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. Author manuscript; available in PMC: 2015 Jun 1.
Published in final edited form as: Dev Biol. 2014 Mar 4;390(1):41–50. doi: 10.1016/j.ydbio.2014.02.021

Exocyst-mediated membrane trafficking is required for branch outgrowth in Drosophila tracheal terminal cells

Tiffani A Jones 1, Linda S Nikolova 1, Ani Schjelderup 1, Mark M Metzstein 1,*
PMCID: PMC4041209  NIHMSID: NIHMS573339  PMID: 24607370

Abstract

Branching morphogenesis, the process by which cells or tissues generate tree-like networks that function to increases surface area or in contacting multiple targets, is a common developmental motif in multicellular organisms. We use Drosophila tracheal terminal cells, a component of the insect respiratory system, to investigate branching morphogenesis that occurs on the single cell level. Here, we show that the exocyst, a conserved protein complex that facilitates docking and tethering of vesicles at the plasma membrane, is required for terminal cell branch outgrowth. We find that exocyst-deficient terminal cells have highly truncated branches and show an accumulation of vesicles within their cytoplasm and are also defective in subcellular lumen formation. We also show that vesicle trafficking pathways mediated by the Rab GTPases Rab10 and Rab11 are redundantly required for branch outgrowth. In terminal cells, the PAR-polarity complex is required for branching, and we find the PAR complex is required for proper membrane localization of the exocyst, thus identifying a molecular link between the branching and outgrowth programs. Together, our results suggest a model where exocyst mediated vesicle trafficking facilitates branch outgrowth, while de novo branching requires cooperation between the PAR and exocyst complexes.

Keywords: Terminal cells, exocyst complex, PAR complex, Drosophila, trachea, branching morphogenesis, lumenogenesis

Introduction

Branching architecture is found in many biological contexts and facilitates numerous biological functions at both the multiple- and single-cell level. For instance, multicellular branching events in the vertebrate lung increase surface area available for gas diffusion (Gehr et al., 1981; Warburton et al., 2010), while branching in neurons allows individual cells to make numerous contacts with targets, which promotes multiplicative signal propagation and processing (Bilimoria and Bonni, 2013; Vetter et al., 2001). There has been significant progress in elucidating the mechanisms of multicellular branching (Carmeliet and Jain, 2011; Conway et al., 2001; Warburton et al., 2005; 2000), but much less is known about the mechanisms underlying subcellular branching. During subcellular branching, membrane bound cytoplasmic extensions emerge from a cell, these extension undergo further bifurcation events to make a network of membrane bound cellular branches. Such subcellular branching presumably depends on membrane addition to specific sites on the plasma membrane. Iterative rounds of such site specification and outgrowth produce a branched cellular morphology. However, the molecular machinery that regulates site specification and membrane addition required for subcellular branching, remains poorly understood.

We use terminal cells, a component of the Drosophila tracheal system, to investigate the molecular machinery required for the development of a branched cellular morphology. Terminal cells are located at the ends of a network of cellular tubes used for insect respiration, where they elaborate processes onto target tissues to supply oxygen and other gases (Ghabrial et al., 2003; Locke, 1957; Samakovlis et al., 1996). Terminal cells are born during embryogenesis and maintain a simple unbranched morphology until hatching (Guillemin et al., 1996). Throughout larval stages, terminal cells grow and branch extensively in response to the fibroblast growth factor (FGF) Branchless (Bnl), which is secreted by hypoxic target tissues (Jarecki et al., 1999). Bnl activates the FGF receptor Breathless (Btl), expressed in terminal cells, to stimulate both outgrowth and branching (Gervais and Casanova, 2011; Jarecki et al., 1999; Lee et al., 1996; Sutherland et al., 1996;). Concurrent with branching, terminal cells form a subcellular lumen through which oxygen is supplied to hypoxic tissue (Gervais and Casanova, 2010; Jarecki et al., 1999; Ruiz et al., 2012; Schottenfeld-Roames and Ghabrial, 2012;). Subcellular lumen formation is thought to be driven by processes of vesicle trafficking guided by the cytoskeleton (Jayanandanan et al., 2014) but the detailed mechanisms by which this occurs, or how the branching of the lumen is coordinated with the cytoplasmic branches is not well understood.

Genetic screens have identified a number of genes required for terminal cell branching morphogenesis and lumen formation (Baer et al., 2007; Ghabrial et al., 2011; Jones and Metzstein, 2011; Levi et al., 2006). One mechanism identified in these screens involves the activity of the PAR-polarity complex (Par-6, Baz, aPKC, and Cdc42). In terminal cells the PAR complex is required for terminal cell branching but not outgrowth, demonstrating that these two processes can be decoupled (Jones and Metzstein, 2011). Here, we focus on the molecular machinery required for branch outgrowth in terminal cells and identify a role for the exocyst complex in subcellular branch outgrowth.

