Structured Review

Proteintech rabbit cor1c polyclonal antibody
Coronin and actin regulatory factors are partitioned to the base of the bud during fission. (A) Representative images of COS-7 cells transfected with GFP-Rab7 (LE, magenta) and mCh-FAM21 (WASH complex, green). Magnified inset (5 × 5 μm) below shows time lapse of a representative fission event. Note: FAM21 signal localizes along entire length of bud, and signal is present on the post-fission bud. Block arrowhead indicates the bud neck, and line arrowhead indicates departing bud . (B) Line scan analysis along the length of endosome bud in pre-fission frame from A shows FAM21 signal labels the length of the bud. Lines are shown in A adjacent to actual area measured so as not to obscure ROI. (C) Fission events as in A were scored for FAM21 enrichment at the bud neck in the pre-fission, fission, and post-fission frames. For post-fission frames, both the bud neck and departed bud were scored for positive FAM21 signal. Table shows data as percentage of fission events with enrichment at each stage ( n = 37 fission events in 16 cells, performed in triplicate). Model indicates where enrichment was assessed at each stage of fission. (D–F) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and ARP3-mEm (ARP2/3 complex, green). Secondary inset shows departed bud that moved out of primary inset. In F, n = 24 fission events in 11 cells, performed in triplicate . (G–I) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and α-actin-mNG (actin structure, green). For I, n = 19 fission events in 14 cells, performed in triplicate . (J–L) As in A–C, for COS-7 cells transfected with GFP-Rab7 (LE, magenta) and <t>COR1C</t> Halo (green). For L, n = 36 fission events in 21 cells, performed in triplicate. Scale bars for whole cell = 5 μm; insets = 1 μm . (M) Summary diagram of how actin recruitment and regulatory factors divide during fission.
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1) Product Images from "Coronin 1C restricts endosomal branched actin to organize ER contact and endosome fission"

Article Title: Coronin 1C restricts endosomal branched actin to organize ER contact and endosome fission

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.202110089

Coronin and actin regulatory factors are partitioned to the base of the bud during fission. (A) Representative images of COS-7 cells transfected with GFP-Rab7 (LE, magenta) and mCh-FAM21 (WASH complex, green). Magnified inset (5 × 5 μm) below shows time lapse of a representative fission event. Note: FAM21 signal localizes along entire length of bud, and signal is present on the post-fission bud. Block arrowhead indicates the bud neck, and line arrowhead indicates departing bud . (B) Line scan analysis along the length of endosome bud in pre-fission frame from A shows FAM21 signal labels the length of the bud. Lines are shown in A adjacent to actual area measured so as not to obscure ROI. (C) Fission events as in A were scored for FAM21 enrichment at the bud neck in the pre-fission, fission, and post-fission frames. For post-fission frames, both the bud neck and departed bud were scored for positive FAM21 signal. Table shows data as percentage of fission events with enrichment at each stage ( n = 37 fission events in 16 cells, performed in triplicate). Model indicates where enrichment was assessed at each stage of fission. (D–F) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and ARP3-mEm (ARP2/3 complex, green). Secondary inset shows departed bud that moved out of primary inset. In F, n = 24 fission events in 11 cells, performed in triplicate . (G–I) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and α-actin-mNG (actin structure, green). For I, n = 19 fission events in 14 cells, performed in triplicate . (J–L) As in A–C, for COS-7 cells transfected with GFP-Rab7 (LE, magenta) and COR1C Halo (green). For L, n = 36 fission events in 21 cells, performed in triplicate. Scale bars for whole cell = 5 μm; insets = 1 μm . (M) Summary diagram of how actin recruitment and regulatory factors divide during fission.
Figure Legend Snippet: Coronin and actin regulatory factors are partitioned to the base of the bud during fission. (A) Representative images of COS-7 cells transfected with GFP-Rab7 (LE, magenta) and mCh-FAM21 (WASH complex, green). Magnified inset (5 × 5 μm) below shows time lapse of a representative fission event. Note: FAM21 signal localizes along entire length of bud, and signal is present on the post-fission bud. Block arrowhead indicates the bud neck, and line arrowhead indicates departing bud . (B) Line scan analysis along the length of endosome bud in pre-fission frame from A shows FAM21 signal labels the length of the bud. Lines are shown in A adjacent to actual area measured so as not to obscure ROI. (C) Fission events as in A were scored for FAM21 enrichment at the bud neck in the pre-fission, fission, and post-fission frames. For post-fission frames, both the bud neck and departed bud were scored for positive FAM21 signal. Table shows data as percentage of fission events with enrichment at each stage ( n = 37 fission events in 16 cells, performed in triplicate). Model indicates where enrichment was assessed at each stage of fission. (D–F) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and ARP3-mEm (ARP2/3 complex, green). Secondary inset shows departed bud that moved out of primary inset. In F, n = 24 fission events in 11 cells, performed in triplicate . (G–I) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and α-actin-mNG (actin structure, green). For I, n = 19 fission events in 14 cells, performed in triplicate . (J–L) As in A–C, for COS-7 cells transfected with GFP-Rab7 (LE, magenta) and COR1C Halo (green). For L, n = 36 fission events in 21 cells, performed in triplicate. Scale bars for whole cell = 5 μm; insets = 1 μm . (M) Summary diagram of how actin recruitment and regulatory factors divide during fission.

Techniques Used: Transfection, Blocking Assay

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 ( and ). (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.
Figure Legend Snippet: 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 ( and ). (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.

Techniques Used: Transfection, Labeling

Immunoblot of type I coronin depletion in COS-7 cells. Immunoblots for combination depletions tested in . Blots show the same samples run three separate times to blot for COR1A, COR1B, and COR1C. Data show that type I coronins can be depleted efficiently individually or in combinations. Source data are available for this figure: .
Figure Legend Snippet: Immunoblot of type I coronin depletion in COS-7 cells. Immunoblots for combination depletions tested in . Blots show the same samples run three separate times to blot for COR1A, COR1B, and COR1C. Data show that type I coronins can be depleted efficiently individually or in combinations. Source data are available for this figure: .

Techniques Used: Western Blot

Immunoblot of type I coronin depletion and rescue in COS-7 cells. (A) Representative immunoblots for type I coronin depletion (COR1A, COR1B, and COR1C) and rescue as in . Data show that type I coronins can be depleted efficiently and that the siRES COR1C constructs express well and at comparable levels for rescue (analysis was performed in triplicate). (B) Immunoblots for type I coronin depletion and rescue as in showing relative expression of rescue constructs and endogenous COR1C. Representative immunoblots probed with antibody to COR1C, GFP, and GAPDH. (C) Quantification of rescue blots in B. Table shows average normalized ratios from three replicates (blots). Values were calculated as indicated in column headers. Briefly, the first column demonstrates clear KD. The second column demonstrates that expression is comparable between exogenously expressed mutants and is also comparable with endogenous. The third column demonstrates that the relative exogenous rescue expression is comparable to WT rescue. The final column is an estimate of CC expression relative to endogenous COR1C based on the average ratio of anti-GFP to anti-COR1C signal, suggesting that the CC is also not expressed above endogenous. This was necessary because the CC is not detectable via the anti-COR1C antibody and so could not be probed for in the same blot. Source data are available for this figure: .
Figure Legend Snippet: Immunoblot of type I coronin depletion and rescue in COS-7 cells. (A) Representative immunoblots for type I coronin depletion (COR1A, COR1B, and COR1C) and rescue as in . Data show that type I coronins can be depleted efficiently and that the siRES COR1C constructs express well and at comparable levels for rescue (analysis was performed in triplicate). (B) Immunoblots for type I coronin depletion and rescue as in showing relative expression of rescue constructs and endogenous COR1C. Representative immunoblots probed with antibody to COR1C, GFP, and GAPDH. (C) Quantification of rescue blots in B. Table shows average normalized ratios from three replicates (blots). Values were calculated as indicated in column headers. Briefly, the first column demonstrates clear KD. The second column demonstrates that expression is comparable between exogenously expressed mutants and is also comparable with endogenous. The third column demonstrates that the relative exogenous rescue expression is comparable to WT rescue. The final column is an estimate of CC expression relative to endogenous COR1C based on the average ratio of anti-GFP to anti-COR1C signal, suggesting that the CC is also not expressed above endogenous. This was necessary because the CC is not detectable via the anti-COR1C antibody and so could not be probed for in the same blot. Source data are available for this figure: .

