Strains, growth conditions, and pharmacological inhibitor
Standard genetic methods and growth conditions were used. Cells were grown in Edinburgh minimal media (EMM) supplemented with appropriate amino acids (ALU) or YE5S as indicated. Particular care was taken to avoid accumulation of suppressors in poorly growing strains by backcrossing, by rapid stocking of newly generated strains, and by streaking them freshly at each experiment.
strains used in this study are listed in Supplemental Table S1. Tagged and truncation strains were constructed by using either a PCR or an integrative plasmid–based approach (Bahler et al., 1998
; Martin and Chang, 2006
) and confirmed by PCR. The myo51tail-3GFP
(or -12myc) strain produces a Myo51 product truncated at amino acid 1087. The myo52tail-tdTomato
strain produces a Myo52 product truncated at amino acid 1162.
MBC (Sigma-Aldrich, St. Louis, MO) was used at final concentration of 25 μg/ml from a stock of 2.5 mg/ml in dimethyl sulfoxide (DMSO). MBC treatment was performed for 30 min at 30°C unless otherwise indicated.
Latrunculin A (Phillip Crews, University of California, Santa Cruz, Santa Cruz, CA) was used at final concentration of 10 or 200 μM as indicated from a stock of 20 mM in DMSO. LatA treatment was performed for 15 min either at 25° or 30°C unless otherwise indicated. Control experiments with DMSO had no effect on F-actin organization or fluorescent fusion protein localization.
Molecular biology methods
All plasmids were constructed using standard molecular biology techniques. In general, genes or gene fragments were cloned after PCR using as template genomic DNA or plasmids and primers containing 5′ extensions with specific restriction sites. Details of the primers and restriction sites used are available upon request.
Details about Tea2N-GFP-Myo52C and control constructs can be found in Lo Presti and Martin (2011
). The chimera Myo52N-GFP-Nup146 was cloned under control of the weak nmt
promoter in pRIP82 vector and encodes, in this order, amino acids 1–1162 of Myo52, a SGRA linker, GFP, a GSSGP linker, and full-length Nup146 (UniProtKB accession number Q09847). For the construction of the control chimera Myo52m4A
-YFP was used as template for the amplification of myo52N
(Motegi et al., 2001
). The cassette myo52N-GFP-nup146
and the control cassettes were linearized and integrated into the ura4
Microscopy was performed with either a spinning-disk confocal microscope or a wide-field fluorescence microscope.
For3-3GFP and Myo52N-GFP-Nup146 imaging was mainly performed using a DeltaVision system (Applied Precision, Issaquah, WA) composed of a customized Olympus IX-71 Inverted Microscope Stand (Olympus, Tokyo, Japan) fitted with a PlanApo 100× oil, 1.42 numerical aperture (NA) objective, a CoolSNAP HQ2 camera (Photometrics, Tucson, AZ), and an Insight SSI 7 Color Combined Unit Illuminator. Images were acquired with softWoRx software (Applied Precision, a GE Healthcare Company), using the fast acquisition mode.
FRAP experiments were performed on a laser scanning confocal microscope (LSM510 Meta; Carl Zeiss, Jena, Germany). Photobleaching was obtained by 25 iterative scans of a selected region encompassing the very tip of a cell at maximal laser power. Images were recorded before photobleaching, immediately after, and subsequently every 2 s, with 5% laser power, as described (Martin and Chang, 2006
All other images were acquired on a spinning-disk system, using a Leica DMI4000B inverted microscope (Leica, Wetzlar, Germany) equipped with an HCX PL APO ×100/1.46 NA oil objective and a PerkinElmer UltraView Confocal system (including a Yokagawa CSU22 real-time confocal scanning head, an argon/krypton laser, and a cooled 14-bit frame transfer electron-multiplying charge-coupled device C9100-50 camera; PerkinElmer, Waltham, MA). Stacks of z-series confocal sections were acquired at 0.3-μm intervals with the UltraView or Volocity software (PerkinElmer), and images were rendered by two-dimensional maximum-intensity projection unless otherwise indicated.
Actin staining was performed as described using Alexa Fluor 488–phalloidin (Invitrogen, Carlsbad, CA) with a fixation time of 40–60 min (Bendezu and Martin, 2011
). Phalloidin staining was generally performed on cells grown at 30°C in YES5 (yeast extract medium + 5 supplements: 225 mg/l adenine, histidine, leucine, uracil, and lysine hydrochloride), except for strains expressing chimeras, for which cells were grown for 16–24 h at 30°C in EMM-AL to induce expression. For actin staining of GFP-tagged strains, Alexa Fluor 488–phalloidin was also used because the GFP signal was not resistant to the fixation and staining procedure, and thus it did not interfere with imaging of the actin cytoskeleton.