The exocyst is an octomeric protein complex consisting of the proteins Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84, and was originally identified for its role in polarized membrane addition that precedes bud outgrowth and secretion in S. cerevisiae (TerBush et al., 1996). The exocyst complex also function in other cellular context. For instance, the complex has been shown to participate in neurite outgrowth and synapse formation in Drosophila (Mehta et al., 2005; Murthy et al., 2003), cilia formation in mammalian cells (Rogers et al., 2004; Zuo et al., 2009), and axon outgrowth and receptor positioning in mammalian neurons (Hazuka et al., 1999;Vega and Hsu, 2001), amongst many other processes. On a molecular level, the exocyst functions by facilitating tethering, docking, and fusion at the plasma membrane (Heider and Munson, 2012; Whyte and Munro, 2002) of vesicles derived from diverse cellular origins, including the Golgi and recycling endosome (He and Guo, 2009; Ponnambalam and Baldwin, 2003). Localization of the exocyst to the plasma membrane is dependent on Rho-family GTPases (Estravís et al., 2011; Kanzaki and Pessin, 2003; Kawase et al., 2006; Ory and Gasman, 2011; X. Zhang, 2001), while trafficking of exocytic vesicles is controlled by Rab-family GTPases (Das and Guo, 2011; Novick et al., 2006; Pfeffer, 2012). In particular, Rab8, Rab10, and Rab11 have been shown to function with the exocyst in delivery of vesicles to the plasma membrane (Babbey et al., 2010; Chen et al., 1998; Feng et al., 2012; Satoh et al., 2005; Takahashi et al., 2012). Rab10 and Rab11 have also been shown to physically interact with the exocyst through directly binding Sec15 (S. Wu et al., 2005; X.-M. Zhang et al., 2004).

Here, we show the exocyst complex is required for branching and branch outgrowth in terminal cells and for formation of mature intracellular lumens. We focus on the role of the exocyst in branching morphogenesis, and provide evidence that the PAR complex controls terminal cell branching by regulating exocyst localization in developing terminal cells. Ultrastructural analysis reveals exocyst deficient terminal cells have defects in vesicle trafficking, implicating polarized membrane addition as a mechanism of branch outgrowth. Finally, we show that redundant vesicle trafficking pathways converge on the exocyst to contribute to the outgrowth of terminal cell branches. These finding demonstrate how the interplay of several molecular mechanisms contribute to subcellular branching morphogenesis.

Materials and Methods

Fly stocks and genetics

Flies were reared on standard cornmeal/dextrose food and larvae were raised at 25°C. The control chromosomes used were: y w FRT19A, FRT82B (Xu and Rubin, 1993) and FRTG13 (Chou, 1996), unless otherwise stated. Alleles analyzed were sec5E10 (Murthy et al., 2003), sec6KG08199 (Zhou et al., 2007), sec10f03085 (Bloomington Drosophila Stock Center), sec151 (Mehta et al., 2005), par-629VV (Jones and Metzstein, 2011), aPKCk06403 (Wodarz et al., 2000), bazFA50 (Wodarz et al., 2000), shits1 (Masur et al., 1990), Rab5k08232 (Wucherpfennig et al., 2003). For mosaic analysis, we used the stocks: y w P{w+,btl-Gal80} FRT19A, hsFLP122; btl-Gal4 UAS-GFP (Jones and Metzstein, 2011), y w hsFLP122; FRTG13 P{w+, tub-Gal80} ; btl-Gal4 UAS-GFP (gift from S. Luschnig), and y w hsFLP122; btl-Gal4 UAS-GFP ; FRT82B P{w+ tub-Gal80}/TM6B (gift from A. Ghabrial). To perform mosaic analysis, shits, sec6, and sec10 were recombined onto FRT19A, FRTG13 and FRT82B, respectively, using standard methods. UAS-Sec5 RNAi (27526), UAS-Sec6 RNAi (27314), UAS-Sec10 RNAi (27483), UAS-Sec15 RNAi (27499), UAS-Chc RNAi (27530), UAS-Rab11 RNAi (27730), UAS-YFP-Rab11 DN (23261), UAS-YFP-Rab10 DN (9786), UAS-Rab8 RNAi (34373) and UAS-Cdc42N17 (6288) were obtained from the Bloomington Drosophila Stock Center, and UAS-Sec3 RNAi (108085) and UAS-Sec8 RNAi (105653) were obtained from the Vienna Drosophila RNAi Center (stock numbers shown in parentheses). Rab5 (stock number 111–239) was obtained from the Drosophila Genetic Resource Center. Homozygous mutant cells were generated using the mosaic analysis with a repressible cell marker (MARCM) technique (Lee and Luo, 1999). To generate mosaics, 0–6 hr embryos were collected in fly food vials at 25° C and treated to a 45 minute heat shock at 38° C in a circulating water bath, then reared at 25° C until the L3 stage (Jones and Metzstein, 2013). Temperature sensitive shi alleles were heat shocked and maintained at room temperature overnight then reared at the restrictive temperature of 29° C until they reached L3.

Light microscopy of terminal cells

Wandering 3rd instar larvae were collected and heat-fixed according to a standard protocol developed in our lab (Jones and Metzstein, 2013). Images were taken on Zeiss AxioImager M1 equipped with an AxioCam MRm.

