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The mechanism of chromosome segregation during mitosis and meiosis

The evolution of eukaryotic cells

My lab is making use of phylogenomic and cell biological approaches for investigating cytoskeletal and chromatin evolution in basal microbial eukaryotes. There is little scientific precedent for comparative genomic approaches in understanding both patterns and mechanisms of cytoskeletal evolution, particularly in terms of the microtubule cytoskeleton as compared to the microfilament (actin) cytoskeleton, or in terms of the evolution of mitosis, meiosis and chromatin structure. When one considers the vast diversity and evolutionary history of eukaryotes, it is clear that questions concerning the early eukaryotic cell would benefit from the phylogenomic and cell biological analyses of eukaryotic microbes that diverged early in the evolution of life. Little is known about the molecular components, functions, and dynamics of the cytoskeleton in basal eukaryotes other than microscopic descriptions.

To assess the evolutionary history of the cytoskeleton in a basal eukaryote, an ongoing project is a comprehensive phylogenomic annotation of cytoskeletal genes in the Giardia intestinalis genome. We are expanding this computational and proteomic strategy to compare the genomes of Spironucleus vortens and the amoeba-fl

agellate Naegleria gruberi with the hope of constructing a genomic “map” of the cytoskeleton and its evolution. The results of our comparative genomic analysis are quite surprising. First, the microtubule system in Giardia appears to contain a complete array of component proteins, including all tubulin isoforms, and many kinesins and dyneins. Yet in stark contrast, the actin or microfilament system is missing the majority of known proteins other than actin itself. Specifically, we find no evidence for myosin or proteins involved in actin assembly, and organization. These results challenge assumptions concerning the strong conservation of these proteins, and have intriguing implications for cytoskeletal evolution – namely we think that the diversification of the microtubule cytoskeleton occurred earlier than the microfilament system. Confirmation of this trend requires the analysis of genomes of free-living relatives of Giardia such as Spironucleus, as well as other basal eukaryotes and is consequently an aim of our sequencing the genomes of Spironucleus vortens and N. gruberi.

Giardia is a common intestinal parasite of humans and animals, and due to its basal phylogenetic position, studies of Giardia afford a unique perspective on the function of the cytoskeleton during the early evolution of eukaryotic cells. Giardia is easily grown in the laboratory, has a sequenced genome, and is amenable to reverse genetic methodologies. We have identified, cloned, and phylogenetically classified twenty-four kinesins from Giardia, roughly triple the number of kinesins as in yeasts. Many of these kinesins, although found in metazoans, are lacking in yeasts. We are investigating their function in pathogenesis, flagellar assembly and mitosis. Novel kinesins may be prime targets for development of drugs for treating giardiasis.

Understanding chromosome segregation in Giardia, a binucleate cell has important implications for understanding mitotic evolution, the genetic fates of two

identical nuclei, and cell polarity. Our results challenge previous hypotheses of cell division in Giardia that describe multiple division planes, and clearly demonstrate the role of two microtubule spindles in chromosome segregation. A crucial event in the evolution of eukaryotes was the evolution of eukaryotic chromatin and the cellular machinery responsible for their segregation. Giardia can also provide an insight towards defining the nature of heterochromatin and the evolution of centromeres. We have initiated phylogenomic and functional analyses of chromatin-associated proteins (including histones, histone-associated and histone modifying proteins, and RNAi machinery.
 

A deconvolved 3D projection of a maize pachytene nucleus showing FISH-localized telomeres. Chromosomes are stained with DAPI.

Meiosis

Maize is the only model system where there is a large collection of mutants that affect meiosis, and where it is possible to do superb cytology. We are using high-resolution fluorescence in situ hybridization (FISH) to study the distribution of selected DNA probes such as telomere and centromere probes as chromosomes pair in male meiocytes. We have demonstrated that the clustering of telomeres on the nuclear envelope is a dynamic process and plays a role in bringing homologous chromosomes into close proximity for further interactions. A major portion of our research effort is to find, characterize and clone meiotic genes in maize. Using a Mutator-tagged allele we cloned a gene poor homologous synapsis (phs1) that is crucial for proper pairing and synapsis of homologous chromosomes during meiosis, ameiotic 1 (am1), a gene required for commitment to the meiotic cell cycle, and absence of first division 1(afd1), a gene involved in regulation of leptotene chromosome structure and sister chromatid cohesion.

Fission yeast has only three chromosomes and their behavior during meiosis can be readily monitored using such tools as GFP-Swi6, a chromodomain protein that binds to telomeres and centromeres. We have developed a cytologically based screen to identify mutants defective in various aspects of meiosis including telomere clustering during meiotic prophase, and chromosome segregation during anaphase I and II, and meiotic chromatin structure. We are analyzing Spindle Pole Body (SPB) replication and function during meiosis and have characterized a class of mutants that have disorganized SPB components and abnormal telomere clustering.

Madrid AS, Mancuso J, Cande WZ, Weis K. The role of the integral membrane nucleoporins Ndc1p and Pom152p in nuclear pore complex assembly and function. J Cell Biol. 2006 May 8;173(3):361-71.

Hamant O, Ma H, Cande WZ. Genetics of Meiotic Prophase I in Plants. Annu Rev Plant Biol. 2006 Jun 2;57:267-302.

Hansen WR, Tulyathan O, Dawson SC, Cande WZ, Fletcher DA. Giardia lamblia attachment force is insensitive to surface treatments. Eukaryot Cell. 2006 Apr;5(4):781-3.

Wang CJ, Harper L, Cande WZ. High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9. Plant Cell. 2006 Mar;18(3):529-44. Epub 2006 Feb 3.

Macrae IJ, Zhou K, Li F, Repic A, Brooks AN, Cande WZ, Adams PD, Doudna JA. Structural basis for double-stranded RNA processing by Dicer. Science. 2006 Jan 13;311(5758):195-8.

Pawlowski WP, Cande WZ. Coordinating the events of the meiotic prophase. Trends Cell Biol. 2005 Dec;15(12):674-81. Epub 2005 Oct 27. Review.

Tu X, Mancuso J, Cande WZ, Wang CC. Distinct cytoskeletal modulation and regulation of G1-S transition in the two life stages of Trypanosoma brucei. J Cell Sci. 2005 Oct 1;118(Pt 19):4353-64. Epub 2005 Sep 6.

Hamant O, Golubovskaya I, Meeley R, Fiume E, Timofejeva L,Schleiffer A, Nasmyth K, Cande WZ. A REC8 dependent plant Shugoshin is required for maintenance of centromeric cohesion during meiosis, and has no mitotic functions. Current Biology. 2005 15:948-954

Jin Y, Mancuso J, Uzawa S, Cromnebold D, Cande WZ. The fission yeast homolog of human transcription factor EAP30 blocks meiotic Spindle Pole Body amplification. Dev Cell. 2005 Jul;9(1):63-73. Erratum in: Dev Cell. 2005 Sep;9(3):439.

Li F, Goto DB, Zaratiegui M, Tang X, Martienssen R, Cande WZ. Two novel proteins, Dos1 and Dos2, interact with Rik1 to regulate heterochromatic RNA interference and histone modification. Curr Biol. 2005 Aug 23;15(16):1448-57.

Pawlowski WP, Golubovskaya IN, Timofejeva L, Meeley RB, Sheridan WF, Cande WZ. Coordination of meiotic recombination, pairing and synapsis by PHS1. (2004) Science. 2004 Jan 2;303(5654):89-92.

Uzawa S, Li F, Jin Y, McDonald KL, Braunfeld MB, Agard DA, Cande WZ. Spindle pole body duplication in fission yeast occurs at the G1/S boundary but maturation is blocked until exit from S by an event downstream of cdc10+. Mol Biol Cell. 2004 Dec;15(12):5219-30. Epub 2004 Sep 22.

Sagolla MJ, Uzawa S, Cande WZ. Individual microtubule dynamics contribute to the function of mitotic and cytoplasmic arrays in fission yeast. J Cell Sci. 2003 Dec 15;116(Pt 24):4891-903.
 

C134 - Chromosome Biology/Cytogenetics
299 - Graduate Research