Roles of Polo-like Kinase 1 in the Assembly of Functional Mitotic Spindles

BACKGROUND The stable association of chromosomes with both poles of the mitotic spindle (biorientation) depends on spindle pulling forces. These forces create tension across sister kinetochores and are thought to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint. Polo-like kinase 1 (Plk1) has been implicated in regulating centrosome maturation, mitotic entry, sister chromatid cohesion, the anaphase-promoting complex/cyclosome (APC/C), and cytokinesis, but it is unknown if Plk1 controls chromosome biorientation. RESULTS We have analyzed Plk1 functions in synchronized mammalian cells by RNA interference (RNAi). Plk1-depleted cells enter mitosis after a short delay, accumulate in a preanaphase state, and subsequently often die by apoptosis. Spindles in Plk1-depleted cells lack focused poles and are not associated with centrosomes. Chromosomes attach to these spindles, but the checkpoint proteins Mad2, BubR1, and CENP-E are enriched at many kinetochores. When Plk1-depleted cells are treated with the Aurora B inhibitor Hesperadin, which silences the spindle checkpoint by stabilizing microtubule-kinetochore interactions, cells degrade APC/C substrates and exit mitosis without chromosome segregation and cytokinesis. Experiments with monopolar spindles that are induced by the kinesin inhibitor Monastrol indicate that Plk1 is required for the assembly of spindles that are able to generate poleward pulling forces. CONCLUSIONS Our results imply that Plk1 is not essential for mitotic entry and APC/C activation but is required for proper spindle assembly and function. In Plk1-depleted cells spindles may not be able to create enough tension across sister kinetochores to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint.

[1]  Stephen S. Taylor,et al.  Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores , 2003, The Journal of cell biology.

[2]  Kyung S. Lee,et al.  A Spindle Checkpoint Arrest and a Cytokinesis Failure by the Dominant-negative Polo-box Domain of Plk1 in U-2 OS Cells* 210 , 2002, The Journal of Biological Chemistry.

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

[4]  E. Salmon,et al.  Microtubule-dependent changes in assembly of microtubule motor proteins and mitotic spindle checkpoint proteins at PtK1 kinetochores. , 2001, Molecular biology of the cell.

[5]  J. Pines,et al.  GFP tagging reveals human Polo-like kinase 1 at the kinetochore/centromere region of mitotic chromosomes , 1998, Chromosoma.

[6]  S. Haggarty,et al.  Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. , 1999, Science.

[7]  C. Sunkel,et al.  polo, a mitotic mutant of Drosophila displaying abnormal spindle poles. , 1988, Journal of cell science.

[8]  M. Kirschner,et al.  Dynamic instability of microtubule growth , 1984, Nature.

[9]  A. Murray,et al.  Visualization of Mad2 Dynamics at Kinetochores, along Spindle Fibers, and at Spindle Poles in Living Cells , 2000, The Journal of cell biology.

[10]  M. Hixon,et al.  The control of mitosis. , 2000, Frontiers in bioscience : a journal and virtual library.

[11]  J. Peters,et al.  The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore–microtubule attachment and in maintaining the spindle assembly checkpoint , 2003, The Journal of cell biology.

[12]  A. Musacchio,et al.  The spindle checkpoint: structural insights into dynamic signalling , 2002, Nature Reviews Molecular Cell Biology.

[13]  J. Pines,et al.  Ordered proteolysis in anaphase inactivates Plk1 to contribute to proper mitotic exit in human cells , 2004, The Journal of cell biology.

[14]  David M. Glover,et al.  Metaphase Arrest with Centromere Separation in polo Mutants of Drosophila , 2001, The Journal of cell biology.

[15]  R. Nicklas,et al.  Tension on chromosomes increases the number of kinetochore microtubules but only within limits. , 2000, Journal of cell science.

[16]  E. Nigg,et al.  Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis , 2003, The Journal of cell biology.

[17]  R. Wolthuis,et al.  Polo-like Kinase-1 Is Required for Bipolar Spindle Formation but Is Dispensable for Anaphase Promoting Complex/Cdc20 Activation and Initiation of Cytokinesis* , 2004, Journal of Biological Chemistry.

[18]  Alexey Khodjakov,et al.  Centrosome-independent mitotic spindle formation in vertebrates , 2000, Current Biology.

[19]  Xiaoqi Liu,et al.  Polo-like kinase (Plk)1 depletion induces apoptosis in cancer cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[20]  C. Sunkel,et al.  The Drosophila POLO kinase localises to multiple compartments of the mitotic apparatus and is required for the phosphorylation of MPM2 reactive epitopes. , 1998, Journal of cell science.

[21]  A Khodjakov,et al.  The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores , 1995, The Journal of cell biology.