Immunofluorescence

Wandering 3rd instar larvae were dissected in 1X PBS to make fillets exposing the tracheal system. Fillets were fixed for 30 minutes in 4% PFA in 1X PBS, rinsed 3 times for 15 minutes in 1X PBST (1X PBS + 0.1% TX100), blocked for 30 minutes at room temperature in PBSTB (1X PBST + 0.02% BSA), then incubated with primary antibody overnight at 4°C. Fillets were then rinsed 3 times for 15 minutes in 1X PBSTB and incubated with secondary antibody for 2 hours at room temperature. Fillets were then rinsed and mounted on glass slides in ProLong® Gold antifade reagent (Invitrogen). Antibodies were used in the following concentrations: goat anti-sec8 (Beronja et al., 2005), at 1:250 and mouse anti-GFP, at 1:1000 (Clontech, #632375). Secondary antibodies, conjugated to Alexa-488 or Alexa-568 (Molecular Probes), were used at 1:1000. Imaging was performed using a Leica TCS SP2 confocal microscope. A Z-stack of 10–25 slices was imaged for each setting sequentially. An average intensity projection was generated in Image-J.

Terminal cell branching and outgrowth quantification

Terminal cell branch number and outgrowth were determined using methods described previously (Jones and Metzstein, 2011). Outgrowth was quantified as the ratio of the length of class I branches to the number of class I branches. For statistical comparisons we used the two-tailed Mann-Whitney U test.

Transmission electron microscopy

High Pressure Freezing

We fixed samples for TEM analysis using a protocol developed in our lab, the details of which will be published later. Briefly, larvae were picked at late L1 or early L2 and kept for a short time in a drop of 1X PBS prior to freezing. Larvae were loaded into Type A specimen carriers (Technotrade, cat. # 24150) and carriers were filled with E. coli as a cryoprotectant. Loaded Type A carriers were closed with the flat side of a Type B specimen carrier (Technotrade, cat. # 24250). Carriers were immediately subjected to high pressure-freezing using a BAL-TEC HPM 010 freezer (BAL-TEC, Inc., Carlsbad, CA). Carriers containing frozen larvae were quickly transferred to cryovials that contained a pre-cooled (− 90°C) mix of 2% osmium tetroxide (OsO4) and 0.1% Uranyl Acetate in 97% acetone (McDonald and Müller-Reichert, 2002) in a Leica EM AFS (Leica Microsystems, Vienna, Austria). To enhance membrane contrast 3% water was added to the fixative (Walther and Ziegler, 2002). Specimens underwent freeze substitution for 72 hrs at −90°C, were gradually warmed at the rate of 5°C/hrs to −20°C, and were kept at this temperature for 8–16 hrs. The temperature was slowly raised to 20°C at the rate of 10°C/hr, and samples then were removed from the AFS unit to room temperature and rinsed immediately with pure acetone five times as follows: 2×15 min, 1×30min, and 2×1 hour, before infiltration and embedding.

Resin Infiltration and Embedding

Infiltration was performed by incubating the specimens in a gradually increasing concentrations of Durcupan Fluka epoxy resin (Fluka Analytical cat. # 44610) at room temperature as follows: 30% epoxy resin in acetone for 5 hrs; 70% resin in acetone overnight; and 90% resin in acetone for 8 hrs-overnight. Specimens were transferred to 100% resin for 24 hrs with 2 changes, then transferred to fresh 100% resin with two changes over a 3 hours period, after which polymerization was performed at 60°C for 48 hrs.

Sectioning and imaging

Ultrathin (50–60nm) sections were obtained using a diamond knife (Diatome) and Reichert Ultracut E microtome. Sections were collected on coated copper grids and post-stained with 2.5% uranyl acetate for 10 min. Sections were imaged at 120 kV using a FEI Tecnai 12 transmission electron microscope.

Vesicle quantification

Vesicle accumulation was quantified by determining the average number of cytoplasmic vesicles per section, in ultrathin sections. From each genotype, a total of at least 8 sections were evaluated (2–6 sections from 3–4 cells each). Vesicles were defined as roughly circular membrane bound structures of 50 nm or greater in diameter lacking electron dense material in their lumens.

Results

The exocyst complex is required for terminal cell branch outgrowth and lumen formation

To test if the exocyst complex is required for terminal cell branching or outgrowth, we used the MARCM system (Lee and Luo, 1999) to generate mosaic animals with terminal cells mutant for exocyst components. The use of mosaics allowed us to investigate the cell autonomous role of the exocyst in terminal cells, as well as to bypass any other requirement for organismal development. We found that terminal cells homozygous for null alleles of sec5, sec6, sec10, and sec15 showed similar defects, including fewer branches and shorter branch lengths (Fig. 1A–E; quantitated in 1G and 1H). We used RNAi-mediated gene knockdown to test a role for other members of the exocyst complex for which null alleles were not available. We found RNAi-mediated knockdown of exocyst complex members sec3, sec8, exo70, and exo84 resulted in terminal cell defects qualitatively similar to those of exocyst mutant cells (Fig. S1). By contrast, as previously reported (Jones and Metzstein, 2011), PAR-polarity proteins such as Par-6 are required for terminal cell branching, but not outgrowth: terminal cells deficient for par-6 and other PAR polarity genes have defects in the total number of branches, but branches that are present extend as far as wild-type branches (Fig. 1F–H and S2). In addition to the effects of loss of exocyst complex members on cell branching and outgrowth, we also observe the lack gas-filled lumens in exocyst defective terminal cells (Figs. S1 and S3). Thus, our result indicates the exocyst is required for proper development of both the basal plasma membrane (corresponding to the cells branch development) and apical plasma membrane (corresponding to lumen development) and suggests a role for the exocyst in coupling these two processes.

Figure 1. The exocyst complex is required for terminal cell morphogenesis.