To image live actin cables, we induced GFP-CHDRng2 expression for 16 h in EMM-AU at 30°C, unless otherwise specified. For imaging actin cable dynamics, we acquired a stack of 14 z-series confocal sections at 0.3-μm intervals for 90 s with a rate of 0.4–0.8 s per time point. To image chimeric proteins, we induced expression for 24 h in EMM-AL at 30°C. To image both live chimeric proteins and GFP-CHDRng2, expression was induced for 18 h in EMM-AU at 30°C unless otherwise specified.
Image data analysis
For fluorescence intensity measurements, we measured the average fluorescence intensity of sum projections of spinning-disk confocal z-stacks of at least five individual cells for each genotype. Background correction was performed by subtracting the background fluorescence intensity in a region that did not contain cells and the autofluorescence levels of wild-type cells expressing no fluorescent marker imaged and processed in the same conditions. All values were normalized to that of endogenous full-length Myo52-tdTomato or Myo52-CFP.
For quantification of the actin defect, we analyzed two-dimensional maximum-intensity projections of phalloidin-stained cells. For each cell, we visually scored the presence of 1) at least one cable oriented in a direction distinct from the longitudinal axis of cell (misoriented cable) and 2) a thick cable bundle. Note that cells bearing thick cable bundles usually had a single thick bundle rather than multiple ones. To count the number of cables extending beyond the cell middle, we used the ObjectJ tool in the ImageJ software (National Institutes of Health, Bethesda, MD) to measure cell length and precisely locate the cell middle. Cells with fewer than three cables crossing this location were assigned a middle extension defect. For each genotype the percentage of cells displaying any of the three parameters was calculated and plotted. Up to 16 independent experiments for a total of 480 cells were quantified, with 25–30 cells assessed each time. The graphs show data from one of them. Phalloidin staining quality varies from one experiment to the next. In particular, growth of the cells in rich (YE5S) or minimal (EMM) medium has significant impact on the quality of the staining, with actin cables consistently better detected for cells grown in rich medium. Therefore, each graph represents one set of stainings conducted in parallel, and numbers across experiments cannot be directly compared. However, the trends shown in the graphs are the same in all repeats. The precise number of independent experiments/total number of cells quantified are as follows: wild-type, 9/270; myo51
, 7/210; myo52
, 2/60; myoV
, 16/480; myoV
tea2N-myo52C, 6/180; myoV
tea2N, 2/60; myoV
myo52C, 2/60; myoV
tea2N-myo52C, 1/30; myoV
myo52N-nup146, 4/120; myov
myo52N, 3/90; myoV
-nup146, 2/60; myo51
, 2/60; myo52
, 2/60; myoV
cdc25-22, 1/27; myo51
cdc25-22, 1/27; mal3
, 3/90; and mal3
For analysis of the FRAP experiments, the mean fluorescence intensities were measured over time in three regions: 1) the photobleached region, 2) the background, and 3) another nonbleached cell. For each time point, the intensities of the bleached region and of the unrelated cell were adjusted by subtracting background signal. To correct for loss of signal due to imaging, the adjusted bleached region intensity was then divided by the adjusted intensity of the other cell. For each experiment, all values were normalized so that the photobleached value equals 0 and the prephotobleaching value equals 1. Finally, averages and standard errors were derived for fluorescence values at individual time points.
For3-3GFP dots and nuclear displacement rates were quantified using the softWoRx tool Leap Frog and averaged using Excel (Microsoft, Redmond, WA). Quantification of nuclear translocation in cells expressing Myo52N-GFP-Nup146 was done on images acquired at the DeltaVision using the ImageJ plug-in Cell Counter to count the number of interphase cells with a nucleus at the very cell end versus the total number of interphase cells. Figures were prepared with Photoshop Elements 6 and Illustrator CS3 (Adobe, San Jose, CA), and movies were prepared using ImageJ 1.41.
Extracts from yeast grown in EMM-AU medium for 21 h at 30°C were prepared in CXS buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.0, 20 mM KCl, 1 mM MgCl2, 2 mM EDTA, pH 7.5, and protease inhibitor cocktail) by grinding in liquid nitrogen with a mortar and pestle. After thawing, NaCl and Triton X-100 were added to final concentrations of 150 mM and 0.1%, respectively. For immunoprecipitations, 150 μl of soluble extract was added to 20 μl of sheep anti-mouse magnetic Dynabead slurry (Dynal, Invitrogen) prebound to 2 μg of monoclonal anti-Myc antibodies (9E10; Santa Cruz Biotechnology, Santa Cruz, CA) and incubated for 2 h at 4°C. Magnetic Dynabeads were then washed four times in CXS and 0.1% Triton and three times in IPP150 (150 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Nonidet P40, 2 mM EDTA, 1 mM MgCl2). Immunoprecipitated material was then recovered by boiling Dynabeads in 60 μl of SDS sample buffer for 5 min at 95°C. Standard protocols were used for SDS–PAGE and Western blot analysis. Antibodies used for immunoprecipitations and Western blots were mouse monoclonal anti-Myc (9E10; Santa Cruz Biotechnology), mouse monoclonal anti-GFP (Roche, Indianapolis, IN), and rabbit polyclonal serum anti-GFP (A6455; Invitrogen).