Bruce McEwen Laboratory
The Role of Centromere Protein E (CENP-E) in Spindle Attachment
McEwen, B.F., Chan, G.K.T., Zubrowski, B., Savoian, M.S., Sauer, M.T., and Yen, T. (2001)
CENP-E is a kinesin-like protein that when depleted from mammalian kinetochores leads to mitotic arrest with a mixture of aligned and unaligned chromosomes. In the present study, we used immunofluorescent, video, and electron microscopy to demonstrate that depletion of CENP-E from kinetochores via antibody microinjection reduces kinetochore microtubule binding by 23% at aligned chromosomes, and severely reduces microtubule binding at unaligned chromosomes. Disruption of CENP-E function also reduced tension across the centromere, increased the incidence of spindle pole fragmentation, and resulted in monooriented chromosomes approaching abnormally close to the spindle pole. Nevertheless, chromosomes showed typical patterns of congression, fast poleward motion, and oscillatory motions. Furthermore, kinetochores of aligned and unaligned chromosomes exhibited normal patterns of checkpoint protein localization. These data are explained by a model in which redundant mechanisms enable kinetochore microtubule binding and checkpoint monitoring in the absence of CENP-E at kinetochores, but where reduced microtubule binding efficiency, exacerbated by poor positioning at the spindle poles, results in chronically monooriented chromosomes and mitotic arrest. Chromosome position within the spindle appears to be a critical determinant of CENP-E function at kinetochores.
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Figure 1: Phenotype of anti-CENP-E injected HeLa cells. a-c: Fluorescence images of an injected cell (lower left corner) and uninjected cell in metaphase (upper middle). Staining in c is against the injected antibody. Clearly, the antibody injection prevents CENP-E localization to the kinetochore (see also (67)). Bar = 10 µm. d: Electron micrograph of an injected cell showing close approach of a kinetochore to a centriole. Bar = 0.5 µm. e: Kinetochore microtubule binding is reduced on congressed and uncongressed chromosomes of injected cells, when compared to congressed chromosomes from uninjected control cells in metaphase.
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Figure 2: Immunofluorescent images of CF-PAC cells depleted of CENP-E via antibody injection. Synchronized CF-PAC cells were microinjected with HX1 antibodies shortly after release from the G1/S block and fixed for staining 10-12 hours later. To visualize endogenous CENP-E (red), cells were stained with rHX1 (rat polyclonal) primary and Cy2-conjugated anti-rat secondary antibodies. Mts (green) were stained with a monoclonal primary and alexa-green conjugated secondary antibodies, while chromosomes (blue) were stained with Hoechst. a). Uninjected control cells on the same coverslip as injected cells. CENP-E staining was prominent at the kinetochores in prometaphase cells (purple arrows), delocalized and less prominent at the kinetochore in out of focus metaphase cells (green arrows), and concentrated in the mid-body in the late anaphase/telophase cell (orange arrow). b). Three injected cells showed no trace of CENP-E staining despite 6-8 X exposure in the red channel (note staining in neighboring uninjected G2 cell). Injected cells showed an increased incidence of spindle pole fragmentation and accompanying spindle distortion. Monooriented chromosomes extremely close to one pole are indicated by red arrows. c, d). Higher magnification view of injected cells with clear examples of bioriented chromosomes aligned to secondary poles (yellow arrows), and monooriented chromosomes stranded near a pole (red arrows). Scale bars = 20 µm (a,b) and 10 µm (c, d).
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Figure 3: Video live cell imaging of a CF-PAC cell injected with HX1 anti-CENP-E antibody. Synchronized CF-PAC cells were microinjected with antibody shortly after release from G1/S block and later remounted into Rose chambers. Coverslips were scanned for injected cells in prophase (injected cells were located via a scribe mark and co-injection with Oregon Green Dextran). This cell was filmed for a total of 2 hours past NEB with a 10 second filming rate (approximate time in minutes: seconds past nuclear envelope breakdown (NEB) is located in the upper right of each frame). a) Early prometaphase. The unattached chromosome indicated by the arrowhead was about to become monooriented and undergo fast poleward motion (see Video 1 in online supplement [.mov file size approximately 10 KB]). b) Windowed frames showing the chromsome indicated in (a) undergoing fast motion to the upper spindle pole (see Video 2 in online supplement [.mov file size approximately 2.5 KB]). c) Windowed frames illustrating congression. The congressing chromosome exhibited a typical transient reversal of motion before completing congression to the spindle equator (see Video 3 in online supplement [.mov file approximately 1.7 KB). Scale bar = 5.0 µm.
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Figure 4: Unattached chromosomes can be located within the spindle. Selected windowed video frames showing two chromosomes sliding by one another. The chromosomes indicated by the arrowhead obtained monooriented attachment and moved rapidly towards the spindle pole, moving past the chromosome indicated by the arrow. The latter obtained bioriented attachment at approximately the same time and congressed to the spindle equator (see Video 4 in online supplement). Scale bar = 5.0 µm.
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Figure 5: Immunofluorescent staining of checkpoint proteins in anti-CENP-E-injected HeLa cells. Synchronized cells were injected with rat anti-CENP-E antibodies 2 hours after release from the G1/S boundary and sampled 10 hours later. A-D) Cells were stained for hMAD2, hMAD1, hBUB1, and hBUBR1 using appropriate rabbit antibodies and Texas Red conjugated anti-rabbit secondary antibodies. Chromosomes and nuclei were stained with DAPI (left column). Injected cells exhibited the normal dissociation of checkpoint proteins from kinetochores of bioriented, aligned chromosomes. Bar = 10 µm.
Summary
In summary, electron microscopy and high resolution live cell and immunofluorescence imaging has enabled us to clear up a major misconception in the literature that CENP-E function is required for chromosome congression. Rather our data firmly establishes that the primary function of CENP-E is to insure reliable bioriented attachment of chromosomes. Although some chromosomes still obtain bioriented attachment in anti-CENP-E injected cells, others are stuck in unfavorable locations with such a low probability of bioriented attachment that they are effectively locked into a permanent monooriented attachment. This explains why we and other investigators always see a partial phenotype (i.e., congressed and uncongressed chromosomes). Our data also indicate that somewhere between 3-9 Mts are required to dissociate checkpoint proteins from the kinetochore. Tension is not required for recruitment or release of checkpoint proteins because the data also show that depletion of CENP-E from the kinetochore eliminates most of the tension generated across the centromere. Thus, our data also shed light on another controversial issue in the literature: the role of tension vs. Mt binding in the release of checkpoint proteins from the kinetochore.