hNuf2 inhibition blocks stable kinetochore–microtubule attachment and induces mitotic cell death in HeLa cells

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore–microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.

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Faculty Opinions recommendation of hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1004915.168758 ◽

2002 ◽

Author(s):

Duane Compton

Keyword(s):

Cell Death ◽

Hela Cells ◽

Mitotic Cell ◽

Microtubule Attachment

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Faculty Opinions recommendation of hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1004915.167210 ◽

2002 ◽

Author(s):

Claire Walczak

Keyword(s):

Cell Death ◽

Hela Cells ◽

Mitotic Cell ◽

Microtubule Attachment

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BUB1 mediation of caspase-independent mitotic death determines cell fate

The Journal of Cell Biology ◽

10.1083/jcb.200702134 ◽

2007 ◽

Vol 178 (2) ◽

pp. 283-296 ◽

Cited By ~ 67

Author(s):

Yohei Niikura ◽

Amruta Dixit ◽

Ray Scott ◽

Guy Perkins ◽

Katsumi Kitagawa

Keyword(s):

Cell Death ◽

Cell Fate ◽

Cell Lines ◽

Chromosome Instability ◽

Spindle Checkpoint ◽

Mitotic Cell ◽

Endonuclease G ◽

Colon Tumors ◽

Mitotic Death ◽

Apoptosis Inducing Factor

The spindle checkpoint that monitors kinetochore–microtubule attachment has been implicated in tumorigenesis; however, the relation between the spindle checkpoint and cell death remains obscure. In BUB1-deficient (but not MAD2-deficient) cells, conditions that activate the spindle checkpoint (i.e., cold shock or treatment with nocodazole, paclitaxel, or 17-AAG) induced DNA fragmentation during early mitosis. This mitotic cell death was independent of caspase activation; therefore, we named it caspase-independent mitotic death (CIMD). CIMD depends on p73, a homologue of p53, but not on p53. CIMD also depends on apoptosis-inducing factor and endonuclease G, which are effectors of caspase-independent cell death. Treatment with nocodazole, paclitaxel, or 17-AAG induced CIMD in cell lines derived from colon tumors with chromosome instability, but not in cells from colon tumors with microsatellite instability. This result was due to low BUB1 expression in the former cell lines. When BUB1 is completely depleted, aneuploidy rather than CIMD occurs. These results suggest that cells prone to substantial chromosome missegregation might be eliminated via CIMD.

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Dual Function Microtubule- and Mitochondria-Associated Proteins Mediate Mitotic Cell Death

Analytical Cellular Pathology ◽

10.1155/2009/783817 ◽

2009 ◽

Vol 31 (5) ◽

pp. 393-405

Author(s):

Leyuan Liu ◽

Rui Xie ◽

Chaofeng Yang ◽

Wallace L. McKeehan

Keyword(s):

Cell Death ◽

Mitotic Cell ◽

Mitotic Arrest ◽

Dual Function ◽

Associated Proteins ◽

Irreversible Aggregation ◽

The Common ◽

Spindle Microtubules ◽

Segregation Of Chromosomes ◽

Mitotic Cells

Background: Survival and evolution of aneuploid cells after an asymmetric segregation of chromosomes at mitosis may be the common initiating event and underlying cause of the genetic diversity and adaptability of cancers. We hypothesize that mechanisms exist to detect impending aneuploidy and prevent it before completion of an aberrant mitosis.Methods: The distribution of isoforms of C19ORF5, an interactive partner with mitochondria-associated LRPPRC and tumor suppressor RASSF1A, state of spindle microtubules and mitochondrial aggregation was analyzed in synchronized mitotic cells and cells stalled in mitosis after treatment with paclitaxel.Results: C19ORF5 distributed broadly across the mitotic spindle and reversibly accumulated during reversible mitotic arrest. Prolonged stabilization of microtubules caused an accumulation of a C19ORF5 product with dual MAP and MtAP properties that caused irreversible aggregation of mitochondria and death of mitotic cells.Conclusions: Dual function microtubule-associated (MAP) and mitochondria-associated (MtAP) proteins generated by prolonged mitotic arrest trigger mitochondrial-induced mitotic cell death. This is a potential mechanism to prevent minimal survivable aneuploidy resulting from an aberrant cell division and cancers in general at their earliest common origin.

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Hec1 and Nuf2 Are Core Components of the Kinetochore Outer Plate Essential for Organizing Microtubule Attachment Sites

Molecular Biology of the Cell ◽

10.1091/mbc.e04-09-0852 ◽

2005 ◽

Vol 16 (2) ◽

pp. 519-531 ◽

Cited By ~ 176

Author(s):

Jennifer G. DeLuca ◽

Yimin Dong ◽

Polla Hergert ◽

Joshua Strauss ◽

Jennifer M. Hickey ◽

...

