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L Wordeman

Publications and source records attributed to L Wordeman.

At least 19 recordsLinked to original sources
PubMed2001

Molecular dissection of the microtubule depolymerizing activity of mitotic centromere-associated kinesin.

Mitotic centromere-associated kinesin (MCAK) is a microtubule depolymerizer that is consistent with its role in promoting chromosome segregation during mitosis. Here we show that the conserved motor domain of MCAK is necessary but not sufficient for microtubule depolymerization in cells or in vitro. The addition of only 30 amino acids N-terminal to the motor restores depolymerization activity. Furthermore, dimerization studies revealed that the smallest functional MCAK deletion constructs are monomers. These results define a highly conserved domain within MCAK and related (KIN I) kinesins that is critical for depolymerization activity and show that this depolymerization is not dependent on MCAK dimerization.

Amino Acid Sequence↗
PubMed2000

Human centromeres and neocentromeres show identical distribution patterns of >20 functionally important kinetochore-associated proteins.

Using combined immunofluorescence and fluorescence in situ hybridization (FISH) analysis we have extensively characterized the proteins associating with two different homologue human neocentromeres at interphase and prometaphase/metaphase, and compared these directly with those found with normal human centromeres. Antisera to CENP-A, CENP-B, CENP-C, CENP-E, CENP-F, INCENP, CLIP-170, dynein, dynactin subunits p150 (Glued) and Arp1, MCAK, Tsg24, p55CDC, HZW10, HBUB1, HBUBR1, BUB3, MAD2, ERK1, 3F3/2, topoisomerase II and a murine HP1 homologue, M31, were used in immuno-fluorescence experiments in conjunction with FISH employing specific DNA probes to clearly identify neocentromeric DNA. We found that except for the total absence of CENP-B binding, neocentromeres are indistinguishable from their alpha satellite-containing counterparts in terms of protein composition and distribution. This suggests that the DNA base of a potential centromeric locus is of minimal importance in determining the overall structure of a functional kinetochore and that, once seeded, the events leading to functional kinetochore formation occur independently of primary DNA sequence.

Anaphase↗
PubMed2000

The kinetochore of higher eucaryotes: a molecular view.

This review summarizes results concerning the molecular nature of the higher eucaryotic kinetochore. The first major section of this review includes kinetochore proteins whose general functions remain to be determined, precluding their entry into a discrete functional category. Many of the proteins in this section, however, are likely to be involved in kinetochore formation or structure. The second major section is concerned with how microtubule motor proteins function to cause chromosome movement. The microtubule motors dynein, CENP-E, and MCAK have all been observed at the kinetochore. While their precise functions are not well understood, all three are implicated in chromosome movement during mitosis. Finally, the last section deals with kinetochore components that play a role in the spindle checkpoint; a checkpoint that delays mitosis until all kinetochores have attached to the mitotic spindle. Brief reviews of kinetochore morphology and of an important technical breakthrough that enabled the molecular dissection of the kinetochore are also included.

Animals↗
PubMed2000

Expression and partial characterization of kinesin-related proteins in differentiating and adult skeletal muscle.

Using pan-kinesin antibodies to screen a differentiating C2C12 cell library, we identified the kinesin proteins KIF3A, KIF3B, and conventional kinesin heavy chain to be present in differentiating skeletal muscle. We compared the expression and subcellular localization characteristics of these kinesins in myogenic cells to others previously identified in muscle, neuronal, and mitotic systems (KIF1C, KIF3C, and mitotic-centromere-associated kinesin). Because members of the KIF3 subfamily of kinesin-related proteins showed altered subcellular fractionation characteristics in differentiating cells, we focused our study of kinesins in muscle on the function of kinesin-II. Kinesin-II is a motor complex comprised of dimerized KIF3A and KIF3B proteins and a tail-associated protein, KAP. The Xenopus homologue of KIF3B, Xklp3, is predominantly localized to the region of the Golgi apparatus, and overexpression of motorless-Xklp3 in Xenopus A6 cells causes mislocalization of Golgi components (). In C2C12 myoblasts and myotubes, KIF3B is diffuse and punctate, and not primarily associated with the Golgi. Overexpression of motorless-KIF3B does not perturb localization of Golgi components in myogenic cells, and myofibrillogenesis is normal. In adult skeletal muscle, KIF3B colocalizes with the excitation-contraction-coupling membranes. We propose that these membranes, consisting of the transverse-tubules and sarcoplasmic reticulum, are dynamic structures in which kinesin-II may function to actively assemble and maintain in myogenic cells.

