An SMC-Domain Protein in Fission Yeast Links Telomeres to the Meiotic Centrosome  Mark R. Flory, Andrew R. Carson, Eric G. Muller, Ruedi Aebersold  Molecular Cell  Volume 16, Issue 4, Pages 619-630 (November 2004) DOI: 10.1016/j.molcel.2004.10.027

Figure 1 Identification of Ccq1p and Its Molecular Features (A) Reconstructed single ion current chromatogram showing relative quantification of enrichment for a peptide mapping to S. pombe ORF SPCC188.07 (ccq1+). The m/z (mass/charge) range shown is 578–594 for the light peak and 562–608 for the heavy peak. Intensity of signal (I) is 1.4 ×105 for the light peak and 6.6 ×105 for the heavy peak. (B) Ccq1p N-terminal region armadillo/heat repeats (color shading from blue at N terminus to red at C terminus), Ccq1p C-terminal region structural maintenance of chromosome (SMC)/ATPase domain, and Ccq1p C-terminal coiled-coil domain homologous to the Pcp1p coiled-coil region. (C) Meiotic and vegetative mRNA expression patterns of ccq1+ and pcp1+ mRNA transcripts. Meiotic onset (*), DNA synthesis (S), and meiotic divisions I (I) and II (II) are as indicated. Each analysis spans ∼9 hr. Range of microarray ratios measured are shown on the y axis. Molecular Cell 2004 16, 619-630DOI: (10.1016/j.molcel.2004.10.027)

Figure 2 Localization Patterns of Ccq1p in Live Cells (A and B) Live cell localization of Ccq1p-GFP, expressed at the endogenous chromosomal locus and controlled by the ccq1+ promoter in strain MFP104, to punctate spots and the nucleoplasm in interphase cells (A and B) and to an elongating nucleus in a mitotic cell (C). (D–F) Live cell localization of known cellular markers for comparison: centrosomal Cut12p-GFP in an interphase cell of strain 470 (D), telomeric Taz1p-CFP in an interphase cell of strain MFP106 (E), and centrosomal (two dots) and nuclear pore-localized (ring) Cut11p-GFP in a mitotic cell of strain 355 (F). (G) Nuclear colocalization in a live MFP104 cell of a knob-like DNA-staining protrusion (top arrow) with Ccq1p punctate spot in strain MFP104 (bottom arrow). (H) Confirmation of Ccq1p-GFP expression intensity from plasmid pMF202 in strain 99 with punctate spots indicated by arrows (H), and corresponding phase contrast image of cells (I). All bars, 5 μm; bar in (F) is for (A)–(F); bar in (I) for (H) and (I). Molecular Cell 2004 16, 619-630DOI: (10.1016/j.molcel.2004.10.027)

Figure 3 Colocalization of Ccq1p and Taz1p at Telomeres in Live Cells (A–H) Live cell colocalization of Ccq1p-CFP with Taz1p-YFP in interphase cells of strain MFP109. (I–L) Exclusion of Ccq1p from kinetochores labeled with Ndc10p-YFP in a live interphase cell of strain MFP108. (M–P) Localization of multiple Ccq1p-YFP dots exclusive from centrosomal Pcp1p-CFP dot in an interphase cell of strain MFP107. (Q–T) Mitotic MFP107 cell exhibiting two Pcp1p-CFP fluorescent dots and diffuse Ccq1p-YFP fluorescence in the nucleoplasm. Bar in (H), 5 μm, for (A)–(H); bar in (T), 5 μm, for (I)–(T). Molecular Cell 2004 16, 619-630DOI: (10.1016/j.molcel.2004.10.027)

