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Rab5 is necessary for the biogenesis of the endolysosomal system in vivo

Abstract

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.

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Figure 1: Mathematical model of endosome number as function of Rab5 levels.
Figure 2: Time course of Rab5 mRNA depletion and analysis of protein expression in mouse liver in vivo.
Figure 3: Loss of EEA1 structures upon Rab5KD in vivo.
Figure 4: Loss of early endosomes, MVBs and lysosomes upon Rab5KD.
Figure 5: Block of LDL endocytosis upon Rab5KD in primary hepatocytes in vitro.
Figure 6: Rab5KD causes mis-sorting of apical proteins in hepatocytes in vivo.

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Acknowledgements

We acknowledge K. Simons, E. Knust, C. Fetzer, T. Galvez, G. O’Sullivan and M. P. McShane for discussions and comments on the manuscript. We thank W. John and A. Muench-Wuttke from the Biomedical Services Facility for mouse care and injections. We acknowledge J. Peychl for the management of the Light Microscopy Facility and K. Manygoats as well as J.-M. Verbavatz for their support. We thank B. Bettencourt and J. Hettinger for siRNA design and serum biochemistry assays, respectively. This work was financially supported by the Virtual Liver initiative (http://www.virtual-liver.de), funded by the German Federal Ministry of Research and Education (BMBF), the Max Planck Society (MPG) and the DFG. A.Z. was supported by a grant from Marie Curie Action, Intra-European Fellowship (fp7-people-ief-2008) and J.G. from an EMBO long-term fellowship.

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M.Z. and V.K. conceived the project and M.Z. directed it. A.Z. designed and directed the animal injection experiments, and performed the stainings and imaging of liver sections. J.G. and A.Z. co-developed the staining procedures for the tissue sections. J.G.H. helped A.Z. to establish the hepatocyte isolation technique. A.Z. established the primary culture, developed the endocytosis assays, staining protocols and the knockdown technique with LNPs in primary hepatocytes. J.G. performed the electron microscopy analysis and quantifications, and the sectioning of liver tissue. R.L.B. selected siRNAs and validated their efficacy and specificity in vitro and in vivo, and designed, performed and analysed some in vivo experiments. H.E.-B. prepared the siRNAs into lipidoid-based formulations and analysed them. S.K. and C.G.P. performed the synthesis and analysis of siRNAs. V.M.R. designed, performed and analysed the 5′RACE assay. H.N. performed the RT–PCR in primary hepatocytes. S.S. under the supervision of A.Z. and J.G. performed the western blot analysis and performed the hepatocyte isolation and primary culturing under the supervision of A.Z.; G.M. under the supervision of Y.K. adapted the QMPIA for primary hepatocytes and liver tissue and performed the image analysis. Y.K. and P.dC.-Z. developed the mathematical model. M.Z., Y.K., A.Z. and J.G. wrote the manuscript, R.L.B. and V.K. participated in the editing.

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Correspondence to Marino Zerial.

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The authors declare no competing financial interests.

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Zeigerer, A., Gilleron, J., Bogorad, R. et al. Rab5 is necessary for the biogenesis of the endolysosomal system in vivo. Nature 485, 465–470 (2012). https://doi.org/10.1038/nature11133

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