Biomedical subjects

B T Schaar

Publications and source records attributed to B T Schaar.

7 recordsLinked to original sources

PubMed2001

NudC associates with Lis1 and the dynein motor at the leading pole of neurons.

NUDC is a highly conserved protein important for nuclear migration and viability in Aspergillus nidulans. Mammalian NudC interacts with Lis1, a neuronal migration protein important during neocorticogenesis, suggesting a conserved mechanism of nuclear movement in A. nidulans and neuronal migration in the developing mammalian brain (S. M. Morris et al., 1998). To further investigate this possibility, we show for the first time that NudC, Lis1, and cytoplasmic dynein intermediate chain (CDIC) colocalize at the microtubule organizing center (MTOC) around the nucleus in a polarized manner facing the leading pole of cerebellar granule cells with a migratory morphology. In neurons with stationary morphology, NudC is distributed throughout the soma and colocalizes with CDIC and tubulin in neurites as well as at the MTOC. At the subcellular level, NudC, CDIC, and p150 dynactin colocalize to the interphase microtubule array and the MTOC in fibroblasts. The observed colocalization is confirmed biochemically by coimmunoprecipitation of NudC with CDIC and cytoplasmic dynein heavy chain (CDHC) from mouse brain extracts. Consistent with its expression in individual neurons, a high level of NudC is detected in regions of the embryonic neocortex undergoing extensive neurogenesis as well as neuronal migration. These data suggest a biochemical and functional interaction of NudC with Lis1 and the dynein motor complex during neuronal migration in vivo.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2001

Doublecortin interacts with mu subunits of clathrin adaptor complexes in the developing nervous system.

Doublecortin is a microtubule-associated protein required for normal corticogenesis in the developing brain. We carried out a yeast two-hybrid screen to identify interacting proteins. One of the isolated clones encodes the mu1 subunit of the adaptor complex AP-1 involved in clathrin-dependent protein sorting. We found that Doublecortin also interacts in yeast with mu2 from the AP-2 complex. Mutagenesis and pull-down experiments showed that these interactions were mediated through a tyrosine-based sorting signal (YLPL) in the C-terminal part of Doublecortin. The functional relevance of these interactions was suggested by the coimmunoprecipitation of Doublecortin with AP-1 and AP-2 from mouse brain extracts. This interaction was further supported by RNA in situ hybridization and immunofluorescence studies. Taken together these data indicate that a certain proportion of Doublecortin interacts with AP-1 and/or AP-2 in vivo and are consistent with a potential involvement of Doublecortin in protein sorting or vesicular trafficking.

Adaptor Protein Complex 1↗

PubMed1998

Characterization of the kinetochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and hBUBR1.

We have identified a 350-amino acid domain in the kinetochore motor CENP-E that specifies kinetochore binding in mitosis but not during interphase. The kinetochore binding domain was used in a yeast two-hybrid screen to isolate interacting proteins that included the kinetochore proteins CENP-E, CENP-F, and hBUBR1, a BUB1-related kinase that was found to be mutated in some colorectal carcinomas (Cahill, D.P., C. Lengauer, J. Yu, G.J. Riggins, J.K. Wilson, S.D. Markowitz, K.W. Kinzler, and B. Vogelstein. 1998. Nature. 392:300-303). CENP-F, hBUBR1, and CENP-E assembled onto kinetochores in sequential order during late stages of the cell cycle. These proteins therefore define discrete steps along the kinetochore assembly pathway. Kinetochores of unaligned chromosome exhibited stronger hBUBR1 and CENP-E staining than those of aligned chromosomes. CENP-E and hBUBR1 remain colocalized at kinetochores until mid-anaphase when hBUBR1 localized to portions of the spindle midzone that did not overlap with CENP-E. As CENP-E and hBUBR1 can coimmunoprecipitate with each other from HeLa cells, they may function as a motor-kinase complex at kinetochores. However, the complex distribution pattern of hBUBR1 suggests that it may regulate multiple functions that include the kinetochore and the spindle midzone.

Amino Acid Sequence↗

PubMed1997

CENP-E function at kinetochores is essential for chromosome alignment.