Keyword(s):

Spindle Checkpoint ◽

Attachment Site ◽

Outer Plate ◽

Microtubule Attachment ◽

Attachment Sites ◽

Outer Domain ◽

Core Components ◽

Fluorescence Light ◽

Microtubule Formation ◽

Stable Core

A major goal in the study of vertebrate mitosis is to identify proteins that create the kinetochore-microtubule attachment site. Attachment sites within the kinetochore outer plate generate microtubule dependent forces for chromosome movement and regulate spindle checkpoint protein assembly at the kinetochore. The Ndc80 complex, comprised of Ndc80 (Hec1), Nuf2, Spc24, and Spc25, is essential for metaphase chromosome alignment and anaphase chromosome segregation. It has also been suggested to have roles in kinetochore microtubule formation, production of kinetochore tension, and the spindle checkpoint. Here we show that Nuf2 and Hec1 localize throughout the outer plate, and not the corona, of the vertebrate kinetochore. They are part of a stable “core” region whose assembly dynamics are distinct from other outer domain spindle checkpoint and motor proteins. Furthermore, Nuf2 and Hec1 are required for formation and/or maintenance of the outer plate structure itself. Fluorescence light microscopy, live cell imaging, and electron microscopy provide quantitative data demonstrating that Nuf2 and Hec1 are essential for normal kinetochore microtubule attachment. Our results indicate that Nuf2 and Hec1 are required for organization of stable microtubule plus-end binding sites in the outer plate that are needed for the sustained poleward forces required for biorientation at kinetochores.

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Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores

The Journal of Cell Biology ◽

10.1083/jcb.201407074 ◽

2015 ◽

Vol 208 (2) ◽

pp. 181-196 ◽

Cited By ~ 81

Author(s):

Soonjoung Kim ◽

Hongtao Yu

Keyword(s):

Spindle Checkpoint ◽

Aurora B ◽

Dependent Manner ◽

Checkpoint Proteins ◽

Multiple Strategies ◽

Complete Inactivation ◽

Microtubule Attachment ◽

Ndc80 Complex ◽

Spindle Microtubules ◽

Multiple Assembly

During mitosis, the spindle checkpoint senses kinetochores not properly attached to spindle microtubules and prevents precocious sister-chromatid separation and aneuploidy. The constitutive centromere-associated network (CCAN) at inner kinetochores anchors the KMN network consisting of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C) at outer kinetochores. KMN is a critical kinetochore receptor for both microtubules and checkpoint proteins. Here, we show that nearly complete inactivation of KMN in human cells through multiple strategies produced strong checkpoint defects even when all kinetochores lacked microtubule attachment. These KMN-inactivating strategies reveal multiple KMN assembly mechanisms at human mitotic kinetochores. In one mechanism, the centromeric kinase Aurora B phosphorylates Mis12C and strengthens its binding to the CCAN subunit CENP-C. In another, CENP-T contributes to KMN attachment in a CENP-H-I-K–dependent manner. Our study provides insights into the mechanisms of mitosis-specific assembly of the checkpoint platform KMN at human kinetochores.

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Downregulation of KRC Induces Proliferation, Anchorage Independence, and Mitotic Cell Death in HeLa Cells

Experimental Cell Research ◽

10.1006/excr.2000.5029 ◽

2000 ◽

Vol 260 (2) ◽

pp. 346-356 ◽

Cited By ~ 10

Author(s):

Carl E. Allen ◽

Lai-Chu Wu

Keyword(s):

Cell Death ◽

Hela Cells ◽

Mitotic Cell ◽

Anchorage Independence

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Silencing Mitosin Induces Misaligned Chromosomes, Premature Chromosome Decondensation before Anaphase Onset, and Mitotic Cell Death

Molecular and Cellular Biology ◽

10.1128/mcb.25.10.4062-4074.2005 ◽

2005 ◽

Vol 25 (10) ◽

pp. 4062-4074 ◽

Cited By ~ 62

Author(s):

Zhenye Yang ◽

Jing Guo ◽

Qi Chen ◽

Chong Ding ◽

Juan Du ◽

...

Keyword(s):

Mitotic Cell ◽

Cytoplasmic Dynein ◽

Dependent Manner ◽

Microtubule Attachment ◽

Anaphase Onset ◽

Chromosome Congression ◽

Chromosome Alignment ◽

M Phase ◽

Dying Cells ◽

Chromosome Decondensation

ABSTRACT Mitosin (also named CENP-F) is a large human nuclear protein transiently associated with the outer kinetochore plate in M phase. Using RNA interference and fluorescence microscopy, we showed that mitosin depletion attenuated chromosome congression and led to metaphase arrest with misaligned polar chromosomes whose kinetochores showed few cold-stable microtubules. Kinetochores of fully aligned chromosomes often failed to show orientation in the direction of the spindle long axis. Moreover, tension across their sister kinetochores was decreased by 53% on average. These phenotypes collectively imply defects in motor functions in mitosin-depleted cells and are similar to those of CENP-E depletion. Consistently, the intensities of CENP-E and cytoplasmic dynein and dynactin, which are motors controlling microtubule attachment and chromosome movement, were reduced at the kinetochore in a microtubule-dependent manner. In addition, after being arrested in pseudometaphase for approximately 2 h, mitosin-depleted cells died before anaphase initiation through apoptosis. The dying cells exhibited progressive chromosome arm decondensation, while the centromeres were still associated with spindles. Mitosin is therefore essential for full chromosome alignment, possibly by promoting proper kinetochore attachments through modulating CENP-E and dynein functions. Its depletion also prematurely triggers chromosome decondensation, a process that normally occurs from telophase for the nucleus reassembly, thus resulting in apoptosis.

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