Initial ciliary assembly inChlamydomonasrequires Arp2/3 complex-dependent endocytosis

Abstract

Cilia are organelles important for signaling and motility. They are composed of microtubules ensheathed in plasma membrane. The mechanisms related to ciliogenesis also require another cytoskeletal element, actin, which has been shown to be important for organizing the basal bodies and transition zone at the base of cilia and for short- and long-range trafficking. However, most studies of actin’s role in ciliogenesis have taken a broad approach by knocking out all filamentous actin until now. Here, we more delicately dissect the interplay between actin and cilia by specifically focusing on actin networks nucleated by the Arp2/3 complex in Chlamydomonas. We find that knocking out Arp2/3-mediated actin networks dramatically impairs ciliary assembly and maintenance in these cells, and these defects are due to a problem with incorporation and gating of existing ciliary proteins, particularly in the early stages of assembly. We also show that cells lacking the Arp2/3 complex have more dramatic defects in ciliary maintenance using material from non-Golgi sources. Finally, we find relocalization of a ciliary membrane protein from the cell periphery to the cilia by internalization is dependent on actin and the Arp2/3 complex. Based on these results, we propose a new model of ciliary protein targeting during early ciliogenesis in which proteins previously targeted from the Golgi to the plasma membrane are reclaimed from this reservoir by Arp2/3-mediated networks.

The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

“…Membrane trafficking in Chlamydomonas has been found to be important for maintaining ciliary length and for ciliary assembly. Previous studies have shown that the Golgi-derived membrane is important for ciliary assembly and maintenance (Dentler, 2013), and inhibiting internalization through Arp2/3 complex perturbation also results in defective ciliary assembly and maintenance (Bigge et al, 2023). Although EM of the Golgi trial (n = 30, N = 3).…”

ERK pathway activation inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

“…Previously, Bigge et al 2020 showed that actin puncta formation, reminiscent of endocytic pits seen in yeast, were absent in ARPC4 cells. In the presence of BCI, actin puncta increased both in steady state cells ( Fig 5B-C ) and in regenerating cells ( SF 3B ).…”

“…Membrane trafficking in Chlamydomonas has been found to be important for maintaining cilia length and for ciliary assembly. Previous studies have shown that Golgi-derived membrane is important for ciliary assembly and maintenance (Dentler, 2013), and inhibiting internalization through Arp2/3 complex perturbation also results in defective ciliary assembly and maintenance (Bigge et al, 2020). While EM of the Golgi showed no noticeable differences in Golgi structure ( SF 3A ), collapsing the Golgi with BFA (Brefeldin A) intensified ciliary shortening when paired with 20 µ M BCI, suggesting that BCI alters other membrane pathways outside of Golgi derived membrane for ciliogenesis ( Fig 5A ).…”

“…Cells were treated with 0.5% DMSO or 30 µ M BCI for 2 hours and then adhered to coverslips for 2 minutes. Coverslips were moved to ice and 200 µ g/mL membrane dye (FMTM 4-64FX, fixable; Thermo, F34653) for 1 minute covered, fixed with 4% PFA in 1x HBSS for 10 minutes, and washed 3×3 minutes in 1x PBS (Bigge et al, 2020). Coverslips were air dried and mounted to slides with Fluoromount G and imaged on the spinning disk confocal microscope.…”

The exocyst complex and intracellular vesicles mediate soluble protein trafficking to the primary cilium

“…The process of protein trafficking to the primary cilium is supported by many players involved in endocytosis and the vesicle transport machinery [24][25][26] . Prominent among them are small GTPases, which act as molecular switches that allow for the guidance of their associated vesicles [27][28][29] .…”