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

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 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.

FIGURE 1:

The Arp2/3 complex is required for normal ciliary maintenance and assembly. (A) Wild-type and arpc4 mutant cells were treated with 100 or 250 μM CK-666 or the inactive CK-689 for 2 h. Superplots show the mean ciliary lengths from three separate biological replicates with error bars representing SD. n = 30 for each treatment in each biological replicate. (B) Wild-type cell, arpc4 mutant cell, and arpc4:ARPC4-V5 cell steady state cilia were also measured with no treatment. Superplots show the mean of three biological replicates with error bars representing SD. n = 30 for each strain in each biological replicate. (C, D) Wild-type cells and arpc4 mutant cells were deciliated using a pH shock, cilia were allowed to regrow, and ciliary length (C) and percent ciliation (D) were determined. Means are displayed with error bars representing 95% confidence interval (C) or SD (D). n = 30 (C) or n = 100 (D) for each strain and each time point in three separate biological replicates. For every time point except 0 min, P < 0.0001 for both length and percent ciliation. (E, F) nap1 mutant cells were pretreated with 10 μM LatB for 30 min before deciliation or treated with LatB upon the return to neutral pH following deciliation. Ciliary length (E) and percent ciliation (F) were determined for each time point. Error bars represent 95% confidence interval (E) or SD (F). n = 30 (E) or n = 100 (F) for three separate experiments. For every time point P > 0.0001 between DMSO and treated samples, except 30 min (10 µM LatB), which is not significant. (G, H) Wild-type cells were pretreated with CK-666 or the inactive CK-689 (100 µM) for 1 h before deciliation or treated with CK-666 or the inactive CK-689 (100 µM) following deciliation. Ciliary length (G) and percent ciliation (H) were measured. Error bars represent 95% confidence interval (G) or SD (H). n = 30 (G) or n = 100 (H) for three separate experiments.

FIGURE 2:

The Arp2/3 complex is required for incorporation of existing protein during ciliary assembly. (A) Treating cells with CHX inhibits protein synthesis, which means that only incorporation of existing protein into the cilia is observed. (B) Wild-type cells and arpc4 mutants were deciliated and allowed to regrow in 10 μM CHX. The percentages above the lines represent the percent of cells with cilia at the indicated time points. The mean is shown with error bars representing 95% confidence interval. n = 30 for each strain and each time point in three separate experiments. For every time point besides 0 min, P < 0.0001 for both length and percent ciliation. (C) Wild-type cells were deciliated and then treated with a combination of 10 μM CHX and CK-666 (100 or 250 μM) or CK-689 (the inactive control, 250 µM) at the same concentration during regrowth. The mean is shown with error bars representing 95% confidence interval. n = 30 for each strain and each time point in three separate experiments. At both 1 and 2 h time points, P < 0.0001 for cells treated with CK-666 compared with wild-type cells, and not significant for cells treated with CK-689 compared with wild-type cells. (D) The length of cilia after 2 h of treatment and regrowth. Superplot shows the mean of three separate experiments with error bars representing SD. n = 30 for each treatment group in three separate experiments.

FIGURE 3:

The Arp2/3 complex is required for ciliary maintenance in the absence of intact Golgi. (A) Treating cells with BFA causes the Golgi to collapse, meaning that any membranes and proteins used to maintain the cilia must come from other sources. (B) Cells were treated with 36 μM BFA for 3 h at which time the drug was washed out. Wild type is represented by black, while arpc4 mutants are gray. The mean is shown with error bars representing 95% confidence interval. Error bars represent 95% confidence interval of the mean. n = 30 for each time point and each strain in three separate experiments. **** represents P < 0.0001. (C) Percent ciliation of the cells in B. n = 100. (D) Resorption speed for wild-type cells and arpc4 mutant cells as determined by fitting a line to the first four time points before washout and determining the slope of the line. Line represents the mean of three separate experiments. n = 3. P = 0.0314.

FIGURE 4:

Ciliary membrane proteins have multiple paths from the plasma membrane. (A) Cells were biotinylated, deciliated, and allowed to regrow before cilia were isolated and probed for biotinylated protein. (B) Total protein in wild-type and arpc4 mutant ciliary isolate investigated by Western blot and Coomassie. (C) Wild-type and arpc4 mutant cell ciliary isolate was investigated by Western blot and probed using streptavidin. Black arrow shows ciliary protein present to a higher degree in wild-type cells than in arpc4 mutant cells. Gray arrows show ciliary protein that is present to a higher degree in arpc4 mutant cells than in wild-type cells. (D) Bands represented by black and gray arrows are quantified for the wild-type cells. Data acquired from three separate experiments. (E) Bands represented by black and gray arrows are quantified for the arpc4 mutant cells. Data represented as the mean from three separate experiments. Error bars represent SD.