CENP-E is a kinesin-like protein that binds to kinetochores and may provide functions that are critical for normal chromosome motility during mitosis. To directly test the in vivo function of CENP-E, we microinjected affinity-purified antibodies to block the assembly of CENP-E onto kinetochores and then examined the behavior of these chromosomes. Chromosomes lacking CENP-E at their kinetochores consistently exhibited two types of defects that blocked their alignment at the spindle equator. Chromosomes positioned near a pole remained mono-oriented as they were unable to establish bipolar microtubule connections with the opposite pole. Chromosomes within the spindle established bipolar connections that supported oscillations and normal velocities of kinetochore movement between the poles, but these bipolar connections were defective because they failed to align the chromosomes into a metaphase plate. Overexpression of a mutant that lacked the amino-terminal 803 amino acids of CENP-E was found to saturate limiting binding sites on kinetochores and competitively blocked endogenous CENP-E from assembling onto kinetochores. Chromosomes saturated with the truncated CENP-E mutant were never found to be aligned but accumulated at the poles or were strewn within the spindle as was the case when cells were microinjected with CENP-E antibodies. As the motor domain was contained within the portion of CENP-E that was deleted, the chromosomal defect is likely attributed to the loss of motor function. The combined data show that CENP-E provides kinetochore functions that are essential for monopolar chromosomes to establish bipolar connections and for chromosomes with connections to both spindle poles to align at the spindle equator. Both of these events rely on activities that are provided by CENP-E's motor domain.

Animals↗

PubMed1996

Kinetochore function: molecular motors, switches and gates.

Kinetochores are essential for accurate chromosome segregation. Recent studies reveal that vertebrate kinetochores are sophisticated propulsion systems composed of not only force generators but also "navigation' and "fail-safe' mechanisms. Advances toward the understanding of the biochemical composition and activities of the components of the kinetochore have come from the molecular characterization of key proteins of the kinetochore complex.

Animals↗

PubMed1995

Mitotic HeLa cells contain a CENP-E-associated minus end-directed microtubule motor.

A minus end-directed microtubule motor activity from extracts of HeLa cells blocked at prometaphase/metaphase of mitosis with vinblastine has been partially purified and characterized. The motor activity was eliminated by immunodepletion of Centromere binding protein E (CENP-E). The CENP-E-associated motor activity, which was not detectable in interphase cells, moved microtubules at mean rates of 0.46 micron/s at 37 degrees C and 0.24 micron/s at 25 degrees C. The motor activity co-purified with CENP-E through several purification procedures. Motor activity was clearly not due to dynein or to kinesin. The microtubule gliding rates of the CENP-E-associated motor were different from those of dynein and kinesin. In addition, the pattern of nucleotide substrate utilization by the CENP-E-associated motor and the sensitivity to inhibitors were different from those of dynein and kinesin. The CENP-E-associated motor had an apparent native molecular weight of 874,000 Da and estimated dimensions of 2 nm x 80 nm. This is the first demonstration of motor activity associated with CENP-E, strongly supporting the hypothesis that CENP-E may act as a minus end-directed microtubule motor during mitosis.

Cell Movement↗

PubMed1992

CENP-E is a putative kinetochore motor that accumulates just before mitosis.

The mechanics of chromosome movement, mitotic spindle assembly and spindle elongation have long been central questions of cell biology. After attachment in prometaphase of a microtubule from one pole, duplicated chromosome pairs travel towards the pole in a rapid but discontinuous motion. This is followed by a slower congression towards the midplate as the chromosome pair orients with each kinetochore attached to the microtubules from the nearest pole. The pairs disjoin at anaphase and translocate to opposite poles and the interpolar distance increases. Here we identify CENP-E as a kinesin-like motor protein (M(r) 312,000) that accumulates in the G2 phase of the cell cycle. CENP-E associates with kinetochores during congression, relocates to the spindle midzone at anaphase, and is quantitatively discarded at the end of the cell division. CENP-E is likely to be one of the motors responsible for mammalian chromosome movement and/or spindle elongation.

Amino Acid Sequence↗