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Sla1p couples the yeast endocytic machinery to proteins regulating actin dynamics

¹ Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow. G12 8QQ, UK
² Wellcome Trust Biocentre, Division of Molecular Cell Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK

View larger version (47K): [in a new window]   Fig. 1. Interaction of Sla1p with proteins involved in actin dynamics. (A) GST-Sla1p was overexpressed in yeast as described in Materials and Methods and purified on glutathione sepharose beads following ion exchange chromatography. Yeast extracts were incubated with either GST-Sla1p beads or with glutathione beads alone. Beads were spun down, washed and bound proteins eluted in SDS sample buffer. Analysis was by western blotting using antibodies as marked. (Lanes: FT, flow through; W3, third wash; B, bound). (B) Further evidence of the Sla1p-Abp1 interaction was demonstrated using immunoprecipitation. Yeast cell extracts from a strain expressing Sla1-HA (KAY355) were incubated with protein-A sepharose bound with anti-Abp1p antibodies. After washing, the bound proteins were separated by SDS-PAGE and transferred to PVDF. The blot was probed with antibodies to Abp1p or HA to detect Sla1p, Srv2p and Sac6p. E, extract; W3, third wash; B, bound.

View larger version (23K): [in a new window]   Fig. 2. The effect of SLA1 deletion on sensitivity of cells to the actin disrupting drug latrunculin-A. KAY40 (SLA1) and KAY20 (sla1) cells were grown to log phase at 30°C. (A) Cells were incubated with different concentrations of latrunculin-A (0-1000 µM) for 15 minutes before being fixed and processed for rhodamine-phalloidin staining. For each concentration of latrunculin-A, cells were counted to assess the percentage of cells that still contained cortical actin patches. (B) Cells were incubated with 200 µM latrunculin-A for up to 60 minutes. Samples were taken over this time period and fixed and processed for rhodamine-phalloidin staining. For each time point cells were counted to assess the percentage of cells that still contained cortical actin patches. Each experiment was repeated three times and 200-250 cells were counted for each concentration or time point on each occasion. The results plotted are the mean of these experiments. Error bars show standard errors of the mean. (C) Images showing wild-type and sla1 cells before and after treatment with 200 µM LAT-A for 1 hour. Note that although sla1 cells still contain punctate cortical structures they no longer contained visible actin cables after LAT-A treatment. Bar, 10 µM.

View larger version (48K): [in a new window]   Fig. 3. Localisation of Sla1p-YFP and Abp1p-CFP in live cells. Exponentially growing KAY442 cells expressing Sla1p-YFP and Abp1p-CFP were mounted on gelatin slides and imaged using the JP4 filter set for YFP and CFP fluorescence. Images were recorded using the Deltavision Restoration microscope as described in the Materials and Methods. 2D maximum intensity projections of 3D data sets are shown.

View larger version (43K): [in a new window]   Fig. 4. FRET analysis of cells expressing Sla1p-YFP and Abp1p-CFP. FRET Images showing the interaction of Sla1p-YFP and Abp1p-CFP cortical patches. Fluorescence resonance energy transfer (FRET) between CFP and YFP was used to measure the relative proximity of Abp1 and Sla1p in KAY441 cells using the JP4 combination of excitation and emission filters sets and dichroic beamsplitter. (A) Deconvolved data from the FRET analysis showing (i) `YFP' fluorescence, (ii) `CFP' fluorescence, (iii) `FRET' fluorescence and (iv) `FRET control' fluorescence — zero in this case. Sla1p-YFP only populations (arrows), Abp1-CFP-only populations (arrowhead) and `FRET' populations can be observed. 3D histograms of the data are shown to the right indicating the relative levels of the `YFP', `CFP' and `FRET' signals in the raw data. The right hand panel shows a merged image showing the distinct overlapping populations. (B) The corrected FRET (FRETC) image, after image arithmetic has been performed, is shown in quantitative pseudocolour representing arbitrary units of fluorescence intensity.

View larger version (56K): [in a new window]   Fig. 5. Localisation of Sla1-GFP in real time. KAY401 cells were taken from a freshly struck YPAD plate and visualised as described in the Materials and Methods. The left panel shows the entire cell (at t=0) from which the subsequent images have been recorded. The images are 2D projections of 3D acquired data (16x0.4 µM optical sections) with 0.02 second exposure, taken every 3 seconds. Arrowheads denote examples of Sla1p-containing spots that remain at the same intensity or increase intensity over this time course. The arrows mark spots that decrease in intensity and disappear completely over the time course. Bar, 2 µm.

View larger version (54K): [in a new window]   Fig. 6. Interaction of Sla1p at the cell cortex with the endocytic machinery. (A) The effect of deletion of the C-terminal repeat region on Sla1p localisation. Cells expressing full-length 9xmyc-tagged Sla1p, KAY303 (left) or mutant sla1Ct-9xmyc, KAY363 (right) were grown in rich media to log phase and then processed for immunofluorescence as described in the Materials and Methods. Note the lack of cortical localisation of Sla1p in the absence of the C-terminal repeat region. Bar, 10 µM. (B) Localisation of Sla1-GFP in END3 and end3-1 cells following a temperature shift. KAY397 (END3) and KAY462 (end3-1) cells were grown overnight at 30°C in rich medium to log phase. Half of each culture was then shifted to 37°C, the non-permissive temperature for the end3-1 mutation. Cells were incubated at 37°C for 2 hours. Cells were then mounted on slides and images recorded as described in the Materials and Methods. (C) Samples of cells were also taken at this time, fixed and processed for rhodamine-phalloidin staining. Cells showing a complete deletion of END3 were grown at 30°C and analysed by rhodamine-phalloidin staining to observe their actin phenotype (D) and to assess localisation of Sla1GFP (E). Bar, 10 µM.

View larger version (104K): [in a new window]   Fig. 7. The effect of deletion and mutation of SLA1 on fluid-phase endocytosis. Cells in exponential growth phase were incubated with lucifer yellow for 1 hour at room temperature. Vacuole morphology was observed by phase contrast microscopy (left panels) and localisation of lucifer yellow by fluorescence microscopy (right panels). Bar, 10 µM.

View larger version (37K): [in a new window]   Fig. 8. The effect of deletion of SLA1 on receptor-mediated endocytosis. (A,B) KAY316 (SLA1) and KAY391 (sla1) cells were grown to log phase. -factor (2.5 µg/ml) was added and samples taken and processed for immunofluorescence microscopy at the times indicated. (C) Cells were assessed at each time point for whether the Ste2p-myc staining was at the cortex of cells (n200 for each time point). (D) Uptake of radiolabelled pheromone was monitored in KAY316 and KAY391 strains to further quantify the internalisation defect of sla1 cells. The graph plotted is the ratio of radioactivity associated with the cells at each time point relative to the initial time zero level of labelling.

View larger version (23K): [in a new window]   Fig. 9. Model of Sla 1p coupling actin dynamics and endocytosis. Our data indicate that in wild-type cells Sla 1p is able to interact both with proteins regulating actin dynamics and with proteins forming part of the endocytic machinery. Sla 1p localisation at the cell cortex is shown to be largely dependent on the presence of functional End3p, a protein of the endocytic machinery, but a subset of complexes containing Sla 1p can also localise with Abp 1p-actin structures. We propose that it is in this larger complex that Sla 1 binding to Abp 1p and Las 17p/Bee 1p is able to link actin dynamics with the endocytic machinery and thereby facilitate endocytosis.