InteractiveFly: GeneBrief

Rab35: Biological Overview | References

BIOLOGICAL OVERVIEW

The coordination between membrane trafficking and actomyosin networks is essential to the regulation of cell and tissue shape. This study examined Rab protein distributions during Drosophila epithelial tissue remodeling and shows that Rab35 is dynamically planar polarized. Rab35 compartments are enriched at contractile interfaces of intercalating cells and provide the first evidence of interfacial monopolarity. When Rab35 function is disrupted, apical area oscillations still occur and contractile steps are observed. However, contractions are followed by reversals and interfaces fail to shorten, demonstrating that Rab35 functions as a ratchet ensuring unidirectional movement. Although actomyosin forces have been thought to drive interface contraction, initiation of Rab35 compartments does not require Myosin II function. However, Rab35 compartments do not terminate and continue to grow into large elongated structures following actomyosin disruption. Finally, Rab35 represents a common contractile cell-shaping mechanism, as mesoderm invagination fails in Rab35 compromised embryos and Rab35 localizes to constricting surfaces. Various stages of tissue morphogenesis involve the contraction of epithelial surfaces. This study identified the Rab GTPase Rab35 as an essential component of this contractile process, which functions as a membrane ratchet to ensure unidirectional movement of intercalating cells (Jewett, 2017).

How do molecular, cell, and developmental processes drive the production of stereotypical tissue forms and body shapes? This has been a central question in biology, and the pursuit of such answers has driven key discoveries in understanding how cell assemblies coordinate cellular adhesion to enable multicellular life. A common characteristic of many higher organisms is an elongated body axis. This elongated body plan, often oriented along the anterior-posterior (AP) axis, is frequently reiterated at the organ level. Cell intercalation is one of the primary mechanisms that is utilized to direct tissue elongation. Tissue elongation is essential to the shaping of an elongated body axis, as well as the development of many internal organs, such as the palate, cochlea, gut, and kidney (Jewett, 2017).

In epithelial sheets, processes that drive cells to change topological relationships can be harnessed by developmental processes to effect changes in tissue architecture7. The oriented contraction of T1 (or vertical, AP) interfaces followed by the growth of T3 interfaces leads to tissue narrowing and extension. This cellular reshaping requires the function of apical and junctional cytoskeletal and adhesion proteins. Interestingly, force generation and changes in cell shape are not continuous during cell intercalation, but instead occur in pulses with intervening stable periods. In one model, pulsatile actomyosin forces generated in the apical/medial cell regions are believed to initiate intercalary movements through oscillations in apical cell areas. Subsequent enrichment of Myosin II at adherens junctions leads to higher tensile forces at AP interfaces and the destabilization of E-cadherin adhesive complexes. There is thus a system of planar polarized protein distributions during cell intercalation, with F-actin and Myosin II enrichments at AP interfaces and apical domains, and adherens junction-associated proteins such as Bazooka/Par-3, E-cadherin, and Armadillo/β-catenin enriched at dorsal and ventral (DV) neighboring interfaces. This combination of tension-producing actomyosin networks and cadherin-dependent adhesion complexes are believed to be central determinants directing early morphogenesis in the Drosophila embryo; however, the role of membrane trafficking in guiding these events has been less clear. Additionally, how periods of active cytoskeletal contraction are tied to processes that function at the plasma membrane to ensure the consolidation and irreversibility of changes requires clarification (Jewett, 2017).

The Rab family of small GTPase proteins is key mediators of membrane trafficking and cytoskeletal dynamics. Rab proteins regulate membrane compartment behaviors through their association with tethering and trafficking effectors, and mutations in Rab proteins are associated with a variety of diseases and developmental disorders. The Rab trafficking pathways that operate during cell intercalation in the early Drosophila gastrula have remained undefined, although the function of classic Clathrin and Dynamin-dependent early endocytic pathways has been explored. Indeed, it has been demonstrated that Formin and Myosin II proteins direct the endocytic uptake of dextran through specialized CIV (cortical immobile vesicle) structures. Additionally, in Drosophila, Rab35 has been shown to play a critical role in directing the morphogenesis of tracheal tube growth as well as synaptic vesicle sorting at the neuromuscular junction. In tissue culture models, Rab35 functions early in endosomal pathways to drive the generation of newborn endosomes and is essential for the terminal steps of cytokinesis and neurite outgrowth (Jewett, 2017).

This study shows that Rab35 demonstrates remarkable compartmental behaviors at the plasma membrane. Rab35 compartments are initially contiguous with the cell surface and form dynamic structures that grow and shrink on the minute time scale. In the absence of Rab35 function, cell interfaces undergo contractile steps, but these steps rapidly reverse themselves, consistent with Rab35 mediating an essential 'ratcheting' function that directs progressive interface contraction. Rab35 compartments form independently of Myosin II function, but require actomyosin forces to terminate. These compartments further function as endocytic hubs that have transient interactions late in their formation with Rab5 and Rab11 endosomes (Jewett, 2017).

The current results have shown that a trafficking network centered on Rab35 acts as a ratcheting mechanism to ensure that interface contraction is progressive and irreversible. Rab35 functions upstream of junctional actomyosin forces during interface contraction to provide a membranous ratchet, and suggests that cytoskeletal-derived forces are required to terminate compartmental behaviors. Rab35 compartments are more numerous and possess longer compartmental times at contractile interfaces of actively intercalating cells. Rab35 compartments form at the plasma membrane and rapidly grow in size during periods of interface contraction, before shrinking through endocytic-dependent processes. Rab35 compartmental behaviors therefore represent a critical point of convergence at which cytoskeletal and membrane trafficking pathways function to drive changes in cell shape (Jewett, 2017).

Changes in cell shape in many systems are driven by pulsatile processes that initiate directed movement before cycling through periods in which the force generating network reforms. Previous work on ratcheting function has concentrated on different actomyosin regimes governed by Myosin II regulators such as Rho1 and Rok that trigger contraction and cell ratcheting. Importantly, when Rab35 function is disrupted, apical cell areas maintain oscillatory behaviors and AP interface lengths still undergo brief periods of contraction. However, contractile periods are followed by reversals and interfaces re-lengthen, producing a failure in interface shortening. This 'wobble' behavior is consistent with Rab35 functioning as a ratchet ensuring unidirectional movement during interface contraction. These results also suggest that junctional actomyosin-driven behaviors may be responding to an upstream trafficking apparatus centered on Rab35. Indeed, junctional Myosin II localization is disrupted in Rab35 knockdown embryos, and accumulates intracellularly along with integral membrane proteins (Neurotactin) and F-actin. In tissue culture cells, it has been shown that disruption of Rab35 similarly leads to an accumulation of F-actin during abscission, which provides another link between Rab35 and cytoskeletal dynamics. Finally, and again consistent with expectations for a compartment-driven ratcheting function, it is also striking that Rab35 compartments are not present uniformly during cell intercalation, but begin to form specifically as interface contraction occurs (Jewett, 2017).

The AP patterning system is responsible for directing Rab35 compartment formation away from apical/medial sites and functionally engaging Rab35 at AP interfaces. It is interesting to note that high levels of anterior patterning information, such as is present in the head region, inhibit planar polarity, cell intercalation, and Rab35 compartment dynamics, while maintaining interfacial Rab35. The common appearance of apically localized Rab35 compartments in the ventral furrow and in bnt mutant embryos (embryos derived from mothers homozygous for the genes bicoid, nanos and torso-like, 'bnt') suggests a model in which ventral fate genes could mediate apical constriction by re-directing Rab35 compartment formation through the inhibition of AP patterning cues (Jewett, 2017).

Myosin II is found in several different populations within intercalating cells. Transient, web-like Myosin II localizations are found in highly dynamic structures in the apical/medial regions of epithelial cells, while more stable, cable-like structures are present at cell junctions. Junctional Myosin II enrichment is dependent on Rab35 function, and is required for the termination of Rab35 compartments. However, the role of apical/medial actomyosin forces and cell oscillations on Rab35 compartmental behaviors is less clear. It appears that Rab35 compartments may require Myosin II medial 'flows' (Rauzi, 2010) to function as a polarizing cue, as Rab35 compartments become less planar polarized in the absence of Myosin II function. It is hypothesize that cell oscillations produce cycles of high and low tension during which Rab35 compartments can form as infoldings of slackened plasma membrane that, when internalized, prevents interface length from rebounding to the same length as was present prior to area contraction. It is important to note that the termination of Rab35 compartments and the internalization of membrane is critical to ratcheting and productive interface contraction, as merely driving more plasma membrane into Rab35 compartments (as is observed in Y-27632, PitStop2, or chlorpromazine-injected embryos) in the absence of compartment termination does not direct lasting changes in cell topologies (Jewett, 2017).

These results also suggest how oscillatory area contractions can be linked to productive movements. In worm and fly embryos14, 47, actomyosin contractions show both productive and non-productive periods. The functional engagement of area oscillations may depend on the presence of the Rab35 ratchet, as immediately prior to intercalation Rab35 compartments are absent from cell interfaces. However, it is intriguing that Rab35 compartments can form when Myosin II function is compromised in Y-27632-injected embryos. It may be that the small amount of Myosin II function left after Y-27632 injection is enough to drive small oscillations in area, or that thermal fluctuations in cell area are enough for compartment formation. Regardless, this will be an interesting area for further studies (Jewett, 2017).

