Biology and MB&B

Graduate Student Career Retreat 2008

Name:  Tina Motwani Rosemarie Doris & Mark Flory

   Lab:  Mark Flory [MB&B]

Abstract

Telomere Targeting of Condensin Complexes in Fission Yeast

Tina Motwani, MBB Department, Wesleyan University

Eukaryotic mitotic fidelity requires faithful duplication and accurate segregation of chromosomes. Recent work in budding yeast and higher eukaryotes suggest that specialized mechanisms facilitate the segregation of repeat-rich chromosomal regions, including rDNA and telomeres, during anaphase.  While rDNA segregation mechanisms have been dissected to some extent molecularly, telomere-associated segregation mechanisms remain enigmatic.  However, our data here suggest that in the fission yeast Schizosaccharomyces pombe, the telomere-associated protein Ccq1p plays a unique role in promoting faithful and accurate chromosome segregation.  Previous work indicated that during meiotic prophase Ccq1p facilitates the centrosomal clustering of telomeres that underlies meiotic chromosomal bouquet formation, a highly conserved event among eukaryotes.  Ccq1p also is expressed and localizes to chromosome ends during mitotic growth, when telomeres and centrosomes remain spatially separated.  This suggests a different role for Ccq1p during mitotic growth.  Mitotic Ccq1p activity is further indicated by the ccq1∆ (gene deletion) phenotype that includes live but defective cells exhibiting anaphase chromosome bridging, remarkably high chromosome loss rates, telomere shortening, and abnormally long cells exhibiting cell cycle delay.  Our current work focuses on understanding the mitotic role performed by Ccq1p.

We were initially surprised to find that conditional knockdown of ccq1⁺ expression using an artificial promoter results in the remarkably rapid (within one cell cycle) formation of anaphase bridges without telomeric length compromise.  The kinetics of bridge formation in this assay argue against our initial hypothesis in which Ccq1p prevents gradual degradation and subsequent fusion of telomeres, a phenotype which usually manifests only after several generations.  An alternative model arose from our proteomic analysis of Ccq1p binding partners via affinity purification and tandem mass spectrometry that revealed multiple protein subunits of the condensin complex.  In its canonical role, the condensin complex compacts chromosomes into physically separable units in preparation for segregation.  Our data suggested the possibility of an additional, specialized role for condensins, in concert with Ccq1p, at telomeric ends.  In conditional (temperature-sensitive) condensin loss-of-function backgrounds, we find that Ccq1p-GFP is lost from telomere ends at the restrictive temperature, indicating that localization of Ccq1p to telomeres is dependent on condensin complex integrity.  In these same backgrounds, telomere spots as marked by Taz1p-mRFP, a known telomere marker, become more numerous at the restrictive temperature, indicating a loss of normal inter-telomere clustering activity.  Finally, we find that overexpression of ccq1⁺ reduces the frequency of anaphase bridges caused by abnormally elongated telomeres in cells that lack Taz1p.  We hypothesize that rescue in this latter case is facilitated by the enhanced Ccq1p-mediated recruitment of condensins to the abnormally long telomeres, thus preventing chromosomal entanglements.  Together, these data suggest that a telomeric Ccq1p-condensin complex is essential for proper chromosome resolution, likely through maintenance of proper inter-telomeric clustering interactions during interphase.