response, since expression of Xbp1 is sufficient to recapitulate the phenotype. Xbp1 regulates the expression of genes involved in protein folding, glycosylation, trafficking, and lipid metabolism. It is possible that one or a small number of specific genes downstream of Xbp1 are responsible for defective branch formation or stability, but the phenotype could also be a secondary consequence of the physiological effects of the ER stress response itself, for example, a failure to deliver sufficient lipids and membrane from the ER to the apical plasma membrane (Rios-Barrera, 2017).

Collagen, with a length of 300 nm, and ApoB chylomicrons with a diameter of > 250 nm, have both been biochemically validated as Tango1 cargos. These molecules are not expressed in terminal cells, and therefore it was clear that Tango1 must have a different substrate in these cells. Given that Tango1 is known for the transport of bulky cargo, that Dpy is the largest Drosophila protein at 800 nm length, and that Dpy vesicles are associated with Tango1 rings in tracheal cells, it is proposed that Dpy is a further direct target of Tango1. Colocalization of Tango1 with its cargo has also been observed in other tissues: with collagen in Drosophila follicle cells and with ApoB in mammalian cell lines (Rios-Barrera, 2017).

No regions of sequence similarity that could represent Tango1-binding sites have been found in Tango1 cargos. There are several possible explanations for this. First, these proteins may contain binding motifs, but the motifs are purely conformational and not represented in a linear amino acid sequence. There is no evidence for or against this hypothesis, but it would be highly unusual, and there is support for alternative explanations. Thus, as a second possibility, all three proteins may require Tango1 for their secretion, but variable adapters could mediate the interactions. In vertebrates, Tango1 can indeed interact with its cargo through other molecules; for instance, its interaction with collagen is mediated by Hsp47. However, in Drosophila, there is no Hsp47 homolog. In the case of ApoB, it has been suggested that microsomal triglyceride transfer protein (MTP) and its binding partner, protein disulphide isomerase (PDI), might associate with Tango1 and TALI to promote ApoB chylomicrons loading into COPII vesicles. Evidence supporting this is that the lack of MTP leads to ApoB accumulation at the ER. It is not known if secretion of other Tango1 cargos like collagen or Dpy also depends on MTP and PDI, but PDI is known also to form a complex with the collagen-modifying enzyme prolyl 4-hydroxylase. Previous work has shown that terminal cells lacking MTP show air-filling defects and fail to secrete Pio and Uninflatable to the apical membrane, and that loss of MTP in fat body cells also affects lipoprotein secretion, as it does in vertebrates. Since cells lacking MTP or Tango1 have similar phenotypes, it is plausible that the MTP function might be connected to the activity of Tango1 (Rios-Barrera, 2017).

The data is interpreted to mean that in the absence of Tango1, primary cargo accumulates in the ER, and in addition, there are secondary, indirect effects that can be suppressed by reducing the Tango1 cargo that overloads the ER. The secondary effects include activation of the ER stress response and intracellular accumulation of other trafficked proteins like Crb, laminins, and overexpressed proteins and probably also the accumulation of heterologous proteins like secreted HRP or GFP in other systems (Rios-Barrera, 2017).

The data suggest that primary and secondary cargo reach the ERES but fail to be trafficked further along the secretory pathway. In this model, primary cargo, probably recruited by adaptors, would be competing with other secondary cargo for ERES/COPII availability, creating a bottleneck at the ERES. This is consistent with recent experiments that show that in tango1-knockdown HeLa cells, VSVG-GFP trafficking does not stop completely, but is delayed. Furthermore, in these experiments, VSVG-GFP is mostly seen in association with Sec16 and Sec31, supporting the clogging model (Rios-Barrera, 2017).

It is not immediately clear why cargo accumulation in terminal cells lacking Tango1 affects the secretion of Crb but not of βPS integrin. While steady states are looked at in this analyses, Maeda (2016) measured the dynamics of secretion and found that loss of Tango1 leads to a reduced rate of secretion of VSVG-GFP, an effect that would have been missed for any proteins the current study classified as not affected by loss of Tango1. Irrespective, a range of mechanisms can be thought of that might be responsible for this difference, including alternative secretion pathways and differences in protein recycling. Alternative independent secretory pathways have been reported in different contexts. For instance, while both αPS1 and βPS integrin chains depend on Sec16 for their transport, the αPS1 chain can bypass the Golgi apparatus and can instead use the dGRASP-dependent pathway for its transport. It would be possible then that in terminal cells, βPS integrin is also trafficked through an alternative pathway that is not affected by loss of Tango1. Similarly, tracheal cells lacking Sec24-CD accumulate Gasp, Vermiform, and Fasciclin III, but not Crb, supporting a role for alternative secretion pathways for different proteins, as has been already proposed. Following this logic, overexpressed βPS integrin would then also be trafficked through a different route from that of the endogenous βPS integrin, possibly because of higher expression levels or because of the presence of the Venus fused to the normal protein (Rios-Barrera, 2017).

