Scherhan et al. 10.1073/pnas.0704860104. |
Fig. 10. Functional analysis of ZIP1-GFP constructs. Percentage of cells completing sporulation ± SE was determined by counting at least 200 cells by using a hemocytomerer after 24 h in liquid sporulation medium. Percentage of viable spores ±SE obtained by dissecting »200 asci/sample.
Fig. 11. Nuclear deformation in individual nuclei. D[l/w] in individual nuclei under the noted conditions were determined in WT and ndj1 cells as described in Materials and Methods.
Fig. 12. The distribution of actin in pachytene cells is not affected in ndj1 mutants. WT (strain HW122) and ndj1 mutant cells (strain EW105) containing Zip1-GFP and stained with Phalloidin-TRITC to show actin patches and cables photographed 4 h after induction of sporulation. (Left) Merge of GFP (green) and actin-fluorophor (red) and DAPI (blue) images. (Right) Actin-fluorophor only. Methods: Actin was stained with Phalloidin-TRITC (Sigma, St. Louis, MO), as described (1), except cells were washed eight times with PBS and suspended in Vectashield (Vector Labs) containing DAPI before microscopy.
1. Kaiser C, Michaelis S, Mitchell A (1994) in Methods in Yeast Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), p 199.
Movie 1. Wild-type (strain HW122) zygotene nucleus traversing from early to late zygotene or early pachytene. Zip1-GFP foci increase in size and number as the synaptonemal complexes (SCs) grow. Extensive nuclear and SC movements are seen. (Elapsed time 43 min, recording speed - 1 frame/5.0 sec).
Movie 2. Wild-type (strain HW122) pachytene nucleus (cell "a" in Fig. 5) displaying mature Zip1-GFP labeled SCs that progresses into diplotene while diplotene nucleus (cell "b" in Fig. 5) with disassembling Zip1-GFP foci undergoes one or two meiotic divisions during the time viewed. Video is edited to four intervals (4-17 min, 61-74 min, 104-121 min, and 160-173 min), as indicated in the upper left-hand corner. SC movements and nuclear shape changes are evident as are the maturation of three additional asci from cells that were post prophase I at the beginning of the recording and appear as a result of autofluorescence on the lower left-hand side of the field after 104 min (Recording speed - 1 frame/5.0 sec, total elapsed time 173 min).
Movie 3. Wild-type (strain HW122) pachytene nucleus exhibiting continuous SC movements with transient appearance of folded U-shaped SCs and maverick chromosomes moving away from the chromosome mass on the left side of the nucleus. Transient shuttling of SCs to and from perinuclear regions are evident as are continuous nuclear movements (Recording speed 1 frame/0.5 sec, total elapsed time 7 min).
Movie 4. The effect of Latrunculin B on SC movement. Wild-type (strain HW122) pachytene nucleus displaying vigorous SC and nuclear movement that ceases almost immediately following the addition of the actin-disrupting drug Latrunculin B at 1.3 min. (Recording speed 1 frame/0.5 sec, total elapsed time 4 min).
Movie 5. Reversibility of Latrunculin B inhibition. Wild-type (strain HW122) pachytene nucleus that had been washed free of Latrunculin B as described in Materials and Methods. (Recording speed 1 frame/0.5 sec, total elapsed time 4 min).
Movie 6. The effect of formaldehyde on SC movement. Two wild-type (strain HW122) pachytene nuclei that were treated with the cross-linking agent formaldehyde before mounting show that virtually all motion is absent. In addition, Zip1-GFP labeled SCs undergo rapid photo bleaching (Recording speed 1 frame/5 sec, total elapsed time 13min).
Movie 7. The role of NDJ1 in SC movement. An ndj1 mutant nucleus exhibiting greatly reduced chromosome and nuclear mobility. Bending and maverick chromosomes are not observed. Latrunculin B was added at 7.5 sec of viewing, further reducing chromosome mobility (Recording speed 1 frame/0.5 sec, total elapsed time 7 min).
Movie 8. Pachytene nuclei in a presumed WT (strain HW122) cell showing highly motile chromosomes and an adjacent ndj1 mutant (strain EW105) cell with relatively dormant chromosomes. Cells from both strains were mixed and a field containing two differently behaving nuclei was recorded for comparison. Note the characteristic absence of chromosome folding in the presumed ndj1 nucleus (Recording speed 1 frame/5.0 sec, total elapsed time 18 min).
Movie 9. WT cells showing vigorous movement of SCs and nuclei produced using maximum projection of z-stack image series recorded at 1 stack (19 frames) per min for 35 min as described in Materials and Methods.
Movie 10. Mutant ndj1 cells showing reduced SC and nuclear mobility produced as described for Movie 9.