InteractiveFly: GeneBrief

Rab-protein 5: Biological Overview | References

During constitutive endocytosis, internalized membrane traffics through endosomal compartments. At synapses, endocytosis of vesicular membrane is temporally coupled to action potential-induced exocytosis of synaptic vesicles. Endocytosed membrane may immediately be reused for a new round of neurotransmitter release without trafficking through an endosomal compartment. Using GFP-tagged endosomal markers, an endosomal compartment in Drosophila neuromuscular synapses was monitored. In conditions in which the synaptic vesicles pool is depleted, the endosome is also drastically reduced and recovers only from membrane derived by dynamin-mediated endocytosis. This suggests that membrane exchange takes place between the vesicle pool and the synaptic endosome. The small GTPase Rab5 is required for endosome integrity in the presynaptic terminal. Impaired Rab5 function affects endo- and exocytosis rates and decreases the evoked neurotransmitter release probability. Conversely, Rab5 overexpression increases the release efficacy. Therefore, the Rab5-dependent trafficking pathway plays an important role for synaptic performance (Wucherpfennig, 2003).

At the presynaptic terminal, Ca²⁺-triggered neurotransmitter (NT) release by exocytosis is immediately followed by the local recycling of the synaptic vesicle (SV) membrane. SV recycling is necessary to preserve the plasma membrane surface area, to sustain the population of SVs, and to maintain the molecular diversity of the vesicle versus the plasma membrane (Wucherpfennig, 2003).

There are at least two distinct recycling mechanisms: 'kiss and run'. During kiss and run, SVs make brief contact with the plasma membrane forming a transient porelike structure through which the NT is released. In contrast, clathrin-mediated endocytosis occurs after complete fusion of the SV with the plasma membrane. New vesicles are subsequently reformed through a complex process initiated by the formation of an invagination at the plasma membrane mediated by clathrin and its adaptors. In lamprey, snake, and fly neuromuscular synapses, the invagination of the membrane into pits occurs at distinct 'centers of endocytosis' surrounding the active zones of exocytosis. Subsequently, amphiphysin, dynamin, and endophilin are thought to lead to the formation of a clathrin-coated, endocytic vesicle. The subsequent steps are still a matter of debate and it is controversial whether endocytic vesicles mature directly into SVs, recycle through an intermediate endosomal compartment before they become SVs, or whether both pathways are used under different conditions of synaptic demand (Wucherpfennig, 2003 and references therein).

In nonneuronal cells, it is well established that endocytic vesicles fuse with endosomes in a process mediated by the small GTPase Rab5 (Bucci, 1992; Horiuchi, 1997). Through the recruitment of several effector molecules, Rab5 has been suggested to form a specialized membrane domain (Rab5 domain) at the early endosome (Sonnichsen, 2000; De Renzis, 2002). Based on this, Rab5 has been used as a marker for early endosomes. Active Rab5 recruits two phosphatidylinositol-3-kinases (PI[3]-kinases), p85α/p110ß and VPS34/p150, which trigger a local enrichment of phosphatidylinositol-3-phosphate (PI[3]P) in the endosomal membrane (Christoforidis, 1999). PI(3)P specifically binds to the FYVE zinc-finger domain of endosomal factors such as the Rab5 effectors EEA1 and Rabenosyn-5, which ultimately mediate endocytic vesicle tethering and fusion with the endosome (Stenmark, 1995; Simonsen, 1998; Lawe, 2000; Nielsen, 2000). Consistently, blocking the PI(3)-kinases with antibodies or wortmannin impairs the association of FYVE domain proteins with the endosome and, thereby, blocks endosomal trafficking (Mills, 1998; Simonsen, 1998). Furthermore, it has been shown that the FYVE domain binds to PI(3)P only when inserted in a lipid bilayer (Misra, 1999; Sankaran, 2001) and that the localization of a myc-tagged tandem repeat of the FYVE domain (myc-2xFYVE) is restricted to early endosomes and the internal membrane of multivesicular bodies (Gillooly, 2000). Therefore, 2xFYVE is a bona fide marker for the PI(3)P-containing endosomes (Wucherpfennig, 2003).

Rab5 has been found on SVs (de Hoop, 1994; Fischer von Mollard, 1994), suggesting that SVs have the capacity to fuse with an endosomal compartment. Furthermore, both neuroendocrine PC12 cells and hippocampal neurons contain synaptic-like vesicles that traffic in a Rab5-dependent manner through endosomal compartments at least in the absence of synaptic transmission. However, it is unclear whether these Rab5-dependent endocytic pathways act only during nourishment and cell signaling in neurons, or also function in the recycling and maturation of SVs during synaptic transmission (Wucherpfennig, 2003).

In favor of SV recycling through the endosome, it has recently been suggested that a neuron-specific isoform of the AP3 clathrin adaptor complex from brain cytosol is required for SV budding from PC12 cell endosomes. Furthermore, FM1-43 styryl dye recycling experiments in the Drosophila neuromuscular junction (NMJ) uncovered two recycling pathways, a rapid and a slower one, suggesting the existence of an endosome-dependent pathway. However, in rat hippocampal neurons in culture, FM1-43 experiments suggested that SVs retain their identity through the endocytic cycle, implying that the SV membrane does not traffic through an intermediate endosome. In addition, SV recycling in neurons is likely too rapid to allow for constitutive trafficking through an intermediate endosomal compartment. However, not all vesicles participate in the endo-exo recycling at any given time. Remaining vesicles may have sufficient time to exchange membrane with the endosome (Wucherpfennig, 2003 and references therein).

A Rab5-positive, PI(3)P-containing endosomal compartment is present at the presynaptic terminal of Drosophila. As in nonneuronal cells, this compartment depends on Rab5 function. The endosome is depleted under conditions where SVs are depleted, and the endosome is replenished by membrane derived by dynamin-mediated endocytosis. Rab5 also influences the synaptic efficacy: impairment of Rab5 function decreases the NT release probability and the recycling SV pool size, whereas overexpression of Rab5 increases the release probability. A working model suggests that membrane exchange between the vesicle pool and the presynaptic endosome occurs and is of functional importance for the efficiency of SVs to fuse with the plasma membrane during Ca²⁺-triggered endocytosis (Wucherpfennig, 2003).

The mechanism of SV recycling has since long been a matter of debate. It has been proposed that vesicles internalized by clathrin-mediated endocytosis traffic through an intermediate endosomal compartment to become mature SVs. However, in cultured hippocampal neurons, endocytic vesicle membrane does not intermix with an internal intermediate compartment. The current study presents evidence that Rab5-dependent membrane exchange between vesicles and the endosome at the synapse can occur. Furthermore, a Rab5-mediated trafficking step determines, in a rate-limiting manner, the synaptic performance (Wucherpfennig, 2003).

It has been established that Rab5 is involved in the fusion of endocytic vesicles with their target endosomal compartment (Bucci, 1992; Stenmark, 1994). In addition Rab5 has been implicated in the budding of endocytic vesicles from the plasma membrane (McLauchlan, 1998). The current data are consistent with a key role of Rab5 during both endocytic trafficking steps at the presynaptic terminal. This is because exocytosis and endocytosis are temporally and functionally coupled at the Drosophila NMJ, making it difficult to ascertain the primary basis of an endo- or exocytic/recycling phenotype. Because the ultrastructure of the endosome is grossly disrupted in Rab5 loss- and gain-of-function mutants, the possibility is favored that it is the Rab5-dependent endosomal dynamics that play a key role in the SV cycle (Wucherpfennig, 2003).