FIGURE 5:

The Arp2/3 complex is required for the relocalization and internalization of the ciliary protein SAG1 for mating. (A) When mating is induced, SAG1-HA is internalized and relocalized to the apex of the cells and cilia for agglutination. (B) Maximum-intensity projections of z-stacks showing SAG1-HA. Scale bar represents 2 μm. (C) Line scans were taken through the cells in z-stack sum images. (D) Line scans in untreated cells (left), LatB-treated cells (middle), and CK-666–treated cells (right) were normalized and fitted with a gaussian curve. The curves were averaged. Black lines represent mean, and shaded regions represent SD. Red represents uninduced samples, cyan represents induced samples. 0 on the y-axis represents the apical region of the cell. n = 30 from a single representative experiment. (E) Percentage of cells with apical enrichment for uninduced (black) and induced (gray) cells for each treatment group. The mean is shown with the solid line. n = 30 for three separate experiments for each treatment. (F) Western blot showing amount of SAG1-HA in uninduced and induced cells in each treatment group, all treated with 0.01% trypsin. (G) Intensity of the bands in F were normalized to the total protein as determined by Coomassie staining and quantified in ImageJ to subtract uninduced from induced to give a representation of the amount of SAG1-HA internalized with induction. Line represents mean of three separate experiments.

FIGURE 6:

Loss of a functional Arp2/3 complex results in changes in actin distribution. (A) Wild-type and arpc4 mutant cells stained with phalloidin to visualize the actin network along with bright field to show cell orientation. Images were taken as a z-stack using Airyscan imaging and are shown as a maximum-intensity projection. Red arrow is pointing to dots at the apex of the cell, and white arrow is pointing to the pyrenoid near the basal end of the cell. Scale bars represent 2 μm. (B) Percentage of cells with apical dots as shown in A. Percentages taken from three separate experiments where n = 100. Line represents the mean. P < 0.0001. (C) Presence of apical dots in the arpc4 mutant rescue expressing ARPC4-V5. Images were taken as a z-stack using Airyscan imaging and are shown as maximum-intensity projections. Red arrow is pointing to dots at the apex of the cell, and white arrow is pointing to the pyrenoid near the basal end of the cell. Scale bars represent 2 μm.

FIGURE 7:

Arp2/3 complex-dependent endocytosis is conserved in Chlamydomonas. (A) Gene presence was determined using BLAST. Word colors correspond to diagram colors. See Supplemental Table 1 for additional information. (B) Cells treated with 30 μM PitStop2 were incubated with FM 4-64FX and imaged on a spinning-disk confocal. Maximum-intensity projections of z-stacks are shown. Scale bars are 2 μm. (C) The background-corrected fluorescence for each sample, including cells treated with 100 µM Dynasore. The mean is shown with error bars showing SD. n = 30 in three separate experiments. P < 0.0001. (D) Wild-type and arpc4 mutant cells treated with FM 4-64FX and imaged on a spinning-disk confocal. Maximum-intensity projections of z-stacks are shown. Scale bars are 2 μm. (E) The background-corrected fluorescence for each sample. The mean is shown with error bars representing SD. n = 30 in three separate experiments. P < 0.0001. (F) The background-corrected fluorescence for cells treated with CK-666 or CK-689. The mean is shown with error bars representing SD. n = 30 in three separate experiments. P < 0.0001.

FIGURE 8:

Actin dots require the Arp2/3 complex and endocytosis. (A–C) Wild-type cells (A), arpc4 mutant cells (B), and wild-type cells treated with 30 µM PitStop2 (C) stained with phalloidin to visualize the actin network before deciliation, immediately following deciliation, and 10 min following deciliation. Bright-field images are to visualize cell orientation. Images were taken as a z-stack and are shown as a maximum-intensity projections. Scale bar represents 2 μm. Red arrows point to dots at the apex of the cell, and white arrows point to the pyrenoid at the opposite end of the cell. (D) The percentage of cells with one dot, two dots, or three dots in each condition. Quantification based on sum slices of z-stacks taken using a spinning-disk confocal. n = 100 in three separate experiments. For wild type, the total number of cells with dots is significantly different for the 0 min time point (**) and the number of dotted cells with three or more dots is significantly different for the 0 time point (****).

FIGURE 9:

The Arp2/3 complex is required for membrane and protein delivery via a Golgi-independent, endocytosis-like process. (A) Arp2/3-mediated actin networks are required for ciliary assembly in Chlamydomonas, particularly during the initial stages. These actin networks are also required for endocytosis and for the endocytosis-like relocalization of a ciliary protein from the plasma membrane to the cilia. Finally, a large endocytic event occurs immediately following deciliation that is Arp2/3 complex mediated. (B) Proposed model of membrane protein and membrane transport from the plasma membrane to the cilia through endocytosis.