Figure 1

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(A–F) Terminal cells in MARCM mosaic L3 larvae, with homozygous cells labeled with GFP. (A) Wild-type terminal cells show extensive outgrowth and subcellular branching. (B–E) Terminal cells homozygous for exocyst complex members sec5, sec6, sec10, or sec15 show a reduction in branching and branch outgrowth, showing only a few branches that are much shorter than those observed in wild-type. (F) Cells mutant for par-6 have fewer branches than wild-type cells, but normal outgrowth. (G) Quantification of terminal cell branch number, measured by counting the total number of branches per cell. (H) Quantification of terminal cell outgrowth, measured as the average length of class I branches (the first side branches to emerge from a terminal cell). *Significant difference from par-6 (p<0.01); n.s., not significant (p>0.05). Dashed white lines indicate the proximal ends of the GFP-labeled cell. Scale bar, 75 µm. Error bars represent ±2 SEM.

Our data suggests the exocyst complex is required for both branch specification and outgrowth. However, it is possible that the exocyst is primarily required for outgrowth but is less important in branch specification, but this cannot be observed since outgrowth is required to observe specified branches. To test this possibility, we used RNAi to partially inactivate exocyst complex members in terminal cells. We found RNAi-mediated knockdown of sec5, sec6, sec10, or sec15, resulted in similar branching defects to those observed in null alleles, but relatively mild outgrowth defects, suggesting the knockdown was indeed incomplete (Fig. 2A–E; quantitated in Fig. 2P). Interestingly, close examination of branches in these RNAi knockdown cells revealed small membrane protrusions along their lengths (Fig. 2G–J). This morphology differed from that of wild-type (Fig. 2F) or exocyst-complex null cells, in which branches nearly always have a smooth, tapered appearance. We interpret these protrusions to be primitive branch sites that have undergone specification, but fail to extend when exocyst function is reduced. These results suggest that the primary role of the exocyst complex is in branch outgrowth. We also note that these nascent branches are more numerous than established branches, suggesting a mechanism of lateral branch inhibition may help pattern terminal cells.

Figure 2. The exocyst is required for terminal cell branch outgrowth.

Figure 2

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(A–O) MARCM was used to generate mosaic animals with GFP-marked terminal cells expressing RNAi transgenes directed against specific exocyst genes. (A) Wild-type terminal cells have many subcellular branches and individual branches (F) have a smooth tapered appearance. (B–E) Terminal cells expressing RNAi directed against exocyst complex members have fewer branches than wild-type cells. (G–J) Individual branches of exocyst RNAi cells show numerous short extensions along their lengths. (K) Terminal cells homozygous for a null allele of par-6, show branching defects, but normal outgrowth. (L–O) RNAi of exocyst components in a par-6 mutant result in defects similar to those observed with RNAi alone. (P) Quantification of terminal cell branch number measured by counting the total number of branches per cell. † (p=0.05); n.s., not significant (p<0.05). Dashed white lines indicate the proximal ends of the GFP-labeled cell. Scale bars are 75 µm within each column. Error bars represent ±2 SEM.

We next asked whether the PAR complex and the exocyst complex had independent roles in terminal cell branching, or whether they were participating together to facilitate this process. To do this we tested for exacerbation of branching defects in terminal cells that were mutant for a par-6 null allele and simultaneously expressing RNAi directed against exocyst complex members. We found branching defects observed upon RNAi of sec5, sec6, sec10, and sec15 were not exacerbated by loss of Par-6 (Fig. 2K–O); the double mutant branching defects are quantitatively similar to defects observed by RNAi expression alone (Fig. 2P). This result suggests the exocyst complex and the PAR complex likely participate in a common process required for terminal cell branching. Interestingly, the small protrusions were still found to be present in the Par-6 exocyst RNAi cells, suggesting initial sampling of branch sites can occur independently of PAR complex activity.

The PAR-polarity complex is required for proper exocyst localization in terminal cells

The Rho GTPase Cdc42 has been shown to directly interact with exocyst-complex members Sec3 (X. Zhang, 2001) and Exo70 (H. Wu et al., 2010). Cdc42 is a component of the PAR-polarity complex, which is required for terminal cell branching morphogenesis (Jones and Metzstein, 2011). Thus, Cdc42 is a candidate to link polarized membrane addition by the exocyst with branch specification by the PAR complex. To investigate this, we determined if the PAR complex was required for localization of the exocyst in terminal cells. We used immunofluorescence to visualize localization of the exocyst-complex member Sec8 in wild-type and Cdc42 mutant terminal cells (Fig. 3A–F), since localization of Sec8 is representative of the localization of the assembled exocyst complex at the cell membrane (Rivera-Molina and Toomre, 2013). We found that in wild-type terminal cells, Sec8 protein is spread diffusely throughout the cell and enriched on the apical plasma membranes. In wild-type cells, Sec8 is additionally found concentrated in distinct puncta on both the apical and basal plasma membranes (Fig. 3B, B’ and C). However, when Cdc42 activity is inhibited by expression of a dominant-negative construct, the punctate localization is completely lost (Fig. 3E, E’ and F), leaving only the diffuse staining. We obtained similar results when we examined a mutant of another PAR-complex member, aPKC (Fig. 3G–I). Together, these results suggest the PAR-polarity complex is required for localized concentration of the exocyst on terminal cell plasma membranes. Since the PAR complex is specifically required for terminal cell branching, these results imply the exocyst puncta are related to processes involved in new branch formation, probably both of the basal and apical membranes.