The data leaves open the question whether this proposed trafficking step is obligatory during SV recycling or if it involves trafficking of only an SV sub-pool at any given time. However, regardless of what percentage of the SV pool recycles at a given time through the endosome, at the steady state, this recycling pathway must play a key role for the synaptic performance of the full SV pool, because synaptic efficacy increases or decreases in a rate-limiting manner depending on the levels of Rab5 function (Wucherpfennig, 2003).

Interfering with Rab5 function using the dominant-negative version of Rab5 causes a reduction in the number of released quanta during synaptic transmission, whereas elevated levels of Rab5 increase the quantal content. Morphological and electrophysiological analysis of these Rab5 mutants shows that the changes in synaptic performance are not due to a change in the readily releasable pool size, but are rather due to a change in the release probability of the SVs (Wucherpfennig, 2003).

How could the membrane exchange between vesicles and the endosome affect the SV release probability? It is well established in cultured mammalian cells that the Rab5 endosome functions as a sorting station where endocytic cargo is targeted either toward recycling or degradation (Zerial, 2001). A similar scenario may take place within the presynaptic terminal. The protein and lipid composition of the SV membrane could be controlled at the endosome by sorting out aged components and replacing them with newly synthesized ones. This, in turn, will be likely of consequence for SV function, particularly for the efficacy of regulated exocytosis and, therefore, for the SV release probability. Furthermore, sorting of alternative vesicular proteins could occur at the endosome. In this context, it has been recently found that the ratio of different Synaptotagmin isoform (Synaptotagmin I vs. Synaptotagmin IV) in SVs affects the efficacy of Ca²⁺-triggered exocytosis (Wucherpfennig, 2003).

Based on the findings that enhancement or reduction of Rab5 function lead to a parallel increase or decrease in the SV release probability, it is suggested that a Rab5-mediated membrane exchange between vesicles and the endosome affects the synaptic strength in a rate-limiting manner (Wucherpfennig, 2003).

Sequential pulses of apical epithelial secretion and endocytosis drive airway maturation in Drosophila

The development of air-filled respiratory organs is crucial for survival at birth. A combination of live imaging and genetic analysis was used to dissect respiratory organ maturation in the embryonic Drosophila trachea. Tracheal tube maturation was found to entail three precise epithelial transitions. Initially, a secretion burst deposits proteins into the lumen. Solid luminal material is then rapidly cleared from the tubes, and shortly thereafter liquid is removed. To elucidate the cellular mechanisms behind these transitions, gas-filling-deficient mutants were identified showing narrow or protein-clogged tubes. These mutations either disrupt endoplasmatic reticulum-to-Golgi vesicle transport or endocytosis. First, Sar1 was shown to be required for protein secretion, luminal matrix assembly, and diametric tube expansion. sar1 encodes a small GTPase that regulates COPII vesicle budding from the endoplasmic reticulum (ER) to the Golgi apparatus (reviewed in Bonifacino and Glick, 2004). Subsequently, a sharp pulse of Rab5-dependent endocytic activity rapidly internalizes and clears luminal contents. The coordination of luminal matrix secretion and endocytosis may be a general mechanism in tubular organ morphogenesis and maturation (Tsarouhas, 2007).

Branched tubular organs are essential for oxygen and nutrient transport. Such organs include the blood circulatory system, the lung and kidney in mammals, and the tracheal respiratory system in insects. The optimal flow of transported fluids depends on the uniform length and diameter of the constituting tubes in the network. Alterations in the distinct tube shapes and sizes cause pronounced defects in animal physiology and lead to serious pathological conditions. For example, tube overgrowth and cyst formation in the collecting duct are intimately linked to the pathology of Autosomal Dominant Polycystic Kidney Disease. Conversely, stenoses, the abnormal narrowing of blood vessels and other tubular organs, are associated with ischemias and organ obstructions (Tsarouhas, 2007 and references therein).

While the early steps of differentiation, lumen formation, and branch patterning begin to be elucidated in several tubular organs, only scarce glimpses into the cellular events of lumen expansion and tubular organ maturation are available. De novo lumen formation can be induced in three-dimensional cultures of MDCK cells. Recent studies in this system revealed that PTEN activation, apical cell membrane polarization, and Cdc42 activation are key events in lumen formation in vitro (Martin-Belmonte, 2007). In zebrafish embryos and cultured human endothelial cells, capillary vessels form through the coalescence and growth of intracellular pinocytic vesicles (Kamei, 2006). These tubular vacuoles then fuse with the plasma membranes to form a continuous extracellular lumen. Salivary gland extension in Drosophila requires the transcriptional upregulation of the apical membrane determinant Crumbs (Crb), but the cellular mechanism leading to gland expansion remains unclear (Tsarouhas, 2007 and references therein).

The epithelial cells of the Drosophila tracheal network form tubes of different sizes and cellular architecture, and they provide a genetically amenable system for the investigation of branched tubular organ morphogenesis. Tracheal development begins during the second half of embryogenesis when 20 metameric placodes invaginate from the epidermis. Through a series of stereotypic branching and fusion events, the tracheal epithelial cells generate a tubular network extending branches to all embryonic tissues. In contrast to the wealth of knowledge about tracheal patterning and branching, the later events of morphogenesis and tube maturation into functional airways have yet to be elucidated. As the nascent, liquid-filled tracheal network develops, the epithelial cells deposit an apical chitinous matrix into the lumen. The assembly of this intraluminal polysaccharide cable coordinates uniform tube growth. Two luminal, putative chitin deacetylases, Vermiform (Verm) and Serpentine (Serp), are selectively required for termination of branch elongation. The analysis of verm and serp mutants indicates that modifications in the rigidity of the matrix are sensed by the surrounding epithelium to restrict tube length. What drives the diametric expansion of the emerging narrow branches to their final size? How are the matrix- and liquid-filled tracheal tubes cleared at the end of embryogenesis (Tsarouhas, 2007)?

This study used live imaging of secreted GFP-tagged proteins to identify the cellular mechanisms transforming the tracheal tubes into a functional respiratory organ. The precise sequence and cellular dynamics were characterized of a secretory and an endocytic pulse that precede the rapid liquid clearance and gas filling of the network. Analysis of mutants with defects in gas filling reveals three distinct but functionally connected steps of airway maturation. Sar1-mediated luminal deposition of secreted proteins is tightly coupled with the expansion of the intraluminal matrix and tube diameter. Subsequently, a Rab5-dependent endocytotic wave frees the lumen of solid material within 30 min. The precise coordination of secretory and endocytotic activities first direct tube diameter growth and then ensure lumen clearance to generate functional airways (Tsarouhas, 2007).