What is the nature of Rab35 compartments? While there are punctate Rab35 compartments present in the apical cytoplasm that partially colocalize with endosomal markers, the major population of Rab35 during early gastrulation and cell intercalation is present in tubular compartments that are contiguous with the cell surface. The extremely elongated Rab35 plasma membrane tubules observed in either Myosin II-disrupted or endocytosis-inhibited embryos, as well as the immediate filling of Rab35 compartments at the cell surface, are consistent with Rab35 marking endocytic infoldings of the plasma membrane. Additionally, Rab35 compartments are positive for a plasma membrane PIP2 species, PtdIns(4,5)P2, and Rab35 immunogold TEM imaging and the quantification of immunogold particle localization demonstrates that Rab35 is largely present in compartments at the cell surface. Indeed, open infoldings marked by Rab35 immunogold form in subapical zones near the adherens junctions. It is interesting to note that similar open, tubular structures have also been observed by TEM near electron dense junctions during cell intercalation18. When Rab35 function is compromised, there is a shift of endocytic, dextran uptake away from AP interfaces and a dramatic increase in failed endocytic events. In tissue culture, Rab35 has been shown to function early in endosomal pathways and immediately after vesicle scission, to drive the generation of newborn endosomes. The data suggest a subtle shift in Rab35 function to an earlier time point occurs during early Drosophila morphogenesis, but is broadly consistent with a Rab35-driven function in directing the delivery of membrane from the cell surface to endosomal pathways. However, the results do not exclude an added role for Rab35 at endosomal compartments, as F-actin and Myosin II accumulate intracellularly in Rab35 compromised embryos. This accumulation is at substantially lower levels than occurs at the cell cortex in wild-type embryos, but could contribute to the observed cell intercalation dynamics, especially the ~17% decrease in oscillatory area amplitudes (Jewett, 2017).

The association of Rab35 compartments with markers for early (Rab5) or recycling (Rab11) endosomes was also examined. While the large majority of Rab35 compartments do not localize with early or recycling endosomes, there was a small fraction of compartments that do (15%). Interestingly, when observed under live imaging conditions, it became apparent that Rab35 compartments do often have transient interactions with endosomal compartments (98%) that occur specifically toward the end of Rab35 lifetimes, consistent with Rab35 feeding endocytosed membrane through a specialized plasma membrane contiguous compartment. These results are consistent with the broader conclusion of this work that endocytic uptake of plasma membrane components is essential to ensuring irreversible changes in interface length that drive cell neighbor exchange. This endocytic uptake could function through two potential mechanisms: (1) E-cadherin adhesion complex removal, or (2) general membrane removal, which would require that individual interfaces possess isolated membrane domains. Embryos that expressed fluorescently labeled Rab35 and E-cadherin did not express E-cadherin at levels sufficient to be resolvable by confocal microscopy, thus making it difficult to distinguish between these models, but this will be an important open question going forward. Previous work has proposed that MyoII function clusters E-cad complexes for endocytosis, while the current results show that Rab35 functions upstream of junctional Myosin. Thus, Rab35 compartments may serve to direct Myosin II clustering of E-cadherin adhesion complexes for endocytosis and junctional remodeling (Jewett, 2017).

An interesting feature of Rab35 behaviors is the absence of paired compartments at contracting interfaces. Approximately 50% of Rab35 compartments form without the presence of any Rab35 compartments on the opposing side of an interface, and 93% of compartments are present without a matching, synchronous Rab35 compartment. This monopolarity of Rab35 function is the first evidence of asymmetric behaviors across a common, shared interface during early germ-band extension (GBE). As Rab35 compartmental behaviors reflect Myosin II activity, this further suggests that there are likely anisotropic forces on opposing sides of individual interfaces. This anisotropy could drive the generation of shear forces that would cause the dissociation of homophilic extracellular E-cadherin bonds. It is also intriguing that, in terms of area oscillations, Rab35 compartmental formation is best correlated with cycles of area contractions in the opposing cell at a shared interface. This would be consistent with the above model in which changes in apical cell area are transmitted through E-cadherin junctions to a neighboring cell that may be in a differentially tensioned state. This may then again permit slackened plasma membrane to be taken up by Rab35-dependent endocytic processes (Jewett, 2017).

A PtdIns(3,4,5)P(3) dispersal switch engages cell ratcheting at specific cell surfaces

Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating contractile forces before cyclically disassembling. This pulsed nature of cytoskeletal forces implies that there must be ratcheting mechanisms that drive processive transformations in cell shape. Previous work has shown that force generation is coordinated with endocytic remodeling; however, how ratcheting becomes engaged at specific cell surfaces remains unclear. This study reports that PtdIns(3,4,5)P(3) is a critical lipid-based cue for ratcheting engagement. The Sbf RabGEF binds to PIP(3), and disruption of PIP(3) reveals a dramatic switching behavior in which medial ratcheting is activated and epithelial cells begin globally constricting apical surfaces. PIP(3) enrichments are developmentally regulated, with mesodermal cells having high apical PIP(3) while germband cells have higher interfacial PIP(3). Finally, this study shows that JAK/STAT signaling constitutes a second pathway that combinatorially regulates Sbf/Rab35 recruitment. Results elucidate a complex lipid-dependent regulatory machinery that directs ratcheting engagement in epithelial tissues (Miao, 2021).

Cell shaping processes use contractile force generation to drive the active contraction of specific cell surfaces that causes tissues to adopt new morphogenetic forms. This selective contraction of cell surfaces drives a diverse range of processes from tissue invagination to cell intercalation to epithelial cell extrusion and wound healing. A key discovery in the last decade of work on these processes is that they are often pulsatile in nature, with highly transient actomyosin populations that briefly apply a tensioning force to an area of the cell cortex. This has raised a central outstanding question-if actomyosin networks display such pulsed behaviors, how does processivity emerge from cyclic systems? To obtain lasting changes from such systems, there is a requirement for ratcheting mechanisms that gain irreversible changes out of periodic, contractile cycles (Miao, 2021).

Several potential molecular mechanisms of cell ratcheting have been discovered, ranging from processes that direct the turnover and reformation of elastic cortical networks to pathways that rely on direct remodeling of the plasma membrane. Previous work has shown that a membranous ratchet centered on SET-domain-binding factor (Sbf) RabGEF and Rab35 function is a critical part of the pathway used to remodel the cell surfaces during early morphogenesis in Drosophila. This membrane ratcheting module is deployable to specific cell surfaces-during cell intercalation, Sbf and Rab35 tubular invaginations of the plasma membrane are primarily found at planar polarized contracting interfaces, where they mediate the processive loss of AP (anterior-posterior) surfaces required for neighbor exchange through endocytic mechanisms. Alternatively, during the apical constrictions that drive invagination of the ventrally located mesoderm, Sbf and Rab35 are enriched at the apical surface, where they direct the ratcheted loss of apical surface areas. However, how this ratcheting activity is selectively recruited to specific cell surfaces is unknown. Although actomyosin function is required to terminate Sbf/Rab35 compartments, it is not required for Sbf and Rab35 recruitment. Thus, Sbf and Rab35 recruitment is not downstream of actomyosin function, suggesting that yet-to-be-identified mechanisms are in place to control Sbf/Rab35 compartmental formation (Miao, 2021).

One potential important cue for directing the localized activities of plasma-membrane-associated proteins are the lipid-signaling phosphoinositides (PIPs), which are often found in microdomains in the plasma membrane. PIPs have been shown to be potent regulators of both membrane trafficking and cytoskeletal networks. The various PIP phospho-species, especially PI(4,5)P2 and PI(3,4,5)P3, can regulate the activity of a number of endocytic-regulatory proteins, such as AP-2 and Dynamin. PIPs are also implicated in controlling actin assembly and plasma membrane-cytoskeletal linkage by binding directly and tightly to at least 30 regulatory proteins. PI(3,4,5)P3 in particular has been deeply implicated in the regulation of membrane trafficking processes such as regulated endocytosis and exocytosis. Upregulation of PIP3 levels induces recycling of the epidermal growth factor receptor to the cell surface, and PIP phospho-balance can regulate syncytial cleavage furrow lengths in the fly embryo. Further, recently published work suggests that PIPs may lay downstream of Toll receptor activity and Src activation in the germband epithelium. However, how PIPs regulate the dynamic cell shape changes that occur as epithelial sheets change dimensions is unclear, and the function of PIP phospho-species during Drosophila gastrulation remains to be closely examined. This study identified phosphatidylinositol phosphates as providing lipid-based membrane cues for morphogenesis and cellular gastrulation dynamics in the early Drosophila embryo. PI(3,4,5)P3 inhibition leads to a potent disruption of contractile cellular behaviors, and PIP3 function controls a 'constriction switch' that determines if epithelial cells enter into an apical constriction or cell interface contraction regime. This switch in contractile surfaces then determines which of two primary morphogenetic paradigms the developing tissue will follow-a primarily intercalatory process driven by neighbor exchange events, or the loss of apical areas that lead to furrow formation and tissue invagination (Miao, 2021).

