Clathrin-mediated endocytosis and recycling of autocrine motility factor receptor to fibronectin fibrils is a limiting factor for NIH-3T3 cell motility.
Autocrine motility factor receptor (AMF-R) is internalized via a clathrin-independent pathway to smooth endoplasmic reticulum tubules. This endocytic pathway is shown here to be inhibited by methyl-(beta)-cyclodextrin (m(beta)CD) implicating caveolae or caveolae-like structures in AMF internalization to smooth ER. AMF-R is also internalized via a clathrin-dependent pathway to a transferrin receptor-negative, LAMP-1/lgpA-negative endocytic compartment identified by electron microscopy as a multivesicular body (MVB). Endocytosed AMF recycles to cell surface fibrillar structures which colocalize with fibronectin; AMF-R recycling is inhibited at 20 degrees C, which blocks endocytosis past the early endosome, but not by m(beta)CD demonstrating that AMF-R recycling to fibronectin fibrils is mediated by clathrin-dependent endocytosis to MVBs. Microtubule disruption with nocodazole did not affect delivery of bAMF to cell surface fibrils indicating that recycling bAMF traverses the MVB but not a later endocytic compartment. Plating NIH-3T3 cells on an AMF coated substrate did not specifically affect cell adhesion but prevented bAMF delivery to cell surface fibronectin fibrils and reduced cell motility. AMF-R internalization and recycling via the clathrin-mediated pathway are therefore rate-limiting for cell motility. This recycling pathway to the site of deposition of fibronectin may be implicated in the de novo formation of cellular attachments or the remodeling of the extracellular matrix during cell movement.
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Département de pathologie et biologie cellulaire, Université de Montréal, Montréal, Québec, Canada H3C 3J7.
Benlimame
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Lagana
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Raz
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Nabi
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D016475:3T3 Cells; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D005353:Fibronectins / Q000378:metabolism*; D005956:Glucose-6-Phosphate Isomerase / Q000378:metabolism; D006801:Humans; D051379:Mice; D060285:Receptors, Autocrine Motility Factor; D018121:Receptors, Cytokine / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D002966:Clathrin; D005353:Fibronectins; D018121:Receptors, Cytokine; C555000:AMFR protein, human; C555002:Amfr protein, mouse; D060285:Receptors, Autocrine Motility Factor; D044767:Ubiquitin-Protein Ligases; D005956:Glucose-6-Phosphate Isomerase