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.
Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria.
Izabela
I
Sumara
Juan F
JF
Giménez-Abián
Daniel
D
Gerlich
Toru
T
Hirota
Claudine
C
Kraft
Consuelo
C
de la Torre
Jan
J
Ellenberg
Jan-Michael
JM
Peters
D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D002478:Cells, Cultured; D023902:Chromosome Pairing / Q000502:physiology*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000378:metabolism; D018386:Kinetochores / Q000378:metabolism*; D051379:Mice; D008938:Mitosis / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D051381:Rats; D008941:Spindle Apparatus / Q000378:metabolism* / Q000502:physiology; D013449:Sulfonamides / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism
D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D007211:Indoles; D013449:Sulfonamides; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D017346:Protein Serine-Threonine Kinases; C474723:hesperadin