The ability of epithelial cells to remodel specific cellular surfaces is central to determining cell dimensions as well as the neighbor community that they will interact with and adhere to. This ability to either grow or contract certain cell sides will determine overall cell shape, and the cumulative effects of these cell shape changes determines tissue behaviors and morphologies. By regulating the contraction of apical surfaces versus cell-cell interfaces, a tissue can drive events as diverse as furrow formation and cell ingression to cell intercalation and the intermixing of cells along the AP axis. However, it has been unclear whether direct cues reside within the plasma membrane that may guide and control the engagement of contractile forces. This study examined the function of plasma membrane phospholipids in recruiting Sbf-Rab35-driven ratcheting. PI(3,4,5)P3 was shown to regulates a switch in ratcheting engagement-a reduction in PIP3 levels causes a reorientation of Sbf-Rab35 compartment formation to apicomedial surfaces. This relocalization is sufficient to change the reversible oscillations in cell area that occur in the wild-type germband epithelium into a processive regime in which apical cell areas shrink and ectopic furrows are formed. Sbf, the guanine nucleotide exchange factor for Rab35, can directly bind PIP3, and PIP3 levels and sites of enrichment are differentially regulated between the germband and ingressing mesoderm to provide a differential lipid-based cue between these two tissues (Miao, 2021).

It is interesting to note that while ectopic furrows form after PIP3 disruption, these furrows are often disorganized and lack the regular appearance of the main ventral furrow that drives the ingression of mesoderm during gastrulation. In some respects, this is to be expected-whether the major portion of the embryo is transformed to attempt to contract apical surfaces, then cells will be engaged in a contractile tug-of-war against each other. This condition is likely shown by the juxtaposition of small and large cells, which is observed in both the germband as well as the ventral furrow after PIP3 disruption. The uneven contraction of apical surfaces may also reflect the different temporal dynamics of Sbf-Rab35 compartments after PIP3 disruption. The new medial compartments that form after PIP3 disruption are much more stable (indeed, lifetimes up to 10x longer in some measurements) and robust than those that form in either the germband or ventral furrow in control embryos. Previous work has shown the importance of contractile cycles to achieve a uniform overall contraction of the apical surfaces. For example, in circumstances where pulsatility, but not contractility, is compromised, contractile networks have been observed to tear and separate-this results in a similar loss of cell area uniformity as detected after PIP3 levels are downregulated. Thus, the change in Sbf-Rab35 compartment function to much more stable and longer cycles may enhance the tug-of-war element of the cell contractions previously referenced, producing 'winner' cells of much smaller apical areas and 'loser' cells that cannot shrink against the pulling forces of neighboring cells and thus possess larger apical areas. It is intriguing that the pulsatility of contraction appears to be such a fundamental element of contractile processes-pulsatility has been observed across a huge variety of contraction-driven processes ranging from wound healing to compaction of the mouse embryo to neuroblast ingression (Miao, 2021).

Another interesting aspect of this work is that both phosphatidyl inositol phosphate species and JAK/STAT-dependent signaling control where ratcheting engagement occurs. If either of these pathways is disrupted, then a medial signal dominates and Sbf-Rab35 compartment formation occurs in a central, apical location. The current model suggests that PIP3 and JAK/STAT signaling may provide a dispersal signal that guides the compartments from a single apico-central location to the cell periphery (in the case of germband epithelial cells) or to smaller, more dispersed apical locations (in cells of the ventral furrow). Based on the direct binding of PIP3 by the Sbf RabGEF in the PIP binding assay, this dispersal may be through a direct interaction. The experiments did not have the resolution to determine if small PIP3 microdomains exist in the plasma membrane, or if further systems direct the formation of smaller, compartmental assemblies. On the other hand, how does JAK/STAT direct ratcheting engagement? Previous work examining the apical constrictions driven by an absence of JAK/STAT signaling implicated a repression of WASP actin networks that, when activated, may cause the enhanced recruitment of apical myosin II populations. Interestingly, this fits with the current step detection measurements. In addition to the changes in Sbf-Rab35 localization, pulsed contractions are stronger and more sustained in JAK/STAT embryos than in PIP3-disrupted embryos. However, previous results have shown that the generation of Sbf-Rab35 compartments is independent of myosin II function. Thus, from these results, we suggest that, while PIP3 directly regulates ratcheting engagement, JAK/STAT may regulate both the underlying oscillatory machinery as well as the strength of the medial signal that directs ratcheting engagement. It is interesting to note that, through the use of automated step detection measurements based on mean squared displacements, it has been previously shown that the oscillatory machinery appears to be strengthened specifically in the ventral cells that will undergo processive apical constriction to form the ventral furrow, which suggests a commonality with the behaviors observed after JAK/STAT disruption (Miao, 2021).

Phosphatidylinositol phosphates and their function in morphogenetic processes PIP phospho-species are attractive candidates to provide important spatial information as they are directly embedded in target membranes. PIP3 has long been implicated in distinguishing the leading edge in migrating cells and is rapidly upregulated after cells are stimulated with chemoattractant where it promotes F-actin assembly necessary for cell crawling. However, how PIP3 regulates gastrulation events has been relatively unstudied. A recent work has demonstrated the Pi3K92E can bind to active Toll receptors and Src signaling complexes, and is planar polarized at AP interfaces, in keeping with our finding of developmental enrichment of PIP3 in the germband and suggesting an intriguing connection between planar positional information and PIP function (Tamada, 2021). PIP3 is necessary for cell migration events in the mesenchymal gastrulation movements that occur in zebrafish, while data from Drosophila have shown that PIP3 levels are upregulated after wounding and help cells recognize affected surfaces. Disrupting PIP3 levels disrupted dorsal closure in the late embryo, where, once again, PIP3 is found at higher levels specifically at those surfaces that are driving tissue remodeling. This work similarly finds the PIP3 levels are developmentally patterned, where they are enriched at contractile surfaces. Other work has shown that a PIP2/PIP3 balance affects actomyosin contractility during the cellularization process that creates the early embryonic epithelium through the recruitment of an actin stabilizer, bottleneck. Sbf-Rab35 compartments have represented an interesting convergence point between pathways that directly regulate cell membrane remodeling and those that control cortical force generation. Going forward, it will be interesting to examine if this convergence includes similar higher-level regulation of the protein networks that have been implicated in migrating systems (Miao, 2021).

This work demonstrates a fundamental switch in contractile behaviors depending on the activity and localization of the PI(3,4,5)P3 lipid cue. There were several limitations to these studies: first, many of the functional disruptions relied on pharmacological or shRNA knockdown lines which often produce only hypomorphic disruptions. Phenotypes were confirmed with secondary shRNA lines that targeted different regions of the selected mRNA and yet produced similar defects; however, deeper disruption of these genes may produce more severe defects at these stages or earlier in development. Second, a PIP3 biosensor (tGPH-GFP) was used to detect PIP3 localization and levels-this is the standard in the field but represents an indirect binder of PIP3, so future probe development may allow a better resolution of PIP3 behaviors. As was mentioned, it would be very interested to be able to better address if PIP3 microdomains exist in the plasma membrane, and if these may directly trigger site-specific Sbf/Rab35 compartmental formation, but these were not resolvable with the combination of probe and microscopy elements. Finally, gastrulation in the early Drosophila embryo shows a remarkable robustness to disruption, so additional phenotypes may be concealed by compensatory mechanisms that were not detected in our analysis (Miao, 2021).

Tracheal tube fusion in Drosophila involves release of extracellular vesicles from multivesicular bodies

Extracellular vesicles (EVs) comprise diverse types of cell-released membranous structures that are thought to play important roles in intercellular communication. While the formation and functions of EVs have been investigated extensively in cultured cells, studies of EVs in vivo have remained scarce. This study reports that EVs are present in the developing lumen of tracheal tubes in Drosophila embryos. Two distinct EV subpopulations are defined, one of which contains the Munc13-4 homologue Staccato (Stac) and is spatially and temporally associated tracheal tube fusion (anastomosis) events. The formation of Stac-positive luminal EVs depends on the tracheal tip-cell-specific GTPase Arl3, which is also required for the formation of Stac-positive multivesicular bodies, suggesting that Stac-EVs derive from fusion of Stac-MVBs with the luminal membrane in tip cells during anastomosis formation. The GTPases Rab27 and Rab35 cooperate downstream of Arl3 to promote Stac-MVB formation and tube fusion. It is proposed that Stac-MVBs act as membrane reservoirs that facilitate tracheal lumen fusion in a process regulated by Arl3, Rab27, Rab35, and Stac/Munc13-4 (Camelo, 2022).