Techniques Used: Western Blot, Construct, Expressing

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.
Figure Legend Snippet: 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.

Techniques Used: Binding Assay, Labeling, Construct

COR1C regulates ARP2/3 complex activity at the endosome bud. (A) Representative images of COS-7 cells cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with mCh-Rab7 (LE, gray), α actin-Halo (green), and ARP3-mNG (magenta). Buds with extended actin (at arrow) were tracked before and after addition of an ARP2/3 complex inhibitor (150 μM CK-666) in merged magnified insets on right (5 × 5 μm). n = 10 cells. (B) Diagram of the ARP3-V5-TurboID biotinylation experiment. HeLa cells were cotransfected with ARP2/3-V5-TurboID and GFP-Rab7, COR1C-GFP, or COR1C-ACT– ΔCC-GFP and treated with biotin, and then biotinylated proteins were bound and eluted from α-biotin beads. (C) Representative V5 and GFP immunoblot from experiment in B shows high levels of biotinylation for full-length COR1C compared with COR1C mutant or Rab7 control. (D) Quantification of immunoblot, as shown in C. Pulldown numbers were calculated by normalizing elute signal with the load signal, performed in triplicate. 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. Source data are available for this figure: .
Figure Legend Snippet: COR1C regulates ARP2/3 complex activity at the endosome bud. (A) Representative images of COS-7 cells cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with mCh-Rab7 (LE, gray), α actin-Halo (green), and ARP3-mNG (magenta). Buds with extended actin (at arrow) were tracked before and after addition of an ARP2/3 complex inhibitor (150 μM CK-666) in merged magnified insets on right (5 × 5 μm). n = 10 cells. (B) Diagram of the ARP3-V5-TurboID biotinylation experiment. HeLa cells were cotransfected with ARP2/3-V5-TurboID and GFP-Rab7, COR1C-GFP, or COR1C-ACT– ΔCC-GFP and treated with biotin, and then biotinylated proteins were bound and eluted from α-biotin beads. (C) Representative V5 and GFP immunoblot from experiment in B shows high levels of biotinylation for full-length COR1C compared with COR1C mutant or Rab7 control. (D) Quantification of immunoblot, as shown in C. Pulldown numbers were calculated by normalizing elute signal with the load signal, performed in triplicate. 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. Source data are available for this figure: .

Techniques Used: Activity Assay, Western Blot, Mutagenesis

Additional TurboID controls indicating that ARP3 TurboID activity is specific. (A) Representative immunoblot showing that ARP3 V5 TurboID and a cytoplasmic nonspecific TurboID are able to biotinylate endogenous COR1C. Blots show the same samples run twice to blot for either endogenous COR1C or V5. Performed in triplicate. (B) Quantification of blots in A showing the amount of COR1C signal in the elute normalized first to the relevant TurboID V5 signal in the input and then to the max value within each replicate. (C) Representative immunoblot showing that ARP3 V5 TurboID has uniquely specific biotinylation of COR1C as shown by its ability to biotinylate only COR1C-GFP and not COR1C ACT– ΔCC-GFP. In contrast, the Cyto V5 TurboID biotinylates proteins nonspecifically in the cytoplasm as shown by similar biotinylation profiles for both COR1C-GFP and COR1C ACT–-ΔCC-GFP. Performed in triplicate. (D) Quantification of blots in C, from three replicates. Graph shows the ratio of COR1C-GFP to COR1C ACT–ΔCC-GFP signal in the elution. Signals in ratio were normalized to both relevant GFP and V5 input signals and then to the minimum value in each replicate. Statistical analysis was performed via two-tailed Student’s t test; *, P < 0.05. X indicates mean, and line indicates median. Source data are available for this figure: .
Figure Legend Snippet: Additional TurboID controls indicating that ARP3 TurboID activity is specific. (A) Representative immunoblot showing that ARP3 V5 TurboID and a cytoplasmic nonspecific TurboID are able to biotinylate endogenous COR1C. Blots show the same samples run twice to blot for either endogenous COR1C or V5. Performed in triplicate. (B) Quantification of blots in A showing the amount of COR1C signal in the elute normalized first to the relevant TurboID V5 signal in the input and then to the max value within each replicate. (C) Representative immunoblot showing that ARP3 V5 TurboID has uniquely specific biotinylation of COR1C as shown by its ability to biotinylate only COR1C-GFP and not COR1C ACT– ΔCC-GFP. In contrast, the Cyto V5 TurboID biotinylates proteins nonspecifically in the cytoplasm as shown by similar biotinylation profiles for both COR1C-GFP and COR1C ACT–-ΔCC-GFP. Performed in triplicate. (D) Quantification of blots in C, from three replicates. Graph shows the ratio of COR1C-GFP to COR1C ACT–ΔCC-GFP signal in the elution. Signals in ratio were normalized to both relevant GFP and V5 input signals and then to the minimum value in each replicate. Statistical analysis was performed via two-tailed Student’s t test; *, P < 0.05. X indicates mean, and line indicates median. Source data are available for this figure: .

