1. Volume 91, Issue 3, Pages 357-366 (October 1997)
CENP-E Is a Plus End–Directed Kinetochore Motor Required for Metaphase Chromosome Alignment 
Kenneth W Wood, Roman Sakowicz, Lawrence S.B Goldstein, Don W Cleveland 
Cell 
Volume 91, Issue 3, Pages (October 1997)
DOI: /S (00)

2. Figure 1 Identification of Xenopus CENP-E
(A) Structural comparison of Xenopus and human CENP-E. Hatched regions represent regions predicted to form α-helical coiled coils (Lupas et al. 1991). Within the N-terminal regions of both hCENP-E and XCENP-E, there is a domain of ∼324 amino acids corresponding to the kinesin-like motor domain. Within these 324 amino acids, XCENP-E and hCENP-E are 74% identical. Overall XCENP-E and human CENP-E are 37% identical and, with conservative substitutions, 57% similar. Unshaded regions within the rod are those regions sharing 47% identity with human CENP-E. One cDNA clone encoded a protein with a 9 amino acid insertion relative to other cDNAs isolated (marked by the arrowhead, see Figure 1C). XCENP-E contains a putative nuclear localization signal (NLS) located at the C-terminal end of the rod domain not present in hCENP-E.
(B) XCENP-E fusion proteins used for polyclonal antibody production.
(C) Deduced amino acid sequence of Xenopus CENP-E. cDNA sequence was compiled from 6 overlapping cDNA clones. Residues identical in hCENP-E and XCENP-E are shaded. The boxed region is the portion of XCENP-E used to assay motility in vitro (see Figure 6). The underlined sequence NSREHSINA at position 599 is the 9 amino acid relative insertion encoded by one of the cDNAs isolated (see Figure 1A). The putative NLS, RKKTK, is also underlined. Nucleotide sequence is available from EMBL/GenBank (see end of this article).
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3. Figure 6 XCENP-E Is a Plus End–Directed Microtubule Motor
(A) XCENP-E residues 1–473 fused at the C terminus to a c-Myc epitope and a hexahistidine tag (see schematic). Coomassie blue stain of purified XCENP-E fusion protein (arrowhead) used for motility (lane 1); immunoblot of XCENP-E fusion protein probed with α-Myc monoclonal antibody (lane 2).
(B) XCENP-E motility assay. Microtubules marked near their minus ends with brightly fluorescent seeds were added with ATP to a flow chamber containing purified XCENP-E fusion protein tethered to the coverslip with α-Myc monoclonal antibody. Gliding of microtubules was monitored by time-lapse digital fluorescence microscopy. Selected frames from one time lapse series, spaced 90 sec apart, are presented. The positions of the plus ends of microtubules numbered 1, 2, and 3 at the start of continuous gliding are marked with solid white dots, and the position of a stationary microtubule end is marked by the arrowhead. The bright seed of microtubule 3 enters the plane of focus at 1.5 min, and glides 13.6 μm downward with the bright seed leading over the following 3 min. Microtubule 2 moves continuously during the first 3 min, after which point it detaches and reattaches further toward the bottom of the frame. Microtubule 1 glides minus-end leading throughout the entire time course. The average microtubule velocity of all microtubules was 5.1 μm/min ± 1.7 (n = 49). Of those, 33 microtubules were unambiguously polarity marked, and all glided with their bright seeds leading. Scalebar, 5 μm.
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4. Figure 2 XCENP-E Localizes to Kinetochores In Vivo and In Vitro
(A) Localization of XCENP-E on cycled mitotic spindles assemble in vitro. Panel 1, rhodamine-tubulin (red), DAPI-stained chromatin (blue); Panel 2, α-XCENP-ETAIL staining. Scalebar, 10 μm.
(B) Xenopus egg extract (∼60 μg/lane) resolved on a 4% gel, immunoblotted and probed with affinity-purified α-XCENP-ETAIL or α-XCENP-EROD antibody. XCENP-E is indicated by the arrowhead, and the XCENP-E-related protein by the dot.
(C) Localization of XCENP-E in cultured Xenopus XTC cells. XTC cells were fixed and stained with mouse monoclonal anti-α-tubulin antibody (red) and affinity-purified rabbit α-XCENP-ETAIL antibody (green). Chromatin was visualized by staining with DAPI (blue). Cells at progressive stages of the cell cycle are displayed. Similar staining was observed using α-XCENP-EROD antibody. Scalebar, 5 μm.
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5. Figure 3 XCENP-E Is Required for Metaphase Chromosome Alignment
(A) Immunodepletion of XCENP-E. An immunoblot of 1 μl each of Xenopus egg extract depleted of XCENP-E using α-XCENP-ETAIL antibody (lane 2), mock depleted with nonimmune rabbit IgG (lane 3), and unmanipulated extract (lane 1) was probed with α-XCENP-ETAIL antibody (upper panel). To control for specificify of depletion and loading of the gel, the blot was subsequently probed with α-XNuMA tail antibody (Merdes et al. 1996) (lower panel).
(B) Representative structures formed in control extracts (panel 1), and in XCENP-E-depleted extract (panels 2–5). Tubulin is shown in red, chromatin in blue. Sperm nuclei were added, and extracts were cycled through interphase and arrested at the following metaphase. Arrowheads in panels 2 and 5 indicate apparently nondisjoined sister chromatids. Scalebar, 10 μm.
(C) Quantitation of structures formed in undepleted extract (n = 123), extract mock depleted with nonimmune rabbit IgG (n = 156) and extract depleted of XCENP-E (n = 98) scored 80 min after exit from interphase. Data are presented from one representative experiment. Structures were scored as bipolar spindles with chromatin aligned at the metaphase plate, bipolar spindles with misaligned chromosomes, monopolar spindles, including radial asters, half spindles, and chromosomes associated with microtubules with indeterminant organization, and other, including multipolar structures and groups of chromosomes apparently unassociated with microtubules.
(D) Coomassie blue staining of α-XCENP-ETAIL and α-XCENP-EROD immunoprecipitates. Immunoprecipitates were prepared from 100 μl CSF-arrested extract using affinity-purified α-XCENP-ETAIL antibody (lane 3), affinity purified α-XCENP-EROD antibody (lane 2), or nonimmune rabbit IgG (lane 1). Immunoprecipitates were washed and resolved by SDS–PAGE on a 5% gel. Proteins were visualized by staining with Coomassie blue. The arrowhead indicates XCENP-E.
Cell  , DOI: ( /S (00) )

