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didum: Biological Overview | References

Sensory neuron terminal differentiation tasks apical secretory transport with delivery of abundant biosynthetic traffic to the growing sensory membrane. Drosophila Rab11 is essential for rhodopsin transport in developing photoreceptors and it was asked if myosin V (Didum) and the Drosophila Rab11 interacting protein, dRip11 (lethal (1) G0003), also participate in secretory transport. Reduction of either protein impairs rhodopsin transport, stunting rhabdomere growth and promoting accumulation of cytoplasmic rhodopsin. MyoV-reduced photoreceptors also develop ectopic rhabdomeres inappropriately located in basolateral membrane, indicating a role for MyoV in photoreceptor polarity. Binary yeast two hybrids and in vitro protein-protein interaction predict a ternary complex assembled by independent dRip11 and MyoV binding to Rab11. It is proposed that this complex delivers morphogenic secretory traffic along polarized actin filaments of the subcortical terminal web to the exocytic plasma membrane target, the rhabdomere base. A protein trio conserved across eukaryotes thus mediates normal, in vivo sensory neuron morphogenesis (Li, 2007).

Across eukaryotes, a protein trio comprising a Rab protein, a member of the family of small GTPases that regulate exchange between membrane compartments, a myosin motor, notably myosin V (MyoV), and a linker/adaptor protein, powers organelle motility and polarized secretion (Hammer, 2002; Deneka, 2003; Seabra, 2004). For example, HeLa and MDCK cells recycle endocytosed cell surface receptors through a recycling endosome, the return leg mediated by Rab11 together with MyoV and the Rab11 adaptor/linker protein, family interacting protein 2 (FIP2) (Hales, 2002). Drosophila photoreceptors are typical polarized epithelial cells and morphogenesis of their photosensory membrane organelles, rhabdomeres, is driven by a late-pupal surge of secretory traffic that greatly expands the apical plasma membrane in a column of closely packed, rhodopsin-rich photosensitive microvilli. It was recently found that Rab11 mediates membrane transport to developing rhabdomeres (Satoh, 2005), prompting an investigation to see if Drosophila MyoV (Bonafe, 1998; MacIver, 1998) and dRip11, and Drosophila FIP2 (Prekeris, 2000) also participate in morphogenic secretory transport (Li, 2007).

Numerous observations link MyoV to polarized membrane transport (Reck-Peterson, 2000). Budding yeast lacking essential MyoV, Myo2p, accumulate cytoplasmic post-Golgi secretory vesicles; secretion continues in mutants, but is not correctly targeted to the growing bud (Johnston, 1991). Melanocytes of mouse dilute mutants lacking MyoVa fail to properly localize melanosome pigment organelles to the actin-rich cell periphery; expression of a MyoVa C-terminal fragment (MyoVa-CT) that displaces endogenous MyoVa from melanosomes mimics MyoVa loss (Wu, 1998). Expression of MyoVa-CT similarly inhibits Xenopus melanosome motility (Rogers, 1999) and HeLa cell transferrin receptor recycling (Lapierre, 2001; Hales, 2002; Rodriguez, 2002). Notably, in polarized MDCK cells, MyoVb-CT selectively disrupts Rab11-dependent apical, but not basolateral, membrane recycling (Lapierre, 2001) (Li, 2007).

Parallel loss-of-function phenotypes suggest MyoV and Rab11 cooperate in membrane transport. Loss of either activity inhibits recycling of CXCR2 chemokine and M4 muscarinic acetylcholine receptors (Volpicelli, 2002; Fan, 2003; Fan, 2004). Similarly, MyoV or Rab11 reduction prevents biogenesis of apical cannicular membranes in polarized hepatocytes (Wakabayashi, 2005) and decreases glutamate receptor 1 (GluR1) subunit delivery to developing synapses of hippocampal cells in culture (Lise, 2006) (Li, 2007).

Direct interaction between rabbit Rab11a and MyoVb is detected in yeast two-hybrid screens (Lapierre, 2001), and deletion of MyoVb-CT's Rab11 binding sequence neutralizes its dominant-negative impact on GluR1 delivery in hippocampal neurons, suggesting MyoVb binds Rab11 in GluR1 trafficking (Lise, 2006). Genetic interaction between Saccharomyces cerevisiae Myo2p and Sec4p mutants (Schott, 1999) is consistent with direct or close cooperation (Li, 2007).

In addition to MyoV, Rab11 interacts with Rab11-FIPs at a signature Rab11 binding domain (RBD) (Prekeris, 2003). Class I FIPs contain a C2 domain that targets recycling vesicles to the plasma membrane, and truncated FIPs lacking the C2 domain inhibit receptor recycling. Drosophila encodes a single class I FIP, dRip11 (Prekeris, 2000), but its function has not been reported (Li, 2007 and references therein).

