Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay.
Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.
Tanudji
Marcel
M
DNAX Research Institute, Palo Alto, California 94304, USA.
Shoemaker
John
J
L'Italien
Lawrence
L
Russell
Loren
L
Chin
Gregory
G
Schebye
Xiao Min
XM
D002135:Calcium-Binding Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D020090:Chromosome Segregation / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015870:Gene Expression / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000187:drug effects / Q000378:metabolism; D064246:Mad2 Proteins; D008938:Mitosis / Q000187:drug effects / Q000502:physiology*; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D012097:Repressor Proteins; D008941:Spindle Apparatus / Q000187:drug effects / Q000378:metabolism
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012097:Repressor Proteins; C068688:centromere protein E; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; D015739:Nocodazole