title: Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO.
abstract: Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking podosome elimination. Concomitantly, treatments inducing podosome formation increased the level of guanosine triphosphate (GTP)-bound ARF1. ARNO was found to colocalize with the adhesive rings of podosomes, whereas ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly.
authors.affiliation: Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore.|Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore.|Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, England, UK.
authors.forename: Nisha Bte Mohd
authors.identifier: http://orcid.org/0000-0002-4434-0504
authors.initials: NB
authors.lastname: Rafiq
authors.affiliation: Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore.|Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore.
authors.forename: Zi Zhao
authors.initials: ZZ
authors.lastname: Lieu
authors.affiliation: Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore.
authors.forename: Tingting
authors.initials: T
authors.lastname: Jiang
authors.affiliation: Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore.
authors.forename: Cheng-Han
authors.identifier: http://orcid.org/0000-0002-8821-8877
authors.initials: CH
authors.lastname: Yu
authors.affiliation: Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore.|Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore.
authors.forename: Paul
authors.identifier: http://orcid.org/0000-0002-8399-3276
authors.initials: P
authors.lastname: Matsudaira
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, England, UK alexander.bershadsky@weizmann.ac.il gareth.jones@kcl.ac.uk.
authors.forename: Gareth E
authors.identifier: http://orcid.org/0000-0001-5879-3048
authors.initials: GE
authors.lastname: Jones
authors.affiliation: Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore alexander.bershadsky@weizmann.ac.il gareth.jones@kcl.ac.uk.|Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Alexander D
authors.identifier: http://orcid.org/0000-0002-9571-7375
authors.initials: AD
authors.lastname: Bershadsky
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008841:Actin Cytoskeleton / Q000201:enzymology; D000199:Actins / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D024507:Nonmuscle Myosin Type IIA / Q000378:metabolism; D000069261:Podosomes / Q000187:drug effects / Q000201:enzymology*; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D054460:rho-Associated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D000199:Actins; D004791:Enzyme Inhibitors; D020690:GTPase-Activating Proteins; D011993:Recombinant Fusion Proteins; C102474:cytohesin-2; C066737:RHOA protein, human; D006160:Guanosine Triphosphate; D054460:rho-Associated Kinases; D024507:Nonmuscle Myosin Type IIA; D020823:ADP-Ribosylation Factor 1; D020742:rhoA GTP-Binding Protein