Linked regulation of motility and integrin function in activated migrating neutrophils revealed by interference in remodelling of the cytoskeleton.
Neutrophils migrate rapidly by co-ordinating regulation of their beta2-integrin adhesion with turnover of filamentous F-actin. The seven-protein Arp2/3 complex regulates actin polymerisation upon activation by proteins of the WASP-family. To investigate links between actin polymerisation, adhesion, and migration, we used a novel osmotic-shock method to load neutrophils with peptides: (1). WASP-WA and Scar-WA (which incorporate the actin- and Arp2/3-binding regions of WASP and Scar1), to compete with endogenous WASP-family members; (2). proline rich motifs (PRM) from the ActA protein of L. monocytogenes or from vinculin, which bind vasodilator-stimulated phosphoprotein (VASP), a regulator of cytoskeleton assembly. In a flow system, rolling-adherent neutrophils were stimulated with formyl tri-peptide. This caused rapid immobilisation, followed by migration with increasing velocity, supported by activated beta2-integrin CD11b/CD18. Loading ActA PRM (but not vinculin PRM) caused concentration-dependent reduction in migration velocity. At the highest concentration, unstimulated neutrophils had elevated F-actin and were rigid, but could not change their F-actin content or shape upon stimulation. Scar-WA also caused marked reduction in migration rate, but WASP-WA had a lesser effect. Scar-WA did not modify activation-dependent formation of F-actin or change in shape. However, a reduction in rate of downregulation of integrin adhesion appeared to contribute to impaired migration. These studies show that interference in cytoskeletal reorganisation that follows activation in neutrophils, can impair regulation of integrin function as well as motility. They also suggest a role of the Arp2/3 complex and WASP-family in co-ordinating actin polymerisation and integrin function in migrating neutrophils.
Anderson
Stephen I
SI
Department of Physiology, The Medical School, The University of Birmingham, Birmingham B15 2TT, UK.
Behrendt
Barbara
B
Machesky
Laura M
LM
Insall
Robert H
RH
Nash
Gerard B
GB
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000378:metabolism; D001426:Bacterial Proteins / Q000494:pharmacology; D039481:CD11b Antigen / Q000378:metabolism; D018821:CD18 Antigens / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D048429:Cell Size / Q000187:drug effects; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000494:pharmacology; D008840:Microfilament Proteins / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology* / Q000502:physiology; D009997:Osmotic Pressure; D010446:Peptide Fragments / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D011506:Proteins / Q000494:pharmacology; D016596:Vinculin / Q000494:pharmacology; D051316:Wiskott-Aldrich Syndrome Protein; D051300:Wiskott-Aldrich Syndrome Protein Family; D000098662:Vasodilator-Stimulated Phosphoprotein
C495926:ACTR2 protein, human; C495928:ACTR3 protein, human; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D001426:Bacterial Proteins; D039481:CD11b Antigen; D018821:CD18 Antigens; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011108:Polymers; D011506:Proteins; C495767:WAS protein, human; D051316:Wiskott-Aldrich Syndrome Protein; D051300:Wiskott-Aldrich Syndrome Protein Family; D000098662:Vasodilator-Stimulated Phosphoprotein; D016596:Vinculin; C072980:actA protein, Listeria monocytogenes; D009240:N-Formylmethionine Leucyl-Phenylalanine; C042932:formylmethionyl-leucyl-phenylalanine methyl ester