Abstract
Numb is an endocytic protein that plays crucial roles in diverse cellular processes such as asymmetric cell division, cell migration and differentiation. However, the molecular mechanism by which Numb regulates endocytic trafficking is poorly understood. Here, we demonstrate that Numb is a docking regulator for homotypic fusion of early endosomes (EEs). Numb depletion causes clustered but unfused EEs, which can be rescued by overexpressing cytosolic Numb 65 and Numb 71 but not plasma membrane-attached Numb 66 or Numb 72. Time-lapse analysis reveals that paired vesicles tend to tether but not fuse with each other in the absence of Numb. We further show that Numb binds to another docking regulator, Mon1b, and is required for the recruitment of cytosolic Mon1b to the EE membrane. Consistent with this, deletion of Mon1b causes similar defects in EE fusion. Our study thus identifies a novel mechanism by which Numb regulates endocytic sorting by mediating EE fusion.
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References
Uemura T, Shepherd S, Ackerman L, Jan LY, Jan YN . Numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos. Cell 1989; 58:349–360.
Rhyu MS, Jan LY, Jan YN . Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 1994; 76:477–491.
Knoblich JA, Jan LY, Jan YN . Asymmetric segregation of Numb and Prospero during cell division. Nature 1995; 377:624–627.
Li HS, Wang D, Shen Q, et al. Inactivation of Numb and Numblike in embryonic dorsal forebrain impairs neurogenesis and disrupts cortical morphogenesis. Neuron 2003; 40:1105–1118.
Petersen PH, Zou K, Hwang JK, Jan YN, Zhong W . Progenitor cell maintenance requires numb and numblike during mouse neurogenesis. Nature 2002; 419:929–934.
Conboy IM, Rando TA . The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis. Dev Cell 2002; 3:397–409.
Pece S, Serresi M, Santolini E, et al. Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis. J Cell Biol 2004; 167:215–221.
Colaluca IN, Tosoni D, Nuciforo P, et al. NUMB controls p53 tumour suppressor activity. Nature 2008; 451:76–80.
Rasin MR, Gazula VR, Breunig JJ, et al. Numb and Numbl are required for maintenance of cadherin-based adhesion and polarity of neural progenitors. Nat Neurosci 2007; 10:819–827.
Cicalese A, Bonizzi G, Pasi CE, et al. The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells. Cell 2009; 138:1083–1095.
Kyriazis GA, Wei Z, Vandermey M, et al. Numb endocytic adapter proteins regulate the transport and processing of the amyloid precursor protein in an isoform-dependent manner: implications for Alzheimer disease pathogenesis. J Biol Chem 2008; 283:25492–25502.
Chan SL, Pedersen WA, Zhu H, Mattson MP . Numb modifies neuronal vulnerability to amyloid beta-peptide in an isoform-specific manner by a mechanism involving altered calcium homeostasis: implications for neuronal death in Alzheimer's disease. Neuromolecular Med 2002; 1:55–67.
Roncarati R, Sestan N, Scheinfeld MH, et al. The gamma-secretase-generated intracellular domain of beta-amyloid precursor protein binds Numb and inhibits Notch signaling. Proc Natl Acad Sci USA 2002; 99:7102–7107.
Ezratty EJ, Bertaux C, Marcantonio EE, Gundersen GG . Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells. J Cell Biol 2009; 187:733–747.
Dho SE, French MB, Woods SA, McGlade CJ . Characterization of four mammalian numb protein isoforms. Identification of cytoplasmic and membrane-associated variants of the phosphotyrosine binding domain. J Biol Chem 1999; 274:33097–33104.
Verdi JM, Bashirullah A, Goldhawk DE, et al. Distinct human NUMB isoforms regulate differentiation vs. proliferation in the neuronal lineage. Proc Natl Acad Sci USA 1999; 96:10472–10476.
Santolini E, Puri C, Salcini AE, et al. Numb is an endocytic protein. J Cell Biol 2000; 151:1345–1352.
Nishimura T, Kaibuchi K . Numb controls integrin endocytosis for directional cell migration with aPKC and PAR-3. Dev Cell 2007; 13:15–28.