This paper reports the presence of membranous EVs in the lumen of developing tracheal tubes in Drosophila embryos. A subset of these EVs is associated with tracheal tube fusion sites and carries the Munc13-4 ortholog Stac. Stac associates with lysosome-related organelles (LROs) that display features of MVBs, the formation of which, as well as of Stac-EVs, depends on the fusion-cell-specific GTPase Arl3. Moreover, we identified Rab27 and Rab35 as partially redundant regulators of Arl3-dependent Stac-MVB formation and tracheal tube fusion. It is proposed that tracheal anastomosis formation involves fusion of Stac-MVBs with the luminal membranes of FCs, resulting in release of Stac-EVs. Attempts to visualize tracheal EV exocytosis using the pH-sensitive fluorescent proteins pHluorin fused to the lysosomal transmembrane protein Lamp1 were unsuccessful. However, it was possible to detect EVs that emerged at and remained associated with anastomosis sites upon completion of dorsal trunk (DT) fusion. Moreover, the absence of Stac-EVs from fusion-defective tracheal metameres in shot mutant embryos supports the idea that Stac-EVs are released during tube fusion (Camelo, 2022).

How is MVB formation regulated in FCs? Consistent with the finding that Rab27 and Rab35 act downstream of Arl3 to promote Stac-MVB formation, Rab27a, Rab27b and Rab35 regulate MVB-dependent exosome secretion in mammalian cells. Moreover, the Stac homolog Munc13-4 is a key effector of Rab27a in LRO docking in hematopoietic cells and melanocytes, and regulates MVB maturation and exosome release in cancer cells. Whereas Rab27 and Rab35 are required for Stac-MVB formation or for anchoring Stac at MVBs, Rab39 is dispensable for FC-specific Stac-LRO formation, suggesting that it acts in a successive step, such as transport or plasma membrane docking of LROs. While the current findings define a core pathway of MVB maturation and EV release in tracheal cells, additional players, such as ESCRT complex components, are likely to be involved. MVBs also act as intermediates in intracellular lumen formation in tracheal terminal cells, which resemble FCs in forming seamless tubes. Interestingly, Rab35 directs transport of apical components to promote terminal cell lumen growth. Whether Rab35 also participates in MVB formation in tracheal terminal cells remains to be tested (Camelo, 2022).

Despite substantial progress in characterizing EVs and their mode of action in cultured cells, studies of physiological functions of EVs in vivo have remained scarce. Exosomes are released into the seminal fluid in Drosophila testis accessory glands and modulate female reproductive behavior. Glia-derived exosomes stimulate growth of motor neurons and tracheae in Drosophila larvae through exosomal microRNA-dependent gene regulation in target cells. Moreover, cardiomyocyte-derived EVs are taken up by macrophages and endothelial cells in zebrafish embryos, and may play a role in trophic support of endothelial cells during vasculogenesis. It will be exciting to explore possible functions of tracheal EVs, including potential roles in long-range intercellular communication across the tracheal lumen. However, the luminal EVs that this study discovered in late-stage embryos are unlikely to participate in such intercellular communication, as the presence at this stage of a luminal matrix appears to constrain EV mobility, and the cuticle covering the apical tracheal cell surface presumably prevents fusion of luminal EVs with the plasma membrane. Rather, Stac-EVs are likely to constitute remnants of MVB-plasma membrane fusion events during anastomosis formation. Stac-MVBs could serve as membrane reservoirs that facilitate lumen fusion. ILVs might back-fuse with the MVB limiting membrane, resulting in a rapid increase of membrane material available at the luminal membrane interfaces in FCs. Such a mechanism has been described in dendritic cells, where MVBs carrying major histocompatibility complex class II (MHC II) in ILVs are reorganized after stimulation, resulting in tubular membrane extensions and transport of MHCII to the plasma membrane. Recruitment of ILVs to the MVB limiting membrane will increase the surface area and may lead to surface exposure of ILV membrane proteins that could promote membrane fusion. Exploring how MVB exocytosis is regulated in tracheal cells, and whether similar mechanisms contribute to vascular anastomosis formation in vertebrates, will be exciting subjects for future studies (Camelo, 2022).

Whacked and Rab35 polarize dynein-motor-complex-dependent seamless tube growth

Seamless tubes form intracellularly without cell-cell or autocellular junctions (see Labarsky, 2003). Such tubes have been described across phyla, but remain mysterious despite their simple architecture. In Drosophila, seamless tubes are found within tracheal terminal cells, which have dozens of branched protrusions extending hundreds of micrometres. This study has found that mutations in multiple components of the dynein motor complex block seamless tube growth, raising the possibility that the lumenal membrane forms through minus-end-directed transport of apical membrane components along microtubules. Growth of seamless tubes is polarized along the proximodistal axis by Rab35 and its apical membrane-localized GAP, Whacked. Strikingly, loss of whacked (or constitutive activation of Rab35) leads to tube overgrowth at terminal cell branch tips, whereas overexpression of Whacked (or dominant-negative Rab35) causes formation of ectopic tubes surrounding the terminal cell nucleus. Thus, vesicle trafficking has key roles in making and shaping seamless tubes (Schottenfeld-Roames, 2013).

Three tube types -- multicellular, autocellular and seamless -- are found in the Drosophila trachea. Most tracheal cells contribute to multicellular tubes or make themselves into unicellular tubes by wrapping around a lumenal space and forming autocellular adherens junctions, but two specialized tracheal cell types, fusion cells and terminal cells, make 'seamless' tubes. How seamless tubes are made and how they are shaped are largely unknown. One hypothesis holds that seamless tubes are built by 'cell hollowing', in which vesicles traffic to the centre of the cell and fuse to form an internal tube of apical membrane, whereas an alternative model proposes that apical membrane is extended internally from the site of intercellular adhesion. In both models, transport of apical membrane would probably play a key role. As terminal cells make seamless tubes continuously during larval life, they serve as an especially sensitive model system in which to dissect the genetic program (Schottenfeld-Roames, 2013).

Tracheal cells are initially organized into epithelial sacs with their apical surface facing the sac lumen. During tubulogenesis, γ-tubulin becomes localized to the lumenal membrane of each tracheal cell, generating microtubule networks oriented with minus ends towards the apical membrane. Terminal and fusion cells are first selected as tip cells that undergo a partial epithelial-to-mesenchymal transition and initiate branching morphogenesis: they lose all but one or two cell-cell contacts and become migratory. Branchless-FGF signalling induces a subpopulation of tip cells to differentiate as terminal cells. During larval life, terminal cells ramify on tissues spread across several hundred micrometres, with branching patterns that reflect local hypoxia. A single seamless tube forms within each branched extension of the terminal cell (Schottenfeld-Roames, 2013).

How trafficking contributes to seamless tube morphogenesis is unknown. Despite clues that vesicle transport plays a role in the genesis of seamless tubes, the tube morphogenesis genes remain elusive. This study characterized the cytoskeletal polarity of larval terminal cells, shows that a minus-end-directed microtubule motor complex is required for seamless tube growth, and characterizes mutations in whacked (wkd) that uncouple seamless tube growth from the normal spatial cues. Sequence analysis indicates that wkd encodes a RabGAP, and it was shown that Rab35 is the essential target of Wkd, and that together, Wkd and Rab35 can polarize the growth of seamless tubes (Schottenfeld-Roames, 2013).

Apical-basal polarity and cytoskeletal organization was examined in mature larval terminal cells. The lumenal membrane was decorated by puncta of Crumbs, a definitive apical membrane marker. Actin filaments were found enriched in three distinct subcellular domains: surrounding seamless tubes, decorating filopodia and outlining short stretches of basolateral membrane. The microtubule cytoskeleton also seemed polarized, with γ-tubulin lining the seamless tubes and enriched at tube tips. These data are consistent with tracheal studies in the embryo. EB1::GFP analyses of growing (plus-end) microtubules demonstrated that some are oriented towards the soma and others towards branch tips. Stable acetylated microtubules ran parallel to the tubes and extended beyond the lumen at branch tips where they may template tube growth. Consistent with such a role, microtubule-tract-associated fragments of apical membrane were observed distal to the blind ends of the seamless tubes. Filopodia extended past the stable microtubules as expected. These data indicate that mature terminal cells maintain the polarity and organization described for embryonic terminal cells. On the basis of γ-tubulin localization, it is inferred that a subset of microtubules is nucleated at the apical membrane, and that apically targeted transport along such microtubules would require minus-end motor proteins. Indeed, homozygous mutant Lissencephaly-1. As γ-tubulin lines the entire apical membrane, growth through minus-end-directed transport might be expected to occur all along the length of seamless tubes, and indeed, a pulse of CD8::GFP (transmembrane protein tagged with GFP) synthesis uniformly labelled the apical membrane as it first became detectable (Schottenfeld-Roames, 2013).

The cytoplasmic dynein motor complex drives minus-end-directed transport of intracellular vesicles in many cell types; to test for its requirement in seamless tube formation, terminal cells were examined mutant for any of four dynein motor complex genes: Dynein heavy chain 64C (Dhc64C), Dynein light intermediate chain (dlic), Dynactin p150 (Glued) and Lis-1. Mutant terminal cells showed a cell autonomous requirement for these genes. Mutant terminal cells had thin cytoplasmic branches that lacked air-filling, and antibody staining revealed that seamless tubes did not extend into these branches although acetylated microtubules often did. It was also noted that formation of filopodia at branch tips is disrupted in dynein motor complex mutants, which may account for the decreased number of branches in mutant terminal cells. Ectopic seamless tubes that were not air-filled were detected near the nucleus, as described below. Interestingly, discontinuous apical membrane fragments (similar to those in Lis-1 embryos) were found in terminal branches lacking seamless tubes, and were associated with microtubule tracts. Whereas γ-tubulin was enriched on truncated tubes and on these presumptive seamless tube intermediates, diffuse γ-tubulin staining was detected throughout the mutant cells, indicating that assembly of apical membrane is required to establish or maintain γ-tubulin localization. Likewise, Crumbs seemed reduced and aberrantly localized. Reduced levels of acetylated microtubule staining in these cells may reflect loss of apical γ-tubulin. Importantly, these data show that stable microtubules extend through cellular projections that lack seamless tubes. Thus, without minus-end-directed transport, stable microtubules are insufficient to promote seamless tube formation, but stable cellular projections are formed and maintained in the absence of seamless tubes (Schottenfeld-Roames, 2013).

