Polarization of yeast cells in spatial gradients of a mating factor ( chemotxs / entatlon )

The process ofcell fusion during mating ofthe yeast Saccharomyces cerevu is mediated by factors secreted by the mating partners. Spatia gradients ofone ofthese mating factors, a-factor, polrid the growth of projections byMATa ceils. The site of previo buddsin did not affect the direction of polarition, and subsequent buddig was alo polarized if mating factor was removed. Orientation occurred in the presence of nocodaude, suggetingithat microtubues were not critical. At extremely iow concentrations of a-factor, ssa2 mutants (which in genetic studis do not d nate between partners producing dierent amounts of a-factor) were able to polarize thefr projections. The sensivity of this spatial sensing mecanim wild-type cells is such that differences in receptor occupancy etmated to be about 1% are sufcient for orientation. Haploid cells ofSaccharomyces cerevisiae are oftwo mating types, MATa and MATa (for reviews, see refs. 1-3). When the two mating types are mixed, cells of opposite mating types fuse together to form a diploid cell. Haploid cells secrete mating type-specific factors which are recognized by the opposite mating type. For example, MATa cells secrete a-factor, and MATa cells have cell surface receptors which bind a-factor. Binding of a-factor to MATa cells stimulates expression ofspecific genes, arrest ofthe cell cycle in GI, and reorganization of cell growth to form a polarized projection termed a shmoo. Proteins required for fusion, such as FUS1 and the agglutinins, are localized to the growing tip of the shmoo (4, 5). Several types of experiment indicate that fusion during mating is not random in a number of yeasts (6, 7). MATa and MATa cells placed together in pairs form diploids with high efficiency (8-10) indicating that the site of fusion can be determined during the mating process. Genetic studies measuring the frequency ofmating ofMATa cells to either oftwo potential MATa cell partners demonstrate that the cell producing the most a-factor is chosen (11, 12), suggesting that a MATa cell might measure gradients of a-factor in determining its choice of mating partner. By measuring the orientation of the gradient, the MATa cell could polarize mating structures directly toward the appropriate mating partner, increasing mating efficiency. The studies reported here were undertaken to directly test whether MATa cells are able to polarize projections induced by gradients of a mating factor. MATERIALS AND METHODS Materials. S. cerevisiae strain 7413-3-3 [wild-type for mating (13)] was obtained from M. Snyder (Yale University), RC631 [sst2(14)] and FC140 [barlP (15)] from L. Marsh (Albert Einstein College of Medicine), and 3268-8-2 [the sst2-1 allele backcrossed 10 times into the 381G background (16)] from D. Jenness (University of Massachusetts Medical The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. School). Cells were grown and tested at 30°C in YPD medium, which contains 1% yeast extract (Oxoid, Basingstoke, U.K.), 2% peptone (Oxoid L34), and 2% glucose. Lowmelting-point agar, a mating factor, nocodazole, and light mineral oil were obtained from Sigma. Meaument of Polaiztio in Spatia Gradients. Cells were grown to midlogarithmic phase in YPD medium at 30TC. They were centrifuged and resuspended at about 2 x 107 cells per ml in YPD medium containing 1% low-melting-point agar (Sigma A4018) at 300C. Small drops of the suspension were sprayed onto a YPD agar dish and allowed to cool. The suspension was overlaid withYPD medium and placed on the stage of a Nikon Diaphot microscope in a Nikon temperature chamber at 300C. Micropipets were made from cleaned capillary tubing (A-M Systems, no. 6010) pulled on a Narishige PB-7 puller. They were filled with filtered YPD medium containing a-factor and held in a pressure holder (General Valve, Fairfield, NJ) mounted in a Narishige MN151 micromanipulator. Continuous positive pressure of 1.5 psi (10.3 kPa) was used to eject the medium containing a-factor. The surface of the medium was covered with mineral oil to prevent evaporation. The micropipet was lowered to about 10 pm above the surface of the agar in a cluster of cells. The field was observed with a x40 objective and recorded with a video camera and time-lapse S-VHS video recorder. Recordings were typically made at 1/240th of normal speed. For analysis, video recordings were played back and analyzed using Image 1/AT software (Universal Imaging, Media, PA). Images were captured, and then the software was used to determine the angle between the direction ofthe pipet and the direction of shmoo growth. The direction of shmoo growth was determined by the line connecting the original center of the cell with the tip of the shmoo. The cosine ofthe angle was calculated and used as a measurement of cell orientation. Esdmation ofReceptor Binding. Release from the pipet was estimated in two ways. First, a pipet was filled with radioactive cAMP and the amount of radioactivity released as a function oftime was measured. Second, for each experiment, the amount offluid lost during the experiment (since pressure ejection was used) was used to determine the release rate. Although release rates varied from pipet to pipet, the two types of measurements yielded similar results. At a pressure of 1.5 psi, fluid was released at a rate of ""3.3 x 10-8 ml/sec. From the molecular weight of the peptide, the diffusion coefficient in water at 30°C was estimated to be 3.2 x 10-6 cm2/sec. This was done by assuming that the diffusion coefficient, D, varies as Do/(Mr)'/3, where Mr is the molecular weight and Do is an empirically determined constant (17). Do was estimated to be 3.8 x 10-5 cm2/sec by using measured values of Leu-Gly-Gly [Mr = 281; D = 6.1 x 10-6 cm2/sec (18)], and ribonuclease [Mr = 13,683;D = 1.5 x 10-6 cm2/sec (17)], adjusted to 30°C due to changes in water viscosity (19). For a mating factor, Mr 1684, the diffusion constant is then 3.2 x 10-6 cm2/sec. This estimate is accurate to within a factor of 2, which is adequate for the conclusions discussed