McGill MA, McGlade CJ . Mammalian numb proteins promote Notch1 receptor ubiquitination and degradation of the Notch1 intracellular domain. J Biol Chem 2003; 278:23196–23203.
Nishimura T, Fukata Y, Kato K, et al. CRMP-2 regulates polarized Numb-mediated endocytosis for axon growth. Nat Cell Biol 2003; 5:819–826.
Sorensen EB, Conner SD . AAK1 regulates Numb function at an early step in clathrin-mediated endocytosis. Traffic 2008; 9:1791–1800.
Spana EP, Doe CQ . Numb antagonizes Notch signaling to specify sibling neuron cell fates. Neuron 1996; 17:21–26.
McGill MA, Dho SE, Weinmaster G, McGlade CJ . Numb regulates post-endocytic trafficking and degradation of Notch1. J Biol Chem 2009; 284:26427–26438.
Cotton M, Benhra N, Le Borgne R . Numb inhibits the recycling of sanpodo in Drosophila sensory organ precursor. Curr Biol 2013; 23:581–587.
Scita G, Di Fiore PP . The endocytic matrix. Nature 2010; 463:464–473.
Berdnik D, Torok T, Gonzalez-Gaitan M, Knoblich JA . The endocytic protein alpha-Adaptin is required for numb-mediated asymmetric cell division in Drosophila. Dev Cell 2002; 3:221–231.
Salcini AE, Confalonieri S, Doria M, et al. Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module. Genes Dev 1997; 11:2239–2249.
Smith CA, Dho SE, Donaldson J, Tepass U, McGlade CJ . The cell fate determinant numb interacts with EHD/Rme-1 family proteins and has a role in endocytic recycling. Mol Biol Cell 2004; 15:3698–3708.
Tokumitsu H, Hatano N, Yokokura S, Sueyoshi Y, Nozaki N, Kobayashi R . Phosphorylation of Numb regulates its interaction with the clathrin-associated adaptor AP-2. FEBS Lett 2006; 580:5797–5801.
Bucci C, Parton RG, Mather IH, et al. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell 1992; 70:715–728.
Wickner W . Membrane fusion: five lipids, four SNAREs, three chaperones, two nucleotides, and a Rab, all dancing in a ring on yeast vacuoles. Annu Rev Cell Dev Biol 2010; 26:115–136.
McBride HM, Rybin V, Murphy C, Giner A, Teasdale R, Zerial M . Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13. Cell 1999; 98:377–386.
Christoforidis S, McBride HM, Burgoyne RD, Zerial M . The Rab5 effector EEA1 is a core component of endosome docking. Nature 1999; 397:621–625.
Rybin V, Ullrich O, Rubino M, et al. GTPase activity of Rab5 acts as a timer for endocytic membrane fusion. Nature 1996; 383:266–269.
Stenmark H, Vitale G, Ullrich O, Zerial M . Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell 1995; 83:423–432.
Wang CW, Stromhaug PE, Kauffman EJ, Weisman LS, Klionsky DJ . Yeast homotypic vacuole fusion requires the Ccz1-Mon1 complex during the tethering/docking stage. J Cell Biol 2003; 163:973–985.
Zeigerer A, Gilleron J, Bogorad RL, et al. Rab5 is necessary for the biogenesis of the endolysosomal system in vivo. Nature 2012; 485:465–470.
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T . Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411:494–498.
Gauthier NC, Monzo P, Gonzalez T, et al. Early endosomes associated with dynamic F-actin structures are required for late trafficking of H. pylori VacA toxin. J Cell Biol 2007; 177:343–354.
Morel E, Parton RG, Gruenberg J . Annexin A2-dependent polymerization of actin mediates endosome biogenesis. Dev Cell 2009; 16:445–457.
Girao H, Geli MI, Idrissi FZ . Actin in the endocytic pathway: from yeast to mammals. FEBS Lett 2008; 582:2112–2119.
Hirota Y, Kuronita T, Fujita H, Tanaka Y . A role for Rab5 activity in the biogenesis of endosomal and lysosomal compartments. Biochem Biophys Res Commun 2007; 364:40–47.
