Abstract
The small GTPase Rab5 controls the fusogenic properties of early endosomes through GTP-dependent recruitment and activation of effector proteins. Expression of a GTPase-defective mutant, Rab5(Q79L), is known to cause formation of enlarged early endosomes. The ability of Rab5-GTP to recruit multiple effectors raises the question whether the Rab5(Q79L)-induced giant endosomes simply represent enlarged early endosomes or whether they have a more complex phenotype. In this report, we have addressed this issue by generating a HEp2 cell line with inducible expression of Rab5(Q79L) and performing ultrastructural analysis of Rab5(Q79L)-induced endosomes. We find that Rab5(Q79L) not only induces formation of enlarged early endosomes but also causes enlargement of later endocytic profiles. Most strikingly, Rab5(Q79L) causes formation of enlarged multivesicular endosomes with a large number of intraluminal vesicles, and endosomes that contain both early and late endocytic markers are frequently observed. In addition, we observe defects in the sorting of the EGF receptor and the transferrin receptor through this compartment.
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Acknowledgments
We thank Ban-Hock Toh, Jean Gruenberg, Marino Zerial and the Developmental Studies Hybridoma Bank (University of Iowa, USA) for kindly providing antibodies. C.S.W. is a predoctoral fellow of the Norwegian Cancer Society. N.M.P. is a postdoctoral fellow of Regional Health Enterprise South-East. This work was also supported by Functional Genomics (FUGE) grants from the Research Council of Norway to O.B., H.S. and A.B.
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An erratum to this article can be found at http://dx.doi.org/10.1007/s00418-009-0660-7
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Supplementary Figure 1. HEp2 cells stably expressing the myc-Rab5 QL (called HEp2-Rab5 QL) were grown on coverslips. Rab5 QL expression was induced upon CdCl2-treatment over night. Cells were permeabilized, fixed (3% PFA, 15 min) and stained with anti-myc, anti-EEA1 and anti-CD63 (A), anti-LBPA (B), or anti-Lamp1 (C). Expression of myc-Rab5 QL induced formation of enlarged endosomes recognized by anti-myc. Further, extensive colocalization between myc and EEA1 was observed, implying that both anti-myc as well as anti-EEA1 can be used to visualize the enlarged endosomes. Late endosomal markers such as CD63 (A), LBPA (B) and Lamp1 (C) also associated the enlarged endosomes indicating their heterogenic nature; mixture of early and late endosomes. Scale bar = 5 µm (PDF 1,296 kb)
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Wegener, C.S., Malerød, L., Pedersen, N.M. et al. Ultrastructural characterization of giant endosomes induced by GTPase-deficient Rab5. Histochem Cell Biol 133, 41–55 (2010). https://doi.org/10.1007/s00418-009-0643-8
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DOI: https://doi.org/10.1007/s00418-009-0643-8