Rab5 Knockdown In Vivo

This paper from the Zerial Laboratory describes a study in which the three mammalian isoforms of Rab5 are reduced in whole livers or cultured hepatocytes by a lipid nanoparticle mediated knockdown treatment. They then analyse images from these cells and liver sections and determine that there are reduced early endosomes, late endosomes and lysosomes in cells depleted of Rab5.

They also identify two functional defects in these tissues; reduced LDL uptake and impaired apical polarity of the hepatocytes. They also generate a simplified model of Rab5/early endosomal dynamics and suggest a role for Rab5 in vesicle fission as well as fusion. Although this model fits the available data quite well, it ignores several other key components of endosomal formation and maturation.

The authors also present the curious finding that while Rab5 depletion reduces both LDL uptake and endosome numbers, dynamin depletion only affects LDL uptake but not the number of endosomes. One possible explanation for this defect is that they quantify the presence of endosomes in this assay largely by EEA1 puncta. Since EEA1 is a Rab5 effector, it is possible that in the Rab5 depleted cells there is still a population of early endosomes, but that these are not detected by EEA1 staining.

Together this study presents a convincing picture for the role of Rab5 in endosomal dynamics, and highlight the important role of Rab5 in both endocytic pathways and in the establishment of polarity in vivo.

Citation

Zeigerer A, Gilleron J, Bogorad RL, Marsico G, Nonaka H, Seifert S, Epstein-Barash H, Kuchimanchi S, Peng CG, Ruda VM, Del Conte-Zerial P, Hengstler JG, Kalaidzidis Y, Koteliansky V, & Zerial M (2012). Rab5 is necessary for the biogenesis of the endolysosomal system in vivo. Nature, 485 (7399), 465-70 PMID: 22622570

Disclosure: I have received constructs and yeast strains from the Zerial laboratory in the past.



Rab5 Knockdown In Vivo by Dave Bridges is licensed under a Creative Commons Attribution 3.0 Unported License.

PI(3)P and Exocytosis

The classic opinion regarding PI(3)P’s role in intracellular trafficking is that it is synthesized and functions primarily on early endosomes. Several recent publications have highlighted a potential second role for this lipid in exocytosis. Extending previous work by this group and others on the positive role of PI3K-C2a in neurosecretory pathways (1,2) and GLUT4 exoctyosis (3,4) this paper from Tania Maffucci's group interrogates the role of this lipid kinase in insulin secretion in INS1 cells. Combined these results suggest a general role for PI3K-C2a and its product, PI(3)P in exocytotic events.

This group has been investigating the role of the class II PI3K isoforms in exocytosis in a variety of systems. In this paper they investigate the role of PI3K-C2a in insulin secretion using INS1 rat insulinoma cells as a model. Stable knockdown cells did not show any defects in proliferation, calcium signaling, intracellular insulin levels or the expression levels and sub-cellular localization of exocytotic proteins. The PI3K-C2a knockdown cells did however show significant defects in insulin secretion, stimulated by either a secretagogue cocktail or potassium chloride.

Mechanistically, the authors show that there is no defect in insulin granules proximal to the plasma membrane at the resting state. They do detect a decrease in the amount of SNAP25 hydrolysis induced by the secretagogue cocktail. SNAP25 hydrolysis has been proposed to be an important step in the fusion of exocytic vesicles (5). This proteolytic event has not been established as a major mechanism in exocytosis, and it may only correlate with defects in PI3K-C2a signaling. However if reduced SNAP25 proteolysis is the mechanistic defect resulting from PI3K-C2a knockdown, then this suggests a role in protease regulation by PI(3)P or another PI(3)P-derived molecule as a key part of the general exocytotic machinery. It is also possible that there is another, as of yet unstudied role of PI3K-C2a and PI(3)P in exocytosis.



Dominguez, V., Raimondi, C., Somanath, S., Bugliani, M., Loder, M., Edling, C., Divecha, N., da Silva-Xavier, G., Marselli, L., Persaud, S., Turner, M., Rutter, G., Marchetti, P., Falasca, M., & Maffucci, T. (2010). Class II Phosphoinositide 3-Kinase Regulates Exocytosis of Insulin Granules in Pancreatic Cells Journal of Biological Chemistry, 286 (6), 4216-4225 DOI: 10.1074/jbc.M110.200295

References

(1) Meunier, Frederic, Shona Osborne, Gerald Hammond, Frank Cooke, Peter Parker, Jan Domin, and Giampietro Schiavo. “Phosphatidylinositol 3-Kinase C2{alpha} Is Essential for ATP-dependent Priming of Neurosecretory Granule Exocytosis.” Molecular Biology of the Cell 16, no. 10 (2005): 4841-4851. PubMed, DOI.

(2) Wen, Peter J, Shona L Osborne, Isabel C Morrow, Robert G Parton, Jan Domin, and Frederic A Meunier. “Ca2+-regulated pool of phosphatidylinositol-3-phosphate produced by phosphatidylinositol 3-kinase C2alpha on neurosecretory vesicles.” Molecular biology of the cell 19, no. 12 (December 2008): 5593-603. PMC, PubMed, DOI.

(3) Maffucci, Tania, Anna Brancaccio, Enza Piccolo, Robert C Stein, and Marco Falasca. “Insulin induces phosphatidylinositol-3-phosphate formation through TC10 activation.” The EMBO journal 22, no. 16 (August 15, 2003): 4178-89. PubMed, DOI.

(4) Falasca, Marco, William E Hughes, Veronica Dominguez, Gianluca Sala, Florentia Fostira, Michelle Q Fang, Rosanna Cazzolli, Peter R Shepherd, David E James, and Tania Maffucci. “The role of phosphoinositide 3-kinase C2alpha in insulin signaling.” The Journal of biological chemistry 282, no. 38 (September 21, 2007): 28226-36. PubMed, DOI.