Role of tropomyosin, alpha-actinin, and actin binding protein 280 in stabilizing Triton insoluble F-actin in basal and chemotactic factor activated neutrophils.
F-actin is a major component of the neutrophil (PMN) cytoskeleton. In basal PMNs, F-actin exists in two structurally and functionally distinct pools: Triton insoluble F-actin (TIF)--cold insensitive, not depolymerizable by dilution, and distributed in pseudopods and submembranous locations; and Triton soluble F-actin (TSF)--unstable in cold, diffusely distributed, and gelsolin enriched. The element(s) conferring these unique properties to the Triton insoluble F-actin pool are unknown, but logically include distinct actin regulatory proteins. To study the morphologic and functional determinants of the Triton insoluble F-actin pool, the distribution and quantity of three candidate regulatory proteins, alpha-actinin, tropomyosin (TM), and actin binding protein (ABP-280), were compared in F-actin (Triton insoluble and Triton soluble) and G-actin pools isolated from basal and chemotactic factor activated human PMNs in suspension, using immunoblots and ionic extraction. F-actin content was measured by NBDphallacidin binding and gel scans. The results show that: (1) alpha-actinin, actin binding protein 280, and tropomyosin are localized to TIF and excluded from TSF; (2) TM, alpha-actinin, and ABP 280 are required to stabilize fractions of Triton insoluble F-actin in PMNs; and (3) chemotactic factor activation results in release of a fraction of TM from the Triton insoluble F-actin pool in temporal association with F-actin polymerization in the Triton insoluble F-actin pool. Shifts in ABP 280 or alpha-actinin do not occur. The results suggest that TM, alpha-actinin, and ABP 280 provide structure to TIF and that TM release from TIF is involved in chemotactic factor induced actin polymerization in PMNs.
Watts
R G
RG
Department of Pediatrics, University of Alabama at Birmingham 35233.
Howard
T H
TH
D000185:Actinin / Q000502:physiology*; D000199:Actins / Q000737:chemistry*; D001481:Basal Metabolism; D002352:Carrier Proteins / Q000502:physiology*; D002630:Chemotactic Factors / Q000502:physiology*; D003285:Contractile Proteins; D064448:Filamins; D006801:Humans; D007477:Ions; D008840:Microfilament Proteins; D009504:Neutrophils / Q000502:physiology*; D011092:Polyethylene Glycols; D012995:Solubility; D014335:Tropomyosin / Q000502:physiology*
D000199:Actins; D002352:Carrier Proteins; D002630:Chemotactic Factors; D003285:Contractile Proteins; C578661:FLNC protein, human; D064448:Filamins; D007477:Ions; D008840:Microfilament Proteins; D014335:Tropomyosin; D000185:Actinin; D011092:Polyethylene Glycols