Techniques Used: Activity Assay, Western Blot, Two Tailed Test

Vacuole diameter and bud length are not changed significantly in KD rescue experiments. (A) For each bud scored for fission in , vacuole diameter was also measured. Graph shows average vacuole diameter per cell, averaged per condition. No significant changes were observed in any condition, showing that only fission is affected and not other measures of endosome morphology. Data for graph from Halo E-vec: 139 endosomes in n = 15 cells; COR1C: 122 endosomes in n = 15 cells; COR1C ACT–: 213 endosomes in n = 17 cells; COR1C ΔCC: 281 endosomes in n = 18 cells; COR1C ACT– ΔCC: 296 endosomes n = 17 cells; and COR1C CC: 331 endosomes in n = 22 cells. X indicates mean, and line indicates median. Statistical analyses were performed with one-way ANOVA. (B) As in A, except FAM21-positive bud length was measured instead of vacuole diameter. Again, no significant changes were observed in any condition showing that only fission is affected and not other measures of endosome morphology. Data for graph from Halo E-vec: 139 endosomes in n = 15 cells; COR1C: 122 endosomes in n = 15 cells; COR1C ACT–: 213 endosomes in n = 17 cells; COR1C ΔCC: 281 endosomes in n = 18 cells; COR1C ACT– ΔCC: 296 endosomes n = 17 cells; and COR1C CC: 331 endosomes in n = 22 cells. X indicates mean, and line indicates median. Statistical analyses were performed with one-way ANOVA.
Figure Legend Snippet: Vacuole diameter and bud length are not changed significantly in KD rescue experiments. (A) For each bud scored for fission in , vacuole diameter was also measured. Graph shows average vacuole diameter per cell, averaged per condition. No significant changes were observed in any condition, showing that only fission is affected and not other measures of endosome morphology. Data for graph from Halo E-vec: 139 endosomes in n = 15 cells; COR1C: 122 endosomes in n = 15 cells; COR1C ACT–: 213 endosomes in n = 17 cells; COR1C ΔCC: 281 endosomes in n = 18 cells; COR1C ACT– ΔCC: 296 endosomes n = 17 cells; and COR1C CC: 331 endosomes in n = 22 cells. X indicates mean, and line indicates median. Statistical analyses were performed with one-way ANOVA. (B) As in A, except FAM21-positive bud length was measured instead of vacuole diameter. Again, no significant changes were observed in any condition showing that only fission is affected and not other measures of endosome morphology. Data for graph from Halo E-vec: 139 endosomes in n = 15 cells; COR1C: 122 endosomes in n = 15 cells; COR1C ACT–: 213 endosomes in n = 17 cells; COR1C ΔCC: 281 endosomes in n = 18 cells; COR1C ACT– ΔCC: 296 endosomes n = 17 cells; and COR1C CC: 331 endosomes in n = 22 cells. X indicates mean, and line indicates median. Statistical analyses were performed with one-way ANOVA.

Techniques Used:

The COR1C CC limits bud actin to facilitate ER contact, endosome fission, and CI-M6PR sorting. (A) Representative images of LE buds stable for duration of acquisition in conditions that did not rescued fission rate (C). COS-7 cells were cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, magenta), mCh-FAM21 (WASH complex, green), and with either Halo E-vec, siRES COR1C ΔCC-Halo, or siRES COR1C ACT– ΔCC-Halo. Arrows indicate bud of interest. (B) Representative images of LE fission events in conditions that rescued fission rate (C). COS-7 cells were cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, magenta), mCh-FAM21 (WASH complex, green), and with either siRES COR1C-Halo, siRES COR1C ACT–-Halo, or siRES COR1C CC-Halo. Arrows indicate bud of interest. (C) Quantification of data in A and B. Graph shows percentage of FAM21-labeled LE buds that underwent fission per cell during a 2-min time lapse. Note that only constructs containing the CC were able to restore fission. Data for graph from Halo E-vec: 139 endosomes in n = 15 cells; siRES COR1C: 122 endosomes in n = 15 cells; siRES COR1C ACT–: 213 endosomes in n = 17 cells; siRES COR1C ΔCC: 281 endosomes in n = 18 cells; siRES COR1C ACT– ΔCC: 296 endosomes n = 17 cells; and siRES COR1C CC: 331 endosomes in n = 22 cells, performed in triplicate. (D) Representative images of the M6PR trafficking assay. The relative fluorescence intensity of internalized anti-CI-MPR antibody immunostaining reveals trafficking of internalized anti-CI-MPR to TGN in Cos7 cells cotransfected with control siRNA, COR1A/1B/1C siRNAs to deplete all type I coronins, or FAM21 siRNA and with either GFP E-vec, siRES COR1C-GFP, or siRES COR1C ΔCC-GFP (not depicted). Cells were stained to mark CI-M6PR (green) and Giantin (Golgi, magenta). Dispersed vesicular CI-M6PR signal is indicative of failure to recycle, whereas concentrated CI-M6PR signal at the Golgi indicates normal retrograde sorting. (E) Quantification of data in D. Graph shows the background-corrected ratio of CI-M6PR signal localized at the Golgi relative to the vesicular signal in the cytoplasm such that larger values indicate less efficient retrograde recycling. Data for graph from control siRNAs: n = 24 cells; FAM21 siRNAs: n = 23 cells; COR1A/1B/1C siRNAs + E-vec: n = 25; COR1A/1B/1C siRNAs + siRES COR1C: n = 24 cells; COR1A/1B/1C siRNAs + siRES COR1C ΔCC: n = 24 cells; COR1A/1B/1C siRNAs + siRES COR1C CC: n = 26 cells, performed in triplicate. (F) Representative images of COS-7 cells cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, gray), mCh-FAM21 (WASH complex, green), BFP-Sec61β (ER, magenta), and either Halo E-vec, siRES COR1C-Halo, siRES COR1C ACT–-Halo, siRES COR1C ΔCC-Halo, siRES COR1C ACT– ΔCC-Halo, or siRES COR1C CC-Halo (gray, left panel). Magnified inset (5 × 5 μm) on right show representative examples of ER contact with endosomes. Note: Vacuolar contact with ER is always preserved, whereas ER contact with the FAM21 labeled bud is not. (G) Line scan analysis of dashed lines shown in D are positioned from the rear vacuolar contact across the length of the FAM21-labeled buds. Double ER peaks (shaded purple) are observed when bud contact is rescued. The first peak is always present and corresponds to the vacuolar contact. The second ER peak which aligns with the FAM21 indicates proper ER recruitment to the bud. Note that ER contact with bud is dependent on the presence of the CC domain. (H) Quantification of data in D. All FAM21-positive LE buds in areas with resolvable ER were tracked, and ER contact was scored as the percentage of time during a 2-min video that contact is maintained. Data for graph from Halo E-vec: 109 endosomes in n = 14 cells; COR1C: 134 endosomes in n = 17 cells; COR1C ACT–: 135 endosomes in n = 17 cells; COR1C ΔCC: 123 endosomes n = 17 cells; COR1C ACT– ΔCC: 105 endosomes in n = 15 cells; or COR1C CC: 137 endosomes in n = 19 cells, performed in triplicate. 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 < 0.001. Scale bars for whole cell = 5 μm; insets = 1 μm.
Figure Legend Snippet: The COR1C CC limits bud actin to facilitate ER contact, endosome fission, and CI-M6PR sorting. (A) Representative images of LE buds stable for duration of acquisition in conditions that did not rescued fission rate (C). COS-7 cells were cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, magenta), mCh-FAM21 (WASH complex, green), and with either Halo E-vec, siRES COR1C ΔCC-Halo, or siRES COR1C ACT– ΔCC-Halo. Arrows indicate bud of interest. (B) Representative images of LE fission events in conditions that rescued fission rate (C). COS-7 cells were cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, magenta), mCh-FAM21 (WASH complex, green), and with either siRES COR1C-Halo, siRES COR1C ACT–-Halo, or siRES COR1C CC-Halo. Arrows indicate bud of interest. (C) Quantification of data in A and B. Graph shows percentage of FAM21-labeled LE buds that underwent fission per cell during a 2-min time lapse. Note that only constructs containing the CC were able to restore fission. Data for graph from Halo E-vec: 139 endosomes in n = 15 cells; siRES COR1C: 122 endosomes in n = 15 cells; siRES COR1C ACT–: 213 endosomes in n = 17 cells; siRES COR1C ΔCC: 281 endosomes in n = 18 cells; siRES COR1C ACT– ΔCC: 296 endosomes n = 17 cells; and siRES COR1C CC: 331 endosomes in n = 22 cells, performed in triplicate. (D) Representative images of the M6PR trafficking assay. The relative fluorescence intensity of internalized anti-CI-MPR antibody immunostaining reveals trafficking of internalized anti-CI-MPR to TGN in Cos7 cells cotransfected with control siRNA, COR1A/1B/1C siRNAs to deplete all type I coronins, or FAM21 siRNA and with either GFP E-vec, siRES COR1C-GFP, or siRES COR1C ΔCC-GFP (not depicted). Cells were stained to mark CI-M6PR (green) and Giantin (Golgi, magenta). Dispersed vesicular CI-M6PR signal is indicative of failure to recycle, whereas concentrated CI-M6PR signal at the Golgi indicates normal retrograde sorting. (E) Quantification of data in D. Graph shows the background-corrected ratio of CI-M6PR signal localized at the Golgi relative to the vesicular signal in the cytoplasm such that larger values indicate less efficient retrograde recycling. Data for graph from control siRNAs: n = 24 cells; FAM21 siRNAs: n = 23 cells; COR1A/1B/1C siRNAs + E-vec: n = 25; COR1A/1B/1C siRNAs + siRES COR1C: n = 24 cells; COR1A/1B/1C siRNAs + siRES COR1C ΔCC: n = 24 cells; COR1A/1B/1C siRNAs + siRES COR1C CC: n = 26 cells, performed in triplicate. (F) Representative images of COS-7 cells cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, gray), mCh-FAM21 (WASH complex, green), BFP-Sec61β (ER, magenta), and either Halo E-vec, siRES COR1C-Halo, siRES COR1C ACT–-Halo, siRES COR1C ΔCC-Halo, siRES COR1C ACT– ΔCC-Halo, or siRES COR1C CC-Halo (gray, left panel). Magnified inset (5 × 5 μm) on right show representative examples of ER contact with endosomes. Note: Vacuolar contact with ER is always preserved, whereas ER contact with the FAM21 labeled bud is not. (G) Line scan analysis of dashed lines shown in D are positioned from the rear vacuolar contact across the length of the FAM21-labeled buds. Double ER peaks (shaded purple) are observed when bud contact is rescued. The first peak is always present and corresponds to the vacuolar contact. The second ER peak which aligns with the FAM21 indicates proper ER recruitment to the bud. Note that ER contact with bud is dependent on the presence of the CC domain. (H) Quantification of data in D. All FAM21-positive LE buds in areas with resolvable ER were tracked, and ER contact was scored as the percentage of time during a 2-min video that contact is maintained. Data for graph from Halo E-vec: 109 endosomes in n = 14 cells; COR1C: 134 endosomes in n = 17 cells; COR1C ACT–: 135 endosomes in n = 17 cells; COR1C ΔCC: 123 endosomes n = 17 cells; COR1C ACT– ΔCC: 105 endosomes in n = 15 cells; or COR1C CC: 137 endosomes in n = 19 cells, performed in triplicate. 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 < 0.001. Scale bars for whole cell = 5 μm; insets = 1 μm.

Techniques Used: Labeling, Construct, Fluorescence, Immunostaining, Staining

Model of actin/actin regulatory factors position and ER MCS just before fission. The WASH complex recruits and activates ARP2/3 to form a stabilizing actin structure on the endosome bud. COR1C is recruited to these actin structures to bind and counter act ARP2/3 actin nucleation via its CC. This results in an actin structure large enough to stabilize the bud, allowing cargo to sort, but small enough to allow ER recruitment and bud fission.
Figure Legend Snippet: Model of actin/actin regulatory factors position and ER MCS just before fission. The WASH complex recruits and activates ARP2/3 to form a stabilizing actin structure on the endosome bud. COR1C is recruited to these actin structures to bind and counter act ARP2/3 actin nucleation via its CC. This results in an actin structure large enough to stabilize the bud, allowing cargo to sort, but small enough to allow ER recruitment and bud fission.

Techniques Used:

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    Proteintech rabbit cor1c polyclonal antibody
    Coronin and actin regulatory factors are partitioned to the base of the bud during fission. (A) Representative images of COS-7 cells transfected with GFP-Rab7 (LE, magenta) and mCh-FAM21 (WASH complex, green). Magnified inset (5 × 5 μm) below shows time lapse of a representative fission event. Note: FAM21 signal localizes along entire length of bud, and signal is present on the post-fission bud. Block arrowhead indicates the bud neck, and line arrowhead indicates departing bud . (B) Line scan analysis along the length of endosome bud in pre-fission frame from A shows FAM21 signal labels the length of the bud. Lines are shown in A adjacent to actual area measured so as not to obscure ROI. (C) Fission events as in A were scored for FAM21 enrichment at the bud neck in the pre-fission, fission, and post-fission frames. For post-fission frames, both the bud neck and departed bud were scored for positive FAM21 signal. Table shows data as percentage of fission events with enrichment at each stage ( n = 37 fission events in 16 cells, performed in triplicate). Model indicates where enrichment was assessed at each stage of fission. (D–F) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and ARP3-mEm (ARP2/3 complex, green). Secondary inset shows departed bud that moved out of primary inset. In F, n = 24 fission events in 11 cells, performed in triplicate . (G–I) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and α-actin-mNG (actin structure, green). For I, n = 19 fission events in 14 cells, performed in triplicate . (J–L) As in A–C, for COS-7 cells transfected with GFP-Rab7 (LE, magenta) and <t>COR1C</t> Halo (green). For L, n = 36 fission events in 21 cells, performed in triplicate. Scale bars for whole cell = 5 μm; insets = 1 μm . (M) Summary diagram of how actin recruitment and regulatory factors divide during fission.
    Rabbit Cor1c Polyclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Coronin and actin regulatory factors are partitioned to the base of the bud during fission. (A) Representative images of COS-7 cells transfected with GFP-Rab7 (LE, magenta) and mCh-FAM21 (WASH complex, green). Magnified inset (5 × 5 μm) below shows time lapse of a representative fission event. Note: FAM21 signal localizes along entire length of bud, and signal is present on the post-fission bud. Block arrowhead indicates the bud neck, and line arrowhead indicates departing bud . (B) Line scan analysis along the length of endosome bud in pre-fission frame from A shows FAM21 signal labels the length of the bud. Lines are shown in A adjacent to actual area measured so as not to obscure ROI. (C) Fission events as in A were scored for FAM21 enrichment at the bud neck in the pre-fission, fission, and post-fission frames. For post-fission frames, both the bud neck and departed bud were scored for positive FAM21 signal. Table shows data as percentage of fission events with enrichment at each stage ( n = 37 fission events in 16 cells, performed in triplicate). Model indicates where enrichment was assessed at each stage of fission. (D–F) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and ARP3-mEm (ARP2/3 complex, green). Secondary inset shows departed bud that moved out of primary inset. In F, n = 24 fission events in 11 cells, performed in triplicate . (G–I) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and α-actin-mNG (actin structure, green). For I, n = 19 fission events in 14 cells, performed in triplicate . (J–L) As in A–C, for COS-7 cells transfected with GFP-Rab7 (LE, magenta) and COR1C Halo (green). For L, n = 36 fission events in 21 cells, performed in triplicate. Scale bars for whole cell = 5 μm; insets = 1 μm . (M) Summary diagram of how actin recruitment and regulatory factors divide during fission.