Animals↗
PubMed2000

How motor proteins influence microtubule polymerization dynamics.

The interplay between microtubules and microtubule-based motors is fundamental to basic aspects of cellular function, such as the intracellular transport of organelles and alterations in cellular morphology during cell locomotion and division. Motor proteins are unique in that they couple nucleotide hydrolysis to force production that can do work. The force transduction by proteins belonging to the kinesin and dynein superfamilies has been thought only to power movement of these motors along the surface of microtubules; however, a growing body of evidence, both genetic and biochemical, suggests that motors can also directly influence the polymerization dynamics of microtubules. For example, at the vertebrate kinetochore, motors interact directly with microtubule ends and modulate polymerization dynamics to orchestrate chromosome movements during mitosis. Although a role for motors in regulating microtubule length has been established, the mechanisms used by motors to promote microtubule growth or shrinkage are unclear, as is an understanding of why cells might choose motors to control dynamics rather than a variety of non-motor proteins known to affect microtubule stability. Elucidation of the exact mechanisms by which motors alter the exchange of tubulin subunits at microtubule ends in vitro may shed light on how microtubule stability is regulated to produce the array of dynamic behavior seen in cells.

Amino Acid Sequence↗
PubMed1999

Mutations in the ATP-binding domain affect the subcellular distribution of mitotic centromere-associated kinesin (MCAK).

Mitotic centromere-associated kinesin (MCAK) is important for anaphase chromosome segregation. MCAK is diffusely localized to both the cytoplasm and the nucleus during interphase. At prophase MCAK is recruited to mitotic centromeres. It is associated with centromeres throughout mitosis and then returns to exhibiting a diffuse nuclear and cytoplasmic localization during interphase. MCAK has several predicted nuclear localization sequences. The subcellular distribution of expressed deletion constructs of GFP-MCAK suggest that the nucleocytoplasmic ratio of MCAK protein is dependent on a balance between several predicted nuclear localization sequences (NLS) and a putative nuclear exclusion sequence (NES) in the amino-terminal region of MCAK. Amino acid substitutions in the ATP-binding domain of the MCAK motor affect nuclear localization, which, in turn, influences the degree of centromere binding.

Adenosine Triphosphate↗
PubMed1998

Mitotic centromere-associated kinesin is important for anaphase chromosome segregation.

Mitotic centromere-associated kinesin (MCAK) is recruited to the centromere at prophase and remains centromere associated until after telophase. MCAK is a homodimer that is encoded by a single gene and has no associated subunits. A motorless version of MCAK that binds centromeres but not microtubules disrupts chromosome segregation during anaphase. Antisense-induced depletion of MCAK results in the same defect. MCAK overexpression induces centromere-independent bundling and eventual loss of spindle microtubule polymer suggesting that centromere-associated bundling and/or depolymerization activity is required for anaphase. Live cell imaging indicates that MCAK may be required to coordinate the onset of sister centromere separation.

Anaphase↗
PubMed1998

Localization of motor-related proteins and associated complexes to active, but not inactive, centromeres.

