Capping protein regulates endosomal trafficking by controlling F-actin density around endocytic vesicles and recruiting RAB5 effectors

  1. Dawei Wang
  2. Zuodong Ye
  3. Wenjie Wei
  4. Jingting Yu
  5. Lihong Huang
  6. Hongmin Zhang
  7. Jianbo Yue  Is a corresponding author
  1. City University of Hong Kong Shenzhen Research Institute, China
  2. Department of Biomedical Sciences, City University of Hong Kong, China
  3. Core Research Facilities, Southern University of Science and Technology, China
  4. 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
  5. City University of Hong Kong Chengdu Research Institute, China
17 figures, 4 videos, 2 tables and 1 additional file

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—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 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—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 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—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—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—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 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—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 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—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 …

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 resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAPRC1B (Rabbit polyclonal)Sigma-AldrichHPA004832IF (1:100)
AntibodyARP2 (Rabbit polyclonal)ProteinTech10922-1-APWB (1:2000)
AntibodyPan Actin (Mouse monoclonal)CytoskeletonAAN02-sIF (1:500)
AntibodyCapZα (Mouse monoclonal)DSHBmAb 5B12.3WB (1:100)
AntibodyCapZβ (Rabbit polyclonal)ProteinTech25043-1-APWB (1:2000)
AntibodyEGFR (Mouse monoclonal)Santa Cruzsc-373746WB (1:100)
AntibodyEEA1 (Rabbit monoclonal)CST3,288IF (1:100)
AntibodyFlag-tag (Mouse monoclonal)Sigma-AldrichF3165WB (1:1000)
AntibodyHA-tag (Rat monoclonal)Sigma-Aldrich11867423001WB (1:3000)
AntibodyHis-tag (Mouse monoclonal)ProteinTech66005-1-IgWB (1:3000)
AntibodyHSC70 (Mouse monoclonal)Santa Cruzsc-7298WB (1:10,000)
AntibodyLAMP1 (Rabbit monoclonal)CST9,091IF (1:200)
AntibodyMYC-tag (Mouse monoclonal)ProteinTech67447-1-IgWB (1:3000)
AntibodyN-WASP (Rabbit polyclonal)Novus BiologicalsNBP1-82512IF (0.25–2 µg/ml)
Antibodyp16-Arc (Rabbit monoclonal)Abcamab51243IF (1:100)
AntibodyRabaptin-5 (Rabbit polyclonal)ProteinTech14350-1-APWB (1:1000)
AntibodyRabex-5 (Mouse monoclonal)Santa Cruzsc-166049WB (1:500)
AntibodyStrepMAB-Classic HRP conjugate (Mouse monoclonal)IBA2-1509-001WB (1:3000)
AntibodyVPS8 (Rabbit polyclonal)ProteinTech15079-1-APWB (1:1000)
AntibodyRAB5A (Rabbit polyclonal)ProteinTech11947-1-APWB (1:1000)
AntibodyRAB5A (Rabbit monoclonal)CST3547IF (1:200)
AntibodyRAB5A (Mouse monoclonal)CST46449IF (1:200)WB (1:1000)
Chemical compound, drugCK-636SelleckS7497
Chemical compound, drugLatrunculin ASanta Cruzsc-202691
Chemical compound, drugEGFBio-RadPHP030A
Chemical compound, drugGDP-β-SSigma-AldrichG7637
Chemical compound, drugGTP-γ-SJena BioscienceNU-412-2
OtherTransferrin-Alexa Fluor 594InvitrogenT13343(25 μg/mL)
OtherEGF-Alexa Fluor 488InvitrogenE13345(1 μg/ml)
OtherPhalloidin-iFluor 647Abcamab176759(1:1000) in 30 μl stock solution
OtherTexas Red-X PhalloidinInvitrogenT7471(1:40) in 1.5 ml stock solution
Cell line (Homo sapiens)HeLa cellsATCCCRL-CCL-2
Cell line (H. sapiens)HEK293 cellsATCCCRL-1573
Table 1
Plasmids used in this study.
pCDNA3.1-Flag-Rabaptin-5pRK5-mRFP-FYVEpCDNA3.1-HA-Rabex-5
pCDNA3.1-MYC-RAB5Aplenti-CapZβ-GFPpCDNA3.1-CapZβ-His6
pRP-CapZβ-TwinStreppGEX-4-R5BDpCDNA3.1-Flag-VPS34
pCDNA3.1-Twinstrep-RAB5AiRFP-FRB-RAB5ACFP-FKBP-CapZβ
mRFP-RAB5ApAGW-GFP-RAB5Aplenti-CapZβ-mcherry
pGEX4T-1-RAB5ApGEX4T-1plenti-Flag-Rabex-5
Plenti-GFP-ML1N2*plenti-CapZβpRSFDuet-1-CapZβ/CapZα1
plenti-CapZβ-TwinstreppsPAX2pMD2.G
pGEX4T-1-Rabaptin-5pCDNA3.1-β-actin-Twinstrepplenti-CapZβ(1-147)-Twinstrep
plenti-CapZβ(148-277)-TwinstrepmCherry-ARPC1B

Additional files

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