Lithium-induced ciliary lengthening sparks Arp2/3 complex-dependent endocytosis.

Ciliary length is highly regulated, but can be disrupted by lithium, which causes ciliary elongation across cell types and organisms. We used the algal system Chlamydomonas reinhardtii to investigate the mechanism behind lithium-induced ciliary elongation. Protein synthesis is not required for lengthening, and the target of lithium, GSK3, has substrates that can influence membrane dynamics. Further, ciliary assembly requires a supply of ciliary membrane as well as protein. Lithium-treated cilia elongate normally with brefeldin treatment, but dynasore treatment produced defective lengthening suggesting a source of membrane from the cell surface rather than the Golgi. Genetic or chemical perturbation of the Arp2/3 complex or dynamin, required for endocytosis, blocks lithium-induced ciliary lengthening. Finally, we found an increase in Arp2/3 complex- and endocytosis-dependent actin filaments near the ciliary base upon lithium treatment. Our results identify a mechanism for lithium-mediated cilium lengthening and demonstrate the endocytic pathway for cilium membrane supply in algae is likely a conserved mechanism given lithium's conserved effects across organisms.

Initial ciliary assembly in Chlamydomonas requires Arp2/3 complex-dependent endocytosis.

Ciliary assembly, trafficking, and regulation are dependent on microtubules, but the mechanisms of ciliary assembly also require the actin cytoskeleton. Here, we dissect subcellular roles of actin in ciliogenesis by focusing on actin networks nucleated by the Arp2/3 complex in the powerful ciliary model, Chlamydomonas. We find that the Arp2/3 complex is required for the initial stages of ciliary assembly when protein and membrane are in high demand but cannot yet be supplied from the Golgi complex. We provide evidence for Arp2/3 complex-dependent endocytosis of ciliary proteins, an increase in endocytic activity upon induction of ciliary growth, and relocalization of plasma membrane proteins to newly formed cilia.

Comparative Phenotyping of Two Commonly Used Chlamydomonas reinhardtii Background Strains: CC-1690 (21gr) and CC-5325 (The CLiP Mutant Library Background).

The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to investigate many essential cellular processes in photosynthetic eukaryotes. Two commonly used background strains of Chlamydomonas are CC-1690 and CC-5325. CC-1690, also called 21gr, has been used for the Chlamydomonas genome project and several transcriptome analyses. CC-5325 is the background strain for the Chlamydomonas Library Project (CLiP). Photosynthetic performance in CC-5325 has not been evaluated in comparison with CC-1690. Additionally, CC-5325 is often considered to be cell-wall deficient, although detailed analysis is missing. The circadian rhythms in CC-5325 are also unclear. To fill these knowledge gaps and facilitate the use of the CLiP mutant library for various screens, we performed phenotypic comparisons between CC-1690 and CC-5325. Our results showed that CC-5325 grew faster heterotrophically in dark and equally well in mixotrophic liquid medium as compared to CC-1690. CC-5325 had lower photosynthetic efficiency and was more heat-sensitive than CC-1690. Furthermore, CC-5325 had an intact cell wall which had comparable integrity to that in CC-1690 but appeared to have reduced thickness. Additionally, CC-5325 could perform phototaxis, but could not maintain a sustained circadian rhythm of phototaxis as CC1690 did. Finally, in comparison to CC-1690, CC-5325 had longer cilia in the medium with acetate but slower swimming speed in the medium without nitrogen and acetate. Our results will be useful for researchers in the Chlamydomonas community to choose suitable background strains for mutant analysis and employ the CLiP mutant library for genome-wide mutant screens under appropriate conditions, especially in the areas of photosynthesis, thermotolerance, cell wall, and circadian rhythms.

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

The green alga Chlamydomonas reinhardtii is a leading model system to study photosynthesis, cilia, and the generation of biological products. The cytoskeleton plays important roles in all of these cellular processes, but to date, the filamentous actin network within Chlamydomonas has remained elusive. By optimizing labeling conditions, we can now visualize distinct linear actin filaments at the posterior of the nucleus in both live and fixed vegetative cells. Using in situ cryo-electron tomography, we confirmed this localization by directly imaging actin filaments within the native cellular environment. The fluorescently labeled structures are sensitive to the depolymerizing agent latrunculin B (Lat B), demonstrating the specificity of our optimized labeling method. Interestingly, Lat B treatment resulted in the formation of a transient ring-like filamentous actin structure around the nucleus. The assembly of this perinuclear ring is dependent upon a second actin isoform, NAP1, which is strongly up-regulated upon Lat B treatment and is insensitive to Lat B-induced depolymerization. Our study combines orthogonal strategies to provide the first detailed visual characterization of filamentous actins in Chlamydomonas, allowing insights into the coordinated functions of two actin isoforms expressed within the same cell.