XB-ART-37633J Cell Biol. May 5, 2008; 181 (3): 411-9.
CENP-E combines a slow, processive motor and a flexible coiled coil to produce an essential motile kinetochore tether.
The mitotic kinesin centromere protein E (CENP-E) is an essential kinetochore component that directly contributes to the capture and stabilization of spindle microtubules by kinetochores. Although reduction in CENP-E leads to high rates of whole chromosome missegregation, neither its properties as a microtubule-dependent motor nor how it contributes to the dynamic linkage between kinetochores and microtubules is known. Using single-molecule assays, we demonstrate that CENP-E is a very slow, highly processive motor that maintains microtubule attachment for long periods. Direct visualization of full-length Xenopus laevis CENP-E reveals a highly flexible 230-nm coiled coil separating its kinetochore-binding and motor domains. We also show that full-length CENP-E is a slow plus end-directed motor whose activity is essential for metaphase chromosome alignment. We propose that the highly processive microtubule-dependent motor activity of CENP-E serves to power chromosome congression and provides a flexible, motile tether linking kinetochores to dynamic spindle microtubules.
PubMed ID: 18443223
PMC ID: PMC2364708
Article link: J Cell Biol.
Grant support: GM29513 NIGMS NIH HHS , R01 GM029513 NIGMS NIH HHS
Genes referenced: cenpe
Article Images: [+] show captions
|Figure 1. CENP-E is a slow, processive motor that maintains microtubule attachment for long periods. (A) Purified CE473-GFP. The arrow points to the purified protein band. (B) Experimental setup for imaging CENP-E single molecules moving along microtubules with TIRF microscopy. X-rhodamine–labeled GMPCPP microtubules were immobilized on a coverslip using antitubulin antibody, and a low concentration of CE473-GFP (0.5–1 nM) was flowed into a flow chamber. (C) Frames from time-lapse imaging of CENP-E motor–GFP moving along microtubules. A fluorescent speckle on the microtubule is indicated with a red line. Two CENP-E molecules moving at different speeds are indicated with arrows. Green, CE473-GFP; red, X-rhodamine–labeled GMPCPP microtubules. (D) Kymographs showing processive movements of the CENP-E motor domain. X-rhodamine speckles on stable microtubules produced vertical red lines. Single molecules of CE473-GFP are shown in green. Yellow arrowheads indicate starts and stops of processive movements. The actual durations of kymographs 1, 2, and 3 were 1,205 s, 1,190 s, and 1,205 s, respectively. (E) Velocity distribution of single CENP-E motor molecules. The median velocity is 8 nm/s (equal to 0.48 μm/min; red line; n = 320; four independent preparations). (F) MSD (ρ(t)) of CENP-E fitted with ρ(t) = v2t2 + 2Dt (n = 90). Error bars indicate SEM. (G) The run time of CENP-E motor was distributed exponentially. The mean run time was determined by fitting the data into a cumulative distribution function. The inset shows the one-cumulative probability of CENP-E run time plotted on a log scale. The mean run time is 195 ± 13 s (mean ± SEM; n = 320). (H) The run length of CENP-E motor was determined by fitting the data into a cumulative distribution function. The inset shows the one-cumulative probability of CENP-E run length plotted on a log scale. The mean run length is 1.5 ± 0.1 μm (mean ± SEM; n = 320). (G and H) Red lines are the exponential fits to the bar graphs. Bars, 2 μm.|
|Figure 2. CENP-E is a highly flexible, dimeric kinesin with a 230-nm discontinuous coiled coil. (A) Coomassie- and silver-stained Xenopus full-length CENP-E (340 kD) purified to near homogeneity from baculovirus-induced insect cells. (B) Electron micrographs of individual CENP-E molecules. Two motor heads are clearly visible, as indicated by yellow arrows. (C) Length distribution of CENP-E. Contour lengths of molecules were measured, and the mean length of CENP-E was 230 ± 25 nm (mean ± SD; n = 20). (D) Coiled-coil prediction of Xenopus CENP-E. Coiled-coil scores were generated using Protean software (DNAstar) and are graphed below the amino acid scale bar. The number 1.3 is the default value indicating the minimum score for known coiled coils, and resulting predicted coiled-coil domains are shown as solid orange rectangles.|
|Figure 3. Full-length CENP-E is a slow plus end–directed kinetochore motor. (A) Coomassie-stained full-length CENP-E–GFP (366 kD) purified from baculovirus-induced insect cells. (B) Microtubule gliding assay with full-length CENP-E. CENP-E–GFP proteins were tethered to a GFP antibody-coated surface of a flow chamber, and polarity-marked microtubules were subsequently introduced. Minus ends of the microtubules are brightly marked. Colored arrowheads indicate the starting positions of three microtubules, and colored dots indicate the minus ends. The mean gliding velocity was 30 ± 7.6 nm/s (mean ± SD; n = 112). (C) Purified full-length CENP-E–GFP was added into the Xenopus extract before spindle assembly. (D) Added CENP-E–GFP was localized to the kinetochore in Xenopus extract spindle. A frame from a time-lapse video of a metaphase spindle in the Xenopus extracts is shown. Red, X-rhodamine tubulin; green, CENP-E–GFP. Bars: (B) 2 μm; (D) 10 μm.|
|Figure 4. CENP-E motor activity is essential for metaphase chromosome alignment. (A) Experimental scheme for Xenopus extract manipulation. (B) Immunoblot of CENP-E (340 kD) and Rod (220 kD; a loading control) in mock-depleted, CENP-E–depleted, wild-type CENP-E–supplemented, and rigor CENP-ET91N–supplemented Xenopus extracts. (C) Recombinant full-length CENP-E partially rescued chromosome alignment, whereas rigor CENP-E failed at rescue. Red, X-rhodamine tubulin; green, DAPI. (D) Quantification of structures formed in Xenopus extracts. More than 200 spindles were scored each in three independent depletion/add-back experiments. Error bars represent SD. Bar, 10 μm.|
|Figure 5. A model for CENP-E as a motile, flexible tether for kinetochore microtubule capture and maintenance of linkage to dynamic spindle microtubules. (A) Using its slow processive motor activity and a weak diffusive binding mode to microtubules, CENP-E walks toward the plus ends of kinetochore microtubules or diffuses along the lattice without dissociating for extended periods. (B) The 230-nm-long coiled coil of CENP-E functions as a safety catch for disassembling microtubules detached from the core kinetochore attachment components, thereby stabilizing the microtubule and enabling rescue. (C) CENP-E is likely to be a part of the kinetochore slip clutch that is engaged on fluxing kinetochore microtubules with its slow plus end–directed motility (Maddox et al., 2003a). CENP-E bound to the microtubule surface may affect kinetochore microtubule plus ends, thereby promoting growth and allowing recapture. (D) Unlike other shorter and more rigidly structured kinetochore capture components, multiple CENP-E molecules are likely to work together by allowing the simultaneous attachment at many different microtubule orientations relative to the kinetochore axis without forcing each other into unproductive conformations. (E) The highly flexible extended coiled coil of CENP-E mediates the initial capture of microtubules by searching a large volume in cells. (F) Its slow, processive motility powers monooriented chromosomes to congress using an adjacent kinetochore fiber (Kapoor et al., 2006).|