PDZRhoGEF and myosin II localize RhoA activity to the back of polarizing neutrophil-like cells.
Chemoattractants such as formyl-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent pathways that promote formation of a protrusive front and contracting back and sides. RhoA, a Rho GTPase, stimulates assembly of actomyosin contractile complexes at the sides and back. We show here, in differentiated HL60 cells, that PDZRhoGEF (PRG), a guanine nucleotide exchange factor (GEF) for RhoA, mediates RhoA-dependent responses and determines their spatial distribution. As with RNAi knock-down of PRG, a GEF-deleted PRG mutant blocks fMLP-dependent RhoA activation and causes neutrophils to exhibit multiple fronts and long tails. Similarly, inhibition of RhoA, a Rho-dependent protein kinase (ROCK), or myosin II produces the same morphologies. PRG inhibition reduces or mislocalizes monophosphorylated myosin light chains in fMLP-stimulated cells, and myosin II ATPase inhibition reciprocally disrupts normal localization of PRG. We propose a cooperative reinforcing mechanism at the back of cells, in which PRG, RhoA, ROCK, myosin II, and actomyosin spatially cooperate to consolidate attractant-induced contractility and ensure robust cell polarity.
Wong
Kit
K
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.
Van Keymeulen
Alexandra
A
Bourne
Henry R
HR
D016764:Cell Polarity; D002630:Chemotactic Factors / Q000378:metabolism; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000032:analysis / Q000037:antagonists & inhibitors / Q000502:physiology*; D018922:HL-60 Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D024462:Myosin Type II / Q000378:metabolism / Q000502:physiology*; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D064067:Rho Guanine Nucleotide Exchange Factors; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
C577234:ARHGEF11 protein, human; D002630:Chemotactic Factors; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; C066737:RHOA protein, human; D024462:Myosin Type II; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020742:rhoA GTP-Binding Protein