    Journal: The Journal of Cell Biology

    Article Title: Coronin 1C restricts endosomal branched actin to organize ER contact and endosome fission

    doi: 10.1083/jcb.202110089

    Figure Lengend Snippet: Coronin and actin regulatory factors are partitioned to the base of the bud during fission. (A) Representative images of COS-7 cells transfected with GFP-Rab7 (LE, magenta) and mCh-FAM21 (WASH complex, green). Magnified inset (5 × 5 μm) below shows time lapse of a representative fission event. Note: FAM21 signal localizes along entire length of bud, and signal is present on the post-fission bud. Block arrowhead indicates the bud neck, and line arrowhead indicates departing bud . (B) Line scan analysis along the length of endosome bud in pre-fission frame from A shows FAM21 signal labels the length of the bud. Lines are shown in A adjacent to actual area measured so as not to obscure ROI. (C) Fission events as in A were scored for FAM21 enrichment at the bud neck in the pre-fission, fission, and post-fission frames. For post-fission frames, both the bud neck and departed bud were scored for positive FAM21 signal. Table shows data as percentage of fission events with enrichment at each stage ( n = 37 fission events in 16 cells, performed in triplicate). Model indicates where enrichment was assessed at each stage of fission. (D–F) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and ARP3-mEm (ARP2/3 complex, green). Secondary inset shows departed bud that moved out of primary inset. In F, n = 24 fission events in 11 cells, performed in triplicate . (G–I) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and α-actin-mNG (actin structure, green). For I, n = 19 fission events in 14 cells, performed in triplicate . (J–L) As in A–C, for COS-7 cells transfected with GFP-Rab7 (LE, magenta) and COR1C Halo (green). For L, n = 36 fission events in 21 cells, performed in triplicate. Scale bars for whole cell = 5 μm; insets = 1 μm . (M) Summary diagram of how actin recruitment and regulatory factors divide during fission.

    Article Snippet: For immunoblotting, rabbit COR1C polyclonal antibody (14749-1-AP; Proteintech) was used at 1:2,000; rabbit COR1B polyclonal antibody (ab119714; Abcam) at 1:2,000; rabbit COR1A polyclonal antibody (ab123574; Abcam) at 1:2,000; muse V5 monoclonal antibody (R960CUS; Thermo Fisher Scientific) at 1:2,000; mouse GFP monoclonal antibody (Clontech Labs 3P Living Colors A.v.

    Techniques: Transfection, Blocking Assay

    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 ( and ). (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.

    Journal: The Journal of Cell Biology

    Article Title: Coronin 1C restricts endosomal branched actin to organize ER contact and endosome fission

    doi: 10.1083/jcb.202110089

    Figure Lengend Snippet: 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 ( and ). (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.

    Article Snippet: For immunoblotting, rabbit COR1C polyclonal antibody (14749-1-AP; Proteintech) was used at 1:2,000; rabbit COR1B polyclonal antibody (ab119714; Abcam) at 1:2,000; rabbit COR1A polyclonal antibody (ab123574; Abcam) at 1:2,000; muse V5 monoclonal antibody (R960CUS; Thermo Fisher Scientific) at 1:2,000; mouse GFP monoclonal antibody (Clontech Labs 3P Living Colors A.v.

    Techniques: Transfection, Labeling

    Immunoblot of type I coronin depletion in COS-7 cells. Immunoblots for combination depletions tested in . Blots show the same samples run three separate times to blot for COR1A, COR1B, and COR1C. Data show that type I coronins can be depleted efficiently individually or in combinations. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Coronin 1C restricts endosomal branched actin to organize ER contact and endosome fission

    doi: 10.1083/jcb.202110089

    Figure Lengend Snippet: Immunoblot of type I coronin depletion in COS-7 cells. Immunoblots for combination depletions tested in . Blots show the same samples run three separate times to blot for COR1A, COR1B, and COR1C. Data show that type I coronins can be depleted efficiently individually or in combinations. Source data are available for this figure: .

    Article Snippet: For immunoblotting, rabbit COR1C polyclonal antibody (14749-1-AP; Proteintech) was used at 1:2,000; rabbit COR1B polyclonal antibody (ab119714; Abcam) at 1:2,000; rabbit COR1A polyclonal antibody (ab123574; Abcam) at 1:2,000; muse V5 monoclonal antibody (R960CUS; Thermo Fisher Scientific) at 1:2,000; mouse GFP monoclonal antibody (Clontech Labs 3P Living Colors A.v.

    Techniques: Western Blot

    Immunoblot of type I coronin depletion and rescue in COS-7 cells. (A) Representative immunoblots for type I coronin depletion (COR1A, COR1B, and COR1C) and rescue as in . Data show that type I coronins can be depleted efficiently and that the siRES COR1C constructs express well and at comparable levels for rescue (analysis was performed in triplicate). (B) Immunoblots for type I coronin depletion and rescue as in showing relative expression of rescue constructs and endogenous COR1C. Representative immunoblots probed with antibody to COR1C, GFP, and GAPDH. (C) Quantification of rescue blots in B. Table shows average normalized ratios from three replicates (blots). Values were calculated as indicated in column headers. Briefly, the first column demonstrates clear KD. The second column demonstrates that expression is comparable between exogenously expressed mutants and is also comparable with endogenous. The third column demonstrates that the relative exogenous rescue expression is comparable to WT rescue. The final column is an estimate of CC expression relative to endogenous COR1C based on the average ratio of anti-GFP to anti-COR1C signal, suggesting that the CC is also not expressed above endogenous. This was necessary because the CC is not detectable via the anti-COR1C antibody and so could not be probed for in the same blot. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Coronin 1C restricts endosomal branched actin to organize ER contact and endosome fission

    doi: 10.1083/jcb.202110089

    Figure Lengend Snippet: Immunoblot of type I coronin depletion and rescue in COS-7 cells. (A) Representative immunoblots for type I coronin depletion (COR1A, COR1B, and COR1C) and rescue as in . Data show that type I coronins can be depleted efficiently and that the siRES COR1C constructs express well and at comparable levels for rescue (analysis was performed in triplicate). (B) Immunoblots for type I coronin depletion and rescue as in showing relative expression of rescue constructs and endogenous COR1C. Representative immunoblots probed with antibody to COR1C, GFP, and GAPDH. (C) Quantification of rescue blots in B. Table shows average normalized ratios from three replicates (blots). Values were calculated as indicated in column headers. Briefly, the first column demonstrates clear KD. The second column demonstrates that expression is comparable between exogenously expressed mutants and is also comparable with endogenous. The third column demonstrates that the relative exogenous rescue expression is comparable to WT rescue. The final column is an estimate of CC expression relative to endogenous COR1C based on the average ratio of anti-GFP to anti-COR1C signal, suggesting that the CC is also not expressed above endogenous. This was necessary because the CC is not detectable via the anti-COR1C antibody and so could not be probed for in the same blot. Source data are available for this figure: .

    Article Snippet: For immunoblotting, rabbit COR1C polyclonal antibody (14749-1-AP; Proteintech) was used at 1:2,000; rabbit COR1B polyclonal antibody (ab119714; Abcam) at 1:2,000; rabbit COR1A polyclonal antibody (ab123574; Abcam) at 1:2,000; muse V5 monoclonal antibody (R960CUS; Thermo Fisher Scientific) at 1:2,000; mouse GFP monoclonal antibody (Clontech Labs 3P Living Colors A.v.

    Techniques: Western Blot, Construct, Expressing

    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.

    Journal: The Journal of Cell Biology

    Article Title: Coronin 1C restricts endosomal branched actin to organize ER contact and endosome fission

    doi: 10.1083/jcb.202110089

    Figure Lengend Snippet: 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.

    Article Snippet: For immunoblotting, rabbit COR1C polyclonal antibody (14749-1-AP; Proteintech) was used at 1:2,000; rabbit COR1B polyclonal antibody (ab119714; Abcam) at 1:2,000; rabbit COR1A polyclonal antibody (ab123574; Abcam) at 1:2,000; muse V5 monoclonal antibody (R960CUS; Thermo Fisher Scientific) at 1:2,000; mouse GFP monoclonal antibody (Clontech Labs 3P Living Colors A.v.

    Techniques: Binding Assay, Labeling, Construct

    COR1C regulates ARP2/3 complex activity at the endosome bud. (A) Representative images of COS-7 cells cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with mCh-Rab7 (LE, gray), α actin-Halo (green), and ARP3-mNG (magenta). Buds with extended actin (at arrow) were tracked before and after addition of an ARP2/3 complex inhibitor (150 μM CK-666) in merged magnified insets on right (5 × 5 μm). n = 10 cells. (B) Diagram of the ARP3-V5-TurboID biotinylation experiment. HeLa cells were cotransfected with ARP2/3-V5-TurboID and GFP-Rab7, COR1C-GFP, or COR1C-ACT– ΔCC-GFP and treated with biotin, and then biotinylated proteins were bound and eluted from α-biotin beads. (C) Representative V5 and GFP immunoblot from experiment in B shows high levels of biotinylation for full-length COR1C compared with COR1C mutant or Rab7 control. (D) Quantification of immunoblot, as shown in C. Pulldown numbers were calculated by normalizing elute signal with the load signal, performed in triplicate. 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. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Coronin 1C restricts endosomal branched actin to organize ER contact and endosome fission

    doi: 10.1083/jcb.202110089

    Figure Lengend Snippet: COR1C regulates ARP2/3 complex activity at the endosome bud. (A) Representative images of COS-7 cells cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with mCh-Rab7 (LE, gray), α actin-Halo (green), and ARP3-mNG (magenta). Buds with extended actin (at arrow) were tracked before and after addition of an ARP2/3 complex inhibitor (150 μM CK-666) in merged magnified insets on right (5 × 5 μm). n = 10 cells. (B) Diagram of the ARP3-V5-TurboID biotinylation experiment. HeLa cells were cotransfected with ARP2/3-V5-TurboID and GFP-Rab7, COR1C-GFP, or COR1C-ACT– ΔCC-GFP and treated with biotin, and then biotinylated proteins were bound and eluted from α-biotin beads. (C) Representative V5 and GFP immunoblot from experiment in B shows high levels of biotinylation for full-length COR1C compared with COR1C mutant or Rab7 control. (D) Quantification of immunoblot, as shown in C. Pulldown numbers were calculated by normalizing elute signal with the load signal, performed in triplicate. 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. Source data are available for this figure: .

    Article Snippet: For immunoblotting, rabbit COR1C polyclonal antibody (14749-1-AP; Proteintech) was used at 1:2,000; rabbit COR1B polyclonal antibody (ab119714; Abcam) at 1:2,000; rabbit COR1A polyclonal antibody (ab123574; Abcam) at 1:2,000; muse V5 monoclonal antibody (R960CUS; Thermo Fisher Scientific) at 1:2,000; mouse GFP monoclonal antibody (Clontech Labs 3P Living Colors A.v.

    Techniques: Activity Assay, Western Blot, Mutagenesis

    Additional TurboID controls indicating that ARP3 TurboID activity is specific. (A) Representative immunoblot showing that ARP3 V5 TurboID and a cytoplasmic nonspecific TurboID are able to biotinylate endogenous COR1C. Blots show the same samples run twice to blot for either endogenous COR1C or V5. Performed in triplicate. (B) Quantification of blots in A showing the amount of COR1C signal in the elute normalized first to the relevant TurboID V5 signal in the input and then to the max value within each replicate. (C) Representative immunoblot showing that ARP3 V5 TurboID has uniquely specific biotinylation of COR1C as shown by its ability to biotinylate only COR1C-GFP and not COR1C ACT– ΔCC-GFP. In contrast, the Cyto V5 TurboID biotinylates proteins nonspecifically in the cytoplasm as shown by similar biotinylation profiles for both COR1C-GFP and COR1C ACT–-ΔCC-GFP. Performed in triplicate. (D) Quantification of blots in C, from three replicates. Graph shows the ratio of COR1C-GFP to COR1C ACT–ΔCC-GFP signal in the elution. Signals in ratio were normalized to both relevant GFP and V5 input signals and then to the minimum value in each replicate. Statistical analysis was performed via two-tailed Student’s t test; *, P < 0.05. X indicates mean, and line indicates median. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Coronin 1C restricts endosomal branched actin to organize ER contact and endosome fission

    doi: 10.1083/jcb.202110089

    Figure Lengend Snippet: Additional TurboID controls indicating that ARP3 TurboID activity is specific. (A) Representative immunoblot showing that ARP3 V5 TurboID and a cytoplasmic nonspecific TurboID are able to biotinylate endogenous COR1C. Blots show the same samples run twice to blot for either endogenous COR1C or V5. Performed in triplicate. (B) Quantification of blots in A showing the amount of COR1C signal in the elute normalized first to the relevant TurboID V5 signal in the input and then to the max value within each replicate. (C) Representative immunoblot showing that ARP3 V5 TurboID has uniquely specific biotinylation of COR1C as shown by its ability to biotinylate only COR1C-GFP and not COR1C ACT– ΔCC-GFP. In contrast, the Cyto V5 TurboID biotinylates proteins nonspecifically in the cytoplasm as shown by similar biotinylation profiles for both COR1C-GFP and COR1C ACT–-ΔCC-GFP. Performed in triplicate. (D) Quantification of blots in C, from three replicates. Graph shows the ratio of COR1C-GFP to COR1C ACT–ΔCC-GFP signal in the elution. Signals in ratio were normalized to both relevant GFP and V5 input signals and then to the minimum value in each replicate. Statistical analysis was performed via two-tailed Student’s t test; *, P < 0.05. X indicates mean, and line indicates median. Source data are available for this figure: .

    Article Snippet: For immunoblotting, rabbit COR1C polyclonal antibody (14749-1-AP; Proteintech) was used at 1:2,000; rabbit COR1B polyclonal antibody (ab119714; Abcam) at 1:2,000; rabbit COR1A polyclonal antibody (ab123574; Abcam) at 1:2,000; muse V5 monoclonal antibody (R960CUS; Thermo Fisher Scientific) at 1:2,000; mouse GFP monoclonal antibody (Clontech Labs 3P Living Colors A.v.