The Drosophila genome includes a single MyoV gene, myoV (didum) (Bonafe, 1998; MacIver, 1998). Drosophila embryos receive substantial maternal MyoV and the protein is ubiquitously expressed throughout development, including the adult retina, where it localizes to the base of the rhabdomere (Mermall, 2005). Mutants lacking MyoV show strong developmental delays and substantial late larval lethality. Surprisingly, rare homozygous mutant escapers showed normal embryogenesis and cellular architecture, suggesting MyoV is dispensable for the wide range of membrane trafficking that supports normal development (Mermall, 2005). Actin staining of myoV mutant eyes showed apparently normal rhabdomeres and adult mutants were normally phototaxic, suggesting that MyoV does not play an obvious role in rhabdomere development or photoreception (Li, 2007 and references therein).

This paper investigated the role of MyoV and dRip11 in the polarized membrane transport that builds Drosophila rhabdomeres. Both were found to be essential. In MyoV mutants, rhodopsin 1 (Rh1) is not delivered to the growing rhabdomere, but instead accumulates in photoreceptor cytoplasm; rhodopsin-bearing vesicles, and the Rab11 and dRip11 they carry, do not approach the rhabdomere base. dRip11 loss similarly impairs secretory transport, delocalizing MyoV and Rab11 and promoting cytoplasmic Rh1. MyoV mutant photoreceptors also develop supernumerary rhabdomeres ectopically positioned within basolateral plasma membrane, suggesting MyoV-mediated transport suppresses formation of inappropriate rhabdomere primordia. Drosophila photoreceptors harness an evolutionarily conserved protein trio to deliver polarized apical membrane traffic in cellular morphogenesis (Li, 2007).

Drosophila photoreceptors, like many polarized epithelial cells, greatly amplify their apical membranes during terminal differentiation via targeted membrane delivery. This study shows that a protein trio (Rab11, dRip11, and MyoV) mediates this morphogenic secretory traffic. MyoV normally concentrates at the base and its loss causes three notable phenotypes of compromised apical transport: Rab11 and dRip11 delocalize from the base, Rh1 accumulates in photoreceptor cytoplasm, and ectopic rhabdomeres are formed. dRip11, the sole Drosophila class I Rab-FIP (Prekeris, 2003), is also required for normal Rh1 transport; its loss delocalizes Rab11 and MyoV. Together with the demonstration that Rab11 is essential for photoreceptor secretory traffic (Satoh, 2005), it is proposed MyoV pulls post-Golgi secretory vesicles, marked for rhabdomere delivery by Rab11 and dRip11, through an exclusionary subcortical cytoskeletal web along polarized microfilaments leading directly to the exocytic targeting patch at the rhabdomere base (Li, 2007).

Cytoplasm adjacent to the rhabdomere base is permeated by a dense microfilament brush, the rhabdomere terminal web (RTW), which extends from the rhabdomere base deep into photoreceptor cytoplasm (Arikawa, 1990; Chang, 2000). Microfilaments are poorly preserved in chemically fixed tissue, but distinct 'RTW cytoplasm' is manifest as organelle-poor cytoplasm behind the rhabdomere. RTW cytoplasm excludes even ribosomes, whose absence contributes to the light, clear appearance of RTW cytoplasm. Biosynthetic ER and Golgi are distributed the length of the cell, in close proximity to the RTW's cytoplasmic terminus (Li, 2007).

The rhabdomere base differentiates in mid-pupal photoreceptors as the photoreceptor apical membrane resolves to a central Moesin-rich rhabdomere primordium surrounded by a Crumbs-rich supporting domain. Once founded, the rhabdomere base organizes the RTW and receives morphogenic traffic. The stalk accepts little traffic, focusing exocytosis to the rhabdomere. The stalk links the rhabdomere to the retina's junctional network and projects it into an apical lumen, the IRS, aligned to the eye's optical axis (Li, 2007).

Membrane transport in light-adapted late pupal photoreceptors is dynamic, with biosynthetic and endocytic traffic reflected in numerous, complex membrane compartments. Post-Golgi secretory traffic is carried in tubular vesicles, approximately 100 nm across; endocytosed membrane gathers in multivesicular bodies. Complex membrane forms are common at the rhabdomere base, likely a consequence of extensive membrane fusion (Li, 2007).

