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 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 endocytosis for initial ciliary assembly.
SUMMARY Using the ciliary model system Chlamydomonas, we find Arp2/3 complex-mediated endocytosis is needed to reclaim cell body plasma membrane for early ciliary assembly.
Competing Interest Statement
PA is a paid consultant for Arcadia Science
Footnotes
The paper was reorganized to better highlight the main points of the paper. We also added text throughout to address comments from reviewers and to clarify some of the conclusions. To figure 1, we added arpc4 mutants treated with CK-666 to demonstrate the specificity of CK-666 in this organism. This was previously in the supplement, but we felt that it should be moved to the main figure. The previous figure 5 (now figure 7) was updated. The clathrin light chain staining was removed as we felt it was likely that is antibody had non-specific targets and therefore may have been confounding the data. To this figure we also included another version of the membrane dye experiment this time treating cells with CK-666 to show that the phenotype was conserved whether we blocked the Arp2/3 complex with a genetic mutation or a chemical inhibitor. To previous figure 8 (now figure 4) we added no biotin controls. We also added a diagram to supplemental figure 2 showing the sequence of the ARPC4 gene and how it was affected by the CIB cassette.
