Figure 1.
Altering the CENH3 N-terminal tail domain leads to defects in meiotic chromosome segregation.
a) CENH3 transgenes tested for fertility in a cenh3-1 homozygous mutant background. The male fertility was examined by Alexander staining. Viable pollen stains pink/red. Female fertility was judged by differential intereference contrast (DIC) microscopy of embryo sacs from at least 100 cleared mature ovules per genotype (Figure S1A). Single cell arrested ovules and ovules without an embryo sac (Figure S1B) were counted as non-viable, and ovules with 7–8 celled embryo sacs (Figure S1B) were counted as viable. Viable ovules may be haploid or aneuploid. b) Male meiotic chromosome spreads from wild type and GFP-tailswap plants. Metaphase I bivalents in the mutant are oval/round in shape, lacking the rhombus shape that indicates tension in wild type (compare A and F). Some metaphase I cells showed chromosomes that failed to congress to the spindle midzone (arrowed in K). Chromosome segregation at anaphase I is random in GFP-tailswap (G to I, L to N). Asynchronous homolog separation was seen at anaphase I (arrowed in G), and premature sister chromatid separation was also seen in meiosis I (arrowed in N). Decondensation at interkinesis was frequently delayed, especially for lagging chromosomes near the spindle midzone (arrowed in J, O). Metaphase II cells in the mutant show random chromosome alignment (U, Z). U shows one univalent (arrowed) and four bivalents plus the remaining univalent on the other side of the cell. Anaphase II chromosome segregation is random (V–X, AA–AC). Tetrad equivalent stages in GFP-tailswap (Y, AD) show several small nuclei instead of the expected four uniform nuclei seen in wild type. Scale bars −1 µm.
Figure 2.
Lack of centromere function in meiosis causes micronuclei to form in GFP-tailswap pollen.
Immunolocalization of alpha-tubulin outlines the nuclear envelope in microspores of GFP-CENH3 and GFP-tailswap pollen (A–F). GFP-tailswap pollen contains multiple micronuclei. Centromere DNA FISH shows that micronuclei contain 1–2 chromosomes each, as opposed to 5 chromosomes in a normal A. thaliana haploid pollen genome (G–L). The pollen grain shown in J–L has three micronuclei. Two contain one chromosome each, while the third contains two chromosomes.
Figure 3.
Reduced inter-kinetochore distance and meiotic spindle defects suggest lack of kinetochore function in GFP-tailswap.
a) Centromere DNA FISH from metaphase I in wild type and in GFP-tailswap. Blue = DNA (DAPI), green = centromere DNA FISH (FITC). GFP-tailswap bivalents lack the centromere stretch exerted by the spindle in wild type, and have reduced inter-kinetochore distance. Representative bivalents are magnified in D and H. Scale bars −1 µm. b) Centromere DNA FISH shows random orientation of bivalent chromosomes in GFP-tailswap meiosis I. Blue = DNA (DAPI), green = centromere DNA FISH (FITC). Metaphase I chromosomes are frequently aligned at unusual angles in the mutant (B). Anaphase I chromosomes show random alignment and premature sister chromatid separation (D). Arrows in A and B show presumed orientation of sister centromeres. Arrows in D indicate separated univalents, while arrowheads show intact bivalents. Scale bars −1 µm.
Figure 4.
GFP-tailswap protein is depleted from kinetochores during pre-meiotic interphase.
Meiocytes from anthers of GFP-CENH3 and GFP-tailswap were imaged using identical exposure times. Flattened projections of several stacked images are shown. The GFP-CENH3 protein showed bright fluorescence at kinetochores in all meiocytes (class I). GFP-tailswap protein always showed reduced fluorescence relative to GFP-CENH3. Three classes of GFP fluorescence were observed in GFP-tailswap: faintly visible (class II, 7%), barely detectable (class III, 47%), and undetectable (class IV, 46%).
Figure 5.
Depletion of GFP-tailswap protein from kinetochores continues progressively during meiosis.
a) Dynamics of kinetochore GFP-CENH3 and GFP-tailswap proteins during meiosis were visualized in anthers. Flattened projections of several stacked images are shown. GFP-CENH3 is visible at uniform intensity at kinetochores throughout meiosis. GFP-tailswap is barely detectable or undetectable in leptotene and zygotene (comparable to class III and class IV in Figure 4). In pachytene and subsequent stages, we did not detect kinetochore GFP fluorescence in GFP-tailswap meiocytes.
Figure 6.
Percentage of GFP-CENH3 and GFP-tailswap meiocytes showing particular classes of GFP fluorescence at kinetochores.
Figure 7.
Depletion of GFP-tailswap protein from meiotic kinetochores causes removal of MIS12.
GFP-CENH3, GFP-tailswap and MIS12 proteins were immunolocalized in anthers during the pachytene stage of meiosis with anti-GFP and anti-MIS12 antibodies. Somatic cells from the same anther are shown as a control. GFP-CENH3 and MIS12 were visualized at both meiotic and somatic kinetochores of GFP-CENH3 plants. In GFP-tailswap plants, GFP-tailswap and MIS12 were both undetectable in GFP-tailswap meiotic kinetochores but can be seen in somatic kinetochores. Scale bars −5 µm.
Figure 8.
Immunolocalization of α-tubulin in wild type and GFP-tailswap meiocytes.
Anther meiocytes from wild type and GFP-tailswap plants were stained with anti-tubulin antibodies. Metaphase I spindles are disorganized and longer in GFP-tailswap, and may contain fewer microtubules (E and I). Meiosis II cells in GFP-tailswap often contain more than two spindles (F–G, J–K). Spindle appearance and orientation are disordered, and may fail to include some chromosomes (arrowed in G). Tetrad equivalent cells (H, L) lack the radial microtubule system that surrounds the four haploid nuclei in wild type. Scale bars −5 µm.
Figure 9.
Removing the meiosis-specific cohesin REC8 does not restore meiotic kinetochore function in GFP-tailswap.
Chromosome spreads from male meiosis in rec8 spo11-1 and rec8 spo11-1 cenh3-1 GFP-tailswap. Anaphase I in rec8 spo11-1 shows orderly separation of sister chromatids that is similar to mitosis, because the rec8 mutation converts kinetochores to a mitosis-like behavior (A, D). Anaphase I in rec8 spo11-1 cenh3-1 GFP-tailswap shows random segregation of univalent chromosomes (B, C, E, F). This is consistent with the observation that removing REC8 does not restore loading of the GFP-CENH3 protein (Figure S6).
Figure 10.
GFP-tailswap protein reloads onto centromeres after meiosis, when mitosis resumes.
a) Microspores of wild type, GFP-CENH3 and GFP-tailswap. Tetrad equivalent stages in GFP-tailswap do not show any GFP fluorescence at meiotic kinetochores (Figure S4). However, GFP-tailswap protein reloads onto kinetochores in a small fraction of microspores, when haploid mitotic divisions are expected to resume. b) Frequency of GFP-positive and -negative microspores from GFP-CENH3 and GFP-tailswap plants. c) The number of GFP foci in each GFP-positive spore is shown.