Growth conditions control the size and order of actin bundles in vitro

The bonding rules for actin filament bundles do not lead to a particular packing symmetry, but allow for either regular or disordered filament packing. Indeed, both hexagonal and disordered types of packing are observed in vivo. To investigate factors which control bundle order, as well as size, we examined the effect of protein concentration on the growth of actin-fascin bundles in vitro. We found that bundles require 4-8 d to achieve both maximum size and order. The largest and best ordered bundles were grown at low fascin and high actin concentrations (an initial fascin/actin ratio of 1:200). In contrast, a much larger number of poorly ordered bundles were formed at ratios of 1:25 and 1:50, and most surprisingly, no bundles were formed at 1:300 or 1:400. Based on these observations we propose a two-stage mechanism for bundle growth. The first stage is dominated by nucleation, which requires relatively high concentrations of fascin and which is therefore accompanied by rapid growth. Below some concentration threshold, nucleation ceases and bundles enter the second stage of slow growth, which continues until the supply of fascin is exhausted. By analogy with crystallization, we hypothesize that slower growth produces better order. We are able to use this mechanism to explain our observations as well as previous observations of bundle growth both in vitro and in vivo.