[22]  L. Wordeman,et al.  The kinetochore of higher eucaryotes: a molecular view. , 2000, International review of cytology.

[23]  P. Hieter,et al.  PKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progression. , 1998, Molecular cell.

[24]  J. Peters,et al.  Two Distinct Pathways Remove Mammalian Cohesin from Chromosome Arms in Prophase and from Centromeres in Anaphase , 2000, Cell.

[25]  Jan-Michael Peters,et al.  The anaphase-promoting complex: proteolysis in mitosis and beyond. , 2002, Molecular cell.

[26]  C. Sunkel,et al.  polo encodes a protein kinase homolog required for mitosis in Drosophila. , 1991, Genes & development.

[27]  T. Kapoor,et al.  Correcting improper chromosome–spindle attachments during cell division , 2004, Nature Cell Biology.

[28]  B. Oakley,et al.  Gamma-tubulin at ten: progress and prospects. , 1999, Cell structure and function.

[29]  W. Earnshaw,et al.  Formation of Spindle Poles by Dynein/Dynactin-Dependent Transport of Numa , 2000, The Journal of cell biology.

[30]  M. Pagano,et al.  Role of Polo-like kinase in the degradation of early mitotic inhibitor 1, a regulator of the anaphase promoting complex/cyclosome. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[31]  R. Nicklas,et al.  Elements of error correction in mitosis: microtubule capture, release, and tension , 1994, The Journal of cell biology.

[32]  Catherine G. Takizawa,et al.  Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C. , 2000, Current opinion in cell biology.

[33]  E. Nishida,et al.  Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase , 2001, Nature.

[34]  T. Kanda,et al.  Histone–GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells , 1998, Current Biology.

[35]  Karl Mechtler,et al.  Mitotic regulation of the human anaphase‐promoting complex by phosphorylation , 2003, The EMBO journal.

[36]  H. Lane,et al.  Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes , 1996, The Journal of cell biology.

[37]  D. Compton,et al.  Chromosome Movement in Mitosis Requires Microtubule Anchorage at Spindle Poles , 2001, The Journal of cell biology.

[38]  E. Salmon,et al.  Checkpoint signals in grasshopper meiosis are sensitive to microtubule attachment, but tension is still essential. , 2001, Journal of cell science.

[39]  Timothy J. Mitchison,et al.  Probing Spindle Assembly Mechanisms with Monastrol, a Small Molecule Inhibitor of the Mitotic Kinesin, Eg5 , 2000, The Journal of cell biology.

[40]  Erich A. Nigg,et al.  Polo-like kinases and the orchestration of cell division , 2004, Nature Reviews Molecular Cell Biology.

[41]  I. Vernos,et al.  The Mitotic Spindle: A Self-Made Machine , 2001, Science.

[42]  M. Bornens.  Centrosome composition and microtubule anchoring mechanisms. , 2002, Current opinion in cell biology.

[43]  A. Kumagai,et al.  Purification and Molecular Cloning of Plx1, a Cdc25-Regulatory Kinase from Xenopus Egg Extracts , 1996, Science.

[44]  D. Compton,et al.  The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles , 2001, The Journal of cell biology.

[45]  A. Amon.  The spindle checkpoint. , 1999, Current opinion in genetics & development.

[46]  B. Schaar,et al.  Localization of CENP-E in the fibrous corona and outer plate of mammalian kinetochores from prometaphase through anaphase , 1997, Chromosoma.

[47]  J. Maller,et al.  Activated Polo-Like Kinase Plx1 Is Required at Multiple Points during Mitosis in Xenopus laevis , 1998, Molecular and Cellular Biology.

[48]  D. Glover,et al.  The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells. , 1995, Genes & development.

[49]  H. Lane,et al.  Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo , 1995, Cell.

[50]  J. Ellenberg,et al.  Regulation of Sister Chromatid Cohesion between Chromosome Arms , 2004, Current Biology.

[51]  Avram Hershko,et al.  The Cyclin-Ubiquitin Ligase Activity of Cyclosome/APC Is Jointly Activated by Protein Kinases Cdk1-Cyclin B and Plk* , 2002, The Journal of Biological Chemistry.

[52]  J. Pines,et al.  Active cyclin B1–Cdk1 first appears on centrosomes in prophase , 2003, Nature Cell Biology.

[53]  J. McIntosh,et al.  The mitotic spindle. , 1989, Scientific American.

[54]  E. Salmon,et al.  Chromosome Dynamics: New Light on Aurora B Kinase Function , 2002, Current Biology.

[55]  D. Glover,et al.  Polo kinase and Asp are needed to promote the mitotic organizing activity of centrosomes , 2001, Nature Cell Biology.