Two strong, hypomorphic sar1 alleles were identified in screens for mutants with tracheal tube defects. In wild-type embryos, the bulk of luminal markers 2A12, Verm, and Gasp has been deposited inside the DT lumen by stage 15. However, in zygotic sar1^P1 mutants (hereafter referred to as sar1), luminal secretion of 2A12, Verm, and Gasp was incomplete. The tracheal cells outlined by GFP-CAAX partially retained those markers in the cytoplasm. sar1 zygotic mutant embryos normally deposited the early luminal marker Piopio by stage 13. Luminal chitin was also detected in sar1 mutants at stage 15. However, the luminal cable was narrow, more dense, and distorted compared to wild-type. To test if the sar1 secretory phenotype in the trachea is cell autonomous, Sar1 was reexpressed specifically in the trachea of sar1 mutants by using btl-GAL4. Such embryos showed largely restored secretion of 2A12, Verm, and Gasp. Thus, it is concluded that tracheal sar1 is required for the efficient secretion of luminal markers, which are predicted to associate with the growing intraluminal chitin matrix (Tsarouhas, 2007).

sar1 mRNA has been reported to be abundantly maternally contributed (Zhu, 2005). At later stages, zygotic expression of sar1 mRNA is initiated in multiple epithelial tissues (Abrams, 2005). To monitor Sar1 zygotic expression in the trachea, a Sar1-GFP protein trap line (Wilhelm, 2005) was used. Embryos carrying only paternally derived Sar1-GFP show a strong zygotic expression of GFP in the trachea. An anti-Sar1 antibody was used to analyze Sar1 expression in the trachea of wild-type, zygotic sar1^P1, and sar1^EP3575Δ28 null mutant embryos (Zhu, 2005) were generated. Both zygotic mutants showed a clear reduction, but not complete elimination, of Sar1 expression in the trachea. To test the effects of a more complete inactivation of Sar1, transgenic flies were generated expressing a dominant-negative sar1^T38N form in the trachea. In btl > sar1^T38N-expressing embryos, early defects were observed in tracheal branching and epithelial integrity as well as a complete block in Verm secretion. In contrast to btl > sar1^T38N-expressing embryos, zygotic sar1^P1 mutant embryos show normal early tracheogenesis with no defects in branching morphogenesis and epithelial integrity (Tsarouhas, 2007).

In summary, tracheal expression of Sar1 is markedly reduced in zygotic sar1 mutant embryos. While maternally supplied Sar1 is sufficient to support early tracheal development, zygotic Sar1 is required for efficient luminal secretion (Tsarouhas, 2007).

Given the conserved role of Sar1 in vesicle budding from the ER, its subcellular localization in the trachea was determined by using anti-Sar1 antibodies. Sar1 localizes predominantly to the ER (marked by the PDI-GFP trap). Continuous COPII-mediated transport from the ER is required to maintain the Golgi apparatus and ER structure. To test if zygotic loss of Sar1 compromises the integrity of the ER and Golgi in tracheal cells, sar1 mutant embryos were stained with antibodies against KDEL (marking the ER lumen) and gp120 (highlighting Golgi structures). In sar1 mutant embryos, a strongly disrupted ER structure and loss of Golgi staining was observed in dorsal trunk (DT) cells at stage 14. Additionally, TEM of stage-16 wild-type and sar1 mutant embryos showed a grossly bloated rough ER structure in DT tracheal cells. Consistent with its functions in yeast and vertebrates, Drosophila Sar1 localizes to the ER and is not only required for efficient luminal protein secretion, but also for the integrity of the early secretory apparatus (Tsarouhas, 2007).

To analyze tracheal maturation defects in sar1 mutant embryos, sar1 strains were generated and imaged that carry either btl > ANF-GFP btl-mRFP-moe or btl > Gasp-GFP. In sar1 mutants, luminal deposition of both ANF-GFP and Gasp-GFP is reduced. Like endogenous Gasp in the mutants, Gasp-GFP was clearly retained in the cytoplasm of sar1 embryos. ANF-GFP was also retained in the tracheal cells of sar1 mutants, but to a lesser extent. Strikingly, sar1 mutants failed to fully expand the luminal diameter of the DT outlined by the apical RFP-moe localization. This defect was quantified by measuring diametric growth rates in metamere 6 for wild-type and sar1 mutant embryos. While early lumen expansion commences in parallel in both genotypes, the later diametric growth of sar1 mutants falls significantly behind compared to wild-type embryos. The DT lumen in sar1 mutants reaches only an average of 70% of the wild-type diameter at early stage 16. Identical diametric growth defects were detected in fixed sar1 mutant embryos expressing btl > GFP-CAAX by analysis of confocal yz sections or TEM. Reexpression of sar1 in the trachea of sar1 mutant embryos not only rescued secretion, but also the lumen diameter phenotype at stage 16. In contrast to the diametric growth defects, DT tube elongation in sar1 embryos was indistinguishable from that in wild-type. This demonstrates distinct genetic requirements for tube diameter and length growth. It also reveals that the sar1 DT luminal volume reaches less than half of the wild-type volume. Prolonged live imaging showed that sar1 mutants are also completely deficient in luminal protein and liquid clearance. Up to 80% of the rescued embryos also completed luminal liquid clearance, suggesting that efficient tracheal secretion and the integrity of the secretory apparatus are prerequisites for later tube maturation steps. Taken together, the above-described results show that tracheal Sar1 is selectively required for tube diameter expansion. Additionally, subsequent luminal protein and liquid clearance fail to occur in sar1 mutants (Tsarouhas, 2007).

Do the tracheal defects of sar1 reflect a general requirement for the COPII complex in luminal secretion and diameter expansion? To test this, lethal P element insertion alleles were examined disrupting two additional COPII coat subunits, sec13 and sec23. Mutant sec13 and sec23 embryos were stained for luminal Gasp and for Crb and α-Spectrin to highlight tracheal cells. At stage 15, embryos of both mutants show a clear cellular retention of Gasp. Furthermore, stage-16 sec13 and sec23 embryos show significantly narrower DT tubes when compared to wild-type. The average diameter of the DT branches in metamere 6 was 4.8 μm and 4.4 μm in fixed sec13 and sec23 embryos, respectively, compared to 6.3 μm in wild-type. Therefore, sec13 and sec23 mutants phenocopy sar1. The phenotypic analysis of three independent mutations disrupting ER-to-Golgi transport thus provides a strong correlation between deficits in luminal protein secretion and tube diameter expansion (Tsarouhas, 2007).

The live-imaging approach defines the developmental dynamics of functional tracheal maturation. At the organ level, three sequential and rapid developmental transitions were identified: (1) the secretion burst, followed by massive luminal protein deposition and tube diameter expansion, (2) the clearance of solid luminal material, and (3) the replacement of luminal liquid by gas. Live imaging of each event additionally revealed insights into the startlingly dynamic activities of the tracheal cells. ANF-GFP-containing structures and apical GFP-FYVE-positive endosomes rapidly traffic in tracheal cells during the secretion burst and protein clearance. The direct live comparison between wild-type and mutant embryos further highlights the dynamic nature of epithelial activity during each pulse (Tsarouhas, 2007).

This study identified several mutations that selectively disrupt distinct cellular functions and concurrently interrupt the maturation process at specific steps. This clearly demonstrates the significance of phenotypic transitions in epithelial organ maturation and establishes that secretion is required for luminal diameter expansion and endocytosis for solid luminal material clearance (Tsarouhas, 2007).

