Regulation and functional differentiation of two actins in Chlamydomonas

2020-04-20T21:50:29Z (GMT) by Masayuki onishi

Actin has a deep root in eukaryotic evolution and plays important roles in a wide range of biological processes, including the determination of cell shape and polarization, vesicle transport and endocytosis, cell motility, and cytokinesis. Many model and non-model organisms possess multiple and often divergent actins, but their regulation and presumed functional differentiation are not understood in detail. For example, the green alga Chlamydomonas has both a conventional actin (IDA5) and a highly divergent actin (NAP1); only IDA5 is expressed in normal proliferating cells. We showed previously that the latrunculin toxins (LatA and LatB) cause loss of filamentous (F-) IDA5 and strong upregulation of NAP1, which then provides essential actin function(s) by forming LatA/B-resistant F-NAP1. Thus, Chlamydomonas provides a unique opportunity to study the regulation and functional differences of multiple actin isoforms. In addition, it provides an avenue to identify factors required for normal function of conventional actin, loss of which would normally be lethal but can be rescued by NAP1 in this organism.

By combining RNA-seq analyses, high-throughput quantitative genetic screens, and live-cell imaging of actin dynamics, we have uncovered the general principle of the regulation of two actins and the components involved (the LAT genes), as well as several other genes involved in actin functions:

(a) Perturbance in F-IDA5 structure and function by LatB triggers the “LAT pathway (LAT1, LAT2, LAT3),” which induces expression of NAP1 and other genes in the SCF/ubiquitin-proteasome system.

(b) The SCF (LAT5, LAT6) targets the non-polymerizable LatB-IDA5 complexes for ubiquitination and degradation. Inhibition of this process by mutations cause reduced cellular fitness, presumably by interference with NAP1 polymerization.

(c) Homologs of some of the LAT genes in Arabidopsis showed phenotypes indicative of actin defects, suggesting a basal evolutionary history of this mechanism.

(d) LatB treatment also induces cofilin and profilin. Cofilin may further the clearance of IDA5 by severing F-IDA5, whereas profilin appears to function in protecting monomeric IDA5 from degradation.

(e) A genetic screen for synthetic lethality with nap1 identified a mutant of cyclase-associated protein (CAP1; SRV2 in budding yeast), which, together with cofilin and profilin, is involved in actin recycling in other organisms. In the mutant, F-actin forms multiple small rings in the cytoplasm, and the cell fails to divide properly.

(f) Complete removal of F-actin by combining nap1 and LatB caused only a partial inhibition of cleavage-furrow ingression, suggesting that an actin-independent mechanism drives furrowing in this Chlamydomonas, which has no type-II myosin.