Galperin E, Sorkin A . Visualization of Rab5 activity in living cells by FRET microscopy and influence of plasma-membrane-targeted Rab5 on clathrin-dependent endocytosis. J Cell Sci 2003; 116:4799–4810.
Lakadamyali M, Rust MJ, Zhuang X . Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes. Cell 2006; 124:997–1009.
Barysch SV, Jahn R, Rizzoli SO . A fluorescence-based in vitro assay for investigating early endosome dynamics. Nat Protoc 2010; 5:1127–1137.
Poteryaev D, Fares H, Bowerman B, Spang A . Caenorhabditis elegans SAND-1 is essential for RAB-7 function in endosomal traffic. EMBO J 2007; 26:301–312.
Poteryaev D, Datta S, Ackema K, Zerial M, Spang A . Identification of the switch in early-to-late endosome transition. Cell 2010; 141:497–508.
Barroso M, Sztul ES . Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome. J Cell Biol 1994; 124:83–100.
Dunn KW, McGraw TE, Maxfield FR . Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome. J Cell Biol 1989; 109:3303–3314.
Barysch SV, Aggarwal S, Jahn R, Rizzoli SO . Sorting in early endosomes reveals connections to docking- and fusion-associated factors. Proc Natl Acad Sci USA 2009; 106:9697–9702.
Hutterer A, Knoblich JA . Numb and alpha-Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs. EMBO Rep 2005; 6:836–842.
Benhra N, Lallet S, Cotton M, Le Bras S, Dussert A, Le Borgne R . AP-1 controls the trafficking of Notch and Sanpodo toward E-cadherin junctions in sensory organ precursors. Curr Biol 2011; 21:87–95.
Sato TK, Rehling P, Peterson MR, Emr SD . Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion. Mol Cell 2000; 6:661–671.
de Araujo ME, Huber LA, Stasyk T . Isolation of endocitic organelles by density gradient centrifugation. Methods Mol Biol 2008; 424:317–331.
Osborne A, Flett A, Smythe E . Endocytosis assays in intact and permeabilized cells. Curr Protoc Cell Biol 2005; Chapter 11:Unit 11.18.
Acknowledgements
We thank Fudi Wang for Mon1A antibody, Yun Wang and Yuxing Wang for technical support. This work was supported by the Ministry of Science & Technology, China (2014CB964602, 2012CB966802, 2011CB965100, 2010CB945600, 2009CB941402, 2007CB947202, 2013ZX09509104 and 2009R0002), the National Natural Science Foundation of China (31301125, 31071283, 30771102, 81502537, 81370457 and 81271498), China Postdoctoral Science Foundation (2015M572383), Shenzhen Peacock Plan (KQCX20130628112914292), Shenzhen Science and Technology Program (JCYJ20150401145529014 and JCYJ20150630114942300), Shenzhen Key Laboratory for Molecular Biology of Neural Development (ZDSY20120617112838879), SIAT Innovation Program for Excellent Young Researchers (201404 and 201413), and faculty development support (Shenzhen Institutes of Advanced Technology-CAS & Tongji University Shanghai East Hospital).
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Supplementary information
Supplementary information Figure S1
Numb knockdown induce EE clustering in HEK293T, COS7, and mice embryonic fibroblast (MEF) cells. (PDF 455 kb)
Supplementary information Figure S2
Knockdown of Numb by using another independent shRNA similarly impairs EE fusion. (PDF 151 kb)
Supplementary information Figure S3
Knockdown of Numblike has no effect on EE fusion. (PDF 157 kb)
Supplementary information Figure S4
Numb-mediated EE fusion is Rab5 activity dependent. (PDF 556 kb)
Supplementary information Figure S5
Numb Knockdown causes EE docking defects. (PDF 424 kb)
Supplementary information Figure S6
The short PTB domain is required for Numb to localize to EEs. (PDF 673 kb)
Supplementary information Figure S7
Numb interacts with Mon1 proteins in vivo. (PDF 439 kb)
Supplementary information Figure S8
Numb knockdown impairs vesicle localization of Mon1 proteins. (PDF 275 kb)
Supplementary information Figure S9
Mon1a knockdown has no effect on EE fusion. (PDF 167 kb)
Supplementary information Figure S10
The effects of Numb or Mon1b knockdown on late endosomes and recycling endosomes. (PDF 113 kb)
Supplementary information Movie S1
A representative movie showing the dynamics of Dil-LDL-containing vesicles, related to Figure S5A. (MOV 3184 kb)
Section 1: a representative movie showing a pair of vesicles that are moving randomly, related to the upper panels of Figure S5A.
