Yeast Eps15-like endocytic protein Pan1p regulates the interaction between endocytic vesicles, endosomes and the actin cytoskeleton
- Tokyo University of Technology, Japan
- Tokyo University of Science, Japan
- Institute of Science and Technology Austria, Austria
Figures
Figure 1 with 1 supplement
Construction and characterization of a Pan1p phosphorylation-site mutant.
(A) Structure of a Pan1p phosphorylation mutant. The two amino-terminal Eps15 homology (EH) domains, long repeat (LR) regions, predicted coiled-coil (CC), acidic region (A), and carboxy-terminal …
Figure 1—figure supplement 1
Construction and characterization of a Sla1p phosphorylation-site mutant.
(A) Structure of the Sla1p phosphorylation mutant. The three src homology 3 (SH3) domains, two Sla1 homology domains (SHD1/2), clathrin-binding motif (CBM) and carboxyl-terminal Sla1 repeats (SR) …
Figure 2 with 1 supplement
The localization of endosomal proteins in pan1-18TA cells.
(A) Localization of GFP-tagged endosomal proteins in pan1-18TA. pan1-18TA cells expressing Abp1-mCherry and GFP-tagged endosomal proteins were grown to early to mid-logarithmic phase in YPD medium …
Figure 2—figure supplement 1
Localization of endosomal proteins in wild-type cells.
Cells expressing each GFP-tagged endosomal protein were grown to early to mid-logarithmic phase in YPD medium at 25°C and observed by fluorescence microscopy.
Scale bars, 2.5 μm.
Figure 3 with 2 supplements
Interaction of endosomes expressing Hse1-3GFP with the actin cytoskeleton.
(A and C) Movement of Hse1-3GFP-containing endosomes in living cells. Wild-type cells expressing Vps26-3mCherry and Hse1-3GFP were grown to log phase at 25°C, treated with DMSO (LatA-) (A) or 250 μM …
Figure 3—figure supplement 1
Localization of 3GFP- or mCherry-tagged Hse1p in wild-type cells.
(A) The functionality of 3GFP- and mCherry-tagged Hse1p was confirmed by testing their ability to complement the growth phenotype of hse1△ cells in a dilution series of each plated on YPD containing …
Figure 3—figure supplement 2
Localization and movement of GFP-Vps21-containing endosomes in living cells.
(A) Localization of Hse1-3GFP or GFP-Vps21 in endocytic compartments. Cells were labeled with A594-α-factor as described in the Materials and methods. The images were acquired simultaneously at 10 …
Figure 4 with 1 supplement
Interaction of Pan1p-containing compartments with actin cables and endosomes.
(A) Localization of Abp140-3GFP and Pan1-mCherry in a wild-type cell. The lower panels correspond to a time series of a higher magnification view of the boxed area in the upper right image. (B) The …
Figure 4—figure supplement 1
Actin cable dynamics in wild-type and pan1-18TA cells.
(A) Cells expressing Abp140-3GFP were grown to early to mid-logarithmic phase at 25°C in YPD medium and imaged at 1 s intervals. Right panels indicate a high magnification view of the boxed area in …
Figure 5
Interaction between endocytic vesicles and actin cables upon inhibition of Prk1p.
(A) The left images represent single frames from movies of ark1△ prk1-as3 mutant cells showing merged images of the GFP (Abp140p) and the mCherry (Pan1p) channel. The ark1△ prk1-as3 mutant cells …
Figure 6 with 2 supplements
Interaction between endocytic vesicles and early endosomes in the pan1-18TA mutant.
(A) Localization of Hse1-3GFP-labeled endosomes and actin structures in pan1-18TA double mutant cells. The pan1-18TA and double mutant cells expressing Hse1-3GFP and Abp1-mCherry were grown to early …
Figure 6—figure supplement 1
Actin structures and actin patch dynamics in pan1-18TA double mutant cells.
(A) Maximum intensity projections of Z stacks of pan1-18TA and double mutant cells labeled with Abp1-mCherry. The Z series was acquired through the entire cell at 0.2 μm intervals. (B) …
Figure 6—figure supplement 2
Ultrastructure of endocytic vesicles and endosomes observed in wild-type and pan1-18TA sac6△ cells.
(A and B) Wild-type and pan1-18TA sac6△ cells were grown at 25°C, fixed using propane jet freezing method and processed for electron microscopic analysis. The lower panels show higher magnification …
Figure 7
Model of the actin cable-mediated endocytic pathway.
Unphosphorylated Pan1p on an endocytic vesicle binds to actin to fix the vesicle to the actin cable. After being pinched off from the membrane, the endocytic vesicle moves into the cytosol as a …
Videos
Video 1
Localization of Hse1-3GFP (left; green in merge) and Abp140-tdTomato (center; red in merge) in wild-type cells.
The interval between frames is 1 s.
Video 2
Localization of Hse1-3GFP (left; green in merge) and Abp140-Tomato (center; red in merge) in wild-type cells treated with 25 μM SMIFH2 or 100 μM CK-666.
Arrowheads indicate examples of Hse1p-labeled endosome. The interval between frames is 1 s.
Video 3
Localization of Abp140-3GFP in wild-type and pan1-18TA cells.
The interval between frames is 1 s.
Video 4
Localization of Abp140-3GFP (left; green in merge) and Pan1-18TA-mCherry (center; red in merge) in pan1-18TA cells.
The interval between frames is 1 s.
Video 5
Localization of Hse1-3GFP (left; green in merge) and Pan1-18TA-mCherry (center; red in merge) in pan1-18TA cells.
Arrowheads indicate examples of colocalization. The interval between frames is 1 s.
Video 6
Localization of Abp140-3GFP (left; green in merge) and Pan1-mCherry (center; red in merge) in ark1△ prk1-as3 cells untreated or treated with 100 μM 1NA-PP1.
Arrowheads indicate examples of vesicles associated with actin cables. The interval between frames is 1 s.
Additional files
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Supplementary file 1
Yeast strains used in this study.
- https://doi.org/10.7554/eLife.10276.023
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Supplementary file 2
Primers used in this study.
- https://doi.org/10.7554/eLife.10276.024
