How villin subclasses coordinate actin remodelling

Abstract

Villins, which were originally named after their occurrence in mammalian microvilli, contain a gelsolin domain and a C-terminal villin headpiece domain. Both domains can bind actin; one villin molecule can therefore bind to two actin filaments, which enables it to function in actin bundling. This is in contrast to the related gelsolin, which has only a calcium-dependent actin-severing function. In plants, villins are expressed widely and have diverse actin regulatory roles, such as actin nucleation, capping, severing and bundling. Plant villins have been divided into three subclasses (I, II and III) on the basis of phylogeny and the number of calcium binding sites. In Arabidopsis, VILLIN1 (VLN1) is the only villin of subclass I and acts only as an actin bundler. VLN2 and VLN3 belong to subclass II, and VLN4 and VLN5 belong to subclass III. The relative functionalities of subclass II and III villins in plants have remained unclear.

To investigate this, the authors performed a detailed analysis of mutant combinations. The vln2 vln3 vln4 (vln2/3/4) triple mutants suffered from growth defects that could be rescued by expression of each of the three villins, which suggests that VLN2, VLN3 and VLN4 may act — to some extent — redundantly. Yet, both villin subclasses have some degree of functional specialization: VLN4 (subclass III) has the strongest actin-bundling activity and VLN2 (subclass II) has the weakest. Conversely, VLN2 has the strongest actin-severing activity and VLN4 has the weakest. VLN3 seems to show intermediate behaviour in both cases. On the one hand, VLN2 has a diffuse cytoplasmic distribution, high calcium binding affinity and strong actin depolymerizing and filament-shortening activity. VLN4, on the other hand, localizes to actin filaments, has low calcium binding affinity and weak actin depolymerizing and filament-shortening activity. The vln4 single mutants have a higher rate of actin severing and decreased actin lifetime. These data suggest that VLN4 mainly functions in calcium-independent actin bundling and stabilization, whereas VLN2 is predominantly involved in calcium-dependent severing. Interestingly, vln2/3 — but not vln2/4 and vln3/4 — double mutants have curly organs, and severing-deficient VLN2 could rescue the general growth defect of vln2/3/4 triple mutants, but not the curly organ phenotype. This suggests that actin severing by subclass II villin — but not actin bundling by subclass III villin — is required for oriented growth.