Drosophila Tango1 was initially found to facilitate collagen secretion in the fat body. More recently, the accumulation of other nonbulky proteins at the ER in the absence of Tango1 has led to the proposal of two models to explain these results: one in which Tango1 regulates general secretion, and the second one where Tango1 is specialized on the secretion of ECM components, since loss of Tango1 leads to the accumulation of the ECM molecules SPARC and collagen. The current results suggest a third explanation, where cargo accumulation in the ER might not necessarily be a direct consequence of only the loss of Tango1. Instead, in addition to depending on Tango1, some proteins of the ECM appear also to depend on each other for their efficient secretion. This is the case for laminins LanB1 and LanB2, which require trimerization before exiting the ER, while LanA can be secreted as a monomer. Loss of collagen itself leads to the intracellular accumulation of ECM components in fat body cells, such as the laminins and SPARC. Conversely, SPARC is required for proper collagen and laminin secretion and assembly in the ECM. Furthermore, intricate biochemical interactions take place between ECM components. Hence, due to the complex genetic and biochemical interactions between ECM components, the dependence of any one of them on Tango1 is difficult to determine without further biochemical evidence. The concept of interdependent protein transport from the ER as such is not new, as it has also been observed in other systems, for instance in immune complexes. During the assembly of T-cell receptor complexes and IgM antibodies, subunits that are not assembled are retained in the ER and degraded (Rios-Barrera, 2017).

Nevertheless, these observations in glial cells, which express laminins but not collagen, allow at least these requirements to be partly separate. This study found that laminins are accumulated due to general ER clogging and not because they rely on Tango1 for their export. This is based on observations that once the protein causing the ER block is removed, laminin secretion can continue in the absence of Tango1. It is still unclear why glial cells can secrete laminins in the absence of collagen whereas fat body cells cannot, but presumably laminin secretion can be mediated by different, unidentified cargo receptors expressed in glial cells (Rios-Barrera, 2017).

This study found that Sec16 forms aberrant aggregates in cells lacking Tango1, as in mammalian cell lines, and that the number of Sec16 particles is reduced. Other studies have shown that Tango1 overexpression produces larger ERES, and that Tango1 and Sec16 depend on each other for localization to ERES. In addition, lack of Tango1 also affects the distribution of Golgi markers. Thus, Tango1 influences not only the trafficking of cargos, but also the morphology of the secretory system (Rios-Barrera, 2017).

It had been suggested that the disorganization of ER and Golgi apparatus in cells lacking Tango1 might be an indirect consequence of the accumulation of Tango1 cargo. The work of Maeda (2016) has provided a possible explanation for the molecular basis, and proposed that Tango1 makes general secretion more efficient, but it has not formally excluded the possibility that the primary cause for the observed defects is secretory protein overload. This study has now shown that this is not the case: In the absence of Tango1, an aberrant ER and Golgi morphology is still observed even after the main primary substrates of Tango1 were removed and, thereby, secretion of other molecules was restored and the ER stress response was prevented (Rios-Barrera, 2017).

ERGIC53 accumulates at the ERES in the absence of Tango1, and this can be partly reversed by removing dpy. This is in apparent contradiction to findings in mammalian cells where Tango1 was necessary for the recruitment of membranes containing ERGIC53 to the budding collagen megacarrier vesicle. However, ERGIC53 also has Tango1-independent means of reaching the ER. The current results indicate that its retrieval from the ER to the ERGIC compartment depends directly or indirectly partly on Tango1. As a cargo receptor for glycoproteins, ERGIC53 may be retained at the ERES as a consequence of the accumulation of its own cargo at these sites. This would mean that it cannot be delivered back to the ERGIC or the cis-Golgi apparatus for further rounds of retrograde transport, which may, in turn, be an explanation for the enlargement of the Golgi matrix protein 130 kD (GM130) compartment seen after Tango1 knockdown (Rios-Barrera, 2017).