In contrast to these defects in seamless tube generation, mutations in wkd confer overly exuberant tube growth. Examination of wkd terminal cell tips revealed a 'U-turn' phenotype in which seamless tubes executed a series of 180 degree turns -- the possibility is entertained that branch retraction, similar to that observed in talin mutants, could contribute to the U-turn defect (Schottenfeld-Roames, 2013).

Homozygous wkd animals survived until pharate adult stages, and, other than the seamless tube defects, had normal tracheal tubes at the third larval instar. Mosaic analysis revealed a terminal cell autonomous requirement for wkd. Mutant clones in multicellular tubes, and in unicellular tubes that lumenize by making autocellular adherens junctions, were of normal morphology. Strikingly, fusion cells, which also form seamless tubes, were unaffected by loss of wkd (Schottenfeld-Roames, 2013).

To determine the molecular nature of wkd, a positional cloning approach was taken. Mapping techniques defined a candidate gene interval of ~ 75 kilobases (kb). Focused was placed on CG5344 as it encodes a protein containing a TBC (Tre2/Bub2/Cdc16) domain characteristic of Rab GTPase-activating proteins, and hence was likely to participate in vesicular trafficking, a process that could lie at the heart of seamless tube formation. A single nucleotide change was identified that resulted in mis-sense (PC24) and non-sense (220) mutations in the CG5344 coding sequence. Pan-tracheal knockdown of wkd by RNA-mediated interference (RNAi) caused terminal cell-specific U-turn defects (other defects characteristic of the ethyl methanesulfonate (EMS)-induced alleles of wkd were detected at a low frequency). A genomic rescue construct for CG5344 rescued wkd mutants, confirming gene identity. On the basis of these results, it is concluded that wkd is CG5344 and that it probably regulates vesicular trafficking during seamless tube morphogenesis (Schottenfeld-Roames, 2013).

To determine the Rab target(s) of Wkd regulation, whether tracheal expression of constitutively active 'GTP-locked'; Rab isoforms (henceforth, RabCA) might phenocopy wkd was investigated. RabCA for 31 of the 33 Drosophila Rabs were tested individually in the tracheal system. Rab35CA alone conferred terminal-cell-specific U-turns defects (Schottenfeld-Roames, 2013).

To evaluate Wkd overexpression, UAS-wkd was expressed in wild-type animals in a pan-tracheal pattern. Excess Wkd caused formation of ectopic seamless tubes surrounding the terminal cell nucleus. At higher levels of expression, small spheres of apical membrane were found adjacent to the nucleus and less abundantly at more distal sites. Consistent with Wkd regulation of vesicle trafficking by modulation of Rab35, expression of a dominant-negative Rab35 (henceforth, Rab35DN) caused formation of ectopic proximal tubules (Schottenfeld-Roames, 2013).

Attempts were made to determine whether Rab35 was the essential target of Wkd GAP activity. Wkd primary structure is equally conserved in three human RabGAPs. All three act as Rab35GAPs, although each has been proposed to have additional targets. To further determine if Wkd acts as a Rab35GAP, whether Rab35DN could suppress wkd mutants was examined; tracheal-specific expression of Rab35DN strongly suppressed the 'U-turn'; defects of wkd-null animals and, surprisingly, also rescued the lethality of wkd. Since mutant Rab35 isoforms phenocopy wkd gain and loss of function, Rab35DN bypasses the requirement for wkd and human Wkd orthologues are Rab35GAPs, it is concluded that the critical function of Wkd is as a GAP for Drosophila Rab35 (Schottenfeld-Roames, 2013).

In other systems Rab35 is implicated in polarized membrane addition to plasma membrane compartments -- for example, immune synapse, cytokinetic furrow and so on -- or, in actin regulation. A role for actin in fusion cell seamless tube formation has been proposed, so whether Wkd and Rab35 act by modulation of the terminal cell actin cytoskeleton was examined. As the actin-bundling protein Fascin (Drosophila singed) was recently identified biochemically as a Rab35 effector, a role of singed in terminal cell tubes was examined, but found no evidence was found for one. Furthermore, overexpression of Wkd, or of Rab35DN, did not significantly alter the terminal cell actin cytoskeleton, leading to the conclusion that actin regulation is not a primary function of Wkd/Rab35 during seamless tube morphogenesis (Schottenfeld-Roames, 2013).

The alternative model -- that Rab35 acts in polarized membrane addition -- was found to be attractive, because extra Rab35-GTP activity promoted seamless tube growth at branch tips whereas depletion of Rab35-GTP promoted tube growth at the cell soma. To test this model, advantage was taken of the information that expression of an activated Breathless-FGFR (lambdaBtl in terminal cells induces robust growth of ectopic seamless tubes surrounding the nucleus; whether growth of the ectopic tubes could be redirected from the soma to the branch tips was investigated by eliminating wkd. The activated FGFR phenotype was not altered in wkd heterozygotes, but in wkd mutant animals (or wkd-RNAi animals) the site of ectopic seamless tube growth was strikingly different. In some cells, extra tubes were found throughout the cell -- in the soma and at branch tip -- whereas in others extra tubes were present only at the branch tip. Thus, the position of seamless tube growth is dependent on Wkd activity, although Wkd itself is not essential for tube formation. These data provide evidence against branch retraction (as occurs in talin mutants) as the mechanism for generating a U-turn phenotype, because branch retraction would not redirect ectopic tube growth (Schottenfeld-Roames, 2013).

To better understand how Wkd and Rab35 determined the site of seamless tube growth, their subcellular distribution was examined. Pan-tracheal expression of mKate2-tagged Wkd (Wkd::mKate2) rescued wkd-null animals. The steady-state subcellular localization of Wkd::mKate2 was restricted to the lumenal membrane with higher accumulation at the growing tips of seamless tubes. At lower levels, cytoplasmic puncta of Wkd::mKate2 were noted that could reflect vesicular localization, as well as labeling of filopodia. It was found that YFP::Rab35 was distributed in a diffuse pattern throughout the terminal cell cytoplasm with some apical enrichment, and notable localization to filopodia. Substantial co-localization of Wkd::mKate2with YFP::Rab35 was found at the apical membrane, in cytoplasmic puncta, and in filopodia. Among endosomal Rabs, Rab35 seemed uniquely abundant within filopodia, and showed the greatest overlap with Wkd at the apical membrane. Substantial overlap was noted between Wkd/Rab35 and acetylated microtubules, including at positions distal to the blind end of seamless tubes. The enrichment of Wkd along seamless tubes indicates that Rab35 functions in an apical membrane trafficking event, leading to the speculation that recycling endosomes at filopodia might be targeted to the growing seamless tube by minus-end motor transport (Schottenfeld-Roames, 2013).

In a similar vein, it is speculated that vesicles might be transported from the soma towards branch tips in a process regulated by Wkd and Rab35. Disruption of such transport might explain why overexpression of Wkd leads to ectopic seamless tube growth in the soma. Whether Wkd::mKate2 localization was compromised in dynein motor complex mutants was examined. As these cells have branches that lack apical membrane/seamless tubes, disruption in the localization pattern of Wkd was anticipated, but it was wondered whether co-localization with acetylated tubulin would be intact, indicative of a microtubule association independent of dynein motor transport. It was found that Wkd::mKate2 is broadly distributed throughout the cytoplasm of dynein motor complex mutants, and does not show enrichment on acetylated microtubule tracts; indeed, substantial basal enrichment was detected of Wkd::mKate2. If Wkd/Rab35-dependent trafficking of apical vesicles was dynein motor complex dependent, ectopic seamless tubes would be expected in the soma of dynein motor complex mutants, similar to those seen with Wkd overexpression or expression of Rab35DN. In fact, such ectopic tubes were consistently found in the dynein motor complex mutants, consistent with dynein-dependent trafficking of Rab35 vesicles. It cannot be ruled out that these defects are due to dynein-dependent processes unrelated to Wkd and Rab35; however, whether the ectopic tubes could be redirected distally by expression of Rab35CA, or elimination of Wkd, was examined. The motor complex ectopic tube phenotype could not be altered, indicating that the phenotype does not arise as an indirect consequence of altered Wkd localization or Rab35 activity (Schottenfeld-Roames, 2013).

