Dynamic Changes in the Spatiotemporal Localization of Rab21 in Live RAW264 Cells during Macropinocytosis

Rab21, a member of the Rab GTPase family, is known to be involved in membrane trafficking, but its implication in macropinocytosis is unclear. We analyzed the spatiotemporal localization of Rab21 in M-CSF-stimulated RAW264 macrophages by the live-cell imaging of fluorescent protein-fused Rab21. It was demonstrated that wild-type Rab21 was transiently associated with macropinosomes. Rab21 was recruited to the macropinosomes after a decrease in PI(4,5)P2 and PI(3,4,5)P3 levels. Although Rab21 was largely colocalized with Rab5, the recruitment of Rab21 to the macropinosomes lagged a minute behind that of Rab5, and preceded that of Rab7. Then, Rab21 was dissociated from the macropinosomes prior to the accumulation of Lamp1, a late endosomal/lysosomal marker. Our analysis of Rab21 mutants revealed that the GTP-bound mutant, Rab21-Q78L, was recruited to the macropinosomes, similarly to wild-type Rab21. However, the GDP-bound mutant, Rab21-T33N, did not localize on the formed macropinosomes, suggesting that the binding of GTP to Rab21 is required for the proper recruitment of Rab21 onto the macropinosomes. However, neither mutation of Rab21 significantly affected the rate of macropinosome formation. These data indicate that Rab21 is a transient component of early and intermediate stages of macropinocytosis, and probably functions in macropinosome maturation before fusing with lysosomal compartments

Role of SNX16 in the Dynamics of Tubulo-Cisternal Membrane Domains of Late Endosomes

In this paper, we report that the PX domain-containing protein SNX16, a member of the sorting nexin family, is associated with late endosome membranes. We find that SNX16 is selectively enriched on tubulo-cisternal elements of this membrane system, whose highly dynamic properties and formation depend on intact microtubules. By contrast, SNX16 was not found on vacuolar elements that typically contain LBPA, and thus presumably correspond to multivesicular endosomes. We conclude that SNX16, together with its partner phosphoinositide, define a highly dynamic subset of late endosomal membranes, supporting the notion that late endosomes are organized in distinct morphological and functional regions. Our data also indicate that SNX16 is involved in tubule formation and cholesterol transport as well as trafficking of the tetraspanin CD81, suggesting that the protein plays a role in the regulation of late endosome membrane dynamics

The SNX-PX-BAR Family in Macropinocytosis: The Regulation of Macropinosome Formation by SNX-PX-BAR Proteins

Background: Macropinocytosis is an actin-driven endocytic process, whereby membrane ruffles fold back onto the plasma membrane to form large (> 0.2 mu m in diameter) endocytic organelles called macropinosomes. Relative to other endocytic pathways, little is known about the molecular mechanisms involved in macropinocytosis. Recently, members of the Sorting Nexin (SNX) family have been localized to the cell surface and early macropinosomes, and implicated in macropinosome formation. SNX-PX-BAR proteins form a subset of the SNX family and their lipid-binding (PX) and membrane-curvature sensing (BAR) domain architecture further implicates their functional involvement in macropinosome formation

The Rab5 effector Rabankyrin-5 regulates and coordinates different endocytic mechanisms.

The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells

Transient fs-time-resolved ESCA photoemission of iodophenylphenol molecules adsorbed as a monolayer on a silicon (100) surface

Heinzmann U, Michelswirth M, Dachraoui H, et al. Transient fs-time-resolved ESCA photoemission of iodophenylphenol molecules adsorbed as a monolayer on a silicon (100) surface. In: Photon Impact (Strong Field) [Abstracts] . Journal of Physics: Conference Series. Vol 194. IOP Publishing; 2009

Photoinduced reconfiguration cycle in a molecular adsorbate layer studied by femtosecond inner-shell photoelectron spectroscopy

Dachraoui H, Michelswirth M, Siffalovic P, et al. Photoinduced reconfiguration cycle in a molecular adsorbate layer studied by femtosecond inner-shell photoelectron spectroscopy. Physics Review Letters. 2011;106(10): 107401