Multicentric chromosomes are often found in tumor cells and certain cell lines. How they are generated is not fully understood, though their stability suggests that they are non-functional during chromosome segregation. Growing evidence has implicated microtubule motor proteins in attachment of chromosomes to the mitotic spindle and in chromosome movement. To better understand the molecular basis for the inactivity of centromeres associated with secondary constrictions, we have tested these structures by immunofluorescence microscopy for the presence of motor complexes and associated proteins. We find strong immunoreactivity at the active, but not inactive, centromeres of prometaphase multicentric chromosomes using antibodies to the cytoplasmic dynein intermediate chains, three components of the dynactin complex (dynamitin, Arp1 and p150 Glued ), the kinesin-related proteins CENP-E and MCAK and the proposed structural and checkpoint proteins HZW10, CENP-F and Mad2p. These results offer new insight into the assembly and composition of both primary and secondary constrictions and provide a molecular basis for the apparent inactivity of the latter during chromosome segregation.

Biomarkers↗
PubMed1996

Disruption of CENP antigen function perturbs dynein anchoring to the mitotic kinetochore.

Injection of purified autoantibodies against human centromeric proteins into HeLa cells during interphase disrupts the organization of the kinetochore and interferes with chromosomal movements during the subsequent mitosis even though the chromosomes retain the ability to bind microtubules. We have investigated the hypothesis that this phenotype arises from effects on cytoplasmic dynein, the microtubule motor protein. In previous experiments we found that introduction of anticentromere antibodies into cell nuclei during the G1- or S-phases causes a prometaphase-like arrest, while injections during G2-phase cause a metaphase arrest. We show here that, in both cases, the level of detectable cytoplasmic dynein at kinetochores is significantly decreased. In contrast, when injected cells were permitted to enter mitosis in the absence of microtubules (conditions where trilaminar kinetochores could be detected by electron microscopy), the intensity of dynein labeling on the kinetochores was identical to that seen in uninjected control cells exposed to colcemid. Therefore, the loss of dynein label on mitotic kinetochores was correlated both with the injection of anticentromere antibodies and with the presence of intact spindle microtubules. We suggest that the injection of anticentromere antibodies somehow weakens the association of dynein with the kinetochore, so that when microtubules are present, these motor molecules are pulled away from the kinetochores as they generate force. This model offers an explanation for the failure of chromosomes of injected cells to move normally in mitosis even though they have attached microtubules.

Antibodies↗
PubMed1995

Mechanisms of chromosome segregation in metazoan cells.

Despite over 100 year of research, the mechanisms that cells use to ensure the proper segregation of chromosomes during mitosis are still surprisingly obscure. However, recent high resolution video light microscopic studies of dividing cells are telling us new and important information about chromosome behavior. Molecular genetics is enabling us to build a more complete list of the components involved in chromosome segregation. And in vitro assays for chromosome segregation are providing information about the signals that control the equipartitioning of sister chromatids during cell division.

Animals↗
PubMed1995

Identification and partial characterization of mitotic centromere-associated kinesin, a kinesin-related protein that associates with centromeres during mitosis.

Using antipeptide antibodies to conserved regions of the kinesin motor domain, we cloned a kinesin-related protein that associates with the centromere region of mitotic chromosomes. We call the protein MCAK, for mitotic centromere-associated kinesin. MCAK appears concentrated on centromeres at prophase and persists until telophase, after which time the localization disperses. It is found throughout the centromere region and between the kinetochore plates of isolated mitotic CHO chromosomes, in contrast to two other kinetochore-associated microtubule motors: cytoplasmic dynein and CENP-E (Yen et al., 1992), which are closer to the outer surface of the kinetochore plates. Sequence analysis shows MCAK to be a kinesin-related protein with the motor domain located in the center of the protein. It is 60-70% similar to kif2, a kinesin-related protein originally cloned from mouse brain with a centrally located motor domain (Aizawa et al., 1992). MCAK protein is present in interphase and mitotic CHO cells and is transcribed as a single 3.4-kb message.

Amino Acid Sequence↗
PubMed1995

A protein similar to the 67 kDa laminin binding protein and p40 is probably a component of the translational machinery in Urechis caupo oocytes and embryos.