The finding that Tango1-depleted cells have a functional secretory pathway despite the ER-Golgi disorganization was unexpected. Stress stimuli like amino acid starvation (but not ER stress response itself) lead to Sec16 translocation into Sec bodies and inhibition of protein secretion. However, uncoupling of ER-Golgi organization from functional secretion has also been observed in other contexts. Loss of Sec23 or Sec24-CD leads to a target peptide sequence KDEL appearing in aggregates of varying sizes and intensities similar to those observed for Sec16 and for KDEL-RFP in cells lacking tango1. Also, GM130 is reduced in Sec23 mutant embryos. Despite these structural problems, these embryos do not show generalized secretion defects and also do not affect the functionality of the Golgi apparatus, as determined by glycosylation status of membrane proteins (Rios-Barrera, 2017).

Thus, Tango1 appears to have an important structural function in coordinating the organization of the ER and the Golgi apparatus, and this, in turn, may enhance vesicle trafficking. This fits with the role of Tango1 in recruiting ERGIC membranes to the ERES, and also with the effects of loss of Tango1 in the distribution of ER and Golgi markers. It has been proposed that the ER and Golgi apparatus in insects, which unlike in mammalian cells is not centralized but spread throughout the cytoplasm, is less efficient for secretion of bulky cargo than mammalian cells that can accommodate and transport it more efficiently through the Golgi ribbon. This difference could explain why tango1 knockout mice seem to have only collagen secretion defects and die only as neonates. However, a complete blockage of the ER might also be prevented by the activity of other MIA3/cTAGE5 family homologs. In mammalian cell culture experiments, even if loss of tango1 affects secretion of HRP, the secretion of other overexpressed molecules like alkaline phosphatase is not affected. This could also be because of the presence of other MIA3/cTAGE5 family homologs. By contrast, because there are no other MIA3/cTAGE5 family proteins in Drosophila, loss of tango1 may lead to the accumulation of a wider range of overexpressed proteins and more overt mutant phenotypes than in mammals (Rios-Barrera, 2017).

Accumulation of laminin monomers in Drosophila glia leads to glial endoplasmic reticulum stress and disrupted larval locomotion

The nervous system is surrounded by an extracellular matrix composed of large glycoproteins, including perlecan, collagens, and laminins. Glial cells in many organisms secrete laminin, a large heterotrimeric protein consisting of an α, β, and γ subunit. Prior studies have found that loss of laminin subunits from vertebrate Schwann cells causes loss of myelination and neuropathies, results attributed to loss of laminin-receptor signaling. This study demonstrates that loss of the laminin γ subunit (LanB2) in the peripheral glia of Drosophila melanogaster results in the disruption of glial morphology due to disruption of laminin secretion. Specifically, knockdown of LanB2 in peripheral glia results in accumulation of the β subunit (LanB1), leading to distended endoplasmic reticulum (ER), ER stress, and glial swelling. The physiological consequences of disruption of laminin secretion in glia included decreased larval locomotion and ultimately lethality. Loss of the γ subunit from wrapping glia resulted in a disruption in the glial ensheathment of axons but surprisingly did not affect animal locomotion. It was found that Tango1, a protein thought to exclusively mediate collagen secretion, is also important for laminin secretion in glia via a collagen-independent mechanism. However loss of secretion of the laminin trimer does not disrupt animal locomotion. Rather, it is the loss of one subunit that leads to deleterious consequences through the accumulation of the remaining subunits (Petley-Ragan, 2016).

The role of laminin and the ECM in glial wrapping has been investigated in vertebrate systems by examining the gross morphological and molecular changes induced by knocking out individual laminin subunits in glia. A decrease in glial wrapping, proliferation, and survival was attributed to a lack of laminin binding to various receptors, including integrins, dystroglycan, and syndecans. However, it was found that knock-out of laminin resulted in more severe phenotypes than the additive phenotypes of multiple-receptor knock-out studies. This paper presents evidence that eliminating one laminin subunit causes cellular changes that go beyond the loss of receptor-ligand signaling and contribute to glial mutant phenotypes (Petley-Ragan, 2016).

