title: PAK4 and alphaPIX determine podosome size and number in macrophages through localized actin regulation.
abstract: Podosomes are actin-rich adhesion structures typical for monocytic cells and are implicated in migration and invasion. Major modes of podosome regulation include RhoGTPase signaling and actin regulatory pathways. However, it is not clearly understood how these signals induce highly localized changes in podosome formation and dynamics. Here, we show that the RhoGTPase effector PAK4, a member of the p21 associated kinase family, and its regulator alphaPIX (PAK-interacting exchange factor), are central to podosome formation in primary human macrophages. Immunofluorescence, biochemical and microarray data indicate that PAK4 acts as physiological regulator of podosomes in this system. Accordingly, transfection of a specific shRNA, as well as expression of PAK4 truncation mutants, resulted in reduced numbers of podosomes per cell. Moreover, expression of kinase active or inactive PAK4 mutants enhanced or reduced the size of individual podosomes, respectively, indicating a modulatory influence of PAK4 kinase activity on podosome size. Similar to the results gained with PAK4, cellular/overexpressed PIX was shown to be closely associated with podosomes. Moreover, both overexpression of alphaPIX wt and a mutant lacking the SH3 domain led to coalescence of podosomes. In sum, we propose that PAK4 and alphaPIX can induce highly localized changes in actin dynamics and thereby regulate size and number of podosomes in primary human macrophages.
authors.affiliation: Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Pettenkoferstr, München, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Gringel
authors.forename: Daniel
authors.initials: D
authors.lastname: Walz
authors.forename: Georg
authors.initials: G
authors.lastname: Rosenberger
authors.forename: Audrey
authors.initials: A
authors.lastname: Minden
authors.forename: Kerstin
authors.initials: K
authors.lastname: Kutsche
authors.forename: Petra
authors.initials: P
authors.lastname: Kopp
authors.forename: Stefan
authors.initials: S
authors.lastname: Linder
mesh_terms: D000199:Actins / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D002452:Cell Count; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D048429:Cell Size; D022081:Cell Surface Extensions / Q000378:metabolism*; D002478:Cells, Cultured; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000166:cytology*; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012313:RNA / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D014162:Transfection; D054462:p21-Activated Kinases
chemical_list: D000199:Actins; D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D050505:Mutant Proteins; D010455:Peptides; D064067:Rho Guanine Nucleotide Exchange Factors; D012313:RNA; C116367:PAK4 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases