Capping protein regulates endosomal trafficking by controlling F-actin density around endocytic vesicles and recruiting RAB5 effectors
- City University of Hong Kong Shenzhen Research Institute, China
- Department of Biomedical Sciences, City University of Hong Kong, China
- Core Research Facilities, Southern University of Science and Technology, China
- Department of Biology, Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research and Shenzhen Key Laboratory of Cell Microenvironment, Southern University of Science and Technology, China
- City University of Hong Kong Chengdu Research Institute, China
Figures
Figure 1 with 1 supplement
CapZ is associated with early endosomes.
(A) CapZβ-GFP-expressing HeLa cells were immunostained with antibodies against EEA1 (top panel) or RAB5A (lower panel). Confocal images taken along the z-axis were projected (Maximum Intensity …
-
Figure 1—source data 1
Original immunoblots.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig1-data1-v2.xlsx
-
Figure 1—source data 2
Data for Figure 1A, B.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
CapZ is associated with early endosomes.
(A) CapZβ-GFP-expressing HeLa cells were treated with or without vacuolin-1 (1 μM) for 12 hr, and were then immunostained with Red-X Phalloidin; the white dashed circles indicate the …
-
Figure 1—figure supplement 1—source data 1
Original immunoblots.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig1-figsupp1-data1-v2.xlsx
-
Figure 1—figure supplement 1—source data 2
Data for 1C, 1E.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig1-figsupp1-data2-v2.xlsx
Figure 2 with 1 supplement
CapZ is required for early endosome maturation.
(A) The expression of CapZβ in control, CapZβ-knockout, or CapZβ-reconstituted HeLa cells. (B) Control, CapZβ-knockout, or CapZβ-reconstituted HeLa cells were treated with EGF-488 for the indicated …
-
Figure 2—source data 1
Original immunoblots.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig2-data1-v2.xlsx
-
Figure 2—source data 2
Data for 2B–2E.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
CapZ is required for early endosome maturation.
(A) Phase-contrast images of control or CapZβ-knockout HeLa cells, and the size of the cells was measured by Image J. (B) Control or CapZβ-knockout HeLa cells were plated in 24-well plates, and were …
-
Figure 2—figure supplement 1—source data 1
Original immunoblots.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig2-figsupp1-data1-v2.xlsx
-
Figure 2—figure supplement 1—source data 2
Data for 1B, 1D-1F, 1 H, 1I.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig2-figsupp1-data2-v2.xlsx
Figure 3 with 5 supplements
CapZ controls the actin density around early endosomes.
(A, B) HeLa cells were stably transfected with EGFP/mCherry or CapZα1-EGFP/CapZβ-mCherry constructs. These cells were either immunostained with phalloidin (A), or with an anti-RAB5A antibody (B). …
-
Figure 3—source data 1
Data for 3A–3D, 3F.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
The role of actin density around endocytic vesicles in the maturation of early endosomes.
(A) F-actin levels in control, CapZβ-knockout, or CapZβ-reconstituted HeLa cells were visualized by phalloidin staining. (B) The expression of CapZα in control and CapZα1/2-knockout HeLa cells was …
-
Figure 3—figure supplement 1—source data 1
Original immunoblots.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig3-figsupp1-data1-v2.xlsx
-
Figure 3—figure supplement 1—source data 2
Data for 1A, 1C–1F, 1I.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig3-figsupp1-data2-v2.xlsx
Figure 3—video 1
The reconstituted 3-D N-Sim images of endogenous Arp2/3 complex and endogenous RAB5 in wild-type HeLa cells.
Figure 3—video 2
The reconstituted 3-D N-Sim images of endogenous Arp2/3 complex and endogenous RAB5 in CapZβ-knockout cells.
Figure 3—video 3
RAB5-positive endosomes movements in wild-type HeLa cells.
Figure 3—video 4
RAB5A-positive endosomes movements in CapZβ-knockout HeLa cells.
Figure 4 with 1 supplement
The actin density around early endosomes regulates its maturation.
(A) Control or CapZβ-knockout HeLa cells were treated with or without CK-636 (100 μM) overnight and were then immunostained with an anti-RAB5A antibody. The number and size of the early endosomes in …
-
Figure 4—source data 1
Original immunoblots.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig4-data1-v2.xlsx
-
Figure 4—source data 2
Data for 4A–4D.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig4-data2-v2.xlsx
Figure 4—figure supplement 1
The role of actin density around endocytic vesicles in the maturation of early endosomes.
(A) The control or CapZβ-knockout HeLa cells were transiently transfected with RFP-RAB5A and ARPC1B-GFP, and were then treated with or without CK-636 (100 μM) overnight, followed by confocal …
-
Figure 4—figure supplement 1—source data 1
Data for 1A–1C.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig4-figsupp1-data1-v2.xlsx
Figure 5 with 1 supplement
CapZ facilitates the recruitment of RAB5 effectors to early endosomes.