This study has demonstrated that glial cells expressing RNAi against three different laminin subunits result in morphological defects. Knockdown of the laminin γ subunit LanB2 results in extensively swollen glia, accumulation of LanB1 in the ER, and expanded ER, leading to ER stress with disrupted intracellular morphology, including vacuole-like structures. In laminin assembly, the β-γ dimer is formed followed by α chain recruitment where trimer formation is necessary for transport through the Golgi and secretion. Studies in vitro and in C. elegans found intracellular accumulation of the γ laminin subunit in the absence of the β subunit. However, the subcellular localization and physiological consequences of the intracellular laminin accumulations was not determined. Analysis of the knockdown of the γ subunit (LanB2) in all glia extended this line of study and found that loss of LanB2 led to β subunit (LanB1) accumulations, ER expansion, and ER stress. Glial swelling due to LanB2 knockdown was correlated with excess LanB1, and the swollen glial phenotype was partially rescued by introducing a heterozygous LanB1 deficiency in the background, effectively halving the amount of LanB1 available for translation. The partial rescue in glial swelling due to LanB1 heterodeficiency supports the hypothesis that unbound LanB1 is acting as a misfolded protein in the ER to induce the unfolded protein response and ER stress. In further support, overexpression of LanB1 alone was found to result in ER aggregates and vacuoles in glia. Knockdown of Tango1, a protein known to mediate ER exit of collagen, resulted in LanB1 and LanB2 accumulations in the ER. Tango1 knockdown results in laminin retention in Drosophila ovary follicle cells. However, the subcellular localization of laminin was not demonstrated. The current results refine these observations as both LanB2 and Tango1 knockdown result in LanB1 accumulations specifically in the expanded ER. However, while LanB2-RNAi led to severe glial swelling and locomotion defects, expression of Tango1 RNAi did not. In the case of Tango1 RNAi, this is possibly due to Tango1 knockdown inhibiting secretion of assembled laminin heterotrimers. Previously, Tango1 has been implicated primarily in collagen secretion, and this study found Tango1 mediates laminin secretion in the absence of collagen IV in perineurial glia. The findings imply that there may be a Tango1-specific binding site on laminin or that Tango1 may play a broader role in protein secretion than previously thought. Together, these findings suggest that the glial defects observed after LanB2 knockdown are due to excess amounts of unbound LanB1 monomers in the ER causing ER stress. Alternatively, the increased LanB1 and ribosomal content in these areas may indicate increased translation due to glia sensing inadequate amounts of external laminin (Petley-Ragan, 2016).

The glial defects observed are likely due to disruption of laminin secretion, but it is possible that the defects observed were in part due to loss of laminin from the extracellular space and loss of ECM receptor signaling, such as integrin signaling. The first explanation is favored as blocking the secretion of the laminin trimer by Tango1 RNAi did not result in glial swelling, suggesting that the reduction in laminin secretion did not lead to glia disruption. Loss of integrins and metalloproteinase-mediated degradation of the surrounding ECM does not lead to glial swelling or formation of vacuoles, but rather triggers a loss of perineurial glia wrapping of the axon. In Drosophila, the fat body and hemocytes contribute to the deposition of ECM proteins in the basement membrane surrounding the nervous system, explaining why the loss of laminin secretion from glia in Tango1 does not lead to the same phenotypes as integrin knockdown (Petley-Ragan, 2016).

This raises the interesting question of why Drosophila glia secrete laminin and not other ECM proteins. Similarly, why do Drosophila wrapping glia, which do not contact a basement membrane, express laminin? Wrapping glia were morphologically affected by LanB2 knockdown. However. accumulations of LanB1 within wrapping glia were not seen, implying that wrapping glia express low levels of laminin. With the available antibodies, it is difficult to detect and assess the levels of laminin between axons and wrapping glia or between wrapping and subperineurial glia. The presence of laminin interior to the nerve is supported by previous experiments where integrin knockdown in wrapping glia resulted in a lack of process extension similar to that seen after LanB2 knockdown, suggesting that integrin may bind to laminin present between axons and wrapping glia or between wrapping and subperineurial glia. Although LanB2 knockdown in wrapping glia resulted in morphological defects, including decreased wrapping, third-instar larvae exhibited no mobility defects and even survived to adulthood and this is also true of integrin knockdown in the wrapping glia (data not shown). The surprising finding that full contact between wrapping glia and peripheral axons is unnecessary for locomotion is consistent with mutants in the Na-K-Cl cotransporter, Ncc69. Ncc69 mutant larvae have significant osmotic swelling that inhibits direct contact between axons and the wrapping glia and yet these larvae had normal action potential activity and survived to adult stages. Apoptosis-induced death of the wrapping glia did, however, result in a total lack of mobility and lethality, indicating that either the wrapping glia or other nrv2-GAL4-expressing cells are essential to survival and locomotion (Petley-Ragan, 2016).

Larval survival and mobility were affected by knockdown of LanB2 in perineurial glia, a glial subtype that does not directly contact axons. Perineurial glia are born late in embryogenesis, surround the CNS and PNS during larval stages, and have been implicated in ECM remodeling during nervous system growth. This study demonstrates that perineurial glia secrete laminin, but not collagen IV, and contribute to nervous system function. The decreased mobility seen in 46F>LanB2-RNAi larvae provides support for the role of the outer layer of perineurial glia and the ECM in structurally supporting the larval nervous system as these glia do not contact either neuronal cell bodies or axons (Petley-Ragan, 2016).