The sudden initiation of an apical secretory burst tightly precedes diametric tube expansion. The completion of both events depends on components of the COPII complex, further suggesting that the massive luminal secretion is functionally linked to diametric growth. How does apical secretion provide a driving force in tube diameter expansion? In mammalian lung development, the distending internal pressure of the luminal liquid on the epithelium expands the lung volume and stimulates growth. Cl⁻ channels in the epithelium actively transport Cl⁻ ions into luminal liquid. The resulting osmotic differential then forces water to enter the lung lumen, driving its expansion (Olver, 2004). By analogy, the tracheal apical exocytic burst may insert protein regulators such as ion channels into the apical cell membrane or add additional membrane to the growing luminal surface. Since the ER is a crucial cellular compartment for intracellular traffic and lipogenesis, its disruption in sar1 mutants may disrupt the efficient transport of so far unknown specific regulators or essential apical membrane addition required for diametric expansion. Alternatively, secreted chitin-binding proteins (ChB) may direct an increase of intraluminal pressure and tube dilation. Overexpression of the chitin-binding proteins Serp-GFP or Gasp-GFP was insufficient to alter the diametric growth rate of the tubes, suggesting that lumen diameter expansion is insensitive to increased amounts of any of the known luminal proteins. In sar1 mutants, the secretion of at least two chitin-binding proteins, Gasp and Verm, is reduced. Chitin, however, is deposited in seemingly normal quantities, but assembles into an aberrantly narrow and dense chitinous cable. This phenotype suggests that the correct ratio between chitin and multiple interacting proteins may be required for the correct assembly of the luminal cable. Interestingly, sar1, sec13, and sec23 mutant embryos form a severely defective and weak epidermal cuticle (Abrams, 2005). The luminal deposition of ChB proteins during the tracheal secretory burst may orchestrate the construction and swelling of a functional matrix, which, in turn, induces lumen diameter dilation. While this later hypothesis is favored, it cannot be excluded that other mechanisms, either separately or in combination with the dilating luminal cable, drive luminal expansion (Tsarouhas, 2007).

During tube expansion, massive amounts of luminal material, including the chitinous cable, fill the tracheal tubes. This study found that Dynamin, Clathrin, and the tracheal function of Rab5 are required to rapidly remove luminal contents, indicating that endocytosis is required for this process. Several lines of evidence argue that the tracheal epithelium activates Rab5-dependent endocytosis to directly internalize luminal material. First, the tracheal cells of rab5 mutants show defects in multiple endocytic compartments. These phenotypes of rab5 mutants become apparent during the developmental period matching the interval of luminal material clearance in wild-type embryos. Second, tracheal cells internalize two luminal markers, the endogenously encoded Gasp and the dextran reporter, exactly prior and during luminal protein clearance. The number of intracellular dextran puncta reaches its peak during the clearance process and ceases shortly thereafter. Lastly, intracellular puncta of both Gasp and dextran colocalize with defined endocytic markers inside tracheal cells. The colocalization of Gasp and dextran with GFP-Rab7 and of Gasp with GFP-LAMP1 suggests that the luminal material may be degraded inside tracheal cells. Taken together, these data show that the tracheal epithelium activates a massive wave of endocytosis to clear the tubes (Tsarouhas, 2007).

Endocytic routes are defined by the nature of the internalized cargoes and the engaged endocytic compartments. What may be the features of the endocytic mechanisms mediating the clearance of luminal material? The phenotype of chc mutants and the presence of intracellular Gasp in CCVs indicate that luminal clearance at least partly relies on Clathrin-mediated endocytosis (CME). In addition to CME, Dynamin and Rab5 have also been implicated in other routes of endocytosis, suggesting that multiple endocytic mechanisms may be operational in tracheal maturation. The nature of the endocytosed luminal material provides an additional perspective. While cognate uptake receptors may exist for specific cargos such as Gasp, Verm, and Serp, the heterologous ANF-GFP, degraded chitin, and the fluid-phase marker dextran may be cleared by either fluid-phase internalization or multifunctional scavenger receptors. Interestingly, Rab5 can regulate fluid-phase internalization in cultured cells by stimulating macro-pinocytosis and the activation of Rabankyrin-5 (Bucci, 1992; Schnatwinkel, 2004; Stenmark, 1994). The defective tracheal internalization of dextran in rab5 mutants provides further loss-of-function evidence for Rab5 function in fluid-phase endocytosis in vivo. The above-described arguments lead to the speculation that additional Rab5-regulated endocytic mechanisms most likely cooperate with CME in the clearance of solid luminal material (Tsarouhas, 2007).

How is liquid cleared from the lumen? While very little is known about this fascinating process, some developmental and mechanistic arguments suggest that this last maturation step is mechanistically distinct. First, the interval of luminal liquid clearance is clearly distinct from the period of endocytic clearance of solids. Second, the dynamic internalization of dextran and the abundance of GFP-marked endocytic structures decline before liquid clearance. Finally, assessment of liquid clearance further suggests that it requires a distinct cellular mechanism (Tsarouhas, 2007).

Viewing the entire process of airway maturation in conjunction, some general conclusions may be drawn. First, the three epithelial pulses are highly defined by their sequence and exact timing, suggesting that they may be triggered by intrinsic or external cues. Second, the analysis of mutants that selectively reduce the amplitude of the secretory or endocyic pulses demonstrates the requirement for each epithelial transition in the completion of the entire maturation process. These pulses are induced in the background of basal secretory and endocytic activities that operate throughout development. Third, specific cellular activities exactly precede each morphological transition. Finally, the separate transitions are interdependent in a sequential manner. Efficient secretion is a prerequisite for the endocytic wave. Similarly, protein endocytosis is a condition for luminal liquid clearance. This suggests a hierarchical coupling of the initiation of each pulse to completion of the previous one in a strict developmental sequence (Tsarouhas, 2007).

This study provides a striking example of how pulses of epithelial activity drive distinct developmental events and mold the nascent tracheal lumen into an air delivery tube. These findings are likely to be relevant beyond the scope of tracheal development. The uniform growth of salivary gland tubes in flies and the excretory canal and amphid channel lumen in worms also require the assembly of a luminal matrix for uniform tube growth (Abrams, 2006; Perens, 2005). Luminal material is also transiently present during early developmental stages in the distal nephric ducts of lamprey. Thus, the coordinated, timely deposition and removal of transient luminal matrices may represent a general mechanism in tubulogenesis (Tsarouhas, 2007).

The endocytic control of JAK/STAT signalling in Drosophila

Domeless (Dome) is an IL-6-related cytokine receptor that activates a conserved JAK/STAT signalling pathway during Drosophila development. Despite good knowledge of the signal transduction pathway in several models, the role of receptor endocytosis in JAK/STAT activation remains poorly understood. Using both in vivo genetic analysis and cell culture assays, it was shown that ligand binding of Unpaired 1 (Upd1) induces clathrin-dependent endocytosis of receptor-ligand complexes and their subsequent trafficking through the endosomal compartment towards the lysosome. Surprisingly, blocking trafficking in distinct endosomal compartments using mutants affecting either Clathrin heavy chain, rab5, Hrs or deep orange led to an inhibition of the JAK/STAT pathway, whereas this pathway was unchanged when rab11 was affected. This suggests that internalization and trafficking are both required for JAK/STAT activity. The requirement for clathrin-dependent endocytosis to activate JAK/STAT signalling suggests a model in which the signalling 'on' state relies not only on ligand binding to the receptor at the cell surface, but also on the recruitment of the complex into endocytic vesicles on their way to lysozomes. Selective activation of the pool of receptors marked for degradation thus provides a way to tightly control JAK/STAT activity (Devergne, 2007).

