Purification and Properties of a Ca²⁺-Independent Barbed-End Actin Filament Capping Protein, CapZ, from Human Polymorphonuclear Leukocytes

Abstract

In human polymorphonuclear leukocytes (PMN), changes in the actin architecture are critical for the shape changes required for chemotaxis and phagocytosis. Barbed-end capping proteins are likely to regulate actin assembly in PMN. The previously identified barbed-end blocking proteins in PMN, gelsolin and CapG, require Ca(2+) to initiate capping of actin filaments. Because chemoattractants can stimulate PMN actin assembly by a calcium-independent signal transduction pathway, we sought to purify a calcium-independent barbed-end capping activity from PMN cytoplasmic extracts. A Ca(2+) -insensitive actin polymerization inhibitory activity was partially purified from human PMN [Southwick & Stossel (1981) J. Biol. Chem 256, 3030]. Using five column chromatography steps, we purified the protein to homogeneity as assessed by silver staining. Purification was associated with an increase in specific activity of greater than 40 X. Western blot analysis identified the protein as the nonmuscle isoform of the heterodimeric capping protein capZ. Human PMN capZ has an apparent disassociation constant of 3 nM for capping in the presence or absence of micromolar Ca(2+), as assessed by both pyrenylactin elongation and depolymerization assays. Similar to the activity reported for the actin polymerization inhibitor, activity of PMN capZ was inhibited by increasing the KC1 concentration from 0.1 M to 0.6 M. The capping function was also inhibited by phosphatidylinositol 4,5-bisphosphate (PIP(2)) micelles, with half-maximal inhibition occurring at 5.5 micrograms mL(-1). PMN capZ did not nucleate actin assembly, sequester actin monomers, or sever actin filaments. Quantitative Western blot analysis revealed that capZ levels corresponded to 0.7-1.0% of the total human PMN cytoplasmic protein. Given its abundance and high affinity for barbed filament ends, capZ is likely to play an important role in the calcium-independent regulation of actin filament assembly associated with PMN chemotaxis.

Arabidopsis Capping Protein (AtCP) Is a Heterodimer That Regulates Assembly at the Barbed Ends of Actin Filaments

“…Biochemical data from other systems show that the combination of CP and profilin increase the density of branched networks generated by the Arp2/3 complex (43) and are consistent with CP functioning to sustain a large pool of actin subunits. Cellular concentrations of CP often correlate with numbers of filament ends such that all filament ends are thought to be capped (50,51). If a similar situation exists in plants with [CP] in the low M range, then the expectation is that average filament lengths should depend on the extent of capping.…”

Chemoattractant-induced lamellipod extension

“…In neutrophils, for example, two major calcium-dependent capping proteins (gelsolin and capG), and one calcium-independent capping protein with a high affinity for filament ends (the nonmuscle isoform of capZ), are present in large amounts (Maun et al, 1996). In Dictyostelium, the capping protein cap32/34 accounts for the major Ca 2ϩ insensitive capping activity (Eddy et al, 1997).…”

Regulation of Sodium Channel Activity by Capping of Actin Filaments

“…In this way, CapZ would both prevent channel inactivation and restore partially declined channel activity. Capping proteins with properties similar to those of muscle CapZ have been detected in many cells, including platelets (Barkalow et al, 1996), neutrophils (Maun et al, 1996;Huang et al, 1999), erythrocytes (Kuhlman et al, 1997), and various cultivated cells (Schafer et al, 1998). In platelets, the ratio of capping protein to actin was estimated as 1:90 (Barkalow et al, 1996).…”