Ultimately, the current findings suggest that loss of receptor-ligand signaling is not the sole cause of glial morphological and physiological defects due to loss of laminins. Due to the conserved structure and function of glia across the animal kingdom, it is likely that Schwann cells lacking one subunit of laminin or with a mutation in one subunit affecting laminin trimerization could also demonstrate morphological and functional defects due to the accumulation of unbound laminin subunits leading to ER stress. This could have significant implications as ER stress is important in the pathogenesis of various diseases affecting myelination in the CNS and PNS. This hypothesis has not yet been investigated in vertebrate glia, but it may provide insight into the reasons decreased glial wrapping, proliferation, and survival are seen in vertebrates after knock-out or with inherited mutations in laminin subunits. Schwann cell-specific deletion of Lamc1 (the γ-1 subunit) leads to a loss of α and β subunit expression and disruption of basement membrane secretion. Loss of γ-1 leads to defects in radial sorting and myelination by myelinating Schwann cells, as well as to loss of proliferation and blocked differentiation in the premyelination stage. Similarly loss of γ-1 leads to the absence of nonmyelinating Schwann cells and the loss of Remak bundles. The wrapping glia of Drosophila are most similar to nonmyelinating Schwann cells and in these cells loss of the γ subunit led to wrapping defects and ER stress. Whether the loss of the vertebrate γ-1 subunit leads to ER stress that results in the loss of nonmyelinating Schwann cells has not been determined. However, the current findings may represent a broader trend in the cellular response to unequal expression of related subunits that together comprise a multimeric protein, such as laminin. Future studies should focus on the effects of ER stress due to mutations in individual subunits of laminin in Schwann cells and other tissues (Petley-Ragan, 2016).

A tendon cell specific RNAi screen reveals novel candidates essential for muscle tendon interaction

Tendons are fibrous connective tissue which connect muscles to the skeletal elements thus acting as passive transmitters of force during locomotion and provide appropriate body posture. Tendon-derived cues, albeit poorly understood, are necessary for proper muscle guidance and attachment during development. This study used dorsal longitudinal muscles of Drosophila and their tendon attachment sites to unravel the molecular nature of interactions between muscles and tendons. A genetic screen using RNAi-mediated knockdown in tendon cells was performed to find out molecular players involved in the formation and maintenance of myotendinous junction; 21 candidates were found out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. Integrin-βPS and Talin, picked as candidates in this screen, are known to play important role in the cell-cell interaction and myotendinous junction formation validating the screen. Candidates were found with enzymatic function, transcription activity, cell adhesion, protein folding and intracellular transport function. Tango1, an ER exit protein involved in collagen secretion was identified as a candidate molecule involved in the formation of myotendinous junction. Tango1 knockdown was found to affect development of muscle attachment sites and formation of myotendinous junction. Tango1 was also found to be involved in secretion of Viking (Collagen type IV) and BM-40 from hemocytes and fat cells (Tiwari, 2015).

O-glycosylation regulates polarized secretion by modulating Tango1 stability

Polarized secretion is crucial in many tissues. The conserved protein modification, O-glycosylation, plays a role in regulating secretion. However, the mechanisms by which this occurs are unknown. This study demonstrates that an O-glycosyltransferase functions as a novel regulator of secretion and secretory vesicle formation in vivo by glycosylating the essential Golgi/endoplasmic reticulum protein, Tango1 (Transport and Golgi organization 1), and conferring protection from furin-mediated proteolysis. Loss of the O-glycosyltransferase PGANT4 resulted in Tango1 cleavage, loss of secretory granules, and disrupted apical secretion. The secretory defects seen upon loss of pgant4 could be rescued either by overexpression of Tango1 or by knockdown of a specific furin (Dfur2) in vivo. These studies elucidate a novel regulatory mechanism whereby secretion is influenced by the yin/yang of O-glycosylation and proteolytic cleavage. Moreover, the data have broader implications for the potential treatment of diseases resulting from the loss of O-glycosylation by modulating the activity of specific proteases (Zhang, 2014).

This study demonstrates that O-glycosylation regulates polarized secretion and secretory vesicle formation in vivo by modulating the stability of an essential component of the secretory apparatus. While previous studies have documented the effects of loss of O-glycosylation on constitutive secretion and secretion of extracellular matrix (ECM) proteins in vivo, the current results elucidate a mechanism by which these effects may occur. This study shows that Tango1 stability is modulated by the presence of O-glycans, which serve to protect it from furin-mediated cleavage. This represents the first example of O-glycans modulating secretion by stabilizing an essential component of the secretory apparatus (Zhang, 2014).

