Characterization of the Rab8-specific membrane traffic route linked to protrusion formation

Abstract

Rab8 has a drastic effect on cell shape, but the membrane trafficking route it regulates is poorly defined. Here, we show that endogenous and ectopically expressed Rab8 is associated with macropinosomes generated at ruffling membrane domains. These macropinosomes fuse or transform into tubules that move toward the cell center, from where they are recycled back to the leading edge. The biogenesis of these tubules is dependent on actin and microtubular dynamics. Expression of dominant-negative Rab8 mutants or depletion of Rab8 by RNA interference inhibit protrusion formation, but promote cell-cell adhesion and actin stress fiber formation, whereas expression of the constitutively active Rab8-Q67L has the opposite effect. Rab8 localization overlaps with both Rab11 and Arf6, and is functionally linked to Arf6. We also demonstrate that Rab8 activity is needed for the transport of transferrin and the transferrin receptor to the pericentriolar region and to cell protrusions, and that Rab8 controls the traffic of cholera toxin B to the Golgi compartment. Finally, Rab8 colocalizes and binds specifically to a synaptotagmin-like protein (Slp1/JFC1), which is involved in controlling Rab8 membrane dynamics. We propose that Rab8 regulates a membrane-recycling pathway that mediates protrusion formation.

Control of cell mechanics by RhoA and calcium fluxes during epithelial scattering

“…46 Dominantnegative Rab8 mutants and silencing of Rab8 results in a loss of protrusions and increased cell-cell adhesions in HT1080 fibrosarcoma cells. 55 Meanwhile, overexpression of active Rab8 in BHK cells causes an increase in actin-based cell protrusions, including lammelipodia and filopodia. 56 Furthermore, Rab8 drives cellular invasion by trafficking the matrix metalloproteinase MT1-MMP to the plasma membrane.…”

Rab8 Regulates Basolateral Secretory, But Not Recycling, Traffic at the Recycling Endosome

“…Studies of vesicular stomatitis virus protein G (VSV-G) delivery from the trans-Golgi network (TGN) have suggested that this process is regulated by Rab8, as dominant-negative (DN) mutants of Rab8 interfere with the delivery of basolateral cargo (Huber et al, 1993b). Rab8 is not localized to the TGN; rather it is localized at or near the recycling endosome (RE) in both polarized and nonpolarized cells (Hattula et al, 2006;Roland et al, 2007). The function of Rab8 at the RE is likely to regulate recycling of membrane traffic, although its role in polarized sorting is not clear.…”

Intermediates in the Guanine Nucleotide Exchange Reaction of Rab8 Protein Catalyzed by Guanine Nucleotide Exchange Factors Rabin8 and GRAB

“…To exert their function, Rab proteins need to be activated by a process requiring guanine nucleotide exchange factors (GEFs). 6 These enzymes accelerate GDP release from and allow the binding of GTP to a Rab protein. Rab proteins can interact with effector proteins that preferentially bind the active GTP but not the GDP state.…”

“…Rab8 also regulates cell shape, and interaction with its GEF Rabin8 appears to be crucial to this function (6). Rabin8 and Rab8 appear to be important in cilium formation by acting in concert with the Bardet-Biedl syndrome complex (7)(8)(9).…”