(A) HEK293 cells were transiently transfected with HA-Rabex-5, and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to …
-
Figure 5—source data 1
Original immunoblots.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig5-data1-v2.xlsx
-
Figure 5—source data 2
Data for 5D–5G, 5J.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig5-data2-v2.xlsx
Figure 5—figure supplement 1
The role of CapZ in the recruitment of RAB5 effectors to early endosomes.
(A, B) Twinstrep-CapZβ with Flag-Rabaptin-5 (A) or Flag-VPS34 (B) were transfected into HEK293 cells. The cell lysates were immunoprecipitated with an anti-Flag antibody, and the pulldowns were …
-
Figure 5—figure supplement 1—source data 1
Original immunoblots.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig5-figsupp1-data1-v2.xlsx
-
Figure 5—figure supplement 1—source data 2
Data for 1D, 1E.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig5-figsupp1-data2-v2.xlsx
Figure 6 with 1 supplement
Recruitment of RAB5 effectors to the early endosomes by CapZ depends on its actin capping activity.
(A) The left panel shows the actin-binding motif in the CapZ structure generated from the protein data bank (PDB ID: 1IZN). Cyan color indicated CapZα structure, and brown color indicated CapZβ …
-
Figure 6—source data 1
Original immunoblots.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig6-data1-v2.xlsx
-
Figure 6—source data 2
Data for 6C–6E.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig6-data2-v2.xlsx
Figure 6—figure supplement 1
The role of the actin-binding motif of CapZ in endocytosis.
(A) HEK293 cells were transfected with Twinstrep-actin, and Myc-RAB5A, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis …
-
Figure 6—figure supplement 1—source data 1
Original immunoblots.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig6-figsupp1-data1-v2.xlsx
-
Figure 6—figure supplement 1—source data 2
Data for 1B.
- https://cdn.elifesciences.org/articles/65910/elife-65910-fig6-figsupp1-data2-v2.xlsx
Author response image 1
(A) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A antibody.
The number and size of the early endosomes in these cells were quantified and weighted by the cell size. (B) Control or CapZβ-knockout HeLa cells were immunostained with Red-X Phalloidin; the white …
Author response image 2
The lysates of control or CapZβ-knockout HeLa cells were subjected to sucrose gradient fractions, followed by respective immunoblot analysis.
Author response image 3
Control, CapZβ-knockout, ARP2-knockout, or CapZβ/ARP2 double-knockout HeLa cells were transiently transfected with RFP-RAB5A, and cells were then imaged without delay for 3 min by time-lapse Nikon high-speed confocal microscopy.
2D track analysis was performed using NIS-Elements AR software.
Author response image 4
Control or CapZβ-knockout HeLa cells were treated with EGF (100 ng/ml) in the presence of cycloheximide (10 μg/ml) (A).
They were then collected at the indicated time points and subjected to western blot analysis against EGFR, ERK, pERK, and Hsp70. The p-ERKs in EGF-treated control or CapZβ-knockout cells were also …
Author response image 5
The ARPC1B-mCherry- or ARPC1B-GFP-expressing HeLa cells were stained with anti-p16-Arc antibody, followed by confocal imaging.
Author response image 6
The control or CapZβ-knockout HeLa cells were transiently transfected with ARPC1B-mCherry or CapZβ-GFP, followed by confocal imaging.
Author response image 7
The control or CapZβ-knockout HeLa cells were immunostained with antibodies against RAB5 and N-WASP, followed by confocal imaging.
Author response image 8
Control or CapZβ-knockout HeLa cells were treated with EGF-488 for the indicated times before fixation and staining with phalloidin-647 and DAPI (without permeabilization), and then processed for immunofluorescence analysis.
Author response image 9
CapZ helps recruit Rabex5 and Rapaptin-5 to RAB5-positive early endosomes, thus establishing a positive feedback loop to further activate RAB5 for the early endosome fusion.
Author response image 10
The expression levels of RAB5A, RAB7A, Rabex5, Rabaptin5, and VPS34 in control or CapZβ-knockout HeLa cells were determined by western blot analysis.
Author response image 11
The dot plot, a box and whisker plot, and a frequency histogram distribution of the endosome size data in control, CapZβ-knockout, or CapZβ-reconstituted HeLa cells.
Videos
Author response video 1
RAB5-positive endosomes movements in wild-type HeLa cells.
Author response video 2
RAB5-positive endosomes movements in CapZb-knockout HeLa cells.
Author response video 3
RAB5-positive endosomes movements in ARP2-knockout HeLa cells.
