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

doi:10.1083/jcb.200208159

Published online 18 November 2002. doi:10.1083/jcb.200208159

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© The Rockefeller University Press, 0021-9525/2002/11/549 $5.00
The Journal of Cell Biology, Volume 159, Number 4, 549-555

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

Jennifer G. DeLuca¹, Ben Moree¹, Jennifer M. Hickey¹, John V. Kilmartin² and E.D. Salmon¹

¹ University of North Carolina at Chapel Hill, Department of Biology, Chapel Hill, NC 27599
² Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK

Address correspondence to Jennifer DeLuca, Department of Biology, 607 Fordham Hall, CB#3280, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Tel.: (919) 962-2354. Fax: (919) 962-1625. E-mail: jgdeluca{at}email.unc.edu

Identification of proteins that couple kinetochores to spindlemicrotubules is critical for understanding how accurate chromosomesegregation is achieved in mitosis. Here we show that the proteinhNuf2 specifically functions at kinetochores for stable microtubuleattachment in HeLa cells. When hNuf2 is depleted by RNA interference,spindle formation occurs normally as cells enter mitosis, butkinetochores fail to form their attachments to spindle microtubulesand cells block in prometaphase with an active spindle checkpoint.Kinetochores depleted of hNuf2 retain the microtubule motorsCENP-E and cytoplasmic dynein, proteins previously implicatedin recruiting kinetochore microtubules. Kinetochores also retaindetectable levels of the spindle checkpoint proteins Mad2 andBubR1, as expected for activation of the spindle checkpointby unattached kinetochores. In addition, the cell cycle blockproduced by hNuf2 depletion induces mitotic cells to undergocell death. These data highlight a specific role for hNuf2 inkinetochore–microtubule attachment and suggest that hNuf2is part of a molecular linker between the kinetochore attachmentsite and tubulin subunits within the lattice of attached plusends.

Key Words: hNuf2; microtubules; kinetochores; mitosis; siRNA

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