6. Figure 4
Addition of α-XENP-E Antibody Disrupts Metaphase Chromosome Alignment, but Not Bipolar Spindle Assembly around Chromatin Beads
(A) Representative structures formed in the presence of 0.5 mg/ml rabbit IgG and in the absence of added antibody (panels 1–3), and in the presence of 0.5 mg/ml α-XCENP-ETAIL antibody (panels 4–8). Tubulin is shown in red; chromosomes, in blue. Extracts containing demebranated sperm nuclei and antibody were cycled through interphase and arrested at the following metaphase.
(B) Left panel: Quantitation of structures formed from sperm nuclei in extract containing no antibody (n = 125), extract containing 0.5 mg/ml nonimmune rabbit IgG (rIgGn = 172), and extract containing 0.5 mg/ml α-XCENP-ETAIL (n = 101) scored 80 min after exit from interphase. Data are presented from one representative experiment. Structures were scored as described in Figure 3C. Right panel: Quantitation of microtubule-containing structures formed around bead-bound chromatin in the presence and absence of 0.5 mg/ml α-XCENP-ETAIL antibody.
(C) Representative spindles assembled around bead-bound plasmid DNA in the presence (panel 2) and absence (panel 1) of 0.5 mg/ml α-XCENP-ETAIL antibody (Heald et al. 1996).
Cell  , DOI: ( /S (00) )

7. Figure 5
Disruption of XCENP-E Function Yields Misaligned Chromosomes that Have Not Undergone Premature Anaphase
Spindles assembled in the extracts incompletely depleted of XCENP-E (85–90% depletion), stained with α-XCENP-ETAIL antibody. Images collected with exposure times sufficient to yield saturated kinetochore staining of control spindles (e.g.Figure 2A) yielded no visible kinetochore staining (A). However, longer exposure revealed residual XCENP-E, present in paired spots characteristic of nondisjoined prometaphase sister chromatids ([B], panels 1 and 2, corresponding to boxes 1 and 2 in [A]). From the fluorescence intensity, we estimate that kinetochore-associated XCENP-E has been reduced 6-fold or greater, consistent with the extent of depletion.
Cell  , DOI: ( /S (00) )

8. Figure 7
A Model for CENP-E Function in Chromosome Alignment: Tethering of Kinetochores to Microtubule Plus Ends
At both leading and trailing kinetochores, CENP-E (yellow ovals) binds to microtubules and moves toward the plus ends, where it remains attached. At the leading kinetochore, CENP-E molecules detach and reattach as kinetochore microtubules disassemble (perhaps induced by XKCM1, depicted as orange ovals), thus moving the kinetochore poleward by maintaining kinetochore attachment to microtubules. At the trailing kinetochore, the plus end–directed CENP-E motor pushes the kinetochore to the newly assembled microtubule plus end. Plus end growth at the trailing kinetochore might occur in response to a promoter of plus end assembly (lavender circles). CENP-E would push only to the extent that microtubule plus ends grew, and as long as the rate of plus end growth did not exceed the rate of CENP-E movement, trailing kinetochore attachment would be maintained. A CENP-E-dependent tether to microtubule plus ends, in combination with regulators of microtubule plus end dynamics functionally linked by tension-sensors, would be sufficient to mediate oscillatory movement of congressing chromosomes.
Cell  , DOI: ( /S (00) )