Partially Redundant Actin Genes in Chlamydomonas Control Transition Zone Organization and Flagellum-Directed Traffic

Abstract

SUMMARY The unicellular green alga Chlamydomonas reinhardtii is a biflagellated cell with two actin genes: one encoding a conventional actin (IDA5) and the other encoding a divergent novel actin-like protein (NAP1). Here, we probe how actin redundancy contributes to flagellar assembly. Disrupting a single actin allows complete flagellar assembly. However, when disrupting both actins using latrunculin B (LatB) treatment on the nap1 mutant background, we find that actins are necessary for flagellar growth from newly synthesized limiting flagellar proteins. Under total actin disruption, transmission electron microscopy identified an accumulation of Golgi-adjacent vesicles. We also find that there is a mislocalization of a key transition zone gating and ciliopathy protein, NPHP-4. Our experiments demonstrate that each stage of flagellar biogenesis requires redundant actin function to varying degrees, with an absolute requirement for these actins in transport of Golgi-adjacent vesicles and flagellar incorporation of newly synthesized proteins.

Initial ciliary assembly inChlamydomonasrequires Arp2/3 complex-dependent endocytosis

“…Next, we investigated the requirement for Arp2/3-nucleated actin in the more complicated process of ciliary assembly where there is a high demand for protein and membrane both from pools of protein already existing in the cell and from new protein synthesis (Wingfield et al 2017;Nachury, Seeley, and Jin 2010;Rohatgi and Snell 2010;Jack et al 2019;Diener, Lupetti, and Rosenbaum 2015). In order to investigate assembly, cells were deciliated by low pH shock and then allowed to synchronously regenerate cilia after being returned to normal pH (Paul A.…”

“…In wild-type cells, NPHP4 localizes in two dots at the base of cilia (Figure 6D-E). When cells are treated with LatB for a short period of time (30 minutes), the conventional actin IDA5 depolymerizes and the localization of NPHP4 is lost ( Figure 6D-E) (Jack et al 2019). After 2 hours, the divergent actin NAP1 is upregulated and the localization of NPHP4 is restored ( Figure 6D-E) (Jack et al 2019).…”

“…In Chlamydomonas, disruption of actin networks with Cytochalasin D (CytoD) resulted in shorter steady-state cilia (W. L. Dentler and Adams 1992), and disruption with Latrunculin B (LatB), which sequesters monomers leading to eventual filament depolymerization, resulted in shortened cilia and impaired regeneration (Avasthi et al 2014;Jack et al 2019). Chlamydomonas actin networks are required for accumulation of IFT machinery at the base of cilia and for entry of IFT material into cilia (Avasthi et al 2014), as well as for trafficking of post-Golgi vesicles to cilia, the synthesis of ciliary proteins, and the organization of the gating region at the base of cilia (Jack et al 2019). Many key advances in our understanding of the relationship between cilia and actin have been discovered using Chlamydomonas, which is proving to be a useful model for studying the actin cytoskeleton and its ciliary functions.…”

“…The actin cytoskeleton of Chlamydomonas contains two actin genes: IDA5, a conventional actin with 91% sequence identity to human b-actin; and NAP1, a divergent actin that shares only 63% of its sequence with human b-actin (Hirono et al 2003;Kato-Minoura et al 1998). We consider NAP1 to be an actin-like protein as opposed to an actin related protein (ARP) because it has a higher sequency identity when compared to actin than to conventional ARPs (Supplemental Figure 1), and because it is able to functionally compensate for the conventional filamentous actin (Jack et al 2019;M. Onishi et al 2018;M.…”

“…Onishi, Pringle, and Cross 2016;Hirono et al 2003). This separability of the two actins has led to the discovery that they can compensate for each other in ciliary maintenance and assembly (Jack et al 2019). While knocking out IDA5 and NAP1 networks results in ciliary defects, studies of the role of actin in ciliary assembly have used global disruption by knocking out either one of the filamentous actins or both, yet actin networks have diverse compositions and topologies that lead to specific subfunctions within cells.…”