Author response video 4
RAB5-positive endosomes movements in CapZb-ARP2 double knockout HeLa cells.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | APRC1B (Rabbit polyclonal) | Sigma-Aldrich | HPA004832 | IF (1:100) |
| Antibody | ARP2 (Rabbit polyclonal) | ProteinTech | 10922-1-AP | WB (1:2000) |
| Antibody | Pan Actin (Mouse monoclonal) | Cytoskeleton | AAN02-s | IF (1:500) |
| Antibody | CapZα (Mouse monoclonal) | DSHB | mAb 5B12.3 | WB (1:100) |
| Antibody | CapZβ (Rabbit polyclonal) | ProteinTech | 25043-1-AP | WB (1:2000) |
| Antibody | EGFR (Mouse monoclonal) | Santa Cruz | sc-373746 | WB (1:100) |
| Antibody | EEA1 (Rabbit monoclonal) | CST | 3,288 | IF (1:100) |
| Antibody | Flag-tag (Mouse monoclonal) | Sigma-Aldrich | F3165 | WB (1:1000) |
| Antibody | HA-tag (Rat monoclonal) | Sigma-Aldrich | 11867423001 | WB (1:3000) |
| Antibody | His-tag (Mouse monoclonal) | ProteinTech | 66005-1-Ig | WB (1:3000) |
| Antibody | HSC70 (Mouse monoclonal) | Santa Cruz | sc-7298 | WB (1:10,000) |
| Antibody | LAMP1 (Rabbit monoclonal) | CST | 9,091 | IF (1:200) |
| Antibody | MYC-tag (Mouse monoclonal) | ProteinTech | 67447-1-Ig | WB (1:3000) |
| Antibody | N-WASP (Rabbit polyclonal) | Novus Biologicals | NBP1-82512 | IF (0.25–2 µg/ml) |
| Antibody | p16-Arc (Rabbit monoclonal) | Abcam | ab51243 | IF (1:100) |
| Antibody | Rabaptin-5 (Rabbit polyclonal) | ProteinTech | 14350-1-AP | WB (1:1000) |
| Antibody | Rabex-5 (Mouse monoclonal) | Santa Cruz | sc-166049 | WB (1:500) |
| Antibody | StrepMAB-Classic HRP conjugate (Mouse monoclonal) | IBA | 2-1509-001 | WB (1:3000) |
| Antibody | VPS8 (Rabbit polyclonal) | ProteinTech | 15079-1-AP | WB (1:1000) |
| Antibody | RAB5A (Rabbit polyclonal) | ProteinTech | 11947-1-AP | WB (1:1000) |
| Antibody | RAB5A (Rabbit monoclonal) | CST | 3547 | IF (1:200) |
| Antibody | RAB5A (Mouse monoclonal) | CST | 46449 | IF (1:200)WB (1:1000) |
| Chemical compound, drug | CK-636 | Selleck | S7497 | |
| Chemical compound, drug | Latrunculin A | Santa Cruz | sc-202691 | |
| Chemical compound, drug | EGF | Bio-Rad | PHP030A | |
| Chemical compound, drug | GDP-β-S | Sigma-Aldrich | G7637 | |
| Chemical compound, drug | GTP-γ-S | Jena Bioscience | NU-412-2 | |
| Other | Transferrin-Alexa Fluor 594 | Invitrogen | T13343 | (25 μg/mL) |
| Other | EGF-Alexa Fluor 488 | Invitrogen | E13345 | (1 μg/ml) |
| Other | Phalloidin-iFluor 647 | Abcam | ab176759 | (1:1000) in 30 μl stock solution |
| Other | Texas Red-X Phalloidin | Invitrogen | T7471 | (1:40) in 1.5 ml stock solution |
| Cell line (Homo sapiens) | HeLa cells | ATCC | CRL-CCL-2 | |
| Cell line (H. sapiens) | HEK293 cells | ATCC | CRL-1573 |
Table 1
Plasmids used in this study.
| pCDNA3.1-Flag-Rabaptin-5 | pRK5-mRFP-FYVE | pCDNA3.1-HA-Rabex-5 |
| pCDNA3.1-MYC-RAB5A | plenti-CapZβ-GFP | pCDNA3.1-CapZβ-His6 |
| pRP-CapZβ-TwinStrep | pGEX-4-R5BD | pCDNA3.1-Flag-VPS34 |
| pCDNA3.1-Twinstrep-RAB5A | iRFP-FRB-RAB5A | CFP-FKBP-CapZβ |
| mRFP-RAB5A | pAGW-GFP-RAB5A | plenti-CapZβ-mcherry |
| pGEX4T-1-RAB5A | pGEX4T-1 | plenti-Flag-Rabex-5 |
| Plenti-GFP-ML1N2* | plenti-CapZβ | pRSFDuet-1-CapZβ/CapZα1 |
| plenti-CapZβ-Twinstrep | psPAX2 | pMD2.G |
| pGEX4T-1-Rabaptin-5 | pCDNA3.1-β-actin-Twinstrep | plenti-CapZβ(1-147)-Twinstrep |
| plenti-CapZβ(148-277)-Twinstrep | mCherry-ARPC1B |
