Actin Dynamics in Aspergillus nidulans

Abstract

Actin is a major cytoskeletal protein required for the polarized growth of filamentous fungi. Recent studies have characterized the dynamics of actin polymers in growing Neurospora crassa and identified the presence of actin patches, cables and rings. In Aspergillus nidulans actin patch and ring dynamics have been documented using fluorescent proteins tagged to actin. However, fluorescently tagged actin does not reveal the presence of actin cables. Recently, the Lifeact construct has been used to label all three actin structures in fungi. Lifeact is a 17 amino acid peptide derived from the Saccharomyces cerevisiae actin binding protein Abp140p. To better understand actin dynamics in living cells, A. nidulans was transformed with the Lifeact reporter construct.

Lifeact expressing strains grew and developed as wild-type. Lifeact labeled actin localized to three different organizational patterns in mature hyphae: a sub-apical collar of endocytic actin patches that was located approximately 2µm from the apex, an apical actin array, and a sub-apical actin web. The apical actin array (AAA – Apical Actin Array) was present in the apices of forty percent of hyphae observed (n=100). The sub-apical actin web (SAW – Sub-apical Actin Web) was present in fifty percent of hyphae observed and was located at an average of 18.46 µm from the apex. It was hypothesized that this network of actin cables was associated with branch and septation site selection or associated with branch and septa formation. An alternative hypothesis was that the SAW acted as a diffusion barrier for nuclei. It was determined that the SAW was neither associated with branch or septa site selection or formation, nor did it act as a barrier for nuclei.

It was observed that the AAA can retract and form the SAW. It was hypothesized that this change in actin dynamics could be connected to the faster growth rates reported for mature hyphae. Measurements of individual hyphae containing the AAA or SAW revealed that hyphae with SAWs grow 1.67 times faster than hyphae with AAAs. This data supports the hypothesis that the presence of the SAW is associated with faster rates of growth. An accumulation of circular vesicles was also observed posterior to the SAW and are believed to be woronin bodies. The identity of the circular structures was not confirmed, but the retraction of the AAA to form the SAW may act as a mechanism to transport apically formed woronin bodies to distal regions of the cell. The SAW may also act as a barrier to maintain woronin bodies in sub-apical regions of the hyphae.

The Lifeact actin reporter gave clear and defined labeling of filamentous actin in A. nidulans without disturbing natural development. The use of Lifeact allowed for novel insights into actin cable dynamics present in the apical and sub-apical regions of hyphae, branch formation, and septa formation.