    Techniques: Activity Assay, Western Blot, Two Tailed Test

    Vacuole diameter and bud length are not changed significantly in KD rescue experiments. (A) For each bud scored for fission in , vacuole diameter was also measured. Graph shows average vacuole diameter per cell, averaged per condition. No significant changes were observed in any condition, showing that only fission is affected and not other measures of endosome morphology. Data for graph from Halo E-vec: 139 endosomes in n = 15 cells; COR1C: 122 endosomes in n = 15 cells; COR1C ACT–: 213 endosomes in n = 17 cells; COR1C ΔCC: 281 endosomes in n = 18 cells; COR1C ACT– ΔCC: 296 endosomes n = 17 cells; and COR1C CC: 331 endosomes in n = 22 cells. X indicates mean, and line indicates median. Statistical analyses were performed with one-way ANOVA. (B) As in A, except FAM21-positive bud length was measured instead of vacuole diameter. Again, no significant changes were observed in any condition showing that only fission is affected and not other measures of endosome morphology. Data for graph from Halo E-vec: 139 endosomes in n = 15 cells; COR1C: 122 endosomes in n = 15 cells; COR1C ACT–: 213 endosomes in n = 17 cells; COR1C ΔCC: 281 endosomes in n = 18 cells; COR1C ACT– ΔCC: 296 endosomes n = 17 cells; and COR1C CC: 331 endosomes in n = 22 cells. X indicates mean, and line indicates median. Statistical analyses were performed with one-way ANOVA.

    Journal: The Journal of Cell Biology

    Article Title: Coronin 1C restricts endosomal branched actin to organize ER contact and endosome fission

    doi: 10.1083/jcb.202110089

    Figure Lengend Snippet: Vacuole diameter and bud length are not changed significantly in KD rescue experiments. (A) For each bud scored for fission in , vacuole diameter was also measured. Graph shows average vacuole diameter per cell, averaged per condition. No significant changes were observed in any condition, showing that only fission is affected and not other measures of endosome morphology. Data for graph from Halo E-vec: 139 endosomes in n = 15 cells; COR1C: 122 endosomes in n = 15 cells; COR1C ACT–: 213 endosomes in n = 17 cells; COR1C ΔCC: 281 endosomes in n = 18 cells; COR1C ACT– ΔCC: 296 endosomes n = 17 cells; and COR1C CC: 331 endosomes in n = 22 cells. X indicates mean, and line indicates median. Statistical analyses were performed with one-way ANOVA. (B) As in A, except FAM21-positive bud length was measured instead of vacuole diameter. Again, no significant changes were observed in any condition showing that only fission is affected and not other measures of endosome morphology. Data for graph from Halo E-vec: 139 endosomes in n = 15 cells; COR1C: 122 endosomes in n = 15 cells; COR1C ACT–: 213 endosomes in n = 17 cells; COR1C ΔCC: 281 endosomes in n = 18 cells; COR1C ACT– ΔCC: 296 endosomes n = 17 cells; and COR1C CC: 331 endosomes in n = 22 cells. X indicates mean, and line indicates median. Statistical analyses were performed with one-way ANOVA.

    Article Snippet: For immunoblotting, rabbit COR1C polyclonal antibody (14749-1-AP; Proteintech) was used at 1:2,000; rabbit COR1B polyclonal antibody (ab119714; Abcam) at 1:2,000; rabbit COR1A polyclonal antibody (ab123574; Abcam) at 1:2,000; muse V5 monoclonal antibody (R960CUS; Thermo Fisher Scientific) at 1:2,000; mouse GFP monoclonal antibody (Clontech Labs 3P Living Colors A.v.

    Techniques:

    The COR1C CC limits bud actin to facilitate ER contact, endosome fission, and CI-M6PR sorting. (A) Representative images of LE buds stable for duration of acquisition in conditions that did not rescued fission rate (C). COS-7 cells were cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, magenta), mCh-FAM21 (WASH complex, green), and with either Halo E-vec, siRES COR1C ΔCC-Halo, or siRES COR1C ACT– ΔCC-Halo. Arrows indicate bud of interest. (B) Representative images of LE fission events in conditions that rescued fission rate (C). COS-7 cells were cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, magenta), mCh-FAM21 (WASH complex, green), and with either siRES COR1C-Halo, siRES COR1C ACT–-Halo, or siRES COR1C CC-Halo. Arrows indicate bud of interest. (C) Quantification of data in A and B. Graph shows percentage of FAM21-labeled LE buds that underwent fission per cell during a 2-min time lapse. Note that only constructs containing the CC were able to restore fission. Data for graph from Halo E-vec: 139 endosomes in n = 15 cells; siRES COR1C: 122 endosomes in n = 15 cells; siRES COR1C ACT–: 213 endosomes in n = 17 cells; siRES COR1C ΔCC: 281 endosomes in n = 18 cells; siRES COR1C ACT– ΔCC: 296 endosomes n = 17 cells; and siRES COR1C CC: 331 endosomes in n = 22 cells, performed in triplicate. (D) Representative images of the M6PR trafficking assay. The relative fluorescence intensity of internalized anti-CI-MPR antibody immunostaining reveals trafficking of internalized anti-CI-MPR to TGN in Cos7 cells cotransfected with control siRNA, COR1A/1B/1C siRNAs to deplete all type I coronins, or FAM21 siRNA and with either GFP E-vec, siRES COR1C-GFP, or siRES COR1C ΔCC-GFP (not depicted). Cells were stained to mark CI-M6PR (green) and Giantin (Golgi, magenta). Dispersed vesicular CI-M6PR signal is indicative of failure to recycle, whereas concentrated CI-M6PR signal at the Golgi indicates normal retrograde sorting. (E) Quantification of data in D. Graph shows the background-corrected ratio of CI-M6PR signal localized at the Golgi relative to the vesicular signal in the cytoplasm such that larger values indicate less efficient retrograde recycling. Data for graph from control siRNAs: n = 24 cells; FAM21 siRNAs: n = 23 cells; COR1A/1B/1C siRNAs + E-vec: n = 25; COR1A/1B/1C siRNAs + siRES COR1C: n = 24 cells; COR1A/1B/1C siRNAs + siRES COR1C ΔCC: n = 24 cells; COR1A/1B/1C siRNAs + siRES COR1C CC: n = 26 cells, performed in triplicate. (F) Representative images of COS-7 cells cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, gray), mCh-FAM21 (WASH complex, green), BFP-Sec61β (ER, magenta), and either Halo E-vec, siRES COR1C-Halo, siRES COR1C ACT–-Halo, siRES COR1C ΔCC-Halo, siRES COR1C ACT– ΔCC-Halo, or siRES COR1C CC-Halo (gray, left panel). Magnified inset (5 × 5 μm) on right show representative examples of ER contact with endosomes. Note: Vacuolar contact with ER is always preserved, whereas ER contact with the FAM21 labeled bud is not. (G) Line scan analysis of dashed lines shown in D are positioned from the rear vacuolar contact across the length of the FAM21-labeled buds. Double ER peaks (shaded purple) are observed when bud contact is rescued. The first peak is always present and corresponds to the vacuolar contact. The second ER peak which aligns with the FAM21 indicates proper ER recruitment to the bud. Note that ER contact with bud is dependent on the presence of the CC domain. (H) Quantification of data in D. All FAM21-positive LE buds in areas with resolvable ER were tracked, and ER contact was scored as the percentage of time during a 2-min video that contact is maintained. Data for graph from Halo E-vec: 109 endosomes in n = 14 cells; COR1C: 134 endosomes in n = 17 cells; COR1C ACT–: 135 endosomes in n = 17 cells; COR1C ΔCC: 123 endosomes n = 17 cells; COR1C ACT– ΔCC: 105 endosomes in n = 15 cells; or COR1C CC: 137 endosomes in n = 19 cells, performed in triplicate. 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 < 0.001. Scale bars for whole cell = 5 μm; insets = 1 μm.