Using genetic analysis this study shows that several regulators of the endocytic pathway are required for normal JAK/STAT signalling in vivo. The membrane-bound Dome receptors undergo ligand-dependent internalization in clathrin-coated vesicles, which are then targeted to the sorting endosome via Rab5. The function of Hrs is required for JAK/STAT activation and to direct most of the active receptors to the MVBs, targeting them to the lysosome for degradation (Devergne, 2007).

One important question is to know whether the trafficking of ligand-bound receptors has any effect on signalling. This question was addressed by looking at Stat nuclear localization, which represents a robust readout to assess JAK/STAT activity, and at pnt-lacZ expression (Devergne, 2007).

The effect of Hrs is opposite on the JAK/STAT pathway compared with its effect on other pathways. Indeed, in the egg chamber, Hrs plays a positive role on JAK/STAT activity, whereas it has been shown to downregulate the EGFR, Notch and TGF-β pathways in the same tissue. Interestingly, HRS has been shown to interact with STAM in the same mono-ubiquitylated recognition complex (Lohi, 2001). STAM is a known JAK/STAT activator (Pandey, 2000), suggesting that HRS could control STAT signalling through its interaction with STAM. So, Hrs could play two crucial roles: first, allowing the sequestration and the sorting of the receptor to the lysosome and, second, activating the ligand-receptor complex in collaboration with STAM (Devergne, 2007).

The data challenge the simple view whereby binding of the ligand to the receptor at the membrane would be sufficient to activate the pathway. Indeed, it was found that equally essential is the need of clathrin for the activation of JAK/STAT signalling. Thus, activation can occur only when the ligand-receptor complex is assembled into clathrin-coated vesicles. In this view, activation would proceed in two steps, requiring both binding of the ligand and interaction with clathrin. The role of clathrin could be to concentrate/cluster receptors and/or bring them together with other signal transducers in the endosomal compartment. This finding is in agreement with a recent work showing that, in mammals, clathrin is required to transduce JAK/STAT signals through the IFNα-receptor, but not the IFNγ-receptor, suggesting a conserved function for clathrin in JAK/STAT signalling (Marchetti, 2006). Interestingly, like in mammals, JAK/STAT signalling in Drosophila might be controlled in a cell-type-specific manner by Chc-dependent endocytosis. Indeed, in Drosophila eyes, Vps25 and TSG101 mutations lead to Upd upregulation and JAK/STAT activation in a Notch-dependent manner (Devergne, 2007).

What is the significance of clathrin function and, more generally, of the requirement for internalization, in JAK/STAT signalling? It has been shown for several signalling pathways that internalization brings together membrane receptors and intracellular pathway components in the endosomal compartment, which thus serves as a platform for signalling. The fact that Dome internalization and activation are coupled to degradation has important consequences. Making signalling complexes only active in the endosomal compartment is a powerful mechanism to control the number of active complexes in the cell. Their targeting to the lysosome allows the control of their lifetime as active receptors, providing a temporal -- hence quantitative -- control on signalling (Devergne, 2007).

Activation of JAK/STAT follows an off/on/off model in which two conditions are required for correct JAK/STAT activation: (1) formation of a ligand-receptor complex (as proposed in the classical model), followed by (2) the internalization of the complex via Chc-containing budding vesicles. The sole formation of the ligand-receptor interaction would lead to an inactive complex (off). However, interaction with Chc and subsequent internalization activate the complex (on), thus ensuring that only the complexes targeted for degradation are activated. Arrival of the complex in the MVB/lysosome turns it into the off state (Devergne, 2007).

Internalization is required for proper Wingless signaling in Drosophila melanogaster

The Wingless pathway regulates development through precisely controlled signaling. This study shows that intracellular trafficking in the Wg target cell regulates Wg signaling levels. In Drosophila cells stimulated with Wg media, dynamin or Rab5 knockdown causes reduced reporter (Super8XTOPflash) activity, suggesting that internalization and endosomal transport facilitate Wg signaling. In the wing, impaired dynamin function reduces Wg transcription. However, when Wg production is unaffected, extracellular Wg levels are increased. Despite this, target gene expression is reduced, indicating that internalization is also required for efficient Wg signaling in vivo. When endosomal transport is impaired, Wg signaling is similarly reduced. Conversely, the expression of Wg targets is enhanced by increased transport to endosomes or decreased hepatocyte growth factor-regulated tyrosine kinase substrate- mediated transport from endosomes. This increased signaling correlates with greater colocalized Wg, Arrow, and Dishevelled on endosomes. Since these data indicate that endosomal transport promotes Wg signaling, these findings suggest that the regulation of endocytosis is a novel mechanism through which Wg signaling levels are determined (Seto, 2006).

This analysis has revealed the surprising finding that intracellular transport affects the efficiency of Wg signaling. In cell culture, knockdown of dynamin, a protein essential for clathrin-mediated internalization, reduces the TOPFlash/RL ratio (RL is Renilla luciferase), which is suggestive of decreased Wg signaling. Similarly, Rab5 knockdown causes reduced TOPFlash/RL ratios under most conditions, suggesting that internalization and endosomal transport are important for Wg signaling. Interestingly, transfection with polIII-RL, a control vector used in a recent screen for modifiers of Wg signaling, produces conflicting results for Rab5 compared with other RL controls, indicating that cell culture-based Wg signaling assays are very sensitive to experimental conditions. Thus, although the cell culture results indicate an endocytic regulation of Wg signaling, in vivo validation is critically important (Seto, 2006).

In the wing, further evidence was found that Wg signaling levels are highly dependent on intracellular transport. When endocytosis is altered, ligand levels and signaling levels are uncoupled such that high Wg levels do not necessarily enhance signaling. Therefore, limited usage has been made of the term morphogen gradient, which could refer to either ligand or signaling levels. Instead describe Wg distribution and signaling readouts are described. When internalization is inhibited in a domain that does not affect Wg production, high levels of Wg(ex) were found, likely as a result of reduced degradation. However, Wg target gene expression is diminished, indicating that impaired internalization decreases Wg signaling in vivo as well as in cell culture. When early endosomal transport is impaired, Senseless (Sens) and Distal-less (Dll) expression are also reduced despite abundant Wg levels. In both cases, markers of high signaling levels are especially affected, indicating that intracellular signaling is important to achieve robust Wg signaling levels. The differential decrease also argues that changes in Sens and Dll expression are not merely the result of cell death or global changes in transcription. Further supporting this, normal expression of other genes was found in the wing pouch. Additionally, when endosomal transport is enhanced or when transport from the endosome is impaired, Wg signaling is increased. These data suggest that protein localization to the endosome facilitates Wg signaling. Conversely, increased transport to MVBs decreases the expression of Wg readouts. This causes an adult wing phenotype that can be suppressed by Wg signaling components. Thus, it is proposed that in addition to low levels of cell surface signaling, intracellular Wg signaling is critical for proper signaling levels (Seto, 2006).

Because endocytosis is tightly regulated, intracellular Wg signaling may allow for the rapid modulation of signaling levels. For example, endosomal transport can be regulated merely by changing the GDP/GTP state of Rab5. This work indicates that impaired endosomal transport by GDP-bound Rab5 reduces Wg signaling, whereas enhanced endosomal fusion by GTP-bound Rab5 increases signaling. Because the GDP/GTP-binding state of Rab5 is controlled posttranslationally by GTPase-activating proteins and guanine nucleotide exchange factors, endocytic regulation likely allows more of a rapid adjustment of signaling than regulatory mechanisms requiring transcription and translation. Furthermore, because endocytic rates vary between cell types, this regulation may allow signaling to be adjusted in particular parts of the body or cells of a tissue. Thus, regulated endocytosis allows for precise temporal and spatial control of Wg signaling (Seto, 2006).

Endocytosis is hypothesized to regulate signaling through several mechanisms. For example, lysosomal degradation of internalized active receptor tyrosine kinases serves to attenuate signaling. However, the data suggest that Wg signaling is enhanced by endocytosis. One theory by which intracellular transport facilitates signaling is that the internalization of ligand-receptor complexes promotes interactions with other signaling members recruited to or already present on endosomes. In MAPK signaling, ERK1 receptors form protein complexes with endosomal MP1 and p14, leading to greater activation of signaling. Similarly, TGFβ signaling may be enhanced by receptor internalization to endosomes where the Smad2 anchor protein SARA is enriched. Although this work and that of others suggests that Wg undergoes receptor-mediated internalization in the wing, these data alone cannot explain the enhanced Wg signaling observed. However, not only are Wg and Arrow colocalized in large endosomal accumulations in hrs mutants, but they also colocalize with the cytoplasmic signaling component Dsh. The colocalization of Wg, Arr, and Dsh correlates with the increased expression of Wg readouts. These data suggest that internalization and endosomal transport may promote Wg signaling by facilitating associations between the Wg-receptor complex and downstream signaling components like Dsh. Interestingly, Dsh is reportedly present on intracellular vesicles, and mutations that impair vesicular localization do disrupt canonical Wg signaling (Seto, 2006).

Axin, a protein that inhibits Wg signaling by down-regulating Arm levels, has also been shown to colocalize with Dsh on intracellular vesicles. Upon Wg signaling, Axin relocalizes from intracellular puncta to the plasma membrane. This correlates with Arm stabilization and increased Wg signaling. Because Axin associates with Dsh and the cytoplasmic tail of Arr, it is proposed that internalized Wg forms an endosomal signaling complex that may relocalize Axin, thereby stabilizing Arm and facilitating signaling (Seto, 2006).

A model of intracellular Wg signaling is presented. Based on the data obtained from altering endocytosis, Wg at the cell surface produces only low levels of Wg signaling in the wing. Wg associates with its receptors and is internalized. When endocytic vesicles fuse with the early endosome, the cytoplasmic domains of the Wg receptors Frizzled and Arr are able to associate with downstream signaling components like Dsh, thereby facilitating Wg signaling. Subsequent endosomal sorting into MVB inner vesicles sequesters the Wg-receptor complex from other signaling components, and the activation of signaling transduction is halted (Seto, 2006).

The endocytic pathway acts downstream of Oskar in Drosophila germ plasm assembly

Cell fate is often determined by the intracellular localization of RNAs and proteins. In Drosophila oocytes, oskar (osk) RNA localization and the subsequent Osk synthesis at the posterior pole direct the assembly of the pole plasm, where factors for the germline and abdomen formation accumulate. osk RNA produces two isoforms, long and short Osk, which have distinct functions in pole plasm assembly. Short Osk recruits downstream components of the pole plasm, whose anchoring to the posterior cortex requires long Osk. The anchoring of pole plasm components also requires actin cytoskeleton, and Osk promotes long F-actin projections in the oocyte posterior cytoplasm. However, the mechanism by which Osk mediates F-actin reorganization remains elusive. Furthermore, although long Osk is known to associate with endosomes under immuno-electron microscopy, it was not known whether this association is functionally significant. This study shows that Rabenosyn-5 (Rbsn-5), a Rab5 effector protein required for the early endocytic pathway, is crucial for pole plasm assembly. rbsn-5^- oocytes fail to maintain microtubule polarity, which secondarily disrupts osk RNA localization. Nevertheless, anteriorly misexpressed Osk, particularly long Osk, recruits endosomal proteins, including Rbsn-5, and stimulates endocytosis. In oocytes lacking rbsn-5, the ectopic Osk induces aberrant F-actin aggregates, which diffuse into the cytoplasm along with pole plasm components. It is proposed that Osk stimulates endosomal cycling, which in turn promotes F-actin reorganization to anchor the pole plasm components to the oocyte cortex (Tanaka, 2008).

The polarized targeting and anchoring of specific molecules and organelles to particular subcellular regions are crucial for many cellular processes, including cell-polarity establishment and cell-fate determination. In many animals, germline fate is controlled by maternal factors localized to a specialized cytoplasmic region within the egg, called the germ plasm. Germ plasm contains germ granules, which are electron-dense, and non-membranous structures consisting of maternal RNAs and proteins required for the formation of germ cells. Drosophila germ plasm, also called pole plasm, forms at the posterior pole of the embryo and is inherited by the germline precursors, or pole cells. Because the cytoplasmic transplantation of the pole plasm into recipient embryos causes the ectopic formation of pole cells, the pole plasm contains sufficient factors for germ-cell formation. This observation also highlights the importance of retaining the pole plasm at the posterior cortex of the embryo to ensure the germ cells form at the appropriate location (Tanaka, 2008).

In Drosophila, the pole plasm is assembled during oogenesis, which is divided into 14 morphologically distinct stages of egg chamber development. The egg chamber is composed of a single oocyte and 15 nurse cells, surrounded by a monolayer of somatic follicle cells. During oogenesis, most components of pole plasm are synthesized in the nurse cells and transported into the oocyte via ring canals, which are cytoplasmic bridges interconnecting the oocyte with nurse cells. Within the oocyte, these factors become concentrated at the posterior pole and are assembled into the polar (germ) granules. These factors are transported by a polarized microtubule (MT) array that is initially nucleated at the oocyte posterior and extends into the nurse cells through the ring canals. During stages 6-7, the MT array is reorganized by the transforming growth factor alpha-like Gurken (Grk) signal. In the stage-6 oocyte, posteriorly restricted Grk induces neighboring follicle cells to adopt the posterior fate. These cells send back as-yet unknown signals to the oocyte to trigger the reorganization of the MT cytoskeleton. Consequently, the MT array within the oocyte becomes polarized along the anteroposterior (AP) axis, with the minus ends abundant at the anterior of the oocyte and the plus ends extending toward the posterior. This MT organization promotes the migration of the oocyte nucleus and associated grk RNA to the future anterior-dorsal corner, where Grk signals the follicle cells to define the dorsoventral axis. The polarized MT array also directs the localization of bicoid (bcd) RNA to the anterior and oskar (osk) RNA to the posterior within the oocyte. The anterior accumulation of bcd RNA is required for the proper development of the embryonic head and thoracic structures. The posterior localization of osk RNA is essential for the formation of the germ cells and abdomen (Tanaka, 2008).

osk RNA localization is tightly coupled to translational control: only the posteriorly localized osk message is translated. The localized Osk protein, in turn, recruits downstream components of the pole plasm, such as Vasa (Vas) and Tudor (Tud) proteins, and the nanos, germ cell-less and polar granule component RNAs. Misexpression of Osk at the anterior of the oocyte causes ectopic pole plasm assembly and the formation of germ cells at the new site, indicating that Osk organizes pole plasm assembly (Tanaka, 2008).