Figure 4 Physical and Genetic Interactions between Ccq1p and Pcp1p (A–D) Colocalization of Pcp1p-CFP and Ccq1p-YFP in live, mating factor (p-factor)-arrested cells of strain MFP111. (E–K) MFP101 (nmt1-pcp1) cells expressing Ccq1p-GFP from plasmid pMF202 exhibit multiple growth defects including abnormal and supernumerary areas of Ccq1p-GFP fluorescence. Arrow: punctate, nuclear spot of Ccq1p-GFP fluorescence. (K): Growth of MFP101 cells with control plasmid pSP1 (top streak) or with Ccq1p-GFP expression plasmid pMF202 (bottom streak) on solid supplemented YES agar, 3 days, 30°C. (L–N) Localization of Pcp1p-CFP in strain MFP112 (ccq1::ura4+) (L-M) and Pcp1p-GFP in ccq1 hemizygous diploid cells (N) (red, Pcp1p-C/GFP; green, phase contrast of cell surface). All bars, 5 μm; bar in (D) for (A)–(D); bar in (J) for (E)–(J), bar in (N) for (L)–(N). Molecular Cell 2004 16, 619-630DOI: (10.1016/j.molcel.2004.10.027)

Figure 5 Telomeric Localization Patterns of Ccq1p and Taz1p during Normal Meiotic Prophase and in a Strain Containing Abnormally Reduced Levels of Ccq1p (A–L) Ccq1p-CFP and Taz1-YFP distribution in diploid cells transiting meiotic prophase demonstrating normal fusion of telomere clusters during karyogamy (A-D), movement of the resulting telomeric cluster to a peripheral end of the nucleus (E-H), and formation of the “horsetail” chromosome morphology characteristic of fission yeast meiotic prophase. (M–X) Ccq1p-CFP and Taz1-YFP colocalization to abnormal telomere cluster structures in transient diploids lacking one ccq1 allele. Two telomere clusters are evident, including closely juxtaposed foci (M-P) and more widely separated foci in horsetail nuclei of normal (Q-T) and perturbed morphology (U-X). Bar, 5 μm. Molecular Cell 2004 16, 619-630DOI: (10.1016/j.molcel.2004.10.027)

Figure 6 Phenotype of Diploid and Haploid Strains Lacking One Ccq1+ Allele during Vegetative Growth (A–C) DNA segregation defects in live hemizygous MFP103 cells (lacking one ccq1+ allele) stained with DAPI. (C): MFP103 cells containing plasmid pMF201 exhibiting abnormal incorporation of GFP-α-tubulin and abnormally stretched DNA (arrow). (D–F) MFP103 cells containing plasmid pMF200 (ccq1+) exhibiting normal cell length and normal chromosome (blue) and tubulin (green) morphologies at interphase (E) and mitosis (F). (G) Growth of MFP103 cells expressing Ccq1p or GFP-Atb2 (tubulin) as indicated on unsupplemented EMM agar, 3 days, 30°C. (H) Southern blot analysis of telomere length defects in diploid (Dip., top) and haploid (Hap., bottom) strains. Strains were prepared and DNA was isolated and treated as described in Experimental Procedures. Top lanes 1 and 2 (replicate analyses): strain MFP103 containing rescue plasmid pMF200 (ccq1+); top lane 3: diploid fission yeast created via intragenic complementation of wild-type strains 99 and 100; top lanes 4 and 5 (replicate analyses): strain MFP103 containing control parent plasmid pSP1. Bottom lane 1: wild-type diploid strain for comparison to haploid strains in lanes 2–7 (see top lane 3 for description of wild-type diploid strain); bottom lanes 2 and 3 (replicate analyses): strain MFP102 containing rescue plasmid pMF200; bottom lanes 4 and 5 (replicate analyses): strain MFP102 containing control parent plasmid pSP1; bottom lane 6: haploid wild-type fission yeast strain 99; bottom lanes 7 and 8 (replicate analyses): strain MFP19 moderately overexpressing GFP-Pcp1p; bottom lane 9: strain MFP105 moderately overexpressing GFP-Ccq1p. (I) Molecular model for normal centrosome-telomere complex formation driven by a Ccq1p-Pcp1p physical interaction. Gray balls represent possible adaptor proteins facilitating Ccq1p-Taz1p interaction at telomeres. All bars, 5 μm; bar in (B) is for (A) and (B); bar in (F) is for (E) and (F). Molecular Cell 2004 16, 619-630DOI: (10.1016/j.molcel.2004.10.027)