    Journal: The Journal of Cell Biology

    Article Title: Coronin 1C restricts endosomal branched actin to organize ER contact and endosome fission

    doi: 10.1083/jcb.202110089

    Figure Lengend Snippet: The COR1C CC limits bud actin to facilitate ER contact, endosome fission, and CI-M6PR sorting. (A) Representative images of LE buds stable for duration of acquisition in conditions that did not rescued fission rate (C). COS-7 cells were cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, magenta), mCh-FAM21 (WASH complex, green), and with either Halo E-vec, siRES COR1C ΔCC-Halo, or siRES COR1C ACT– ΔCC-Halo. Arrows indicate bud of interest. (B) Representative images of LE fission events in conditions that rescued fission rate (C). COS-7 cells were cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, magenta), mCh-FAM21 (WASH complex, green), and with either siRES COR1C-Halo, siRES COR1C ACT–-Halo, or siRES COR1C CC-Halo. Arrows indicate bud of interest. (C) Quantification of data in A and B. Graph shows percentage of FAM21-labeled LE buds that underwent fission per cell during a 2-min time lapse. Note that only constructs containing the CC were able to restore fission. Data for graph from Halo E-vec: 139 endosomes in n = 15 cells; siRES COR1C: 122 endosomes in n = 15 cells; siRES COR1C ACT–: 213 endosomes in n = 17 cells; siRES COR1C ΔCC: 281 endosomes in n = 18 cells; siRES COR1C ACT– ΔCC: 296 endosomes n = 17 cells; and siRES COR1C CC: 331 endosomes in n = 22 cells, performed in triplicate. (D) Representative images of the M6PR trafficking assay. The relative fluorescence intensity of internalized anti-CI-MPR antibody immunostaining reveals trafficking of internalized anti-CI-MPR to TGN in Cos7 cells cotransfected with control siRNA, COR1A/1B/1C siRNAs to deplete all type I coronins, or FAM21 siRNA and with either GFP E-vec, siRES COR1C-GFP, or siRES COR1C ΔCC-GFP (not depicted). Cells were stained to mark CI-M6PR (green) and Giantin (Golgi, magenta). Dispersed vesicular CI-M6PR signal is indicative of failure to recycle, whereas concentrated CI-M6PR signal at the Golgi indicates normal retrograde sorting. (E) Quantification of data in D. Graph shows the background-corrected ratio of CI-M6PR signal localized at the Golgi relative to the vesicular signal in the cytoplasm such that larger values indicate less efficient retrograde recycling. Data for graph from control siRNAs: n = 24 cells; FAM21 siRNAs: n = 23 cells; COR1A/1B/1C siRNAs + E-vec: n = 25; COR1A/1B/1C siRNAs + siRES COR1C: n = 24 cells; COR1A/1B/1C siRNAs + siRES COR1C ΔCC: n = 24 cells; COR1A/1B/1C siRNAs + siRES COR1C CC: n = 26 cells, performed in triplicate. (F) Representative images of COS-7 cells cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, gray), mCh-FAM21 (WASH complex, green), BFP-Sec61β (ER, magenta), and either Halo E-vec, siRES COR1C-Halo, siRES COR1C ACT–-Halo, siRES COR1C ΔCC-Halo, siRES COR1C ACT– ΔCC-Halo, or siRES COR1C CC-Halo (gray, left panel). Magnified inset (5 × 5 μm) on right show representative examples of ER contact with endosomes. Note: Vacuolar contact with ER is always preserved, whereas ER contact with the FAM21 labeled bud is not. (G) Line scan analysis of dashed lines shown in D are positioned from the rear vacuolar contact across the length of the FAM21-labeled buds. Double ER peaks (shaded purple) are observed when bud contact is rescued. The first peak is always present and corresponds to the vacuolar contact. The second ER peak which aligns with the FAM21 indicates proper ER recruitment to the bud. Note that ER contact with bud is dependent on the presence of the CC domain. (H) Quantification of data in D. All FAM21-positive LE buds in areas with resolvable ER were tracked, and ER contact was scored as the percentage of time during a 2-min video that contact is maintained. Data for graph from Halo E-vec: 109 endosomes in n = 14 cells; COR1C: 134 endosomes in n = 17 cells; COR1C ACT–: 135 endosomes in n = 17 cells; COR1C ΔCC: 123 endosomes n = 17 cells; COR1C ACT– ΔCC: 105 endosomes in n = 15 cells; or COR1C CC: 137 endosomes in n = 19 cells, performed in triplicate. 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 < 0.001. Scale bars for whole cell = 5 μm; insets = 1 μm.

    Article Snippet: For immunoblotting, rabbit COR1C polyclonal antibody (14749-1-AP; Proteintech) was used at 1:2,000; rabbit COR1B polyclonal antibody (ab119714; Abcam) at 1:2,000; rabbit COR1A polyclonal antibody (ab123574; Abcam) at 1:2,000; muse V5 monoclonal antibody (R960CUS; Thermo Fisher Scientific) at 1:2,000; mouse GFP monoclonal antibody (Clontech Labs 3P Living Colors A.v.

    Techniques: Labeling, Construct, Fluorescence, Immunostaining, Staining

    Model of actin/actin regulatory factors position and ER MCS just before fission. The WASH complex recruits and activates ARP2/3 to form a stabilizing actin structure on the endosome bud. COR1C is recruited to these actin structures to bind and counter act ARP2/3 actin nucleation via its CC. This results in an actin structure large enough to stabilize the bud, allowing cargo to sort, but small enough to allow ER recruitment and bud fission.

    Journal: The Journal of Cell Biology

    Article Title: Coronin 1C restricts endosomal branched actin to organize ER contact and endosome fission

    doi: 10.1083/jcb.202110089

    Figure Lengend Snippet: Model of actin/actin regulatory factors position and ER MCS just before fission. The WASH complex recruits and activates ARP2/3 to form a stabilizing actin structure on the endosome bud. COR1C is recruited to these actin structures to bind and counter act ARP2/3 actin nucleation via its CC. This results in an actin structure large enough to stabilize the bud, allowing cargo to sort, but small enough to allow ER recruitment and bud fission.

    Article Snippet: For immunoblotting, rabbit COR1C polyclonal antibody (14749-1-AP; Proteintech) was used at 1:2,000; rabbit COR1B polyclonal antibody (ab119714; Abcam) at 1:2,000; rabbit COR1A polyclonal antibody (ab123574; Abcam) at 1:2,000; muse V5 monoclonal antibody (R960CUS; Thermo Fisher Scientific) at 1:2,000; mouse GFP monoclonal antibody (Clontech Labs 3P Living Colors A.v.

    Techniques: