Asymmetric Chromosome Oscillation during Mitosis and Protein Architecture of the Human Kinetochore Measured by K-SHREC (Kinetochore-Speckle High Resolution Co-Localization)

XIAOHU WAN: Asymmetric Chromosome Oscillation during Mitosis and Protein Architecture of the Human Kinetochore Measured by K-SHREC (Kinetochore-Speckle High Resolution Co-Localization) (Under the direction of Dr. E.D. Salmon) Mitotic chromosomes are known to oscillate during prometaphase and metaphase. This study demonstrated that kinetochores move faster in poleward (P) motion than in awayfrom-the-pole (AP) motion. P and AP motions also showed different position versus time curves, suggesting distinct mechanisms behind the phenomenon. Sister kinetochores oscillate with different phases relative to each other. The leading kinetochore usually switches first, from P to AP motion, followed by the trailing one switching from AP to P motion. Such asymmetry and phase lag produces oscillation in centromere stretch at twice the frequency of individual kinetochores. The leading kinetochore switches after sister chromosomes reach maximum centromere stretch, suggesting tension may trigger the kinetochore switching. To further investigate kinetochore dynamics, K-SHREC (Kinetochore-Speckle High Resolution Co-Localization) was developed to map the relative protein positions within kinetochores using two color fluorescent speckle microscopy, where centroids, orientations and geometries of fluorescent proteins were identified by asymmetric 3D Gaussian fitting in 3D image stacks. The accuracy of this method can reach +/-5nm. The

[1]  S Inoué,et al.  1. EARLY HISTORY: THE DYNAMIC EQUILIBRIUM MODEL , 1995 .

[2]  Tetsuya Hori,et al.  The CENP-H–I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres , 2006, Nature Cell Biology.

[3]  Gaudenz Danuser,et al.  Kinesin 5–independent poleward flux of kinetochore microtubules in PtK1 cells , 2006, The Journal of cell biology.

[4]  E. Salmon,et al.  The dynamic kinetochore-microtubule interface , 2004, Journal of Cell Science.

[5]  H. Maiato,et al.  Drosophila CLASP is required for the incorporation of microtubule subunits into fluxing kinetochore fibres , 2005, Nature Cell Biology.

[6]  D. Cimini,et al.  Anaphase Spindle Mechanics Prevent Mis-Segregation of Merotelically Oriented Chromosomes , 2004, Current Biology.

[7]  E. Salmon,et al.  Spinning disk confocal microscope system for rapid high-resolution, multimode, fluorescence speckle microscopy and green fluorescent protein imaging in living cells. , 2003, Methods in enzymology.

[8]  T. Hori,et al.  CENP-O class proteins form a stable complex and are required for proper kinetochore function. , 2007, Molecular biology of the cell.

[9]  E D Salmon,et al.  Directional instability of kinetochore motility during chromosome congression and segregation in mitotic newt lung cells: a push-pull mechanism , 1993, The Journal of cell biology.

[10]  P. Sorger,et al.  Automatic fluorescent tag detection in 3D with super‐resolution: application to the analysis of chromosome movement , 2002, Journal of microscopy.

[11]  P Wadsworth,et al.  Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-alpha tubulin. , 2001, Molecular biology of the cell.

[12]  John R. Yates,et al.  The human CENP-A centromeric nucleosome-associated complex , 2006, Nature Cell Biology.

[13]  Jessica K. Polka,et al.  Implications for Kinetochore-Microtubule Attachment from the Structure of an Engineered Ndc80 Complex , 2008, Cell.

[14]  J. B. Rattner,et al.  CENP-F is a .ca 400 kDa kinetochore protein that exhibits a cell-cycle dependent localization. , 1993, Cell motility and the cytoskeleton.

[15]  Peter K Sorger,et al.  Molecular organization of the Ndc80 complex, an essential kinetochore component. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Yumi Kim,et al.  CENP-E combines a slow, processive motor and a flexible coiled coil to produce an essential motile kinetochore tether , 2008, The Journal of cell biology.

[17]  E. Salmon,et al.  Hec1 and nuf2 are core components of the kinetochore outer plate essential for organizing microtubule attachment sites. , 2004, Molecular biology of the cell.

[18]  J. Spudich,et al.  Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[19]  C. Rieder.  The formation, structure, and composition of the mammalian kinetochore and kinetochore fiber. , 1982, International review of cytology.

[20]  Andrea Musacchio,et al.  Architecture of the Human Ndc80-Hec1 Complex, a Critical Constituent of the Outer Kinetochore* , 2005, Journal of Biological Chemistry.

[21]  A. Murray,et al.  Localization of Mad2 to Kinetochores Depends on Microtubule Attachment, Not Tension , 1998, The Journal of cell biology.

[22]  W. Earnshaw,et al.  CENP-C binds the alpha-satellite DNA in vivo at specific centromere domains. , 2002, Journal of cell science.

[23]  E. Salmon,et al.  The spindle-assembly checkpoint in space and time , 2007, Nature Reviews Molecular Cell Biology.

[24]  R. Zinkowski,et al.  Structure and molecular organization of the centromere-kinetochore complex. , 1992, Trends in cell biology.

[25]  Mitsuhiro Yanagida,et al.  Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1. , 2007, Developmental cell.

[26]  A. Desai,et al.  KNL1 and the CENP-H/I/K complex coordinately direct kinetochore assembly in vertebrates. , 2007, Molecular biology of the cell.

[27]  B. McEwen,et al.  The outer plate in vertebrate kinetochores is a flexible network with multiple microtubule interactions , 2007, Nature Cell Biology.

[28]  Andrea Musacchio,et al.  Kinetochore Microtubule Dynamics and Attachment Stability Are Regulated by Hec1 , 2006, Cell.