Roles for Tango1 in constitutive secretion, Golgi structure, and COPII vesicle formation have been identified previously, but the factors that regulate its activity and stability are not completely understood. This study demonstrates that Tango1 stability is modulated by the competing activities of a specific O-glycosyltransferase (PGANT4) and a specific furin (Dfur2) in secretory cells of the Drosophila digestive tract. Tango1 is a ubiquitously expressed protein that regulates not only constitutive secretion, but also the formation of large secretory vesicles that transport bulky ECM proteins in certain cells. Tango1/Mia3 is proposed to bind secretory cargo via its luminal domain and COPII coat subunits via its cytoplasmic domain, thereby coordinating the size of secretory vesicles to accommodate large ECM proteins. However, not all cells have the same secretory demands nor produce large vesicular carriers. This study demonstrates that the expression of PGANT4 specifically in the secretory PR cells of the digestive tract confers increased stability to Tango1 by protecting it from Dfur2-mediated cleavage, thereby allowing the formation of large mucin-containing secretory vesicles within these cells. As the enzymes controlling the initiation of O-glycosylation are typically abundantly expressed in cells under high secretory burden, it raises the possibility that O-glycosylation may modulate the stability of Tango1 in other tissues, ensuring that Tango1 activity is commensurate with the secretory demands of the cell (Zhang, 2014).

How are O-glycans specifically influencing Tango1 stability? Based on in vitro studies, O-glycans may influence protease sensitivity by blocking access to vicinal protease cleavage sites. Additionally, O-glycans may affect protein conformation, thus altering protease sensitivity at more distant sites. Finally, O-glycosylation may also influence the binding of partner or cargo proteins, thereby affecting protease access. Although the data suggest that O-glycans added by PGANT4 may influence vicinal protease cleavage, the possibility cannot be ruled out that O-glycans may influence Tango1 conformation and/or cargo binding. Future studies mapping all sites of glycosylation of Tango1 by PGANT4 will aid in determining the mechanisms involved (Zhang, 2014).

PGANT4 is one member of a large family of enzymes (PGANTs in Drosophila, GALNTs in humans, and ppGalNAcTs in mice) that control the initiation of O-glycosylation. These family members are expressed in unique spatial and temporal patterns during development and in adult tissues. Additionally, these enzymes display unique substrate specificities, with some members preferring to add the initial GalNAc (peptide transferases) and others preferring to add GalNAc to previously glycosylated substrates (glycopeptide transferases). The concerted activity of these many members is thought to result in the elaborate glycosylation patterns typically seen in mucin-like molecules. As members of this family are abundantly expressed in many secretory cells and tissues, it raises the possibility that the yin/yang provided by the opposing forces of O-glycosylation and proteolytic cleavage may serve as a more widespread, dynamic system to regulate the stability and bioactivity of many proteins. In support of this theory, recent glycoproteomic studies performed in mammalian cell culture have mapped sites of O-glycosylation to be in close proximity to potential furin cleavage sites on many proteins. Additionally, these studies also identified O-glycans on mammalian Tango1/Mia3, suggesting that O-glycans may perform similar protective functions to control secretion in mammalian systems. Indeed, alterations in both Tango1/Mia3 and O-glycosylation have been associated with diseases of the mammalian gastrointestinal tract, which are typically characterized by loss of secretion, loss of mucous membrane formation, and increased diffusion barrier permeability. Interestingly, PGANT4 is most similar to the mammalian ppGalNAc-T10, which is abundantly expressed in the digestive system. It will be interesting to determine if mammalian Tango1/Mia3 is similarly regulated by O-glycosylation and proteolytic cleavage (Zhang, 2014).

More broadly, the results provide in vivo evidence for models where competing activities of O-glycosyltransferases and proteases may modulate protein stability, with imbalances in these activities contributing to disease. Genome-wide association studies demonstrating a link between O-glycosylation and blood lipid levels have led to the proposal that O-glycans on proteins involved in lipid metabolism may confer protection from proteolysis, thereby influencing HDL-cholesterol and triglyceride levels, and thus cardiovascular disease risk. Similarly, in the case of familial tumoral calcinosis, it is proposed that mutations in an O-glycosyltransferase (GALNT3 in humans and Galnt3 in mice) may lead to decreased FGF23 glycosylation, increased FGF23 cleavage, abnormal phosphate levels, and calcified tumor development in patients. However, although cell culture and in vitro assays have shown that FGF23 can be glycosylated by Galnt3, the glycosylation status of endogenous FGF23 in the presence or absence of Galnt3 has not been examined (in mice or humans). Similarly, although O-glycans can confer protection from protease cleavage of peptides in vitro, a demonstration that cleavage of endogenous FGF23 can be rescued by altering levels of an endogenous protease in vivo has not been previously shown. The current results provide in vivo evidence for the interplay between O-glycosylation and protease sensitivity, supporting the model of how loss of O-glycosylation may contribute to human disease and disease associations. More importantly, the ability to rescue the effects of loss of O-glycosylation by altering the levels of a specific furin in vivo suggests that inhibition/modulation of specific proteases may be a viable treatment option for certain diseases associated with aberrant O-glycosylation (Zhang, 2014).

