Figure 3.
The COR1C CC is necessary and sufficient for COR1C recruitment. (A) Domain diagrams of the mutations made to test domain functionality in COR1C. ACT– indicates point mutations in actin-binding residues (R28D, K418E/K419E, K427E/K428E). ΔCC indicates a truncation removing the CC (COR1C residues 1–444). CC indicates predicted CC along with 30 upstream AAs (COR1C residues 414–474). (B) Representative images of COS-7 cells cotransfected with COR1A/1B/1C siRNA to deplete all type I coronins and with mCh-Rab7 (LE, gray), α actin-mNG (green), and either siRES COR1C-Halo, siRES COR1C ACT–-Halo, siRES COR1C ΔCC-Halo, siRES COR1C ACT– ΔCC-Halo, or siRES COR1C CC-Halo (magenta) to identify which domains are required to clear the extended actin structure from the distal bud. Magnified insets (5 × 5 μm) show representative examples of actin-positive endosome buds (at arrow). (C) Quantification of data in B. Graph shows percentage of actin-labeled buds with extended actin structures per cell from siRES COR1C-Halo: 480 endosomes in n = 22 cells; siRES COR1C ACT–-Halo: 578 endosomes in n = 21 cells; siRES COR1C ΔCC-Halo: 575 endosomes in n = 24 cells; siRES COR1C ACT– ΔCC-Halo: 549 endosomes in n = 22 cell; and siRES COR1C CC-Halo: 616 endosomes in n = 23 cells, performed in triplicate. (D) Representative images of COS-7 cells cotransfected with COR1C siRNA (for depletion), GFP-Rab7 (LE, gray), mCh-FAM21 (WASH complex, magenta), and siRES COR1C-Halo, siRES COR1C ACT–-Halo, siRES COR1C ΔCC-Halo, siRES COR1C ACT– ΔCC-Halo, or siRES COR1C CC-Halo (green) to measure the relative levels of recruitment to FAM21 marked buds for different COR1C mutants. Magnified insets (5 × 5 μm) of representative endosomes with FAM21 marked buds shown on right. Dashed line indicates where line scan analysis in D was done. (E) Line scan analysis of dashed lines shown in D are positioned to cross perpendicular to bud neck. Matching COR1C peaks indicate enrichment at the FAM21-labeled bud. Note that constructs lacking the CC do not form clear peaks. (F) Graph of data from experiment D; Halo signal enrichment at FAM21 buds relative to background is scored for the following samples: siRES COR1C-Halo: n = 79 endosomes in nine cells; siRES COR1C ACT–-Halo: n = 87 endosomes in nine cells; siRES COR1C ΔCC-Halo: n = 80 endosomes in nine cells; siRES COR1C ACT– ΔCC-Halo: n = 75 endosomes in 10 cells; and siRES COR1C CC-Halo: n = 67 endosomes in 11 cells, performed in triplicate. Note that a value of 1 indicates no enrichment over cytoplasmic background as in CC deletion. X indicates mean, and line indicates median. Statistical analyses were performed with one-way ANOVA, P value from Tukey’s test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars for whole cell = 5 μm; insets = 1 μm.

The COR1C CC is necessary and sufficient for COR1C recruitment. (A) Domain diagrams of the mutations made to test domain functionality in COR1C. ACT– indicates point mutations in actin-binding residues (R28D, K418E/K419E, K427E/K428E). ΔCC indicates a truncation removing the CC (COR1C residues 1–444). CC indicates predicted CC along with 30 upstream AAs (COR1C residues 414–474). (B) Representative images of COS-7 cells cotransfected with COR1A/1B/1C siRNA to deplete all type I coronins and with mCh-Rab7 (LE, gray), α actin-mNG (green), and either siRES COR1C-Halo, siRES COR1C ACT–-Halo, siRES COR1C ΔCC-Halo, siRES COR1C ACT– ΔCC-Halo, or siRES COR1C CC-Halo (magenta) to identify which domains are required to clear the extended actin structure from the distal bud. Magnified insets (5 × 5 μm) show representative examples of actin-positive endosome buds (at arrow). (C) Quantification of data in B. Graph shows percentage of actin-labeled buds with extended actin structures per cell from siRES COR1C-Halo: 480 endosomes in n = 22 cells; siRES COR1C ACT–-Halo: 578 endosomes in n = 21 cells; siRES COR1C ΔCC-Halo: 575 endosomes in n = 24 cells; siRES COR1C ACT– ΔCC-Halo: 549 endosomes in n = 22 cell; and siRES COR1C CC-Halo: 616 endosomes in n = 23 cells, performed in triplicate. (D) Representative images of COS-7 cells cotransfected with COR1C siRNA (for depletion), GFP-Rab7 (LE, gray), mCh-FAM21 (WASH complex, magenta), and siRES COR1C-Halo, siRES COR1C ACT–-Halo, siRES COR1C ΔCC-Halo, siRES COR1C ACT– ΔCC-Halo, or siRES COR1C CC-Halo (green) to measure the relative levels of recruitment to FAM21 marked buds for different COR1C mutants. Magnified insets (5 × 5 μm) of representative endosomes with FAM21 marked buds shown on right. Dashed line indicates where line scan analysis in D was done. (E) Line scan analysis of dashed lines shown in D are positioned to cross perpendicular to bud neck. Matching COR1C peaks indicate enrichment at the FAM21-labeled bud. Note that constructs lacking the CC do not form clear peaks. (F) Graph of data from experiment D; Halo signal enrichment at FAM21 buds relative to background is scored for the following samples: siRES COR1C-Halo: n = 79 endosomes in nine cells; siRES COR1C ACT–-Halo: n = 87 endosomes in nine cells; siRES COR1C ΔCC-Halo: n = 80 endosomes in nine cells; siRES COR1C ACT– ΔCC-Halo: n = 75 endosomes in 10 cells; and siRES COR1C CC-Halo: n = 67 endosomes in 11 cells, performed in triplicate. Note that a value of 1 indicates no enrichment over cytoplasmic background as in CC deletion. X indicates mean, and line indicates median. Statistical analyses were performed with one-way ANOVA, P value from Tukey’s test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars for whole cell = 5 μm; insets = 1 μm.

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