The roles of RabGAP proteins have started to become clear only in recent years. Historically, it has been difficult to determine which Rab proteins are substrates of specific RabGAPs. Tests of in vitro GAP activity produced conflicting results, and in some cases did not seem indicative of in vivo function. Indeed, the specificity of Carabin (also known as Wkd orthologue TBC1D10C) has been controversial: it was first shown to act as a RasGAP, whereas later studies indicate a Rab35-specific GAP activity. The in vivo genetic data for wkd, together with recent studies characterizing the function of all three human Wkd-like TBC protein, make a compelling case that this family of proteins acts as GAPs for Rab35. Furthermore, this study establishes a role for classical vesicle trafficking proteins in seamless tube growth. As seamless tubes, but not multicellular or autocellular tracheal tubes, are affected by mutations in wkd and Rab35, this study also establishes an in vivo cell-type-specific requirement for trafficking genes in tube morphogenesis (Schottenfeld-Roames, 2013).

It is concluded that Wkd and Rab35 regulate polarized growth of seamless tubes, and it is speculated that Wkd and Rab35 direct transport of apical membrane vesicles to the distal tip of terminal cell branches (when equilibrium is shifted towards active Rab35-GTP), or to a central location adjacent to the terminal cell nucleus (when equilibrium is shifted towards inactive Rab35-GDP). Analogous to its previously described roles in targeting vesicles to the immune synapse in T cells, the cytokinetic furrow in Drosophila S2 cells and the neuromuscular junction in motor neurons, Rab35 would promote transport of vesicles from a recycling endosome compartment to the apical membrane. It is further speculated that Breathless-FGFR activation at branch tips may couple terminal cell branching with seamless tube growth within that new branch (Schottenfeld-Roames, 2013).

Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins

Exchange of proteins at sorting endosomes is not only critical to numerous signaling pathways but also to receptor-mediated signaling and to pathogen entry into cells; however, how this process is regulated in synaptic vesicle cycling remains unexplored. This work presents evidence that loss of function of a single neuronally expressed GTPase activating protein (GAP), Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity. Analyses of genetic interactions with the ESCRT machinery as well as chimeric ubiquitinated synaptic vesicle proteins indicate that endosomal trafficking facilitates the replacement of dysfunctional synaptic vesicle components. Consequently, sky mutants harbor a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release. Thus, the trafficking of vesicles via endosomes uncovered using sky mutants provides an elegant mechanism by which neurons may regulate synaptic vesicle rejuvenation and neurotransmitter release (Uytterhoeven, 2011).

Synaptic vesicles recycle locally at the synapse, and this study now provides genetic evidence that the synapse holds the capacity to regulate the sorting of synaptic vesicle proteins at 2xFYVE and Rab5 positive endosomes. 2xFYVE-GFP positive endosomes involved in signaling pathways exist at nerve endings. Likewise, membrane invaginations and endosomal-like cisternae form as a result of bulk membrane uptake during intense nerve stimulation, and in various endocytic mutants. However, the current data indicate that endosomes that accumulate in sky mutants are fundamentally different from these 'endocytic cisternae.' First, endocytic cisternae are not enriched for the endosomal markers 2xFYVE and Rab5. Second, endosomal structures in sky mutants do not appear to directly form at the plasma membrane. Third, endocytic cisternae may directly fuse with the membrane, however, based on mEJC and TEM analyses, sky induced endosomes appear to function as an intermediate station. Note that FM 1-43 dye can enter and leave endosomes in sky mutants, suggesting small synaptic vesicles can form at these structures. Together with time-course experiments in sky mutants, endosomes specifically accumulate upon stimulation and dissipate during rest. Therefore, the data suggest that endosomes in sky mutants constitute an intermediate step in the synaptic vesicle cycle. Such a compartment may also be operational in wild-type synapses because a 2xFYVE-GFP endosomal compartment can be largely depleted from synaptic vesicles upon endocytic blockade and because synaptic vesicles harbor proteins that are also commonly found on endosomes (Uytterhoeven, 2011).

Rab GTPases are involved in numerous intracellular trafficking events. In particular in synaptic vesicle traffic, Rab3, and its close isoform Rab27 have been implicated in vesicle tethering and/or priming, while Rab5 has been also implicated in endocytosis. However, an involvement of Rab proteins in other aspects of the synaptic vesicle cycle, including recycling, remains enigmatic. This systematic analysis of CA Rabs now implicates several Rabs in synaptic recycling. First, consistent with previous results, the data suggest that Rab5 mediates transport to synaptic endosomes. Second, Rab7^CA and Rab11^CA appear to inhibit new vesicle formation, likely by controlling post endosome trafficking. Finally, this study found that Rab23^CA and Rab35^CA mediate transport to or retention of synaptic vesicles at sub-boutonic structures. Neither of these Rabs had yet been implicated in synaptic vesicle traffic and hence, these in vivo studies identified different Rab proteins involved in aspects of the synaptic vesicle cycle (Uytterhoeven, 2011).

The expression of Rab5^CA and Rab35^CA results in increased trafficking of vesicles to -or retention of vesicles at- sub-boutonic structures, and unlike expression of Rab23^CA, Rab5^CA and Rab35^CA also result in a facilitation of neurotransmitter release similar to sky mutants. Rab35 has been found to localize to the plasma membrane (Sato, 2008) and this study shows enrichment at NMJ boutons close to the membrane as well. Rab35 has been implicated in endosomal, clathrin-dependent traffic, phagocytic membrane uptake in non-neuronal cells and actin filament assembly during Drosophila bristle development (Allaire, 2010, Sato, 2008, Shim, 2010, Zhang, 2009). Given these roles, Rab35 is ideally posed to also play a role in the endosomal traffic of synaptic vesicles, and the current in vitro and in vivo genetic interaction studies indicate Sky to be a Rab35 GAP in synaptic vesicle trafficking. Although Rab5 mediates endosomal traffic in different cell types, the data indicate that Sky is not a GAP for Rab5 in vivo at the synapse. While this study does not exclude Sky activating the GTPase activity of alternative Rabs in different contexts, the results suggest a Sky-Rab35 partnership that restricts endosomal trafficking of synaptic vesicles (Uytterhoeven, 2011).

In most cell types the ESCRT complex mediates sorting of ubiquitinated proteins into multivesicular bodies; however, such a function has not been characterized for synaptic vesicle components. This study provide evidence that the ESCRT complex mediates synaptic vesicle protein sorting in sky mutants where synaptic vesicles cycle excessively via endosomes. This study found genetic interactions with ESCRT genes. In addition, more efficient clearance of an artificially ubiquitinated synaptic vesicle protein, Ub-nSybHA, was also found in sky mutants, and this effect is ESCRT dependent. Combined, the data indicate endosomal sorting to control the quality of proteins in the synaptic vesicle cycle in sky mutants (Uytterhoeven, 2011).

Increased transmitter release as a result of endosomal sorting e.g. in sky mutants, may appear beneficial in some instances, however in inhibitory neurons for example, such an effect could result in reduced neuronal signaling while in excitatory neurons this feature could over activate postsynaptic cells. Clearly, fine-tuned neurotransmission in neuronal populations may ensure optimal information flow, and misregulation of Sky activity in neuronal populations may cause systemic defects including larval paralysis and death. Further underscoring this notion, in humans, mutations in the Sky orthologue TBC1D24 are causative of focal epilepsy (Uytterhoeven, 2011).

The sorting mechanism uncovered by loss of Sky function or upon expression of Rab35^CA may address the use-dependent decline in protein- or lipid-function at synapses, and the continuous need to rejuvenate the synaptic vesicle protein pool. In addition, it may also constitute a mechanism to remove inappropriately endocytosed membrane components from the vesicle pool. The trafficking pathway inhibited by Sky may also yield the ability of synapses to adapt the functional properties of their synaptic vesicles, thus controlling the nature or abundance of proteins involved in vesicle fusion and neuronal signaling (Uytterhoeven, 2011).

Rab35 mediates transport of Cdc42 and Rac1 to the plasma membrane during phagocytosis

Phagocytosis of invading microbes requires dynamic rearrangement of the plasma membrane and its associated cytoskeletal actin network. The polarization of Cdc42 and Rac1 Rho GTPases to the site of plasma membrane protrusion is responsible for the remodeling of actin structures. However, the mechanism of Rho GTPase recruitment to these sites and the identities of accessory molecules involved in this process are not well understood. This study uncovered several new components involved in innate immunity in Drosophila melanogaster. The data demonstrate that Rab35 is a regulator of vesicle transport required specifically for phagocytosis. Moreover, recruitment of Cdc42 and Rac1 to the sites of filopodium and lamellipodium formation is Rab35 dependent and occurs by way of microtubule tracks. These results implicate Rab35 as the immune cell-specific regulator of vesicle transport within the actin-remodeling complex (Shim, 2010).

