Partially redundant actin genes in Chlamydomonas control flagellum-directed traffic and transition zone organization

Abstract

Flagella of the unicellular green alga Chlamydomonas reinhardtii are nearly identical to cilia of mammalian cells and provide an excellent model to study ciliogenesis. These biflagellated cells have two actin genes: one encoding a conventional actin (IDA5) and the other encoding a divergent novel actin-like protein (NAP1). Previously, we described a role for actin in regulation of flagella-building intraflagellar transport machinery and now probe how actin redundancy contributes to this process using a nap1 mutant Chlamydomonas strain. Treatment with Latrunculin B, a potent actin polymerization inhibitor on the nap1 mutant background acutely disrupts all filamentous actins in the cell. We find that actins are an absolute requirement for flagellar growth when the preexisting pool of flagellar precursors is depleted. Nap1 mutants treated with Latrunculin B also showed reduced protein synthesis during regeneration. Finally, loss of functional actins reduced the incorporation of existing flagellar proteins as well as caused mislocalization of a key transition zone gating protein, NPHP-4. These experiments demonstrate that each stage of flagellar biogenesis requires redundant actin function to varying degrees, with an absolute requirement for actin in incorporation of newly synthesized flagellar proteins.

The elusive actin cytoskeleton of a green alga expressing both conventional and divergent actins

“…We now believe this is due to compensation by the upregulation of NAP1 in ida5 mutants (Kato Minoura, 2005;Onishi et al, 2016;Onishi et al, 2018). Loss of both actins is lethal (Onishi et al, 2016), and our recent studies demonstrate critical functions for at least one actin at multiple stages of ciliary assembly (Jack et al, 2018).…”

“…Most obvious is the array of filamentous actin localized posterior of the nucleus in vegetative and gametic cell types. The actin-binding and nucleating proteins responsible for coordinating this actin population are not known, but recent findings in our lab strongly suggest a role for actin in trafficking vesicles from the Golgi (Jack et al, 2018). This mid-cell actin revealed by Atto 488 Phalloidin labeling, along with Cryo-ET showing distinct actin filaments near the Golgi (Figure 3A), steers us to investigate the functional role of actin in ciliary protein transport, which we believe is myosin-dependent (Avasthi et al, 2014).…”

“…Outside of the compensatory functions, the requirement for dual expression is not clear since NAP1 expression is undetectable under normal conditions and only upregulated slightly in the absence of IDA5 or strongly upon flagellar reassembly following deflagellation (Hirono et al, 2003). Despite the upregulation of NAP1 upon flagellar regeneration, we previously found that nap1 null mutants have no flagellar assembly defect outside of the inability to buffer perturbations to IDA5 (Jack et al, 2018). However, NAP1 can largely perform the functions of filamentous IDA5, as ida5 mutants show only slow swimming.…”

“…Ciliary growth ultimately proceeds and can reach normal length. However, when both actins are disrupted acutely, Chlamydomonas cells show dramatic defects in ciliary protein synthesis, vesicular trafficking, and organization of a key gating region dictating ciliary protein composition (Jack et al, 2018). Given that ida5 mutants expressing NAP1 alone don't show these defects, it appears NAP1 can largely perform actindependent functions needed for ciliary assembly despite its sequence divergence with IDA5.…”

Rpgrip1l controls ciliary gating by ensuring the proper amount of Cep290 at the vertebrate transition zone

“…For this reason, the assembly and function of the TZ is a hot topic in biomedical research. A lot of proteins participate in TZ assembly and/or function as ciliary gatekeepers at the TZ [12- 14,16,17,19,23,24,27,29,30,33,34,37,41,69,70,[73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90]. However, the relationships between these proteins and hence the mechanisms underlying ciliary gating at the TZ remain largely elusive.…”