Gene Silencing of CENP-E by Small Interfering RNA in HeLa Cells Leads to Missegregation of Chromosomes after a Mitotic Delay

Abstract

, 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.

Functional Analysis of Human Microtubule-based Motor Proteins, the Kinesins and Dyneins, in Mitosis/Cytokinesis Using RNA Interference

“…Consistent with previous studies (Putkey et al, 2002;Weaver et al, 2003;Tanudji et al, 2004), cells depleted of CENP-E exhibit unaligned chromosomes close to spindle poles and misaligned chromosomes positioned between the metaphase plate and spindle poles at metaphase. Kinetochore pairs on misaligned sister chromatids showed no interaction or diminished interaction with spindle MTs.…”

“…Kinetochore pairs on misaligned sister chromatids showed no interaction or diminished interaction with spindle MTs. Previous studies demonstrated that CENP-E binds to kinetochores and MTs and stabilizes their interaction, thereby contributing to silencing the mitotic spindle checkpoint (Putkey et al, 2002;Weaver et al, 2003;Tanudji et al, 2004). CENP-E associates with and activates the mitotic checkpoint kinase BubR1, indicating that the spindle checkpoint is sensed through CENP-E kinetochore activity (Chan et al, 1998(Chan et al, , 1999Mao et al, 2003;Weaver et al, 2003).…”

“…CENP-E associates with and activates the mitotic checkpoint kinase BubR1, indicating that the spindle checkpoint is sensed through CENP-E kinetochore activity (Chan et al, 1998(Chan et al, , 1999Mao et al, 2003;Weaver et al, 2003). However, the observation that there are few unaligned and/or misaligned chromosomes in CENP-E esiRNAtreated cells suggests that functionally redundant mechanisms may operate to control spindle MT-kinetochore attachment and chromosome congression (Tanudji et al, 2004).…”

Cohesin Associates with Spindle Poles in a Mitosis-specific Manner and Functions in Spindle Assembly in Vertebrate Cells

“…The 21-nucleotide siRNA duplexes were designed and synthesized by QIA-GEN against hSMC1 (5Ј-CAC CAT CAC ACT TTA ATT CCA-3Ј), hRad21 (5Ј-CTG GGA GTA GTT CGA ATC TAT-3Ј), SA1 (5Ј-CAC GTA GAA TCA GAT GTT CTA-3Ј), SA2 (5Ј-TCG GTG GTA GAT GAT TGG ATA-3Ј), and CENP-E (5Ј-AAC ACG GAT GCT GGT GAC CTC-3Ј) (Tanudji et al, 2004). The target sequence for SUV39H1 is as described previously (Ait-Si- Ali et al, 2004).…”

“…To address whether the observed spindle abnormality is due to kinetochore dysfunction, CENP-E was depleted by siRNA ( Figure 4A). CENP-E depletion was shown to impair bipolar spindle attachment to kinetochores in human cells (Schaar et al, 1997;Tanudji et al, 2004). Furthermore, CENP-E depletion leads to spindle checkpoint activation (Schaar et al, 1997;Yao et al, 2000;McEwen et al, 2001;Tanudji et al, 2004).…”