JCS01334 Supplemental Material

Files in this Data Supplement:

• Supplemental Table (Adobe PDF)
• Supplemental Figure 1 -

  Fig. S1. Nuclear lamina defects in clone 8 cells delayed in mitosis with MG132. (A-C) Clone 8 cells in untreated (A,B) and  treated (C) cultures stained with antibodies against lamins (green), KLP61F (red) and a chromatin dye (blue). (A) Prophase shows KLP61F localized to centrosomal asters and an intact nuclear lamina with no evidence of nuclear envelope breakdown. (B) Representative of 21 of 697 cells near metaphase of mitosis, showing KLP61F localized to spindles and the nuclear lamina organization similar to those in somatic cells in brains of wild-type animals. (C) Representative of 12 cells near metaphase of 1159 cells in treated cultures showing the nuclear lamina in an disorganized state. Nearby cell in interphase provides internal control for lamin staining. Subconfluent cultures of clone 8 cells were diluted 1:3 in fresh media and moved to 30 mm petri dishes containing a sterile 20 mm square cover slip. After 24 hours’ growth at 25°C, media was exchanged for fresh media with or without 50 mg/ml MG301 and fixed at 2, 4 and 12 hours post media exchange. All time points in treated cultures show lamina defects. Data here was derived from cells fixed 2 hours post media exchange.

• Supplemental Figure 2 -

  Fig. S2. Ncd localization in somatic cells of wild-type larval brains and cultured clone 8 cells. (A-D) Larval brains  of wild-type animals (A), cultured clone 8 cells (B,C) and larval brains of ncd¹ mutants (D) stained with antibodies directed against Ncd (red), g-tubulin (green) and DAPI (blue). (A) Asterisk indicates interphase cell without detectable Ncd staining. Arrows (A, B) indicate interpolar fibers, (C) subnuclear enrichment of Ncd, and (D) metaphase alignment of chromosomes in two mitotic cells. Scale bar, 5 mm. (A) Ncd consistently showed weak immunostaining of fibers that extended between spindle poles at metaphase (arrow). Ncd showed nuclear localization during interphase that appeared to be enriched near heterochromatin, but difficult to image. Immunostaining of nuclei was not detected in all interphase cells, Ncd was detected in all cells in prophase, suggesting that Ncd may be up regulated in preparation for mitosis and/or down regulated in non-proliferating cells. (B) To confirm the localization of our antibodies, Ncd was localized in cultured clone 8 cells derived from imaginal discs (Peel and Milner, 1992) and of somatic origin. Ncd was nuclear during interphase and enriched near heterochromatin (arrow). (C) Ncd localized to fibers extending pole to pole during mitosis. (D) Neither spindle nor nuclear staining was observed in null ncd¹ mutants, indicating that immunostaining of spindles and nuclei were due to Ncd and not to the 150 kD protein detected by immunoblot analysis (Fig. 1 in text). Clone 8 cells were cultured as described at 25°C in 25 ml flasks in Shields and Sangs M3 media supplemented with 2% (v/v) heat inactivated fetal bovine serum, 50 mg/ml penicillin/streptavidin, 5 mg/ml insulin (cell culture reagents, Sigma) and 2.5% fly extract prepared as described (Peel and Milner, 1992) except that frozen flies were homogenized in a small dedicated coffee grinder with a sheet of Parafilm underneath the grinder’s cover to limit spillage. For immunostaining, media was removed and cells were washed with PEMS, slips were plunged into methanol at 20°C for 6 minutes and then into a post-fix of 2% paraformaldehyde in PBS at room temperature for 6-10 minutes. Immunostaining was as described in text.

• Supplemental Figure 3 -

  Fig. S3. Wide-field imaging of Skeletor localization in the wild-type and klp61F mutant spermatocytes. Spermatocytes of wild-type (wt) and klp61F³ mutants stained with antibodies against Skeletor (green), g-tubulin (red) and DAPI (blue). Note that Skeletor was associated with some masses of chromatin.