Phagocytes, including macrophages and monocytes, play a critical role in host defense mechanisms. They migrate to sites of infection and eliminate invading microbes by using a number of different methods, including phagocytosis. These processes require morphological changes that are typically mediated by changes in actin structure. Therefore, actin filament rearrangement must be tightly regulated during this process. Previous reports have demonstrated that Rho GTPases such as Cdc42 and Rac1 are essential to actin filament rearrangement during the formation of lamellipodial and filopodial membrane protrusions, respectively. These Rho GTPases are recruited to specific areas where actin rearrangement will occur. For example, Cdc42 and Rac1 are recruited to the phagocytic cup during phagocytosis. The location and distribution of these Rho GTPases are critical for cell migration and morphology. As such, how these proteins polarize within the cell is an important topic under research by many laboratories. A positive feedback loop involving vesicular active transport has been suggested to promote Cdc42 polarization. Alternatively, a combination of transport and lateral diffusion in the plasma membrane of budding yeast appears to be responsible for Cdc42 accumulation at sites adjacent to the bud scar. However, how these Rho GTPases are involved in the regulation of plasma membrane protrusion during phagocytosis is not well understood (Shim, 2010).

This study demonstrates that Cdc42 and Rac1 polarity on the plasma membrane is established through microtubule-based vesicle transport rather than lateral diffusion toward the locus of actin filament rearrangement in Drosophila. The translocation of Cdc42 and Rac1 to the site of dynamic actin rearrangement was completely dependent on Rab35 activity. It is intriguing that Rab35 activity was manifested mainly in Drosophila hemocytes, resulting in specific defects in the immune response against infection. Several reports suggested the possibility that polar distribution of Cdc42 is established through actomyosin-directed vesicle transport via actin cables. However, blockade of Cdc42 polarization by inhibition of microtubule polymerization, as well as proper Cdc42 localization to the plasma membrane despite the absence of actin polymerization, suggests that translocation of this Rho GTPase occurs through the microtubule network (Shim, 2010).

Consistent with the current observation, several studies recently reported that Rab35 appears to regulate cell shape and actin cytoskeletal rearrangement during neurite outgrowth and bristle development. Based on the effect of Rab35 on cell shape, Chevallier (2009) suggested that that Rab35 may activate Cdc42 during actin remodeling. However, the current data indicate that Rab35 does not regulate the activity of Rho GTPase directly. Instead, it appears to control the recruitment of the Rho GTPases to the plasma membrane during cytoskeletal rearrangement. These results indicate that Rab35 plays an important role in actin filament rearrangement in diverse developmental processes (Shim, 2010).

Examination of whether a physical interaction between Rab35 and Cdc42 or Rac1 exists by using immunoprecipitation demonstrated a lack of direct physical association among these molecules. Thus, how the vesicles regulated by Rab35 specifically load Cdc42 and Rac1 as cargo remains unknown. Additional studies are required to further determine how vesicles select Rho GTPases as well as how Rab35 functions during the loading and selection steps of vesicle transport (Shim, 2010).

Rab35 controls actin bundling by recruiting fascin as an effector protein

Actin filaments are key components of the eukaryotic cytoskeleton that provide mechanical structure and generate forces during cell shape changes, growth, and migration. Actin filaments are dynamically assembled into higher-order structures at specified locations to regulate diverse functions. The Rab family of small guanosine triphosphatases is evolutionarily conserved and mediates intracellular vesicle trafficking. This study found that Rab35 regulates the assembly of actin filaments during bristle development in Drosophila and filopodia formation in cultured cells. These effects were mediated by the actin-bundling protein fascin, which directly associated with active Rab35. Targeting Rab35 to the outer mitochondrial membrane triggered actin recruitment, demonstrating a role for an intracellular trafficking protein in localized actin assembly (Zhang, 2009).

A dynamic actin network is required for normal cell morphology, cell locomotion, and cytokinesis. These processes require polymerization of globular actin monomers into filaments and bundling of the filaments under the control of actin-binding proteins (ABPs). Certain ABPs cross-link filamentous actin (F-actin) into ordered parallel bundles that maintain the structural integrity of the cell and are structural components of filopodia, stereocilia, and microvilli. It is unclear how cells initiate the dynamic assembly of actin at the right times and places during development, physiological stresses, injury, and disease (Zhang, 2009).

The importance of F-actin bundling during development is readily apparent during bristle formation in Drosophila. Bristles are mechanosensory organs found in genetically controlled locations on the cuticle. Their shapes and growth are dependent on actin bundles. The largest bristles, macrochaetae, are formed by a 'shaft' cell that extrudes a cytoplasmic extension. This extension contains evenly distributed microtubules and F-actin bundles located just beneath the plasma membrane. Bristle morphologies reflect the organization of actin bundles and can be used to study the regulation of actin in vivo (Zhang, 2009).

Rab proteins constitute the largest subset of Ras-family small guanosine triphosphatases (GTPases). Rab proteins control formation, motility, and docking of vesicles in specific trafficking pathways by recruiting specific effector proteins to different membrane compartments. Rab proteins are evolutionarily conserved: Each of the >70 types of mammalian Rab proteins is related to a particular Drosophila Rab protein. This study tested all 31 Drosophila Rab GTPases systematically for their abilities to influence fly development, with the use of dominant negative (DN) mutant proteins produced in specific cell types. Rab activities are controlled by a cycle of associations with GTP or guanosine diphosphate (GDP). The DN mutants contained a Thr/Ser --> Asn mutation that causes the proteins to bind preferentially to GDP and remain inactive. Overproduced DN proteins presumably tie up interacting proteins such as Rab exchange factors in nonproductive associations (Zhang, 2009).

Only one of the 31 Drosophila Rab proteins had a strong effect on the actin cytoskeleton. Flies producing DN Rab35 (Rab35DN) in the peripheral nervous system (driven by prospero-gal4) exhibited unique and specific bristle morphology defects not seen with any other Rab DN gene. Rab35DN caused the development of adult bristles that had sharp bends, kinks, and forked ends in the thoraxand other body regions including the head. Bristles from Rab35DN-expressing flies, stained with the actin-binding dye phalloidin at 45 to 47 hours after puparium formation, had a wavy, loose, and disconnected actin organization relative to the wild-type control. Similar phenotypes are observed in mutants deficient for certain ABPs, which suggests that Rab35 may function as an ABP or through ABP(s). In the thoracic cuticle, production of Rab11DN or Rab5DN, which block Rab proteins that regulate endocytic trafficking, caused extensive defects in membrane growth and bristle distribution; these phenotypes are distinct from the Rab35DN effect. Rab35 was ubiquitously expressed, with transcripts and proteins especially abundant in the developing nervous system (Zhang, 2009).

To test whether the defective-bristle actin phenotype was due to reduced Rab35 function, UAS-Rab35 hairpin RNA interference (RNAi) was expressed in flies to reduce Rab35 mRNA. The RNAi caused the same phenotypes as did Rab35DN, which confirmed that the DN protein was inhibiting the intended target. The bristle phenotype caused by Rab35RNAi was completely rescued upon expression of a mouse wild-type Rab35 gene. In an otherwise wild-type genetic background, mouse Rab35DN caused the same phenotype as did fly Rab35DN in the peripheral nervous system. Thus, at least some functions of Rab35 protein are conserved from flies to mammals, an evolutionary span of ~500 million years (Zhang, 2009).

Expressing wild-type Rab35 (Rab35WT) in cultured cells induced multiple filopodia-like cellular extensions. No such effects were seen upon expression of Rab35DN. Similarly, Rab35 induces peripheral processes in Jurkat T cells and neurite outgrowth in PC12 and N1E-115 cells. The effect of extra Rab35 on cultured cells might reflect its role in vivo, allowing shaft cells to sprout protrusions during bristle development. Rab35DN, in contrast, stopped filopodia growth in vitro and caused defects in growing bristles in vivo. Ubiquitous Rab35DN expression driven by tubulin-gal4 caused lethality in embryos and, over a period of days, the death of cultured cells (Zhang, 2009).

Treating Drosophila S2 cells with the actin polymerization inhibitor latrunculin A, but not with the microtubule-disrupting agent nocodazole, efficiently blocked the Rab35-driven morphology change. Thus, Rab35 appeared to regulate actin assembly (Zhang, 2009).

To explore how Rab35 influences the actin cytoskeleton, this study set out to identify effector proteins that bind Rab35 directly. Rab effectors bind specific Rab proteins; binding is dependent on the Rab protein being in its GTP-bound, active state. The effectors have diverse functions in vesicle sorting, motor protein binding, vesicle trafficking, membrane fusion, and other roles yet to be defined. Affinity chromatography was used to purify proteins that preferentially bind Rab35-GTP, which has been used to identify other Rab effectors. Purified glutathione S-transferase (GST)-tagged Rab35WT protein was loaded with guanosine 5'-O-(3'-thiotriphosphate) (GTP-γ-S) or with GDP and incubated with bovine brain cytosolic extracts. Several proteins were found to bind Rab35-GTP-γ-S specifically. Mass spectrometry revealed a prominent 55-kD polypeptide that bound Rab35-GTP-γ-S to be fascin. Myc-tagged Rab35 and FLAG-tagged fascin coimmunoprecipitated from cell extracts. Fascin bound more strongly to Rab35WT than to Rab35DN. Purified GST-Rab35 fusion protein bound fascin in vitro. Thus, Rab35 binds fascin directly. Fascin bound more strongly to Rab35WT preloaded with GTP-γ-S than to Rab35WT preloaded with GDP in immunoprecipitations of endogenous Rab35 from fly cells and in GST pull-downs, consistent with the identification of fascin as a Rab35 effector (Zhang, 2009).