Figure 3. The PAR complex is required for exocyst membrane concentration in terminal cells.

Figure 3

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Terminal cells in L3 mosaic larvae were identified by cytoplasmic GFP expression and then probed with anti-GFP (A, D, and G) and anti-Sec8 (B, E, and H) antisera. (C, F and I) merged channels (GFP in green, anti-Sec8 in red). (B and B’) In wild-type cells, Sec8 is found diffusely throughout the cell and in distinct membrane-localized puncta. Terminal cells homozygous for Cdc42 (E and E’) or aPKC (H and H’) lose the punctate but not the diffuse staining. In the image of the aPKC mutant cell, normal Sec8 puncta can be observed in an adjacent, non-GFP labeled wild-type cell (asterisk). B’, E’, and H’ show magnified views of B, E, and H (boxed regions). Scale bars: A–I, 10 µm; B’, E’, and H’, 2.5 µm.

Exocyst and Cdc42 activity is required for normal vesicle trafficking in terminal cells

Given the role of the exocyst complex in vesicle trafficking and fusion at the plasma membrane, one prediction is that loss of exocyst function would result in an accumulation of cytoplasmic vesicles in mutant terminal cells. Such a phenotype has been observed in exocyst defective yeast and mammalian cells (Guo et al., 1999; TerBush et al., 1996). We tested if this is the case in exocyst defective terminal cells using transmission electron microscopy (TEM). We found that in wild-type terminal cells, intracellular vesicles are rarely observed in the cytoplasm (0.13±0.35, vesicle per section; Fig. 4A). However, when RNAi is used to reduce expression of either sec5 (Fig. 4B) or sec15 (Fig. 4C), we found the terminal branch cytoplasm contained many large intracellular vesicles, with sec5-deficient and sec15-deficient terminal cells contained an average of 4.7±1.5 and 1.9±1.0 vesicles per section, respectively. Since each section represents only a small fraction of the cell, these data indicate that exocyst-mutant terminal cells accumulate a large number of vesicles in their cytoplasm.

Figure 4. Terminal cells defective for the exocyst complex or Cdc42 activity accumulate cytoplasmic vesicles.

Figure 4

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(A–D) Terminal cell branch ultrastructure observed in thin cross-section using transmission electron microscopy (TEM). (A) Wild-type terminal cell branches show circular cross-sectional morphology, the lumen is expanded and clear of cytoplasmic material and the cytoplasm is devoid of large vesicles. Note: lysosomes appear as circular structures containing electron dense staining. (B and C) Terminal cells expressing RNAi for exocyst complex member sec5 or sec15, show an accumulation of vesicles in their cytoplasm (examples shown in insets). (D) Terminal cells expressing a dominant-negative Cdc42 also show vesicle accumulation (example shown in inset). The relative circular shape, areas of less electron dense regions surrounded by more electron dense bilayer structures define vesicles. Arrows in A indicate microtubules (MT; close up in inset), which are much smaller than cytoplasmic vesicles; arrow in D indicates what appears to be a swollen and abnormal Golgi structure; asterisks indicate lumens or lumen-like structures. Scale bars, 400 nm.

Since Cdc42 is required for localization of the exocyst to the plasma membrane, we predicted we would observe a vesicle accumulation phenotype similar to exocyst complex mutants in terminal cells inhibited for Cdc42 activity. When we examined the ultrastructure of terminal cells expressing dominant-negative Cdc42, we observed an accumulation of intracellular vesicles similar to that observed in sec5 and sec15 deficient cells. The defects observed in these Cdc42 activity defective cells are even more severe than those observed in exocyst-mutant terminal cells (8.1±2.5 vesicles per section). Our results suggest that vesicle trafficking in terminal cells is abnormal in the absence of exocyst or Cdc42 function.

Finally, consistent with the results found by light microscopy, the lumen of exocyst- or Cdc42-function deficient terminal cells appear defective on the ultrastructural level, and seem either occluded or immature. However, lumen formation appears to have been initiated, with the terminal branches containing a large membrane-bound compartment in most sections examined (asterisks in Fig. 4A–D).

Multiple trafficking pathways contribute to branch outgrowth

Rab GTPases have been shown to work in concert with the exocyst complex to mediate trafficking of vesicles to the plasma membrane (Das and Guo, 2011). Rab10 is involved in trafficking of vesicles derived from the Golgi (Lerner et al., 2013; Sano et al., 2007; Wang et al., 2011), and Rab11 is primarily involved in trafficking of vesicles from recycling endosomes (Chen et al., 1998; Satoh et al., 2005; Takahashi et al., 2012). To determine the role of these Rab proteins in terminal cell development, and to identify the source of vesicles that contribute to branch outgrowth, we expressed dominantnegative forms of Rab10 or Rab11 (J. Zhang et al., 2007) in terminal cells. We found expression of either rab10-DN (Fig. 5A) or rab11-DN (Fig. 5B) resulted in strong defects in total branch number, but only mild outgrowth defects (quantified in Fig. 5F and G). Interestingly, we found terminal cells co-expressing rab10-DN and rab11-DN (Fig. 5C) show outgrowth defects that are much more severe than either of the single mutants and are comparable to those of an exocyst null mutant (Fig. 5F and G). At the ultrastructural level, terminal cells defective for Rab10 or Rab11 activity show cytoplasmic vesicle accumulation similar to that observed in exocyst complex and Cdc42 activity defective cells (Fig. 5D and E). These findings imply that redundant vesicle trafficking pathways contribute to terminal branch outgrowth and these pathways are likely to converge on the exocyst to facilitate this process.

