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.