Partially redundant actin genes in Chlamydomonas control transition zone and flagellum-directed protein dynamics

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 the regulation of flagella-building intraflagellar transport machinery. Here, we probe how actin redundancy contributes to this process using a nap1 mutant Chlamydomonas strain. Disruption of a single actin allows normal or slower incorporation but complete flagellar assembly. However, when we disrupt both actins with Latrunculin B (LatB) treatment on the nap1 mutant background, we find that actins are necessary for flagellar growth from newly synthesized limiting flagellar proteins. In addition, total actin loss also showed reduced protein synthesis during regeneration. We find under actin depleted conditions, there is reduced incorporation of existing flagellar proteins as well as mislocalization of a key transition zone gating protein, NPHP-4. Extended (2 hour) treatment with LatB, a condition under which NAP1 is upregulated, restores NPHP-4 localization. This suggests NAP1 can perform the functions of conventional actin at the transition zone. Our experiments demonstrate that each stage of flagellar biogenesis requires redundant actin function to varying degrees, with an absolute requirement for these actins in incorporation of newly synthesized flagellar proteins. INTRODUCTION Assembly and composition of the eukaryotic flagellum (also known as the cilium) are critical for signaling and development in most cell-types in the human body. The flagella of the green alga Chlamydomonas reinhardtii are essentially identical to the cilia of mammalian cells and provide an excellent model to study cell signaling, cell motility, and regulation of ciliary assembly. To date, the known mechanisms dictating the behavior of these organelles are largely dependent on the microtubule cytoskeleton. The flagellum is composed of microtubules that extend from a microtubule organizing center known as the basal body, and flagellar assembly requires control of microtubule dynamics. Trafficking from sites of cellular protein synthesis to their ultimate destination in flagella is also thought to occur on microtubule tracks (Tai et al., 1999). However, evidence for the role of actin, another major cytoskeletal component, in ciliary regulation is increasing. In mammalian cells, disruption of actin leads to increases in both ciliary length and percentage of ciliated cells (Kohli et al., 2017; Sharma et al., 2011), which may be due to roles for actin in basal body migration, docking, and stabilization (Kim et al., 2010; Pan et al., 2007; Park et al., 2008; Tu et al., 2017; Yeyati et al., 2017). A recent study showed loss of Myosin-Va, an actin-based motor protein involved in trafficking of secretory vesicles from the post-Golgi to the plasma membrane, resulted in reduced ciliation. Disruption of Myosin-Va function stops the formation of the elongated ciliary membrane. This new result suggests actin and Myosin-Va are required for microtubule-dependent trafficking of preciliary and ciliary vesicles to the base of the cilia and therefore necessary for ciliogenesis (Tang, et. al, 2018). Actin is also required for vesicle budding in the endocytic pathway (Girao et al., 2008), which may influence ciliary assembly as there is a trafficking pathway connecting the endocytic compartments to a vesicular compartment involved in ciliary assembly (Kim et al., 2010). Membrane remodeling for both ciliary exocytosis (Nager et al., 2017) and ciliary resorption are actin dependent processes (Saito et al., 2017). In summary, the current understanding is that actin networks (that potentially block cortical access of basal bodies and ciliary proteins) inhibit cilium formation and elongation in mammalian cells but are also required for membrane trafficking to support ciliogenesis. Here, using an algal model system, we show a broader requirement for filamentous actin in flagellar protein synthesis, trafficking, and incorporation of proteins into an assembling flagellum. Using Chlamydomonas reinhardtii as a model to interrogate flagellar dynamics, we are able to induce flagellar severing on demand to allow synchronous and successive rounds of flagellar regeneration. Chlamydomonas has two actin genes, one that encodes a conventional actin (IDA5) and another that encodes a novel actin-like protein (NAP1) with ~65% homology to mammalian actin (Hirono et al., 2003). Chlamydomonas cells treated with cytochlasin D, and actin polymerization inhibitor, exhibit flagellar shortening and regrowth upon washout of the drug. Suggesting a role for actin in flagellar maintenance (Dentler and Adams, 1992). ida5 mutants and myosin inhibited cells had impaired flagellar motor recruitment to basal bodies, impaired entry of motors into the flagellar compartment, and ultimately a reduced initial flagellar growth rate (Avasthi et al., 2014). Despite defects in flagellar protein recruitment and flagellar assembly in ida5 conventional actin mutants, these mutants ultimately grow flagella to wild-type lengths. Since ida5 mutant flagella eventually reach wild type length, the degree to which actin is required is still in question. In this study, we asked if more severe flagellar biogenesis phenotypes were masked by redundant roles of the second Chlamydomonas actin NAP1, as NAP1 expression increases upon IDA5 loss or disruption (Onishi et al., 2016). Using genetic and chemical perturbations of both Chlamydomonas actins, we found that the two actin genes have overlapping functions that include flagellar protein synthesis and composition of the flagellar gate. We also found an absolute requirement for these actins in flagellar incorporation of newly synthesized flagellar proteins. Here, we explore the role of IDA5 and NAP1 as well as their importance for trafficking flagellar proteins and building flagella. RESULTS Simultaneous disruption of all filamentous actins within Chlamydomonas cells To investigate if NAP1 contributes to the actin-dependent flagellar assembly functions previously identified for the conventional actin IDA5 (Avasthi et al., 2014), we obtained a nap1 null mutant strain, which was isolated based on its sensitivity to Latrunculin B (LatB), a conventional actin depolymerizing agent (Onishi et al., 2016). Here we use both a genetic and chemical approach to understand the role of actin in flagellar assembly (Figure 1A). LatB has been shown to disrupt filamentous actin in Chlamydomonas reinhardtii (Avasthi et al., 2014; Onishi et al., 2016). Treatment with LatB for ten minutes is sufficient to disrupt actin filaments in wild-type cells (Onishi et al., 2016). To confirm that LatB disrupts actin filaments under our experimental conditions, we fixed and stained the nap1 mutant strain with phalloidin, a filamentous actin probe (Figure 1). We see actin filaments are present in the nap1 mutants prior to drug treatment (Figure 1B). Previous attempts to visualize actin filaments with phalloidin have not been successful in vegetative Chlamydomonas cells. However, by selecting for cell health, considering the cell cycle, and shortening the incubation time to decrease background signal (Craig et al., in preparation), we were able to improve the signal to noise ratio and achieve specific filamentous actin labeling. Upon LatB treatment, these filaments disappear (Figure 1D). Chlamydomonas cells are known to upregulate NAP1 upon LatB treatment