Figure 5. Activity of Rab GTPases Rab10 and Rab11 are required for terminal cell branch development.

Figure 5

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(A–C) Mosaic animals were generated using MARCM and GFP co-expressed with dominant-negative transgenes for the indicated Rab. (A and B) Terminal cells expressing dominant-negative Rab10 or dominant-negative Rab11 show defects in branching, but only mild defects in outgrowth. (C) Co-expression of Rab10-DN and Rab11-DN leads to severe branching and outgrowth defects. (D and E) TEM of branch ultrastructure in terminal cells expressing dominant-negative Rab10 or RNAi directed against Rab11 show accumulation of vesicles within the cell cytoplasm. Cytoplasmic vesicles highlighted in insets. (F and G) Quantification of branch number and outgrowth. * (p<0.01). Dashed white lines indicate the proximal end of the GFP-labeled cell (A–C). Scale bars, A–C, 75 µm; D and E, 200 nm.

Importantly, the results obtained with Rab10 and Rab11 are specific: expression in terminal cells of multiple other Rab dominant negative transgenes (J. Zhang et al., 2007), including Rab8 (Fig. S4), and Rabs 3, 4, 7, 9, 23, 27 and 32 (data not shown) did not lead to overt branching or lumen formation defects.

Endocytosis is required for terminal cell branching

As Rab11 is primarily involved in trafficking from the recycling endosome to the plasma membrane, we predicted endocytosis might also be important for terminal cell development. To test this, we examined critical components of the clathrin-mediated endocytosis machinery: clathrin heavy chain, a major component of clathrin-coated pits (Conibear, 2010; Swan, 2013) and dynamin, a GTPase known to facilitate scission of endocytic vesicles from the plasma membrane (Ramachandran, 2011). We found terminal cells expressing RNAi directed against Chc (clathrin heavy-chain) or mutant for shibire (shi, the Drosophila homolog of dynamin) have severe defects in total branch number, but only mild defects in branch outgrowth (Fig. S5). Additionally, terminal cells mutant for Rab5, a key regulator of early endosome formation (Pfeffer, 2001), show defects similar to shi or Chc mutant cells in branching and outgrowth (Fig. S5). These results suggest a trafficking pathway of endocytosis to recycling endosomes is required for terminal cell branching. However, we have not ruled out that these defects may be a consequence of indirect effects on vesicle exocytosis caused by depletion of the endosome or other intracellular membrane stores.

Discussion

Drosophila tracheal terminal cells have proven to be a powerful model for investigating molecular mechanisms controlling the formation of a branched cell. In particular, much has been learned about the signaling pathways required for terminal cell specification and initial development (Gervais and Casanova, 2011; Ghabrial et al., 2003; Guillemin et al., 1996). However, much less is known about the mechanisms of terminal cell branching and branch outgrowth. Previously, we showed the PAR-polarity complex is necessary for branching and functions downstream of the FGF signaling pathway that regulates growth of terminal cells towards hypoxic tissue (Jarecki et al., 1999; Jones and Metzstein, 2011). However, the PAR-polarity complex is not in itself required for branch outgrowth. Here, we characterize a role for exocyst-mediated vesicle trafficking in terminal cell branch outgrowth. We find disruption of all tested exocyst-complex components results in severe branch extension defects in terminal cells. Branch outgrowth requires membrane addition at specific sites and the exocyst is known to facilitate docking and fusion of vesicles at target membranes (He and Guo, 2009; Lipschutz and Mostov, 2002). Our ultrastructural analysis of terminal cells defective for exocyst-complex components reveals an accumulation of vesicles within the cytoplasm. These vesicles are likely those that would deliver membrane required for branch extension, but in exocyst defective cells are unable to fuse with their target membranes and remain trapped in the cytoplasm. Thus, we propose that exocystmediated vesicle fusion is a key mechanism of branch outgrowth in terminal cells.