Confocal immunofluorescence localizes Rab11 to puncta throughout photoreceptor cytoplasm, with a prominent concentration at the rhabdomere base . dRip11 immunolocalization resembled Rab11, with cytoplasmic puncta and localization at the rhabdomere base. Note that Rab11 and dRip11 lie within RTW cytoplasm, overlapping the actin brush extending from the rhabdomere's curving base. MyoV concentrates across the rhabdomere base of late pupal photoreceptors, often appearing strongest at the sides. Like Rab11 and dRip11, MyoV staining is strongly within RTW cytoplasm. Cytoplasmic MyoV is lightly diffuse with scattered brighter puncta (Li, 2007).

The RTW's role as both a barrier and a carrier for morphogenic traffic is an instance of a general theme of a dynamic regulatory role of the subcortical cytoskeleton in secretion. Myosin S1 decoration shows RTW filaments are oriented with plus-ends at the membrane, a correct orientation for MyoV-based secretory transport (Arikawa, 1990), and disruption of the actin cytoskeleton prevents the morphogenic traffic that rebuilds crab rhabdomeres at dusk. The RTW's strong polarization and anchorage to a secretory targeting patch resembles the polarized actin cables that mediate budding yeast secretory traffic (Li, 2007 and references therein).

Absorptive and secretory epithelial cell specialists often regulate apical membrane activity by dynamic, Rab11-dependent exchange of plasma membrane with recycling endosomes. For example, gastric parietal cells meet demand for additional acid secretion by Rab11-, Rab11-FIP2-, and MyoV-dependent delivery of additional H+/K+ ATPase pumps to the cell surface from a recycling endosome (Duman, 1999; Hales, 2001; Lapierre, 2001). Like GPCRs generally, Drosophila Rh1 is endocytosed upon stimulation but appears to be degraded rather than recycled back to the rhabdomere. Drosophila photoreceptor Rab11-dependent transport appears to be principally devoted to delivery of newly synthesized cargo from the TGN to the plasma membrane, a conserved Rab11 activity (Chen, 1998) now seen to further parallel recycling transport. Ectopic rhabdomeres in hypomorphs suggest MyoV normally suppresses the establishment of inappropriate rhabdomere primordia; once-founded ectopic rhabdomeres develop in concert with principal rhabdomeres, presumably drawing from the same secretory traffic. It is speculated that MyoV normally drives traffic to the differentiating rhabdomere primordium and that positive feedback driven by the incorporation of morphogenic determinants, perhaps proteins that anchor and promote RTW development, gives the original, 'true' apical membrane an overwhelming growth advantage, starving weak, inappropriate sites. MyoV reduction might diminish this advantage, allowing ectopic foci to capture sufficient morphogenic traffic to assemble a rhabdomere patch (Li, 2007).

The observation that MyoV is required for normal rhabdomere development differs from Mermall's report of normal rhabdomeres in MyoV^Q1052st mutant eyes (Mermall, 2005). However, long ribbons of the principal rhabdomeres dominate phalloidin-stained longitudinal sections, and ectopic rhabdomeres, often patches a few microns across, are not prominent. Mermall's supplementary Fig. 1 L, a tangential section, shows actin-bright profiles apart from the principal rhabdomeres - potentially ectopic rhabdomeres. Massive biosynthetic traffic in late pupal photoreceptors sensitizes cells to compromise of efficient, accurate transport and accumulation of cytoplasmic Rh1 reflects an inability of transport to keep pace with biosynthesis (Li, 2007).

dRip11 loss inhibits secretory transport and misolcalizes Rab11 and MyoV. It is suggested that dRip11 couples two broad streams of membrane transport, Rab11- and MyoV-dependent activities, to drive morphogenic secretory traffic. The results are consistent with previously demonstrated roles for FIPs as contributors to membrane targeting (Meyers, 2002; Lindsay, 2004), and as scaffolds for the growing Rab11 effector ensemble (Pooley, 2006). Similar to chromaffin cells, where MyoV only partially overlaps with secretory vesicles (Rose, 2003), MyoV and Rab11 only partially overlap in developing photoreceptors, and it is likely MyoV transports multiple and changing cargoes (Li, 2007).

Rab11 participates in both constitutive and Ca2+-regulated secretion (Khvotchev, 2003), and both cargo binding and Ca2+ regulate MyoV activity (Krementsov, 2004; Li, 2005; Thirumurugan, 2006). Rhabdomere morphogenesis utilizes constitutive exocytosis, with substantial rhabdomere growth before Rh1 expression and photoresponse Ca2+ influx. Rhabdomeres likewise develop normally in the dark, indicating light-dependent Ca2+ elevation is not required for MyoV morphogenic transport. It is proposed that dRip11, in proximity to MyoV via their mutual binding to Rab11 on post-Golgi secretory vesicles, interprets or conveys non-Ca2+-stimulated MyoV activation, promoting developmental MyoV secretory transport (Li, 2007).

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date revised: 29 February 2008

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