Although osk has no known alternatively spliced variants, the osk message produces two protein isoforms, long and short Osk, by translation from in-frame alternative start codons. Short Osk shares its entire sequence with the long isoform. Nevertheless, genetic evidence shows that the two Osk isoforms have distinct functions in the assembly of the pole plasm. Long Osk is required for all the components of the pole plasm, including Osk itself, to be anchored to the posterior cortex, preventing their diffusion into the cytoplasm. However, the mechanism by which long Osk retains pole plasm components at the posterior cortex remains unknown (Tanaka, 2008).

A recent immuno-electron microscopic study revealed that the two Osk isoforms localize to distinct organelles in the oocyte posterior: long Osk associates with endosomes and short Osk is concentrated in the polar granules (Vanzo, 2007). Long Osk also upregulates endocytosis, which occurs preferentially at the oocyte posterior (Vanzo, 2007). Therefore, the endocytic pathway may be involved in pole plasm assembly downstream of long Osk, although data are lacking to show that the association between long Osk and endosomes is functionally significant. Several reports have suggested that vesicular trafficking is involved in pole plasm assembly and germ cell formation. For example, in mutants for Rab11, which encodes a small GTPase involved in the recycling of endosomes, osk RNA fails to be transported to the oocyte posterior, instead forming aggregates close to the posterior. However, the defects in osk RNA localization in Rab11 mutants are thought to be an indirect consequence of the disrupted MT polarization (Tanaka, 2008).

This study shows that Drosophila Rabenosyn-5 (Rbsn-5), a Rab5 effector protein involved in the early endocytic pathway, is required for osk RNA localization and pole plasm assembly. Although the primary defect of the rbsn-5 mutation is, as in the Rab11 mutant, caused by the failure to maintain MT polarity, which secondarily affects osk RNA localization, evidence is provided that the endocytic pathway also functions downstream of Osk to anchor the pole plasm components to the oocyte cortex (Tanaka, 2008).

Vas is a reliable marker for the germline throughout Drosophila development. A GFP-Vas fusion protein enables the direct visualization of the pole plasm and germ cells in the living organism. During oogenesis, GFP-Vas accumulates at the oocyte posterior from stage 9 onward. Using GFP-Vas as a marker, a germline clonal screen was performed targeting chromosome 2L for mutations that disrupted pole plasm assembly. From 5122 lines mutagenized with EMS, 66 mutants were isolated defective in GFP-Vas localization. Twenty-seven of these were alleles of cappuccino, spire or profilin (chickadee), three genes on 2L that are known to be involved in osk RNA localization, which validates the screening strategy (Tanaka, 2008).

Among the other mutants recovered was a recessive lethal mutation, C241, that mapped to 28C2-29E2. Subsequent deficiency mapping and sequencing of the mutant chromosome revealed that the C241 mutation was a single nucleotide substitution in the CG8506 gene, which resulted in a premature stop codon at position 315 of the 505 amino acid open reading frame (ORF). The introduction of a transgene containing a genomic DNA fragment with the CG8506 transcriptional unit rescued the C241 mutant phenotypes (described below). These data show that CG8506 corresponds to the gene that was mutated at the C241 locus. Rabbit and rat polyclonal antisera raised against full-length CG8506 did not detect a truncated form of CG8506 in ovarian extracts from C241 heterozygotes. Furthermore, neither antibody showed immunoreactivity in C241 homozygous clones, suggesting that the truncated protein was not expressed at detectable levels and/or was unstable. Therefore, C241 appeared to be a strong loss-of-function, presumably a protein-null, allele of CG8506 (Tanaka, 2008).

CG8506 (Rabenosyn - FlyBase) encodes a protein homologous to Rabenosyn-5 (Rbsn-5) (Nielsen, 2000). Rbsn-5 interacts with several Rab proteins, including Rab5, which functions in early endosomal transport (de Renzis, 2002; Eathiraj, 2005). Several Rbsn-5 protein domains are conserved across species, including the FYVE domain, which binds phosphatidylinositol-3-phosphate (Nielsen, 2000). However, invertebrate Rbsn-5 homologs lack the C-terminal domain common to the mammalian homologs of this protein. Since the C-terminal domain of mammalian Rbsn-5 is responsible for its interaction with Rab5 (de Renzis, 2002; Eathiraj, 2005), whether CG8506 interacted with Rab5 was examined. Pull-down assays showed that GST-Rab5 efficiently pulled down in-vitro-synthesized CG8506 protein in the presence of a GTP analog, GTP-γS, but inefficiently in the presence of GDP. The interaction between CG8506 and Rab5-GTP was specific, because the interactions of CG8506 with Rab11 and Rab7 were at background levels. Consistent with a physical interaction between CG8506 and Rab5 in vitro, in CG8506^C241 GLCs, neither auto-fluorescent granules derived from endocytosed yolk proteins nor the incorporation of a fluorescent marker for endocytosis, FM4-64, were observed in the oocytes, suggesting that CG8506 functions cooperatively with Rab5 in the early endocytic pathway. Thus, CG8506 is the Drosophila ortholog of Rbsn-5 and has an evolutionarily conserved function in the endocytic pathway (Tanaka, 2008).

This study shows that that Osk maintains, but does not establish, the posterior accumulation of endosomal proteins and asymmetric endocytosis, and that Osk can recruit endosomal proteins and stimulate endocytosis even at an ectopic site. It is further shown that the anchoring of the pole plasm components to the oocyte cortex requires the Osk-dependent stimulation of endocytic activity. These data reveal an interdependent relationship between Osk anchoring and localized endocytic activity at the oocyte posterior (Tanaka, 2008).

In rbsn-5^- oocytes, the anterior misexpression of Osk induces aberrant F-actin aggregates, which diffuse along with pole plasm components into the cytoplasm. Several lines of evidence suggest that the anchoring of pole plasm components requires the proper organization of F-actin. Since endosomal proteins are recruited by long Osk, the idea is favored that the endocytic pathway functions downstream of long Osk to anchor the pole plasm components at the cortex by regulating F-actin dynamics. Supporting this idea, in addition to its roles in early endosomal sorting, Rab5 acts as a signaling molecule that remodels F-actin networks (Lanzetti, 2004). Rab11, which regulates the recycling of endosomes, is also involved in F-actin organization during cellularization in Drosophila blastoderm embryos (Riggs, 2003). Intriguingly, the recruitment of endosomal proteins by Osk is not sufficient for proper F-actin reorganization to anchor the pole plasm components at the cortex, because their recruitment occurs even in oocytes lacking Rbsn-5, in which cortical anchoring fails. It is therefore proposed that the continuous cycling of endosomes is required for pole plasm components to be anchored to the oocyte cortex. This scenario is compatible with a model in yeasts, which use endocytic cycling coupled with localized exocytosis to maintain their polarity (Valdez-Taubas, 2003), although it is unclear if F-actin reorganization is involved in this process (Tanaka, 2008).