Fascin is an actin cross-linking protein that organizes F-actin into tightly packed parallel bundles in protruding (filopodia) and nonprotruding (microspike) structures at the leading edges of cells. Higher than normal fascin levels have been associated with cancer cell migration, so the protein has been proposed as a marker for cancer diagnosis and a therapeutic target. Fascin is produced in many tissues and is especially abundant in the nervous system. In Drosophila, fascin mutants (called singed) are female sterile and have aberrant mechanosensory bristles due to dysfunctional actin structures. The Drosophila egg chamber is composed of a germline cyst surrounded by a somatic follicular epithelium. Each cyst consists of 15 nurse cells and one oocyte. Cortical cytoskeletal structures are required during late oogenesis when nurse cell cytoplasm is rapidly transferred to the oocyte. The sterility phenotype of singed led to an examination of the influence of Rab35DN in nurse cells (driven by tubulin-gal4 at 22°C) or follicle cells. Both caused female sterility (94%). The interfering Rab35DN caused reduced ovarian actin levels relative to control flies, and ovary structure was abnormal (Zhang, 2009).

Whether the physical interaction between Rab35 and fascin was reflected in a genetic interaction was tested. Rab35RNAi, produced in peripheral neurons, was used to damage bristle development. Altered bristle morphology was suppressed when extra fascin was supplied to the same cells. Increased fascin compensated for reduced Rab35 function, which suggests that fascin is at least one of the major proteins regulated by Rab35 (Zhang, 2009).

Purified GST-fascin and GST-Rab35 were mixed together or separately with purified nonmuscle F-actin in vitro. Actin-bundling activity increased with fascin concentration. No effect of Rab35 on actin bundling was observed, alone or in combination with fascin. Thus, Rab35 has no discernible effect on actin bundling in vitro, but its association with fascin may be a means to control when or where actin is bundled in vivo (Zhang, 2009).

Perhaps activated Rab35 recruits fascin to a subcellular location where fascin stimulates actin bundling. To explore this idea, the relative locations of Rab35 in different cell types were studied and its association with fascin in mammalian cells. Rab35WT was enriched near the plasma membrane and colocalized with fascin at the leading edge of filopodia and within microspikes in lamellipodia. Rab35DN caused severe alteration of cellular structure and accumulated to a greater extent in the cytosol than along the membrane. In the presence of Rab35DN, less membrane-associated fascin was observed. The membrane association of Rab35 and fascin was confirmed by a cell fractionation analysis; 33.4% of Rab35 fused to yellow fluorescent protein (YFP-Rab35) and 17.7% of fascin fractionated with the membrane fraction. The association of fascin with the membrane fraction was at least partially dependent on Rab35, because less Rab35 and less fascin associated with the membrane in cells where Rab35DN was expressed. Thus, Rab35 may bring fascin to the plasma membrane to influence subcortical actin structure and initiate filament bundling (Zhang, 2009).

If fascin mediates the effects of Rab35, then Rab35-induced filopodia formation should be reduced when fascin function is blocked. Fascin was depleted in Rab35-expressing cells by introducing fascin small hairpin RNA. The interfering RNA significantly blocked filopodia formation induced by Rab35. Phosphorylation of fascin at Ser39 is important for its actin-bundling activity and proper localization to filopodia. Point mutants were produced that mimic the active dephosphorylated (Ser39 --> Ala, S39A) or inactive phosphorylated (Ser39 --> Asp, S39D) forms of fascin. Expression of tdTomato-tagged S39A or S39D fascin mutants in NIH 3T3 cells along with Rab35 had opposite effects on filopodia formation. The S39A mutant in combination with Rab35WT caused more protein to accumulate at the tip of cell extensions relative to wild-type fascin plus Rab35WT, but no significant increase in the number of filopodia was observed. In contrast, S39D in combination with Rab35 reduced the number of filopodia. In these cells most of the Rab35WT protein remained near the plasma membrane. These results are consistent with a role for fascin as a downstream effector of Rab35 in filopodia formation (Zhang, 2009).

A gene was constructed encoding a modified form of Rab35 targeted to the outer mitochondrial membrane, a location that normally has modest levels of fascin or assembled actin and no detectable Rab35. When Rab35DN was used in this experiment, the modified protein was on the surface of mitochondria. No discernible change in actin structure was observed in the vicinity of mitochondria. In contrast, when Rab35WT was brought to the mitochondrial outer membrane, the mitochondria were consistently decorated with increased actin meshworks. As a control, Rab5WT targeted to mitochondria in the same manner did not cause actin assembly in the vicinity of mitochondria. Fascin relocation to mitochondria was confirmed by cell fractionations. Mitochondrial enrichment of fascin was observed when Rab35WT-mito was produced. Thus, relocation of Rab35 can drive the location of actin assembly (Zhang, 2009).

The results show that a Rab35 effector protein, fascin, is able to stimulate local actin bundling and thus control bristle and filopodia formation. The exact ways in which such a mechanism may be used probably vary among cell and tissue types. In cultured cells, active Rab35 recruits fascin to drive actin bundling at the leading edge of cell protrusions. During Drosophila bristle development, Rab35 may recruit fascin and induce actin bundling to initiate the cytoplasmic extension required for bristle extension. Inadequate Rab35 function leads to bends and kinks in the bristles (Zhang, 2009).

Conflicting results about Rab35 function have been obtained from different cell types and models. Rab35RNAi revealed a function in cytokinesis in Drosophila S2 cells; in HeLa cells, Rab35 also plays a role in cytokinesis. No cytokinesis phenotype was observed in mutants of Caenorhabditis elegans Rab35, but Rab35 transports yolk receptors in oocytes. In HeLa-CIITA, a cell line in which major histocompatibility complex (MHC) class II is expressed, Rab35 regulates a recycling pathway in a clathrin-, AP2-, and dynamin-independent manner. In Jurkat T cells, Rab35-mediated recycling appears to be clathrin-dependent. In PC12 and N1E-115 cells, activated Rab35 stimulates neurite outgrowth via a Cdc42-dependent pathway. Drosophila Rab35, like mammalian Rab35, is found near the plasma membrane, on intracellular vesicles, and in the cytosol. This broad distribution contrasts with more discrete locations of other Rab proteins, which suggests that Rab35 may have diverse functions (Zhang, 2009).

Interfering with Rab35 in living flies showed its importance for normal bristle morphology and its function in regulating actin assembly. The powerful influence of Rab35 on the cytoskeleton can now be at least partly explained by localization of fascin and its consequent influence on actin filament bundling (Zhang, 2009).

Search PubMed for articles about Drosophila Rab35

Allaire, P. D., Marat, A. L., Dall'Armi, C., Di Paolo, G., McPherson, P. S. and Ritter, B. (2010). The Connecdenn DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes. Mol Cell 37(3): 370-382. PubMed ID: 20159556

Camelo, C., Korte, A., Jacobs, T. and Luschnig, S. (2022). Tracheal tube fusion in Drosophila involves release of extracellular vesicles from multivesicular bodies. J Cell Sci. PubMed ID: 35019140

Chevallier, J., Koop, C., Srivastava, A., Petrie, R. J., Lamarche-Vane, N. and Presley, J. F. (2009). Rab35 regulates neurite outgrowth and cell shape. FEBS Lett 583(7): 1096-1101. PubMed ID: 19289122

Jewett, C. E., Vanderleest, T. E., Miao, H., Xie, Y., Madhu, R., Loerke, D. and Blankenship, J. T. (2017). Planar polarized Rab35 functions as an oscillatory ratchet during cell intercalation in the Drosophila epithelium. Nat Commun 8(1): 476. PubMed ID: 28883443

Rauzi, M., Lenne, P. F. and Lecuit, T. (2010). Planar polarized actomyosin contractile flows control epithelial junction remodelling. Nature 468(7327): 1110-1114. PubMed ID: 21068726

Sato, M., Sato, K., Liou, W., Pant, S., Harada, A. and Grant, B. D. (2008). Regulation of endocytic recycling by C. elegans Rab35 and its regulator RME-4, a coated-pit protein. EMBO J 27(8): 1183-1196. PubMed ID: 18354496

Schottenfeld-Roames, J. and Ghabrial, A. S. (2012). Whacked and Rab35 polarize dynein-motor-complex-dependent seamless tube growth. Nat Cell Biol 14(4): 386-393. PubMed ID: 22407366

Shim, J., Lee, S. M., Lee, M. S., Yoon, J., Kweon, H. S. and Kim, Y. J. (2010). Rab35 mediates transport of Cdc42 and Rac1 to the plasma membrane during phagocytosis. Mol Cell Biol 30(6): 1421-1433. PubMed ID: 20065041

Uytterhoeven, V., Kuenen, S., Kasprowicz, J., Miskiewicz, K. and Verstreken, P. (2011). Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins. Cell 145(1): 117-132. PubMed ID: 21458671

Zhang, J., Fonovic, M., Suyama, K., Bogyo, M. and Scott, M. P. (2009). Rab35 controls actin bundling by recruiting fascin as an effector protein. Science 325(5945): 1250-1254. PubMed ID: 19729655

date revised: 18 February 2024

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