ABSTRACT
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 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 clathrin-mediated endocytosis of ciliary proteins, an increase in endocytic activity upon induction of ciliary growth, and relocalization of plasma membrane proteins to newly formed cilia. Our data support a new model of ciliary protein and membrane trafficking during early ciliogenesis whereby proteins previously targeted to the plasma membrane are reclaimed by Arp2/3 complex-dependent clathrin-mediated endocytosis for initial ciliary assembly.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Panels A and B in Figure 1 were replaced with superplots; Panel D, with regeneration in the rescue strain, added to figure 1; Panel B in Figure 2 was replaced with superplot; Panel C, phalloidin staining of the rescue strain, added to figure 4 and brightfield images were added to this figure to orient the cells; Figures 5 and 8 from the previous version were combined and reworked to emphasize the parallels between the loss of Arp2/3 and clathrin mediated endocytosis; Panel F was replaced with a new blot; Brightfield images were added to figure 7 to orient the cells, and previous images taken with a spinning disk confocal were replaced with images taken with airyscan; Figure 9 became Figure 8, and the blots in this image were replaced; Supplemental figure 2 was added to confirm the mutant and rescue; Images in supplemental figure 6 were replaced with new images and brightfield images to orient the cell