Rbsn-5 is primarily required for the maintenance of MT polarity that directs posterior localization of osk RNA. Rab11 is also required for MT polarization in the oocyte (Jankovics, 2001; Dollar, 2002). However, the accumulation of endosomal proteins and upregulation of endocytosis at the oocyte posterior require the oocyte polarization, which promotes the reorganization of the MT array. Thus, MT polarization and asymmetric activation of the endocytic pathway are probably interdependent as well. Furthermore, maintenance of polarized endocytic activity depends on Osk. Intriguingly, Osk is also thought to maintain MT polarity, as posterior accumulation of Kin-βgal is partially defective in the absence of Osk (Zimyanin, 2007). It is therefore likely that the endocytic pathway and Osk form a positive-feedback loop that maintains oocyte polarity: Osk may maintain MT polarity through recruiting endosomal proteins. Based on these results, a model is proposed in which the endocytic pathway is involved in several distinct steps in pole plasm assembly (Tanaka, 2008).

The localization of bcd RNA to the anterior pole of the oocyte requires the ESCRT-II (endosomal sorting complex required for transport II) complex, which sorts mono-ubiquitinated endosomal transmembrane proteins into multivesicular bodies. Furthermore, Vps36p, a component of the ESCRT-II complex, binds bcd 3' UTR in vitro and co-localizes with bcd RNA at the oocyte anterior, suggesting the direct involvement of ESCRT-II in bcd RNA localization. osk RNA, however, appears to use another mechanism for its posterior localization, since its localization is unaffected in the absence of ESCRT-II function. Several lines of evidence suggest that ER organization and RNA localization are linked. However, it is considered unlikely that the ER directs the posterior localization of osk RNA, because ER components and osk RNP distributed differentially in developing oocytes. Interestingly, the osk RNP and the endosomal proteins are in close proximity during their transport to the oocyte posterior. Although their close association may simply be owing to the dynamic rearrangements of the MT array during stages 7-8, these findings suggest that the endocytic pathway may also play a role in the targeting of osk RNP to the posterior pole of the oocyte. Retroviral genomic RNAs are known to hitchhike on endosomal vesicles to reach the plasma membrane. Therefore, it will be interesting to learn if osk RNA is also transported to the posterior pole of the oocyte along with the endosomes (Tanaka, 2008).

Search PubMed for articles about Drosophila rab5

Abrams, E. W. and Andrew, D. J. (2005). CrebA regulates secretory activity in the Drosophila salivary gland and epidermis. Development 132: 2743-2758. PubMed Citation: 15901661

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Christoforidis, S., et al. (1999). Phosphatidylinositol-3-OH kinases are Rab5 effectors. Nat. Cell Biol. 1: 249-252. PubMed Citation: 10559924

de Hoop, M. J., et al. (1994). The involvement of the small GTP-binding protein Rab5a in neuronal endocytosis. Neuron 13: 11-22. PubMed Citation: 8043272

De Renzis, S., Sonnichsen, B. and Zerial, M. (2002). Divalent Rab effectors regulate the sub-compartmental organization and sorting of early endosomes. Nat. Cell Biol. 4: 124-133. PubMed Citation: 11788822

Devergne, O., Ghiglione, C. and Noselli, S. (2007). The endocytic control of JAK/STAT signalling in Drosophila. J. Cell Sci. 120(Pt 19): 3457-64. PubMed Citation: 17855388

Dollar, G., Struckhoff, E., Michaud, J. and Cohen, R. S. (2002). Rab11 polarization of the Drosophila oocyte: a novel link between membrane trafficking, microtubule organization, and oskar mRNA localization and translation. Development 129: 517-526. PubMed Citation: 11807042

Eathiraj, S., Pan, X., Ritacco, C. and Lambright, D. G. (2005). Structural basis of family-wide Rab GTPase recognition by rabenosyn-5. Nature 436: 415-419. PubMed Citation: 16034420

Fischer von Mollard, G., et al. (1994). Localization of Rab5 to synaptic vesicles identifies endosomal intermediates in synaptic vesicle recycling pathway. Eur. J. Cell Biol. 65: 319-326. PubMed Citation: 7720727

Gillooly, D.J., et al. (2000). Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J. 19: 4577-4588. PubMed Citation: 10970851

Horiuchi, H., et al. (1997). A novel rab5 gdp/gtp exchange factor complexed to rabaptin-5 links nucleotide exchange to effector recruitment and function. Cell. 90: 1149-1159. PubMed Citation: 9323142

Jankovics, F., Sinka, R. and Erdélyi, M. (2001). An interaction type of genetic screen reveals a role of the Rab11 gene in oskar mRNA localization in the developing Drosophila melanogaster oocyte. Genetics 158: 1177-1188. PubMed Citation: 11454766

Kamei, M., et al. (2006). Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 442: 453-456. PubMed Citation: 16799567

Lanzetti, L., Palamidessi, A., Areces, L., Scita, G. and Di Fiore, P. P. (2004). Rab5 is a signaling GTPase involved in actin remodelling by receptor tyrosine kinases. Nature 429: 309-314. PubMed Citation: 15152255

Lawe, D. C., et al. (2000). The FYVE domain of early endosome antigen 1 is required for both phosphatidylinositol 3-phosphate and Rab5 binding. Critical role of this dual interaction for endosomal localization. J. Biol. Chem. 275: 3699-3705. PubMed Citation: 10652369

Lohi, O. and Lehto, V. P. (2001). STAM/EAST/Hbp adapter proteins--integrators of signalling pathways. FEBS Lett. 508(3): 287-90. PubMed Citation: 11728436

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Misra, S., and Hurley, J. H. (1999). Crystal structure of a phosphatidylinositol 3-phosphate-specific membrane-targeting motif, the FYVE domain of Vps27p. Cell 97: 657-666. PubMed Citation: 10367894

Nielsen, E., et al. (2000). Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain. J. Cell Biol. 151: 601-612. PubMed Citation: 11062261

Olver, R. E., Walters, D. V. and Wilson, S. M. (2004). Developmental regulation of lung liquid transport, Annu. Rev. Physiol. 66: 77-101. PubMed Citation: 14977397

Perens, E. A. and Shaham S. (2005). C. elegans daf-6 encodes a patched-related protein required for lumen formation. Dev. Cell 8(6): 893-906. PubMed Citation: 15935778

Riggs, B., Rothwell, W., Mische, S., Hickson, G. R., Matheson, J., Hays, T. S., Gould, G. W. and Sullivan, W. (2003). Actin cytoskeleton remodeling during early Drosophila furrow formation requires recycling endosomal components Nuclear-fallout and Rab11. J. Cell Biol. 163: 143-154. PubMed Citation: 14530382

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Seto, E. S. and Bellen, H. J. (2006). Internalization is required for proper Wingless signaling in Drosophila melanogaster. J. Cell Biol. 173(1): 95-106. PubMed Citation: 16606693

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Tanaka, T. and Nakamura, A. (2008). The endocytic pathway acts downstream of Oskar in Drosophila germ plasm assembly. Development 135: 1107-1117. PubMed Citation: 18272590

Tsarouhas, V., et al. (2007). Sequential pulses of apical epithelial secretion and endocytosis drive airway maturation in Drosophila. Dev. Cell 13: 214-225. PubMed Citation: 17681133

Valdez-Taubas, J. and Pelham, H. R. (2003). Slow diffusion of proteins in the yeast plasma membrane allows polarity to be maintained by endocytic cycling. Curr. Biol. 13: 1636-1640. PubMed Citation: 13678596

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date revised: 20 July 2008

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