A Rab10-dependent mechanism for polarized basement membrane secretion during organ morphogenesis

Basement membranes (BMs) are specialized extracellular matrices that are essential for epithelial structure and morphogenesis. However, little is known about how BM proteins are delivered to the basal cell surface or how this process is regulated during development. This study identified a mechanism for polarized BM secretion in the Drosophila follicle cells. BM proteins are synthesized in a basal endoplasmic reticulum (ER) compartment from localized mRNAs and are then exported through Tango1-positive ER exit sites to basal Golgi clusters. Next, Crag targets Rab10 to structures in the basal cytoplasm, where it restricts protein delivery to the basal surface. These events occur during egg chamber elongation, a morphogenetic process that depends on follicle cell planar polarity and BM remodeling. Significantly, Tango1 and Rab10 are also planar polarized at the basal epithelial surface. It is proposed that the spatial control of BM production along two tissue axes promotes exocytic efficiency, BM remodeling, and organ morphogenesis (Lerner, 2013).

Shaping cells and organs in Drosophila by opposing roles of fat body-secreted Collagen IV and perlecan

Basement membranes (BMs) are resilient polymer structures that surround organs in all animals. Tissues, however, undergo extensive morphological changes during development. It is not known whether the assembly of BM components plays an active morphogenetic role. To study in vivo the biogenesis and assembly of Collagen IV, the main constituent of BMs, a GFP-based RNAi method (iGFPi) was used designed to knock down any GFP-trapped protein in Drosophila. Use of this method revealed that Collagen IV is synthesized by the fat body, secreted to the hemolymph (insect blood), and continuously incorporated into the BMs of the larva. Incorporation of Collagen IV determines organ shape, first by mechanically constricting cells and second through recruitment of Perlecan, which counters constriction by Collagen IV. These results uncover incorporation of Collagen IV and Perlecan into BMs as a major determinant of organ shape and animal form (Pastor-Pareja, 2011).

Secretion of collagen in human cells involves components of the CopII coatomer, required for ER-to-Golgi transport in the secretory pathway. It has been shown as well in human cells that Tango1, a CopII cargo adaptor, is required for secretion of Collagen VII. This study knocked down expression of Tango1 and observed that this caused retention of Vkg-GFP in fat body cells. Confocal imaging revealed that Vkg-GFP accumulated in growing intracellular aggregates. The aggregates eventually coalesced and occupied most cytoplasm between the lipid droplets, affecting cell viability. These larvae lacked Vkg-GFP in the BM of other organs, such as the wing disc. The same results was obtained with pumpless-Gal4. Additionally, knockdown of the CopII coat components Sar1 or Sec23 caused similar intracellular accumulation of Vkg-GFP. These results show that fat body cells produce large amounts of Vkg protein and indicate that secretion of Collagen IV in Drosophila requires CopII coated vesicles and Tango1 (Pastor-Pareja, 2011).

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Bard, F., Casano, L., Mallabiabarrena, A., Wallace, E., Saito, K., Kitayama, H., Guizzunti, G., Hu, Y., Wendler, F., Dasgupta, R., Perrimon, N. and Malhotra, V. (2006). Functional genomics reveals genes involved in protein secretion and Golgi organization. Nature 439(7076): 604-607. PubMed ID: 16452979

Ishikawa, Y., Ito, S., Nagata, K., Sakai, L. Y. and Bachinger, H. P. (2016). Intracellular mechanisms of molecular recognition and sorting for transport of large extracellular matrix molecules. Proc Natl Acad Sci U S A 113(41): E6036-E6044. PubMed ID: 27679847

Johnson, A., Bhattacharya, N., Hanna, M., Pennington, J. G., Schuh, A. L., Wang, L., Otegui, M. S., Stagg, S. M. and Audhya, A. (2015). TFG clusters COPII-coated transport carriers and promotes early secretory pathway organization. EMBO J 34(6): 811-827. PubMed ID: 25586378

Ke, H., Feng, Z., Liu, M., Sun, T., Dai, J., Ma, M., Liu, L. P., Ni, J. Q. and Pastor-Pareja, J. C. (2018). Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements. J. Genet. Genomics. PubMed ID: 29935791