Oocytes of the echiuroid, Urechis caupo, contain an abundant maternal mRNA that encodes a protein very similar to LBP/p40, originally identified as a non-integrin 67 kDa laminin binding protein. We have sequenced the Urechis caupo mRNA for LBP/p40, and a similar mRNA from the Hawaiian sea urchin, Tripneustes gratilla. Both of the encoded proteins, as well as LBP/p40 proteins from other sources, share significant homology in the amino 2/3 of the proteins, but diverge extensively at the carboxyl ends. LBP/p40 protein is present in growing and in full-grown U. caupo oocytes. The protein concentration remains constant for the first 48 hours of embryogenesis and then begins to decline. In sucrose gradients run with homogenates from coelomocytes, oocytes, and early embryos, all of the LBP/p40 protein appears to be associated with either polysomes or free 40 S ribosomal subunits. In later embryos, an increasing proportion of the protein is found in the soluble fraction. Immunohistochemistry indicates that LBP/p40 is uniformly distributed in early U. caupo embryos, with no localization at the cell surface. In later embryos LBP/p40 is localized in specific parts of the embryo which may correspond to neural tissue.

Amino Acid Sequence↗
PubMed1993

Inhibition of anaphase spindle elongation in vitro by a peptide antibody that recognizes kinesin motor domain.

Isolated central spindles or spindles in detergent-permeabilized cells from the diatom Cylindrotheca fusiformis can undergo ATP-dependent reactivation of spindle elongation in vitro. We have used a peptide antibody raised against a 10-amino acid portion common to the kinesin superfamily motor domain to look for kinesin-like motor activity during anaphase B of mitosis. The peptide antibody localizes to central spindles. Upon ATP reactivation of spindle elongation, antigens recognized by the antibody are associated exclusively with the central spindle midzone where antiparallel microtubules of each half-spindle overlap. The antibody recognizes several polypeptides by immunoblot using isolated spindle extracts. One of these polypeptides behaves like kinesin with respect to nucleotide-specific binding to and release from taxol-stabilized microtubules. Preincubation of the spindle model with the peptide antibody inhibits subsequent ATP reactivation of spindle elongation. Coincubation of the peptide antibody with peptide antigen rescues spindle function. These results support a role for kinesin-related protein(s) in spindle elongation (anaphase B) of mitosis and suggest that one or several polypeptides that we have identified in spindle extracts may fulfill this function.

Adenosine Triphosphate↗
PubMed1991

Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes.

We have used indirect immunofluorescence in combination with correlative EM to subdivide the mammalian kinetochore into two domains based on the localization of specific antigens. We demonstrate here that the fibrous corona on the distal face of the kinetochore plate contains tubulin (previously shown by Mitchison, T. J., and M. W. Kirschner. 1985. J. Cell Biol. 101:755-765) and the minus end-directed, ATP-dependent microtubule motor protein, dynein; whereas a 50-kD CREST antigen is located internal to these components in the kinetochore. Tubulin and dynein can be extracted from the kinetochore by 150 mM KI, leaving other, as yet uncharacterized, components of the kinetochore corona intact. Microtubules and tubulin subunits will associate with kinetochores in vitro after extraction with 150 mM KI, suggesting that other functionally significant, corona-associated molecules remain unextracted. Our results suggest that the corona region of the kinetochore contains the machinery for chromosome translocation along microtubules.

Animals↗
PubMed1990

Localization of cytoplasmic dynein to mitotic spindles and kinetochores.

What is the origin of the forces generating chromosome and spindle movements in mitosis? Both microtubule dynamics and microtubule-dependent motors have been proposed as the source of these motor forces. Cytoplasmic dynein and kinesin are two soluble proteins that power membranous organelle movements on microtubules. Kinesin directs movement of organelles to the 'plus' end of microtubules, and is found at the mitotic spindle in sea urchin embryos, but not in mammalian cells. Cytoplasmic dynein translocates organelles to the 'minus' end of microtubules, and is composed of two heavy chains and several light chains. We report here that monoclonal antibodies to two of these subunits and to another polypeptide that associates with dynein localize the protein to the mitotic spindle and to the kinetochores of isolated chromosomes, suggesting that cytoplasmic dynein is important in powering movements of the spindle and chromosomes in dividing cells.

Adenosine Triphosphatases↗