The centrosome regulates the Rab11- dependent recycling endosome pathway at appendages of the mother centriole

Abstract

The recycling endosome localizes to a pericentrosomal region via microtubule-dependent transport. We previously showed that Sec15, an effector of the recycling endosome component, Rab11-GTPase, interacts with the mother centriole appendage protein, centriolin, suggesting an interaction between endosomes and centrosomes. Here we show that the recycling endosome associates with the appendages of the mother (older) centriole. We show that two mother centriole appendage proteins, centriolin and cenexin/ODF2, regulate association of the endosome components Rab11, the Rab11 GTP-activating protein Evi5, and the exocyst at the mother centriole. Development of an in vitro method for reconstituting endosome protein complexes onto isolated membrane-free centrosomes demonstrates that purified GTP-Rab11 but not GDP-Rab11 binds to mother centriole appendages in the absence of membranes. Moreover, centriolin depletion displaces the centrosomal Rab11 GAP, Evi5, and increases mother-centriole-associated Rab11; depletion of Evi5 also increases centrosomal Rab11. This indicates that centriolin localizes Evi5 to centriolar appendages to turn off centrosomal Rab11 activity. Finally, centriolin depletion disrupts recycling endosome organization and function, suggesting a role for mother centriole proteins in the regulation of Rab11 localization and activity at the mother centriole.

Figure 1. Recycling endosome components associate with appendages of the mother centriole

(A) Membrane-free centrosomes were isolated from cells expressing centriolin-mVenus (green), spun onto glass coverslips (31) and stained for centrosomes (5051, blue) and Rab11 (red). Rab11 co-stained with centriolin-mVenus, which was concentrated on the mother centriole (4). Merge of 5051, centriolin-mVenus, and Rab11 signals shown in white. Bar, 1 μm. (B) Percentage of Sec15, Rab11, Exo84, Evi5, FIP3, Rab8, centriolin, and 5051 that localize to isolated centrosomes (both centrioles) and concentrate on the mother centriole (as in A), (n=3 independent experiments, Bar is SE, 150 centrosomes/bar). (C) Cell lysates from cells treated with lamin (control) or cenexin siRNAs show cenexin depletion and no significant changes in the levels of other proteins. (D) Isolated centrosomes (as in A) from cells depleted of cenexin or lamin were stained for centrin (green), Sec15 (red), Rab11 (red), Evi5 (red), centriolin (red), and cenexin (red). Bottom images show centrin.

Figure 2. Centriolin regulates the centrosomal localization of the exocyst and Evi5

(A) Cells stably expressing centriolin-mVenus (green) were stained for Sec15 (red). Bar, 10 μm. Below, the percent of cells with centrosome (5051) localized Sec15, Sec6, Exo84, Rab11, Evi5, Centriolin, or Cenexin was calculated in cells treated with siRNAs targeting lamin, Rab11, centriolin, or Evi5. (n=3 experiments, n=50 centrosomes/treatment/experiment. Bar is SE.) (B) Centrosomes isolated from cells expressing GFP, GFP-Nud1, or GFP-C-terminus were stained for centrin (green) and Sec15 (Red). Right, fold-change of Sec15 on isolated centrosomes was calculated (n=3 experiments, p<0.02, n>25 centrosomes/treatment. Bar is SE). (C) Left, exocyst disruption (Sec6 depletion) diminishes Evi5 and Sec15 signal at centrosome. Right, quantification of percent of cells with no centrosome localization (n=3 experiments, * p-values <0.02, bar is SE). Lamin depletion was used as control. (D) Proposed structural model for the hierarchy of molecular anchoring at the mother centriole: 1) cenexin anchors centriolin at the appendages of the mother centriole 2) centriolin anchors its binding partner the exocyst 3) the exocyst anchors Evi5 which is a known binding partner of Rab11-GTP and its proposed GAP.

Figure 3. Membrane-free Rab11-GTP specifically associates with cenexin at mother centrioles

(A) Isolated centrosomes from GAPDH- or cenexin-depleted cells were incubated with purified GST-Rab11-GTPγS or GST-Rab11-GDP, spun onto glass coverslips and stained for centrin (green) and GST (red). Below, GST intensity at centrosome was calculated. Representative of n=3 experiments, n>100 centrosomes/condition. P-value is p<1*10^-4 between Rab11-GDP and Rab11-GTPγS in control cells, Bar is SE. (B) Isolated centrosomes were incubated with purified MBP or MBP-Evi5N, spun onto glass coverslips, stained for Rab11 (green), MBP (blue), or centrin. Below, Rab11 intensity at centrosome was calculated. Representative of n=3 experiments, n>30 centrosomes/condition. P-value is p<1*10^-4. Bar is SE. (C) Isolated centrosomes from cells depleted of centriolin were stained for centrin (red), Evi5 (green), and Rab11 (green). Bar, 1 μm. Below, Rab11 (grey) or Evi5 (green) intensity at the centrosome was calculated. Representative of n=3 experiments with p-value <1×10^-4, n>50 centrosomes/treatment/experiment, Bar is SE. (D) Model for regulating Rab11 activity at the mother centriole. Cenexin organizes centriolin, Exocyst, and Evi5. The mother centriole localization of Evi5 can then act on Rab11-GTP and convert it to inactive Rab11-GDP.

Figure 4. Mother centriole appendage protein depletion disrupts recycling endosome function

(A) TEM of cells after endocytosis of Tfn-HRP-DAB, showing reaction product around the mother centriole and concentrated near appendages (4 small white arrows right panel). Arrowheads (black) depict “trails” of Tfn in left and right panels. Bar, 1 μm. (B) Image of a single centriole with electron-dense Tfn-HRP-DAB filled endosomes emanating from what appear to be centriole appendages (small white arrows indicate appendages; large black arrowheads indicate “endosome trails” of Tfn). Bar, 500 nm. (C-D) Cells were treated with centriolin shRNA, non-targeting (NT) shRNA (control), or cenexin shRNA. (C) Cells were incubated with Alexa Fluor 594-conjugated Tfn (red) and chased for up to 60 minutes with non-conjugated Tfn. Cells were fixed at the indicated times. Insets show cells with GFP-shRNA expression. (D) Biotinylated-Tfn filled cells were chased with non-conjugated Tfn for indicated times. Biotinylated-Tfn levels were quantified by densitometry (The difference between NT-shRNA and centriolin shRNA or cenexin shRNA at 40 minutes is statistically significant, p-value <0.05, n=3 experiments, bar is SE).