Role of tropomyosin, α‐actinin, and actin binding protein 280 in stabilizing triton insoluble f‐actin in basal and chemotactic factor activated neutrophils

Abstract

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, α‐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) α‐actinin, actin binding protein 280, and tropomyosin are localized to TIF and excluded from TSF; (2) TM, α‐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 α‐actinin do not occur. The results suggest that TM, α‐actinin, and ABP 280 provide structure to TIF and that TM release from TIF is involved in chemotactic factor induced actin polymerization in PMNs.