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First published online 9 November 2004
doi: 10.1242/jcs.01493

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Distribution of Can1p into stable domains reflects lateral protein segregation within the plasma membrane of living S. cerevisiae cells

Katerina Malinska^1,*, Jan Malinsky², Miroslava Opekarova³ and Widmar Tanner^1,

¹ Universität Regensburg, Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universitätsstrasse 31, 93040 Regensburg, Germany
² Institute of Experimental Medicine, CAS, and 1st Faculty of Medicine, Charles University, Albertov 4, 12801 Prague 2, Czech Republic
³ Institute of Microbiology, CAS, Videnska 1083, 14220 Prague 4, Czech Republic

View larger version (44K): [in a new window]   Fig. 1. Actin depolymerization does not affect the Can1p distribution in the plasma membrane. Cells expressing CAN1GFP (strain KM30; A-E) were treated with latrunculin A, an inhibitor of actin polymerization (F-J). Differential interference contrast (DIC) images (A,F), fluorescence signals from the corresponding transversal sections (B,G) and surface confocal sections (C,H) are presented. The treatment efficiency was tested by TRITC-phalloidin staining of actin filaments in fixed cells (fluorescence in D,I; DIC in E,J). Notice the absence of the fluorescence signal from I. The identical scanning parameters were set during the acquisition of D and I. Bars, 10 µm (A); 5 µm (E,J).

View larger version (126K): [in a new window]   Fig. 2. Cortical actin does not localize with Can1p. Simultaneous fluorescence detection of Can1p (using GFP; A, green in D) and actin (using TRITC-phalloidin; B, red in D) in strain KM30, and the corresponding differential interference contrast image (C) are shown. Bar, 5 µm.

View larger version (59K): [in a new window]   Fig. 3. Microtubules are not required to maintain the Can1p distribution. Cells expressing CAN1GFP (strain KM30; A-E) were treated with nocodazole in order to disassemble the microtubules (F-J). Differential interference contrast (DIC) images (A,F), fluorescence signals from the corresponding transversal confocal sections (B,G) and surface confocal sections (C,H) are presented. The treatment efficiency was tested by indirect immunofluorescence detection of tubulin in fixed cells (fluorescence in D,I; DIC in E,J). Notice the absence of the fluorescence signal from I. The identical scanning parameters were set during the acquisition of D and I. Bar, 10 µm.

View larger version (81K): [in a new window]   Fig. 4. Can1p plasma-membrane distribution is not related to the cell wall. Cells expressing CAN1GFP (strain KM30) were protoplasted with zymolyase. Differential interference contrast images (A,B,G,I), fluorescence signals from the corresponding transversal confocal sections (C,D), and surface confocal sections (E,F,H) before (A,C,E) and after (B,D,F-I) the treatment are presented. Bar, 10 µm.

View larger version (71K): [in a new window]   Fig. 5. Pma1p and Sur7p plasma-membrane distributions in various protoplasts. Cells expressing Pma1GFP (top left) or Sur7p (bottom left) were protoplasted by a consecutive treatment with zymolyase (middle) and glucuronidase (right). Differential interference contrast images (AC,J-L), fluorescence signals from the corresponding transversal confocal sections (D-F,M-O) and surface confocal sections (G-I,P-R) are presented. Arrowheads in C,I point to a burst cell. Bars, 10 µm.

View larger version (14K): [in a new window]   Fig. 6. Dynamics of Pma1GFP in the plasma membrane. A round spot of 0.5 µm diameter was bleached within a surface optical section of a living cell (strain KM12) and the fluorescence recovery was measured. Fluorescence intensities were corrected for the photobleaching caused by the repeated scans of the sample and normalized to the intensity before the bleaching (level 1 in the graph). Mean values of the relative fluorescence intensities in nine experiments (dots) and the fitted curve are presented.

View larger version (122K): [in a new window]   Fig. 7. Can1p and Fur4p show similar plasma-membrane distributions. Plasma-membrane distributions of Can1p (A,B) and Fur4p (C-E) are compared. Fluorescence signals from transverse confocal sections (A,C,D) and surface views (B,E) are shown. Notice that proteins expressed from the chromosome [strains KM10 (A,B) and KM79 (C)] or from the centromeric plasmid (strain KM162; D,E) show the same fluorescence patterns. Surface views in B,E were constructed as an average of fluorescence signals from eight consecutive confocal sections each (z-axis sampling: 0.3 µm). Bar, 5 µm.

View larger version (112K): [in a new window]   Fig. 8. Fur4p, Sur7 and Can1p colocalize in the plasma membranes of living cells. Simultaneous localizations of Fur4GFP/Can1mRFP (A,D,G,J; strain KM149), Can1GFP/Sur7mRFP (B,E,H,K; strain KM160) and Fur4GFP/Sur7mRFP (C,F,I,L; strain KM158) were performed. GFP (top) and RFP (middle) fluorescence channels, and a merged image (bottom) are shown. Notice the absence of mRFP signal from buds and daughter cells. The fluorescence-intensity profiles along the cell surface (outside the dashed arrows in G-I) were plotted in J-L, respectively. The curves were smoothed using a mean filter to reduce the noise and normalized to the same maximum value. Bar, 10 µm.

View larger version (89K): [in a new window]   Fig. 9. Fur4p and Sur7p do not localize with Pma1p in plasma membranes of living cells. Simultaneous localizations of Fur4GFP/Pma1mRFP (two left columns) and Pma1GFP/Sur7mRFP (two right columns) were performed. Fluorescence signals from transversal confocal sections (first and third columns from the left), and surface confocal sections (second and fourth columns) are shown. GFP (A-D) and RFP (E-H) fluorescence channels and a merged image (I-L) are shown. The fluorescence-intensity profiles along the cell surface (outside the dashed arrows in I,K) and through the surface section (between the lines in J,L) were plotted in M,O and N,P, respectively. The curves were smoothed by mean filter to reduce the noise and normalized to the same maximum value. Bar, 10 µm.

View larger version (43K): [in a new window]   Fig. 10. Sur7p is a raft protein. Crude membranes from strain Sur7GFP were solubilized by cold Triton X-100 and separated on Optiprep density gradients. Sur7GFP and Pma1p were detected in fractions 1-6 (top to bottom).