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The microtubule-dependent motor centromere-associated protein E (CENP-E) is an integral component of kinetochore corona fibers that link centromeres to spindle microtubules.

Yao X, Anderson KL, Cleveland DW - J. Cell Biol. (1997)

Bottom Line: Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis.In congressing chromosomes, CENP-E is preferentially associated with (or accessible at) the stretched, leading kinetochore known to provide the primary power for chromosome movement.Taken together, this evidence strongly supports a model in which CENP-E functions in congression to tether kinetochores to the disassembling microtubule plus ends.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Ludwig Institute for Cancer Research, School of Medicine, University of California, La Jolla, CA 92093-0660, USA.

ABSTRACT
Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Using immunoelectron microscopy, CENP-E is shown to be an integral component of the kinetochore corona fibers that tether centromeres to the spindle. Immediately upon nuclear envelope fragmentation, an associated plus end motor trafficks cytoplasmic CENP-E toward chromosomes along astral microtubules that enter the nuclear volume. Before or concurrently with initial lateral attachment of spindle microtubules, CENP-E targets to the outermost region of the developing kinetochores. After stable attachment, throughout chromosome congression, at metaphase, and throughout anaphase A, CENP-E is a constituent of the corona fibers, extending at least 50 nm away from the kinetochore outer plate and intertwining with spindle microtubules. In congressing chromosomes, CENP-E is preferentially associated with (or accessible at) the stretched, leading kinetochore known to provide the primary power for chromosome movement. Taken together, this evidence strongly supports a model in which CENP-E functions in congression to tether kinetochores to the disassembling microtubule plus ends.

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Characterization of the affinity-purified CENP-E antibody HpX. (A) Schematic drawing of CENP-E denoting the region used  to generate HpX, a fragment of 70 kD recombinant polypeptide expressed in bacteria. (B) Specificity of affinity-purified HpX antibody.  Immunoblots of mitotic whole cell lysates (lane 2, 50 μg) and isolated chromosome scaffold (lane 4, 25 μg). The same materials were  separated in SDS-PAGE and stained with Coomassie blue (lanes 1 and 3). (C) Upper panels: CENP-E is accumulated in the cytoplasm  just before nuclear envelope breakdown. Indirect immunofluorescence image of HeLa cells stained with HpX antibody (upper left),  DAPI (upper middle) and human CREST sera (upper right). CREST sera stained centromeres in both early interphase cell (arrowheads) and late interphase cells (arrows). CENP-E signal appeared only in the late interphase cells (upper left, arrows). Interphase nuclei lack CENP-E staining (arrowheads). Lower panels: CENP-E is located to kinetochores as pairs of clearly resolved double dots  (lower left, arrows), while CREST sera mark centromeres as unresolved dots (lower right, arrowhead). Bars: (upper panels) 20 μm;  (lower panels) 10 μm.
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Figure 1: Characterization of the affinity-purified CENP-E antibody HpX. (A) Schematic drawing of CENP-E denoting the region used to generate HpX, a fragment of 70 kD recombinant polypeptide expressed in bacteria. (B) Specificity of affinity-purified HpX antibody. Immunoblots of mitotic whole cell lysates (lane 2, 50 μg) and isolated chromosome scaffold (lane 4, 25 μg). The same materials were separated in SDS-PAGE and stained with Coomassie blue (lanes 1 and 3). (C) Upper panels: CENP-E is accumulated in the cytoplasm just before nuclear envelope breakdown. Indirect immunofluorescence image of HeLa cells stained with HpX antibody (upper left), DAPI (upper middle) and human CREST sera (upper right). CREST sera stained centromeres in both early interphase cell (arrowheads) and late interphase cells (arrows). CENP-E signal appeared only in the late interphase cells (upper left, arrows). Interphase nuclei lack CENP-E staining (arrowheads). Lower panels: CENP-E is located to kinetochores as pairs of clearly resolved double dots (lower left, arrows), while CREST sera mark centromeres as unresolved dots (lower right, arrowhead). Bars: (upper panels) 20 μm; (lower panels) 10 μm.

Mentions: Previous studies revealed cell cycle–regulated distribution of CENP-E and localization of CENP-E near the kinetochore region of mitotic chromosomes. To define more closely the location of CENP-E during kinetochore maturation into a trilaminar structure, a polyclonal antibody against a bacterially expressed portion of the rod domain of CENP-E (amino acids 955–1571, designated as HpX, illustrated in Fig. 1 A) was generated and affinity purified using an antigen-coupled Sepharose matrix. Protein immunoblot analysis revealed that the affinity-purified CENP-E antibody specifically recognized a single protein band of ∼310 kD in whole mitotic HeLa cell extracts and isolated chromosome scaffolds (Fig. 1 B, lanes 2 and 4, respectively). This 310-kD band was not recognized by preimmune serum. To verify further the specificity of this HpX antibody, CENP-E localization was visualized in HeLa cells using HpX antibody and a fluorescein-conjugated goat anti–rabbit secondary antibody (Fig. 1 C, upper left), while a human CREST anticentromere antibody that reacts primarily with CENP-B followed by a rhodamine-conjugated goat anti–human secondary antibody was used to identify the actual centromere (Fig. 1 C, upper right). This revealed that, in accord with previous reports (Yen et al., 1992; Brown et al., 1995), CENP-E accumulates in the cytoplasm of G2 cells (Fig. 1 C, upper left, arrows) but is absent from most interphase cells (arrowheads). At mitosis, CENP-E staining appears as pairs of clearly resolved double dots (lower left, arrows), while CREST centromere antigens are present as pairs of unresolved dots (Fig. 1 C, lower right, arrowhead).


