Figure 4.

VLN1 Bundles F-Actin in a Calcium- and Calmodulin-Independent Manner.

(A) A low-speed cosedimentation assay was used to determine the bundling activity of VLN1. Actin alone (3 μM) and actin plus 1 μM VLN1 were incubated under polymerization conditions and the reactions were centrifuged at 13,500g. Equivalent amounts of total, supernatant, and pellet were separated by SDS-PAGE. Samples are: lane 1, actin alone total; lane 2, actin alone supernatant; lane 3, actin alone pellet; lane 4, actin plus VLN1 total; lane 5, actin plus VLN1 supernatant; and lane 6, actin plus VLN1 pellet.

(B) To determine whether calcium affects bundling activity, the experiments described in (A) were repeated (n = 3) in the presence of varying free Ca²⁺ concentrations. The resulting gels were scanned to determine the amount of actin in the pellet at different free [Ca²⁺]. The percentage of actin sedimenting at 4.6 nM Ca²⁺ was 56.5%; at 100 nM Ca²⁺ was 60%; at 1 μM Ca²⁺ was 59.1; at 12 μM Ca²⁺ was 55.90; and at 1 mM Ca²⁺ was 63.9%.

(C) To test the effects of calmodulin, 3 μM actin and 200 nM VLN1 were incubated in the presence of 500 μM free [Ca²⁺] and various amounts of calmodulin. The resulting gels were scanned to determine the percentage of actin (black bars) and VLN1 (gray bars) that sedimented under each condition.