Prometaphase

Actively growing root-tip meristems were pretreated with 0.05% aqueous solution of colchicine for 4 h at room temperature, fixed in ethanol : acetic acid (3 : 1) for at least 3 h at room temperature, and stored at −20°C until use.
Chromosome counting and basic karyotype analyses were performed using the standard Feulgen staining technique [55 (link)]. Ideograms (Additional file 2: Figure S2) were constructed based on measurements of at least five well-spread metaphase plates per individual (not shown) and measurements were used to calculate Haploid Karyotype Length (HKL). A single ideogram of each species and cytotype is provided, except for cytotypes B⁷B⁷ and B⁶B⁶ in which structural chromosomal variants were found (Table 2). Idiograms were constructed using Autoidiogram software (courtesy of Dr Wolfgang Harand, formerly University of Vienna; for details see [55 (link)]).
Chromosomal spreads for FISH were prepared by enzymatic digestion and squashing, as described earlier [4 (link),16 (link)] with some modifications. Briefly, material was digested with 1% cellulase Onozuka (Serva, Heidelberg, Germany), 1% cytohelicase (Sigma-Aldrich, Vienna, Austria), and 1% pectolyase (Sigma-Aldrich) for 18 min at 37°C. Cover slips were removed at −80°C and preparations air-dried. FISH followed the established protocol [16 (link),56 (link)]. Probes used for FISH were: 35S (18S/25S) rDNA from Arabidopsis thaliana in plasmid pSK+; 5S rRNA genic region from Melampodium montanum in plasmid pGEM-T Easy. Probes were labeled with biotin or digoxygenin (Roche, Vienna, Austria) either directly by PCR (5S rDNA) or using a nick translation kit (35S rDNA; Roche, Vienna, Austria). Digoxygenin was detected with antidigoxygenin antibody conjugated with FITC (5 μg mL^-1: Roche, Vienna, Austria) and biotin with ExtrAvidin conjugated with Cy3 (2 μg mL^-1: Sigma-Aldrich, Vienna, Austria). Preparations were analyzed with an AxioImager M2 epifluorescent microscope (Carl Zeiss, Vienna, Austria), images captured with a CCD camera, and processed using AxioVision ver. 4.8 (Carl Zeiss, Vienna, Austria) with only those functions that apply equally to the whole image. For rDNA localization, a minimum of 20 well-spread metaphases and prometaphases was analysed for each individual.

HeLa, MCF-7 and COS-1 cells (ATCC) were maintained in DMEM – 10% FBS (Invitrogen). Sf9 cells (Invitrogen) were cultured in SF-900 II medium (Invitrogen). Transfections in COS-1 cells were performed with FuGene 6 (Roche) according to manufacturer instructions. For synchronization, cells were deprived of serum for 24 h and released in the presence of one of the following inhibitors: 16 h, 5 µM cyclosporin to arrest at G0/G1; 24 h with 100 nM wortmannin for late G1, 24 h with 400 µM mimosine for G1/S, 48 h with 5 mM hydroxyurea for S-phase, 24 h with 10 µM etoposide for G2/M and for 17 h with 150 ng/ml nocodazole for M-phase (prometaphase) followed by shake-off to collect mitotic cells. All inhibitors were purchased from Sigma. Mitotic cells were washed and released for the indicated times in the presence or absence of 10 µg/ml cycloheximide (Sigma), 20 µM MG132 (Calbiochem), 0.1–1 µM okadaic acid (Calbiochem) and 20 nM calyculin A (Calbiochem). Alternatively, mitotic cells were resuspended in nocodazole containing media supplemented with 20 µM PD98059 (Sigma) or 10 µM purvalanol A (Calbiochem) alone and/or okadaic acid or calyculin A for 2 h.

To determine the number of ovarioles in wild type and mutant ovaries, ovaries were stained with antibodies against Vasa and Engrailed, a transcription factor that is expressed only in niche cells of the germaria [52 (link)]. The number of niches was determined by counting Engrailed positive cells adjacent to Vasa positive cells.
Several nuclear parameters were assessed. To determine intensity of GFP-BAF, emerin, and lamin at the nuclear envelope or within the GSC nucleus, a line segment was drawn across each nucleus. The rim intensity was defined as the average grey value of the two intersection points between the line and the nuclear envelope. The interior intensity was defined as the mean grey value along the line that is inside of the nucleus. For these experiments, three replicates were performed, corresponding to three different slides made for each genotype. In each case, imaging parameters were kept the same among experiments and samples. The middle section of each GSC was chosen for quantification. Background was subtracted for these quantifications. To measure nuclear roundness, each nucleus was traced based on lamin staining in ImageJ and roundness was determined as 4*area/(π*major_axis^2).
Several mitotic parameters were assessed. Mitotic stages were determined based on staining patterns of α-tubulin, Cnn, and H3S10p using the following criteria (Fig. S2): 1) Prophase was defined as H3S10p staining that was restricted to the periphery and evidence of microtubules nucleation at the periphery of the nuclear envelope, 2) Prometaphase was defined as chromosomes marked by H3S10p that were partially condensed and microtubules that were beginning to connect chromosomes to the spindle poles, 3) Metaphase was defined as chromosomes marked with H3S10p that were fully condensed and aligned at the metaphase plates, and microtubules formed the characteristic fusiform metaphase spindle, 4) Anaphase was defined as chromosomes marked with H3S10p that were separated into two clusters that each had individual chromosome arms that were visible, and 5) Telophase was defined as H3S10p staining that was weaker and the presence of a central spindle at the mid-plane of the cell. To determine alignment of microtubules (MTs), images were visualized in ImageJ and misaligned MTs were defined as MTs that cross each other’s path at the metaphase plate. The percentage of MT coverage of chromosomes was quantified in maximum projection images of metaphase GSCs that were stained with antibodies against H3S10p and α-tubulin. The coverage was defined as a ratio of the chromosome length that was covered by MTs divided by the entire length of the chromosome mass. The intensity of CID and CENP-C in images of metaphase GSCs were quantified in Image J using the sum slices projection method. Only well-separated CID and CENP-C foci were included. For each centromere, background was subtracted with 50 pixels rolling ball radius. Total grey values were reported for both CID and CENP-C staining. Different genotypes were imaged using the same parameters and at the same time.
To quantify DCP-1 intensity in wing discs, discs were hand dissected from third instar larvae of the indicated genotypes and stained with antibodies against cleaved DCP-1 and DAPI. Samples were imaged using a confocal microscopy under the same setting at the same time. DCP-1 intensity was quantified from a summed z-projection with background subtracted using image J (50 pixel rolling ball radius). The total intensity was divided by the area of the wing disc to control for size differences. Resulting DCP-1 mean intensity was graphed. At least three replicates were performed for each genotype.