The microtubule-dependent motor centromere-associated protein E (CENP-E) is an integral component of kinetochore corona fibers that link centromeres to spindle microtubules.

Yao X, Anderson KL, Cleveland DW - J. Cell Biol. (1997)

Characterization of the affinity-purified CENP-E antibody HpX. (A) Schematic drawing of CENP-E denoting the region used  to generate HpX, a fragment of 70 kD recombinant polypeptide expressed in bacteria. (B) Specificity of affinity-purified HpX antibody.  Immunoblots of mitotic whole cell lysates (lane 2, 50 μg) and isolated chromosome scaffold (lane 4, 25 μg). The same materials were  separated in SDS-PAGE and stained with Coomassie blue (lanes 1 and 3). (C) Upper panels: CENP-E is accumulated in the cytoplasm  just before nuclear envelope breakdown. Indirect immunofluorescence image of HeLa cells stained with HpX antibody (upper left),  DAPI (upper middle) and human CREST sera (upper right). CREST sera stained centromeres in both early interphase cell (arrowheads) and late interphase cells (arrows). CENP-E signal appeared only in the late interphase cells (upper left, arrows). Interphase nuclei lack CENP-E staining (arrowheads). Lower panels: CENP-E is located to kinetochores as pairs of clearly resolved double dots  (lower left, arrows), while CREST sera mark centromeres as unresolved dots (lower right, arrowhead). Bars: (upper panels) 20 μm;  (lower panels) 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139792&req=5

Figure 1: Characterization of the affinity-purified CENP-E antibody HpX. (A) Schematic drawing of CENP-E denoting the region used to generate HpX, a fragment of 70 kD recombinant polypeptide expressed in bacteria. (B) Specificity of affinity-purified HpX antibody. Immunoblots of mitotic whole cell lysates (lane 2, 50 μg) and isolated chromosome scaffold (lane 4, 25 μg). The same materials were separated in SDS-PAGE and stained with Coomassie blue (lanes 1 and 3). (C) Upper panels: CENP-E is accumulated in the cytoplasm just before nuclear envelope breakdown. Indirect immunofluorescence image of HeLa cells stained with HpX antibody (upper left), DAPI (upper middle) and human CREST sera (upper right). CREST sera stained centromeres in both early interphase cell (arrowheads) and late interphase cells (arrows). CENP-E signal appeared only in the late interphase cells (upper left, arrows). Interphase nuclei lack CENP-E staining (arrowheads). Lower panels: CENP-E is located to kinetochores as pairs of clearly resolved double dots (lower left, arrows), while CREST sera mark centromeres as unresolved dots (lower right, arrowhead). Bars: (upper panels) 20 μm; (lower panels) 10 μm.
Mentions: Previous studies revealed cell cycle–regulated distribution of CENP-E and localization of CENP-E near the kinetochore region of mitotic chromosomes. To define more closely the location of CENP-E during kinetochore maturation into a trilaminar structure, a polyclonal antibody against a bacterially expressed portion of the rod domain of CENP-E (amino acids 955–1571, designated as HpX, illustrated in Fig. 1 A) was generated and affinity purified using an antigen-coupled Sepharose matrix. Protein immunoblot analysis revealed that the affinity-purified CENP-E antibody specifically recognized a single protein band of ∼310 kD in whole mitotic HeLa cell extracts and isolated chromosome scaffolds (Fig. 1 B, lanes 2 and 4, respectively). This 310-kD band was not recognized by preimmune serum. To verify further the specificity of this HpX antibody, CENP-E localization was visualized in HeLa cells using HpX antibody and a fluorescein-conjugated goat anti–rabbit secondary antibody (Fig. 1 C, upper left), while a human CREST anticentromere antibody that reacts primarily with CENP-B followed by a rhodamine-conjugated goat anti–human secondary antibody was used to identify the actual centromere (Fig. 1 C, upper right). This revealed that, in accord with previous reports (Yen et al., 1992; Brown et al., 1995), CENP-E accumulates in the cytoplasm of G2 cells (Fig. 1 C, upper left, arrows) but is absent from most interphase cells (arrowheads). At mitosis, CENP-E staining appears as pairs of clearly resolved double dots (lower left, arrows), while CREST centromere antigens are present as pairs of unresolved dots (Fig. 1 C, lower right, arrowhead).

Bottom Line: Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis.In congressing chromosomes, CENP-E is preferentially associated with (or accessible at) the stretched, leading kinetochore known to provide the primary power for chromosome movement.Taken together, this evidence strongly supports a model in which CENP-E functions in congression to tether kinetochores to the disassembling microtubule plus ends.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Ludwig Institute for Cancer Research, School of Medicine, University of California, La Jolla, CA 92093-0660, USA.

ABSTRACT
Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Using immunoelectron microscopy, CENP-E is shown to be an integral component of the kinetochore corona fibers that tether centromeres to the spindle. Immediately upon nuclear envelope fragmentation, an associated plus end motor trafficks cytoplasmic CENP-E toward chromosomes along astral microtubules that enter the nuclear volume. Before or concurrently with initial lateral attachment of spindle microtubules, CENP-E targets to the outermost region of the developing kinetochores. After stable attachment, throughout chromosome congression, at metaphase, and throughout anaphase A, CENP-E is a constituent of the corona fibers, extending at least 50 nm away from the kinetochore outer plate and intertwining with spindle microtubules. In congressing chromosomes, CENP-E is preferentially associated with (or accessible at) the stretched, leading kinetochore known to provide the primary power for chromosome movement. Taken together, this evidence strongly supports a model in which CENP-E functions in congression to tether kinetochores to the disassembling microtubule plus ends.

Show MeSH
Related in: MedlinePlus