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

Abstract

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.

Figure 1.

hNuf2 localizes to kinetochores throughout mitosis. HeLa cells were fixed and double stained with antibodies to hNuf2 and α-tubulin and observed using a spinning disc confocal fluorescence microscope.

Figure 2.

hNuf2-depleted cells exhibit a prolonged mitotic block and undergo cell death. (A) Reduction of hNuf2 levels after siRNA transfection. HeLa cells were transfected with hNuf2 siRNA, harvested 24, 48, and 72 h after transfection, and subjected to protein immunoblot analysis with hNuf2 antibodies and actin antibodies to control for gel loading. Time points are indicated above each lane, and time 0 indicates untransfected cells. (B) hNuf2 siRNA transfection results in a mitotic defect. Cells were transfected with either hNuf2 siRNA or a control 21-nucleotide siRNA duplex, and imaged 24, 48, and 72 h after transfection using a 10× phase objective. (C) Quantitation of control and hNuf2 siRNA–transfected cell phenotypes. Cells adherent to the culture dish with an intact nuclear envelope and decondensed chromatin were scored as interphase. Rounded cells with condensed chromosomes and smooth, uniform membranes were scored as mitotic. Cells that were multilobed, dense, and exhibited a nonuniform membrane were scored as dead (Mills et al., 1999; Zhang and Xu, 2002) (n = 6,000 cells per time point averaged from three independent experiments). (D) Mitotic progression of control siRNA– versus hNuf2 siRNA–transfected cells. 48 h after transfection, cells were transferred to live-cell chambers and time lapsed using a 40× phase objective. Top row, typical control siRNA–transfected cell; bottom row, typical hNuf2 siRNA–transfected cell. Time shown in minutes. (E) Time course of change in cellular DNA content after hNuf2 siRNA transfection. Time after transfection along the x axis is in hours, and untransfected cells were used as a control. Cells were analyzed by flow cytometry as described in the Materials and methods. (F) Cellular DNA content as analyzed by flow cytometry of cells treated for 48 h with 10 μM vinblastine. (G) Uptake of Trypan blue in hNuf2 siRNA–transfected cells. Mock-transfected and hNuf2 siRNA–transfected cells were incubated with Trypan blue 72 h after transfection and observed by phase-contrast and epifluorescence microscopy. (H) Nuclear fragmentation of hNuf2-depleted cells. Cells transfected with hNuf2 siRNA were fixed and stained with DAPI 48 h after transfection.

Figure 3.

hNuf2-depleted cells lack stable kinetochore microtubules. (A and B) Immunofluorescent cell images of hNuf2 siRNA–transfected and mock-transfected cells. 48 h after transfection, cells were fixed for immunofluorescence and stained with the indicated antibodies. DAPI was used to visualize the DNA. (C) hNuf2 siRNA–transfected cells lack cold-stable kinetochore microtubules. Cells were incubated on ice for 10 min to induce complete microtubule disassembly of all nonkinetochore microtubules, and were subsequently fixed and processed for immunofluorescence using hNuf2 and tubulin antibodies and DAPI to stain DNA.

Figure 4.

hNuf2-depleted kinetochores retain the spindle checkpoint proteins Mad2 and BubR1 and the motor proteins CENP-E and cytoplasmic dynein. Cells were mock transfected (A) or transfected with an hNuf2 siRNA (B) and fixed for immunofluorescence 48 h after transfection. The cells were stained using the indicated antibodies (left columns) and a kinetochore marker (CREST serum; center columns). Each image shows one kinetochore pair. (C) Kinetochore localization of CENP-E and dynein in nocodazole-treated cells. Cells were subjected to nocodazole treatment and immunofluorescence analysis.