In various cellular contexts, the exocyst facilitates membrane addition required for both general and polarized cell growth (Cole and Fowler, 2006; Heider and Munson, 2012). General cell growth is a process of membrane addition that occurs throughout the entire plasma membrane and leads to an overall increase in cell size and length. Conversely, polarized outgrowth occurs at specific sites and results in extension of small regions of the cell membrane. Terminal cells presumably employ both types of cellular growth: general growth, as established branches get longer and wider during larval development, and polarized outgrowth, required for new branch formation. We find that in terminal cells, the exocyst is localized diffusely throughout the cell, as well as in greater concentrations at specific plasma membrane sites. We find that these sites of concentration are dependent upon the PAR complex, as the punctate localization is lost and we only observed diffuse staining in PAR-complex mutant terminal cells. PAR complex mutants do not show defects in branch outgrowth, indicating that the diffusely localized pool of exocyst is sufficient for growth of the cell and for the extension of established branches. Thus, it appears that PAR complex-dependent membrane concentration of the exocyst is required only for de novo branching. We propose a model that de novo branch outgrowth is driven by a transient increase in exocyst complex concentration at branch sites (Fig. 6). As a potential mechanism for such an increase, we propose that local FGF receptor activation at the plasma membrane promotes a transient increase in exocyst concentration, leading to exocyst-mediated membrane addition at these sites, resulting in new branch formation. This process continues through iterative rounds of specification and outgrowth to generate a branched cellular morphology. A potential molecular link between FGF receptor activation and exocyst localization, is the PAR complex component Cdc42. It is known that receptor tyrosine kinase activation can lead to the recruitment of PI3K (Funamoto et al., 2002) and thus to a local increase in phosphatidylinositol (3,4,5)-triphosphate (PIP3) concentration. PIP3 in turn can recruit GEFs that activate Cdc42 (Yang et al., 2012), and Cdc42 is known to stimulate assembly of the exocyst complex (Estravís et al., 2011; Kanzaki and Pessin, 2003; H. Wu et al., 2010). One part of this model is that the PAR complex is not specifically localized to branch sites, but instead is locally activated to promote branching. This is consistent with our previous observation that the PAR complex, despite being required for branching, is not specifically localized to branch sites in terminal cells. In this way the PAR complex facilitates branching but is not instructive for this process. Finally, it is important to note that concentrated exocyst localization is found at more sites on the membrane then will form branches. Furthermore, the small protrusive structures we observe in partial inactivation of exocyst complex activity by RNAi are independent of the PAR complex. Thus, it is likely that the PAR complex serves to reinforce potential sites of branching, which may also explain why some terminal cell branching occurs even in the complete absence of the PAR complex (Jones and Metzstein, 2011) Since branches in terminal cells are typically spaced apart it is probable that a mechanism of lateral inhibition occurs upon branch specification and outgrowth, as may be observed in our partial exocyst knockdown experiments. Testing whether these mechanism function in terminal cells will likely require the development of techniques that will allow live cell imaging and biochemical approaches to detect changes in local concentrations of the key components (Schottenfeld-Roames and Ghabrial, 2012).

Figure 6. Branching morphogenesis model.

Figure 6

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Activation of the FGF receptor leads to local concentration of the exocyst complex, via activation of the PAR complex. General cell growth and branch elongation is control by trafficking of vesicle from the recycling endosomes or Golgi to the exocyst. Newly formed branches inhibit outgrowth of subsequent branches by a process of lateral inhibition.

The process of de novo branch formation requires addition of membrane to a specific site on the cell surface. Vesicles that deliver membrane to the plasma membrane are primarily derived from two intracellular compartments: the Golgi and the recycling endosome (Bryant et al., 2010; Pfeffer, 2012; Ponnambalam and Baldwin, 2003; Prigent et al., 2003; Whyte and Munro, 2002). To investigate which of these compartments is the likely source of membrane used for branch outgrowth, we examined terminal cells where we had inactivated pathway-specific vesicle-trafficking genes. We found that disruption of either Golgi to plasma membrane trafficking, through interference of Rab10 activity, or disruption of recycling endosome to plasma membrane trafficking, through interference of Rab11 activity, leads to only mild branch outgrowth defects. However, the simultaneous interference of Rab10 and Rab11 leads to very strong outgrowth defects, comparable to loss of the exocyst. These results suggest that the vesicles required for branch outgrowth can be derived either from the Golgi or from the recycling endosome. Such a mechanism of terminal branch outgrowth contrasts with axonal growth, in which membrane is thought to be derived primarily from the Golgi (Tekirian, 2002), but may have parallels with dendritic morphogenesis, in which membranes can come from multiple sources (Sann et al., 2009). We do not yet know if the vesicles derived from these two sources have different functional properties, for instance in the delivery of proteins or other macromolecules required for later steps in terminal cell branch function, such as guidance (Englund et al., 2002; Steneberg and Samakovlis, 2001) or adhesion to underlying substrates (Levi et al., 2006).

The closest parallel to the membrane extension and branching we are studying in terminal cells is in the elaboration of neuronal processes. Significantly, the exocyst complex has been shown to be required for polarized migration and neurite outgrowth and morphology, similar to the processes we describe here (Dupraz et al., 2009; Fujita et al., 2013; Lalli and Hall, 2005), including possible regulation by FGF receptor activity (Chernyshova et al., 2011). Very interestingly, there is also evidence for the PAR complex as a mechanism regulating exocyst-mediated cell migration and outgrowth in neurons (Das et al., 2013; Lalli, 2009) and other cells (Jiu et al., 2014), akin to what we describe here. Thus, our results suggest the PAR complex/exocyst plays an evolutionary conserved role in generating subcellular branching morphology.

Mechanisms of terminal cell branching morphogenesis encompass a number of important cell biological processes including a specific combination of cellular organization, polarity, and trafficking processes. Here, we have shown exocyst-mediated vesicle trafficking is critical for terminal cell branch outgrowth and propose a model where localized PAR complex activity regulates localization of the exocyst. Continued genetic analysis of mutants obtained from this pliable genetic system should reveal more about the general processes necessary for subcellular morphogenesis, which are common to branched cells such as neurons, oligodendrocytes, and megakaryocytes.

Supplementary Material

01. Terminal cells expressing RNAi directed against exocyst complex members show branching, outgrowth, and lumen defects.