Lerner, D. W., McCoy, D., Isabella, A. J., Mahowald, A. P., Gerlach, G. F., Chaudhry, T. A. and Horne-Badovinac, S. (2013). A Rab10-dependent mechanism for polarized basement membrane secretion during organ morphogenesis. Dev Cell 24(2): 159-168. PubMed ID: 23369713

Liu, M., Feng, Z., Ke, H., Liu, Y., Sun, T., Dai, J., Cui, W. and Pastor-Pareja, J. C. (2017). Tango1 spatially organizes ER exit sites to control ER export. J Cell Biol 216(4): 1035-1049. PubMed ID: 28280122

Ma, W. and Goldberg, J. (2016). TANGO1/cTAGE5 receptor as a polyvalent template for assembly of large COPII coats. Proc Natl Acad Sci U S A 113(36): 10061-10066. PubMed ID: 27551091

Maeda, M., Saito, K. and Katada, T. (2016). Distinct isoform-specific complexes of TANGO1 cooperatively facilitate collagen secretion from the endoplasmic reticulum. Mol Biol Cell 27(17): 2688-2696. PubMed ID: 27413011

Pastor-Pareja, J. C. and Xu, T. (2011). Shaping cells and organs in Drosophila by opposing roles of fat body-secreted Collagen IV and perlecan. Dev Cell 21(2): 245-256. PubMed ID: 21839919

Petley-Ragan, L. M., Ardiel, E. L., Rankin, C. H. and Auld, V. J. (2016). Accumulation of laminin monomers in Drosophila glia leads to glial endoplasmic reticulum stress and disrupted larval locomotion. J Neurosci 36(4): 1151-1164. PubMed ID: 26818504

Raote, I., Ortega Bellido, M., Pirozzi, M., Zhang, C., Melville, D., Parashuraman, S., Zimmermann, T. and Malhotra, V. (2017). TANGO1 assembles into rings around COPII coats at ER exit sites. J Cell Biol 216(4): 901-909. PubMed ID: 28280121

Reynolds, H. M., Zhang, L., Tran, D. T. and Ten Hagen, K. G. (2019). Tango1 coordinates the formation of ER/Golgi docking sites to mediate secretory granule formation. J Biol Chem. PubMed ID: 31690624

Rios-Barrera, L. D., Sigurbjornsdottir, S., Baer, M. and Leptin, M. (2017). Dual function for Tango1 in secretion of bulky cargo and in ER-Golgi morphology. Proc Natl Acad Sci U S A 114(48): E10389-E10398. PubMed ID: 29138315

Saito, K., Chen, M., Bard, F., Chen, S., Zhou, H., Woodley, D., Polischuk, R., Schekman, R. and Malhotra, V. (2009). TANGO1 facilitates cargo loading at endoplasmic reticulum exit sites. Cell 136(5): 891-902. PubMed ID: 19269366

Santos, A. J., Nogueira, C., Ortega-Bellido, M. and Malhotra, V. (2016). TANGO1 and Mia2/cTAGE5 (TALI) cooperate to export bulky pre-chylomicrons/VLDLs from the endoplasmic reticulum. J Cell Biol 213(3): 343-354. PubMed ID: 27138255

Tiwari, P., Kumar, A., Das, R. N., Malhotra, V. and VijayRaghavan, K. (2015). A tendon cell specific RNAi screen reveals novel candidates essential for muscle tendon interaction. PLoS One 10: e0140976. PubMed ID: 26488612

Wilson, D. G., Phamluong, K., Li, L., Sun, M., Cao, T. C., Liu, P. S., Modrusan, Z., Sandoval, W. N., Rangell, L., Carano, R. A., Peterson, A. S. and Solloway, M. J. (2011). Global defects in collagen secretion in a Mia3/TANGO1 knockout mouse. J Cell Biol 193(5): 935-951. PubMed ID: 21606205

Yuan, L., Kenny, S. J., Hemmati, J., Xu, K. and Schekman, R. (2018). TANGO1 and SEC12 are copackaged with procollagen I to facilitate the generation of large COPII carriers. Proc Natl Acad Sci U S A 115(52): E12255-E12264. PubMed ID: 30545919

Zhang, L., Syed, Z. A., van Dijk Hard, I., Lim, J. M., Wells, L. and Ten Hagen, K. G. (2014). O-glycosylation regulates polarized secretion by modulating Tango1 stability. Proc Natl Acad Sci U S A 111(20): 7296-7301. PubMed ID: 24799692

date revised: 20 April 2020

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