Figure 2.
Type I coronins confine actin to the bud neck. (A) Domain diagrams of type I coronins showing clear conservation of domain structure indicating the possibility of redundant function. (B) Representative images of COS-7 cells transfected with mCh-Rab7 (LE, gray), α actin-mNG (green), and control siRNAs, COR1C siRNAs, COR1C/1A siRNAs, COR1C/1B siRNAs, or COR1A/1B/1C siRNAs. Magnified inset (5 × 5 μm) below shows actin distribution on LE buds. Lines are shown adjacent to actual area measured in B so as not to obscure ROI. Note the extension of actin structures along the distal bud with the simultaneous depletion of two or more type I coronins (Videos 5 and 6). (C) Line scan analysis of signal distribution along the bud length for magnified inset examples shown in B. Lines are shown adjacent to actual area measured to not obscure area of interest. Note, actin fluorescent signal spreads into the matching bud signal as more type I coronins are depleted. (D) Quantification of data in B. Graph of percentage of actin-labeled buds with extended actin structures in cells treated with either control siRNAs (for 550 endosomes in n = 32 cells), COR1C siRNAs (for 598 endosomes in n = 23 cells), COR1C/1A siRNA (for 668 endosomes in n = 31 cells), COR1C/1B siRNA (for 721 endosomes in n = 29 cells), or COR1A/1B/1C siRNAs (for 578 endosomes in n = 29 cells), performed in triplicate. X indicates mean, and line indicates median. (E) Representative images of COS-7 cells transfected with mCh-Rab7 or GFP Rab7 (LE, gray), α actin-Halo (green), COR1A/1B/1C siRNAs, and with ARP3-mNG (ARP2/3 complex, magenta), mCh-FAM21 (WASH complex, magenta), GFP-VPS35 (retromer complex, magenta), or FLAG-ARDRB2-mNG (membrane cargo, magenta) reveals that the extended actin structures do not disrupt the recruitment of upstream cargo-sorting complexes or sorting of cargo into bud. Arrows indicate endosome bud. Statistical analyses were performed with one-way ANOVA, P value from Tukey’s test: ***, P < 0.001. Scale bars for whole cell = 5 μm; insets = 1 μm. (F) Summary figure showing changes in relative localization of actin and cargo-sorting components along the endosome.

Type I coronins confine actin to the bud neck. (A) Domain diagrams of type I coronins showing clear conservation of domain structure indicating the possibility of redundant function. (B) Representative images of COS-7 cells transfected with mCh-Rab7 (LE, gray), α actin-mNG (green), and control siRNAs, COR1C siRNAs, COR1C/1A siRNAs, COR1C/1B siRNAs, or COR1A/1B/1C siRNAs. Magnified inset (5 × 5 μm) below shows actin distribution on LE buds. Lines are shown adjacent to actual area measured in B so as not to obscure ROI. Note the extension of actin structures along the distal bud with the simultaneous depletion of two or more type I coronins (Videos 5 and 6). (C) Line scan analysis of signal distribution along the bud length for magnified inset examples shown in B. Lines are shown adjacent to actual area measured to not obscure area of interest. Note, actin fluorescent signal spreads into the matching bud signal as more type I coronins are depleted. (D) Quantification of data in B. Graph of percentage of actin-labeled buds with extended actin structures in cells treated with either control siRNAs (for 550 endosomes in n = 32 cells), COR1C siRNAs (for 598 endosomes in n = 23 cells), COR1C/1A siRNA (for 668 endosomes in n = 31 cells), COR1C/1B siRNA (for 721 endosomes in n = 29 cells), or COR1A/1B/1C siRNAs (for 578 endosomes in n = 29 cells), performed in triplicate. X indicates mean, and line indicates median. (E) Representative images of COS-7 cells transfected with mCh-Rab7 or GFP Rab7 (LE, gray), α actin-Halo (green), COR1A/1B/1C siRNAs, and with ARP3-mNG (ARP2/3 complex, magenta), mCh-FAM21 (WASH complex, magenta), GFP-VPS35 (retromer complex, magenta), or FLAG-ARDRB2-mNG (membrane cargo, magenta) reveals that the extended actin structures do not disrupt the recruitment of upstream cargo-sorting complexes or sorting of cargo into bud. Arrows indicate endosome bud. Statistical analyses were performed with one-way ANOVA, P value from Tukey’s test: ***, P < 0.001. Scale bars for whole cell = 5 μm; insets = 1 μm. (F) Summary figure showing changes in relative localization of actin and cargo-sorting components along the endosome.

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