(A–D) GFP labeled terminal cells in MARCM mosaic L3 larvae expressing RNAi directed against exocyst complex members exo70, exo84, sec3, or sec8 have defects in branching and branch outgrowth. (A’–D’) Brightfield microscopy shows the absence of a air-filled lumens in cells with reduced exocyst complex activity. Dashed white lines indicate the proximal ends of the defective cell. Arrowheads highlight portions of the air-filled lumen. Scale bar, 75 µm.

02. The PAR-polarity complex is not required for terminal cell branch outgrowth.

Quantification of terminal cell branch outgrowth measured as the average length of class I branches (the first side branches to emerge from a terminal cell). Error bar represent ±2 SEM.

03. The exocyst complex is required for terminal cell lumenogenesis.

(A–F) Brightfield microscopy shows the air-filled lumen of terminal cells in MARCM mosaic L3 larvae, where homozygous cells were identified by expression of GFP (see Fig. 1). (A) Wild-type terminal cells contain a single air-filled lumen within each branch. (B–E) Terminal cells homozygous for exocyst complex members sec5, sec6, sec10, or sec15 show no air-filled lumen. (F) Terminal cells homozygous for the PAR-polarity protein, Par-6 show only a partial lumen within the central branch. Dashed white lines indicate the proximal ends of the homozygous cell. Arrowheads highlight portions of the air-filled lumen. Scale bar, 75 µm.

04. The Rab GTPase Rab8 is not required for terminal cell branch development.

Terminal cell expressing RNAi directed against Rab8 shows apparently normal branching and outgrowth.

05. The endocytic recycling pathway is required for terminal cell branch development.

Terminal cells in MARCM mosaic L3 larvae with homozygous mutant cells labeled with GFP. (A–C) Terminal cells expressing RNAi directed against Chc (clathrin heavy chain), or homozygous mutant for shi (dynamin), or Rab5 have defects in branching and mild outgrowth defects. (D and E) Quantification of branch number and outgrowth. Error bars represent ±2 SEM. Dashed white lines indicate the proximal end of the GFP-labeled cell (A–C). Scale bar, 75 µm.

  • The exocyst complex is required for branch outgrowth in tracheal terminal cells

  • The PAR polarity complex is required for proper exocyst localization in terminal cells

  • Multiple vesicle trafficking pathways function in terminal cell branching

Acknowledgments

We are thankful to Hugo Bellen, Thomas Schwarz, Amin Ghabrial, Stefan Luschnig, Jun Zhang, Matt Scott, and Ulrich Tepass for fly stocks and antibodies. We are grateful to members of the Metzstein laboratory and the University of Utah Membranes Trafficking Interest Group for useful discussions; and Gillian Stanfield, Diane Ward, Kim Frizzell, and the Metzstein laboratory for comments on this manuscript. Fly stocks were obtained from the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Center, and the National Institute of Genetics Fly Stock Center, Japan. T.A.J. was supported by the University of Utah Genetics Training Grant T32-GM007464 from NIH NIGMS.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01. Terminal cells expressing RNAi directed against exocyst complex members show branching, outgrowth, and lumen defects.

(A–D) GFP labeled terminal cells in MARCM mosaic L3 larvae expressing RNAi directed against exocyst complex members exo70, exo84, sec3, or sec8 have defects in branching and branch outgrowth. (A’–D’) Brightfield microscopy shows the absence of a air-filled lumens in cells with reduced exocyst complex activity. Dashed white lines indicate the proximal ends of the defective cell. Arrowheads highlight portions of the air-filled lumen. Scale bar, 75 µm.

NIHMS573339-supplement-01.tif (6.9MB, tif)
02. The PAR-polarity complex is not required for terminal cell branch outgrowth.

Quantification of terminal cell branch outgrowth measured as the average length of class I branches (the first side branches to emerge from a terminal cell). Error bar represent ±2 SEM.

NIHMS573339-supplement-02.tif (345.2KB, tif)
03. The exocyst complex is required for terminal cell lumenogenesis.

(A–F) Brightfield microscopy shows the air-filled lumen of terminal cells in MARCM mosaic L3 larvae, where homozygous cells were identified by expression of GFP (see Fig. 1). (A) Wild-type terminal cells contain a single air-filled lumen within each branch. (B–E) Terminal cells homozygous for exocyst complex members sec5, sec6, sec10, or sec15 show no air-filled lumen. (F) Terminal cells homozygous for the PAR-polarity protein, Par-6 show only a partial lumen within the central branch. Dashed white lines indicate the proximal ends of the homozygous cell. Arrowheads highlight portions of the air-filled lumen. Scale bar, 75 µm.

NIHMS573339-supplement-03.tif (3.5MB, tif)
04. The Rab GTPase Rab8 is not required for terminal cell branch development.

Terminal cell expressing RNAi directed against Rab8 shows apparently normal branching and outgrowth.

NIHMS573339-supplement-04.tif (660.6KB, tif)
05. The endocytic recycling pathway is required for terminal cell branch development.

Terminal cells in MARCM mosaic L3 larvae with homozygous mutant cells labeled with GFP. (A–C) Terminal cells expressing RNAi directed against Chc (clathrin heavy chain), or homozygous mutant for shi (dynamin), or Rab5 have defects in branching and mild outgrowth defects. (D and E) Quantification of branch number and outgrowth. Error bars represent ±2 SEM. Dashed white lines indicate the proximal end of the GFP-labeled cell (A–C). Scale bar, 75 µm.

NIHMS573339-supplement-05.tif (1.9MB, tif)

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