Excess p50 induces Arp1-dependent dynactin clusters containing the assembly factor VezA

Dynactin, a complex essential for cytoplasmic dynein function, contains an Arp1 mini-filament with a pointed-end sub-complex including Arp11 and a shoulder sub-complex including p50 and p150. We recently identified in Aspergillus nidulans a vezatin homolog VezA that enhances dynactin assembly, but VezA does not co-localize with dynactin, suggesting a transient interaction. It was found that overexpression of p50 separates the shoulder from the mini-filament and causes late-onset neurodegeneration in mice, but its cellular effects in the context of VezA need to be studied. Here, we surprisingly found that p50 overexpression in A. nidulans causes Arp11, p50 (but not p150), and VezA to form co-localized clusters whose presence depends on Arp1. Formation of the VezA cluster also depends on Arp11, and VezA significantly enhances the intensity of Arp11-GFP clusters but not that of p50-GFP clusters. Moreover, an evolutionarily conserved region of p50 (aa17-25) forming a beta-sheet structure with Arp1 as revealed in previous cryo-EM studies is critical for the formation of Arp11, p50 and VezA clusters. These results suggest that excess p50 induces cluster formation driven by p50-Arp1 interactions, and that this assembly intermediate lacking the intact shoulder traps VezA, which participates in the Arp11-Arp1-p50 interactions. [Media: see text] [Media: see text].