JCS009977 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Specificity of cohesin SMC antibodies. (a) Immunoblots containing 10 μg of embryo or whole ovary extracts were stained with anti-cohesin antibodies. Affinity-purified SMC1 and SMC3 antibodies recognize a single predominant band of the predicted molecular mass in embryos, and two to three closely spaced bands in ovary extracts. (b) An FLP/FRT strategy was utilized to generate germ-line clones in the ovary. Absence of LacZ staining marks a region 2B cyst that is homozygous for the smc1 excision allele, smc1^exc46 (outlined in white). Region 2A cysts that are heterozygous for smc1^exc46 are visible on the left. Whole-mount preparations were immunostained for β-galactosidase (orange), SMC1 (green), DNA (blue) and C(3)G (magenta). In the Region 2B cyst lacking β-galactosidase (LacZ) signal, no signal above background is observed for SMC1. Note the bright centromeric foci and thread-like SMC1 signal in the adjacent region 2A cyst. In addition, thread-like C(3)G staining is completely absent in the smc1^−/− cyst. Images are projections of of deconvolved Z-series. Bar, 5 μm.

• Supplemental Figure S2 -

  Fig. S2. SMC1 localization throughout a single germarium. A wild-type whole-mount germarium (outlined in white) is immunostained for SMC1 (green) and GFP-ORD (orange). SMC1 signal is absent in germ-line stem cells at the anterior tip of the germarium (asterisk). SMC1 and ORD colocalize in pre-meiotic cysts (solid arrow) as well as in 16-cell meiotic cysts in regions 2A, 2B and 3. Thread-like SMC1 staining (<) is visible in a number of nuclei throughout the germarium. The oocyte nucleus in region 3 is marked by an open arrow. Images are projections of deconvolved Z-series. Bar, 10 μm.

• Supplemental Figure S3 -

  Fig. S3. Cohesin SMC localization in a late-stage pachytene oocyte. A whole-mount stage 6 oocyte nucleus is shown (dotted line indicates nuclear perimeter) with SMC1/3 (green), CID (orange) and DNA (blue) staining. Thread-like cohesin SMC signal is still visible in this older vitellarial stage as well as non-chromosomal SMC staining throughout the nucleus. Note that the DNA also displays a thread-like pattern. Images are projections of deconvolved Z-series. Bar, 10 μm.

• Supplemental Figure S4 -

  Fig. S4. Soluble nuclear protein is removed in chromosome spread preparations. (a) Whole-mount germaria from y w (no transgene) and transgenic females expressing nuclear GFP (GFP-nls) were fixed and stained with DAPI. GFP fluorescence signal (green) is robust in the nuclei of the germarial cells. Images are projections of deconvolved Z-series. For each channel, the images for transgenic GFP-nls germaria and the y w negative control were captured and processed identically. Bar, 5 μm. (b) Chromosome spread preparations were prepared from ovaries of y w and GFP-nls females and immunostained for SMC1/3 (white), GFP (white), C(3)G (magenta) and DNA (blue). SMC1/3 images were captured using a 250 millisecond exposure and GFP images were captured using 250 msecond and 500 msecond exposures. The GFP signal in spreads from GFP-nls ovaries and y w ovaries is identical at both exposures (and also at higher exposures; data not shown) indicating that soluble nucleoplasmic GFP is removed during the chromosome spread preparation. Bar, 5 μm.

• Supplemental Figure S5 -

  Fig. S5. Disruption of cohesin SMC localization in an ord^null germarium. A whole-mount ord^null germarium immunostained for SMC1/3 (green) and C(3)G (magenta) is shown. In contrast to wild-type, no bright SMC foci are visible at any stage within the germarium. Note the lack of bright foci in nuclei prior to pre-meiotic S-phase (region 1, curly bracket) as well as in region 3 (square bracket). For comparison with wild type, see Fig. S2. Although thread-like SMC1/3 staining overlaps with C(3)G in early region 2A (arrow), SMC1/3 threads become fragmented in later stages (also see Figs 6 and 8). Open arrow indicates disrupted SMC1/3 signal in the region 3 oocyte. Images are projections of a deconvolved Z-series. Bar, 10 μm.

• Supplemental Figure S6 -

  Fig. S6. Centromeric SMC1/3 staining is absent in ord^null germaria. Enlarged images taken from Figs S2 and S5, containing region 1 and 3 nuclei, are shown together for comparison. Cohesin SMC1/3 staining is visible at the centromeres in wild type (top panel); however, no SMC1/3 foci are visible in ord^null germaria (bottom panel). Images are projections of deconvolved Z-series. Bar, 2 μm.

• Supplemental Figure S7 -

  Fig. S7. Thread-like cohesin SMC staining does not depend on C(3)G activity. Individual nuclei from whole-mount preparations of c(3)G mutants immunostained for SMC1/3 (green) and C(3)G (magenta) are shown. Although, no C(3)G signal is observed, threads of SMC staining are visible in region 2A and region 3 in the mutant germaria. Bars, 2 μm.

• Supplemental Figure S8 -

  Fig. S8. Thread-like SMC1/3, ORD and C(3)G staining is absent in c(2)M mutants. A germarium from a c(2)M^EP2115<^/i> female expressing GFP-ORD was immunostained for SMC1/3 (green), GFP-ORD (orange) and C(3)G (magenta). The entire germarium is outlined in white with regions 2B and 3 indicated by ovals. Images are projections of a deconvolved Z-series. Bar, 10 μm.