Rad51 and Dmc1 sr foci are extremely small and clustered at sub-diffraction distances.

(A-D) dSTORM micrographs of Rad51 filaments assembled on a linear 2.7 kbp dsDNA in vitro in the presence of the meiotic protein Hed1. Corresponding widefield micrographs are inset at top right and the scale bars are 1 μm wide (E-H) Example dSTORM and widefield (bottom right insets) micrographs of wild type nuclei stained for (E) Rad51 and (F) for Dmc1; mnd1 nuclei stained for (G) Rad51 and (H) Dmc1. Arrowheads indicate examples of 100 nm paired Rad51 sr foci, magnified in the upper right insets. Scale bar is 1 μm for dSTORM and widefield micrographs, 100 nm in top right insets. (I-K) Distributions of Rad51→Rad51 sr foci nearest neighbor measurements for (I) wild type, (J) mnd1, and (K) zip1 mutants. Micrographs from cultures 3.5 hours after meiotic induction. (L-O) Each Rad51 focus in a pair of Rad51 foci imaged under widefield can contain more than one Rad51 sr focus. (L-N) Micrographs of paired Rad51 foci observed at widefield resolution (left) and with dSTORM (right) containing 1 and 1 (L); 1 and 2 (M); and 2 and 2 (N) Rad51 sr foci. Scale bar is 400 nm. (O) Distribution of the frequencies of Rad51 sr foci in one focus (blue) or both foci (red) in paired Rad51 foci at low resolution. 48 widefield pairs of Rad51 foci were scored. (P) Characterization of the dimensions of Rad51 and Dmc1 sr foci. An ellipse was fit to each sr focus. Lmajor is the length of the ellipse’s long axis; aspect ratio equals Lmajor/Lminor. Scoring of 432, 212, and 304 sr foci from 5 wild type, 5 mnd1, and 5 zip1 nuclei for Rad51 scoring; 359 and 592 sr foci from 3 wild type and 6 mnd1 nuclei for Dmc1 scoring. Errors are S.D.