Minus End-directed Microtubule Motor Research Articles

Cell cycle regulation of dynein association with membranes modulates microtubule-based organelle transport.

Cytoplasmic dynein is a minus end-directed microtubule motor that performs distinct functions in interphase and mitosis. In interphase, dynein transports organelles along microtubules, whereas in metaphase this motor has been implicated in mitotic spindle formation and orientation as well as chromosome segregation. The manner in which dynein activity is regulated during the cell cycle, however, has not been resolved. In this study, we have examined the mechanism by which organelle transport is controlled by the cell cycle in extracts of Xenopus laevis eggs. Here, we show that photocleavage of the dynein heavy chain dramatically inhibits minus end-directed organelle transport and that purified dynein restores this motility, indicating that dynein is the predominant minus end-directed membrane motor in Xenopus egg extracts. By measuring the amount of dynein associated with isolated membranes, we find that cytoplasmic dynein and its activator dynactin detach from the membrane surface in metaphase extracts. The sevenfold decrease in membrane-associated dynein correlated well with the eightfold reduction in minus end-directed membrane transport observed in metaphase versus interphase extracts. Although dynein heavy or intermediate chain phosphorylation did not change in a cell cycle-dependent manner, the dynein light intermediate chain incorporated approximately 12-fold more radiolabeled phosphate in metaphase than in interphase extracts. These studies suggest that cell cycle-dependent phosphorylation of cytoplasmic dynein may regulate organelle transport by modulating the association of this motor with membranes.

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.

Microtubule motors associated with kinetochores in mitotic cells

Chromosomes in dividing tissue culture cells exhibit three types of movement along mitotic spindle microtubules: l)Fast minus-end directed movement (prometaphase), 2)Plus-end directed movement, and 3) Slow minus-end directed movement (anaphase) . In all cases these movements are mediated by the kinetochore region of the centromere of mitotic chromosomes. This region consists of three domains based on both immunocytochemistry and electron microscopy. The outermost or kinetochore domain is composed of the distal fibrous corona and trilamminar plate. The central and pairing domains are located in the chromatin beneath the kinetochore. Both plus- and minus-end directed microtubule motors have been localized to the kinetochore region of mitotic CHO chromosomes. I have used double-label immunocytochemistry to map the location of these motors within the centromere region at the level of the light microscope. Furthermore, I have cloned and expressed a number of novel kinesin-related motors, two of which (Clone 26 and Clone 14) are localized to kinetochores and kinetochore microtubules, respectively.

Isolation and characterization of a gene (CBF2) specifying a protein component of the budding yeast kinetochore.

We have cloned and determined the nucleotide sequence of the gene (CBF2) specifying the large (110 kD) subunit of the 240-kD multisubunit yeast centromere binding factor CBF3, which binds selectively in vitro to yeast centromere DNA and contains a minus end-directed microtubule motor activity. The deduced amino acid sequence of CBF2p shows no sequence homologies with known molecular motors, although a consensus nucleotide binding site is present. The CBF2 gene is essential for viability of yeast and is identical to NDC10, in which a conditional mutation leads to a defect in chromosome segregation (Goh, P.-Y., and J. V. Kilmartin, in this issue of The Journal of Cell Biology). The combined in vitro and in vivo evidence indicate that CBF2p is a key component of the budding yeast kinetochore.

The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor

The Drosophila ncd gene is required for chromosome segregation during female meiosis. Previous analyses suggested that the ncd gene encoded a protein with sequence simillarity to the kinesin motor domain, which suggested that, like kinesin, the ncd protein might be a plus end-directed microtubule motor. Here we describe the expression of ncd protein in E. coli and the initial characterization of the ncd protein's motor properties. The ncd protein is indeed a microtubule motor, but the polarity of movement is minus end directed. The ncd protein also has microtubule bundling activity. These findings limit possible models for the in vivo functions of the ncd protein and suggest that motor proteins with similar sequence can generate movement in opposite directions along a microtubule.

Identification and immunolocalization of cytoplasmic dynein in dictyostelium

A high molecular weight microtubule binding protein has been isolated from homogenates of Dictyostelium. Because of its sedimentation velocity (20s), ATP-sensitive binding to microtubules, UV-vanadate-ATP mediated fragmentation, prominent CTPase activity, and its ability to produce limited microtubule movement in vitro, we consider this protein to be a form of cytoplasmic dynein. A polyclonal antibody monospecific to this protein was produced, and dynein's intracellular distribution in ameboid cells was examined by immunofluorescence. The antibody labels a punctate cytoplasmic pattern, localizes to a spherical region adjacent to the nucleus, and also appears to label the nuclei. The punctate staining pattern is consistent with cytoplasmic dynein's proposed function in organelle transport. The spherical juxtanuclear object stained is coincident with this cell's microtubule organizing center, an obvious termination point for minus-end directed microtubule motors. By immunofluorescence, there does not appear to be a substantial amount of dynein in the intranuclear mitotic spindles of Dictyostelium. These data provide evidence for localization of cytoplasmic dynein in cells, and suggest that Dictyostelium will be a useful system in which to study the molecular biology of microtubule-associated motor enzymes.