Section 2: a representative movie showing a pair of vesicles that are undergoing tethering, related to the middle panels of Figure S5A.
Section 3: a representative movie showing a pair of vesicles that dynamically tether, dock, and finally fuse with each other, related to the bottom panels of Figure S5A.
Supplementary information Movie S2
A representative movie showing the dynamics of Dil-LDL-containing vesicles in control cells, Numb-KD cells, and Numblike-KD MCF7A cells, related to Figure 2A. (MOV 2216 kb)
Section 1: a representative movie showing the dynamic fusion process of Dil-LDL-containing endocytic vesicles in control MCF7A cells, related to the upper panels of Figure 2A.
Section 2: a representative movie showing that Dil-LDL-containing vesicles keep long-time tethering and do not undergo further docking and fusion in Numb-knockdown MCF7A cells, related to the middle panels of Figure 2A.
Section 3: a representative movie showing that Numblike knockdown in MCF7A cells does not affect the dynamic fusion process of Dil-LDL-containing endocytic vesicles, related to the bottom panels of Figure 2A.
Supplementary information Movie S3
A representative movie showing the dynamics of Transferrin-555 containing vesicles in control, Numb-KD, and Numblike KD MCF7A cells, related to Figure S5B. (MOV 2701 kb)
Section 1: a representative movie showing the dynamic fusion process of transferrin-containing endocytic vesicles in control MCF7A cells, related to the upper panels of Figure S5B.
Section 2: a representative movie showing that Transferrin-555-containing vesicles keep long-time tethering and do not undergo further docking and fusion in Numb-knockdown MCF7A cells, related to the middle panels of Figure S5B.
Section 3: a representative movie showing the dynamic fusion process of transferrin-containing endocytic vesicles in Numblike knockdown MCF7A cells is not affected, related to the bottom panels of Figure S5B.
Supplementary information Movie S4
A representative movie showing the dynamic fusion process of Rab5-RFP-positive vesicles in control cells and Numb-KD MCF7A cells, related to Figure 2D. (MOV 2364 kb)
Section 1: a representative movie showing the dynamic fusion process of Rab5-RFP-labeled endocytic vesicles in control MCF7A cells, related to the upper panels of Figure 2D.
Section 2: a representative movie showing that the Rab5-RFP-positive endocytic vesicles took a much longer time from tethering to final fusion in Numb-knockdown MCF7A cells, related to the bottom panels of Figure 2D.
Supplementary information Movie S5
A representative movie showing the dynamics of Dil-LDL-containing vesicles in control cells, Mon1a-KD, and Mon1b-KD MCF7A cells, related to Figure 6H. (MOV 1760 kb)
Section 1: a representative movie showing the dynamic fusion process of Dil-LDL-containing endocytic vesicles in control MCF7A cells, related to the upper panels of Figure 6H.
Section 2: a representative movie showing that the dynamic fusion process of Dil-LDL-containing endocytic vesicles is not affected by Mon1a knockdown, related to the middle panels of Figure 6H.
Section 3: a representative movie showing that Dil-LDL-containing vesicles keep long-time tethering and do not undergo further into docking and fusion in Mon1b-knockdown MCF7A cells, related to the bottom panels of Figure 6H.
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Shao, X., Liu, Y., Yu, Q. et al. Numb regulates vesicular docking for homotypic fusion of early endosomes via membrane recruitment of Mon1b. Cell Res 26, 593–612 (2016). https://doi.org/10.1038/cr.2016.34
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DOI: https://doi.org/10.1038/cr.2016.34
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