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A broadly conserved membrane-associated protein required for the functional interaction of kinesin-I with axonal cargo was identified. Mutations in sunday driver (syd) and the axonal transport motor kinesin-I cause similar phenotypes in Drosophila, including aberrant accumulations of axonal cargoes. GFP-tagged mammalian SYD localizes to tubulovesicular structures that costain for kinesin-I and a marker of the secretory pathway. Coimmunoprecipitation analysis indicates that mouse SYD forms a complex with kinesin-I in vivo. Yeast two-hybrid analysis and in vitro interaction studies reveal that SYD directly binds kinesin-I via the tetratricopeptide repeat (TPR) domain of kinesin light chain (KLC) with K(d) congruent with 200 nM. We propose that SYD mediates the axonal transport of at least one class of vesicles by interacting directly with KLC.

To test whether kinesin-II is important for transport in the mammalian photoreceptor cilium, and to identify its potential cargoes, we used Cre-loxP mutagenesis to remove the kinesin-II subunit, KIF3A, specifically from photoreceptors. Complete loss of KIF3A caused large accumulations of opsin, arrestin, and membranes within the photoreceptor inner segment, while the localization of alpha-transducin was unaffected. Other membrane, organelle, and transport markers, as well as opsin processing appeared normal. Loss of KIF3A ultimately caused apoptotic photoreceptor cell death similar to a known opsin transport mutant. The data suggest that kinesin-II is required to transport opsin and arrestin from the inner to the outer segment and that blocks in this transport pathway lead to photoreceptor cell death as found in retinitis pigmentosa.

Mitosis requires dynamic attachment of chromosomes to spindle microtubules. This interaction is mediated largely by kinetochores. During prometaphase, forces exerted at kinetochores, in combination with polar ejection forces, drive congression of chromosomes to the metaphase plate. A major question has been whether kinetochore-associated microtubule motors play an important role in congression. Using immunodepletion from and antibody addition to Xenopus egg extracts, we show that the kinetochore-associated kinesin-like motor protein CENP-E is essential for positioning chromosomes at the metaphase plate. We further demonstrate that CENP-E powers movement toward microtubule plus ends in vitro. These findings support a model in which CENP-E functions in congression to tether kinetochores to dynamic microtubule plus ends.

The receptor tyrosine phosphatases DPTP69D and DPTP99A are expressed on motor axons in Drosophila embryos. In mutant embryos lacking DPTP69D protein, motor neuron growth cones stop growing before reaching their muscle targets, or follow incorrect pathways that bypass these muscles. Mutant embryos lacking DPTP99A are indistinguishable from wild type. Motor axon defects in dptp69D dptp99A double mutant embryos, however, are much more severe than in embryos lacking only DPTP69D. Our results demonstrate that DPTP69D and DPTP99A are required for motor axon guidance and that they have partially redundant functions during development of the neuro-muscular system.

The Drosophila no distributive disjunction (nod) gene encodes a kinesin-like protein that has been proposed to push chromosomes toward the metaphase plate during female meiosis. We report that the nonmotor domain of the nod protein can mediate direct binding to DNA. Using an antiserum prepared against bacterially expressed nod protein, we show that during prometaphase nod protein is localized on oocyte chromosomes and is not restricted to either specific chromosomal regions or to the kinetochore. Thus, motor-based chromosome-microtubule interactions are not limited to the centromere, but extend along the chromosome arms, providing a molecular explanation for the polar ejection force.

The in vivo function of the microtubule motor protein kinesin was examined in Drosophila using genetics and immunolocalization. Kinesin heavy chain mutations (khc) cause abnormal behavior and lethality. Mutant larvae exhibit loss of mobility and tactile responsiveness in the most posterior segments, followed by general paralysis and death during larval or pupal development. Adults homozygous for a temperature-sensitive allele also exhibit a loss in mobility and sensory responses. The data indicate that kinesin function is essential and suggest that kinesin has an important role in the neuromuscular system, perhaps as a motor for axonal transport. The possibility of more general cellular functions remains open, but observation of embryogenesis and morphogenesis in khc mutants suggests that mitosis and the cell cycle can proceed in spite of impaired kinesin function. Immunolocalization suggests that kinesin may have some general cellular functions but that it is not a major component of mitotic spindles.

The Drosophila ncd gene is required for chromosome segregation during female meiosis. Previous analyses suggested that the ncd gene encoded a protein with sequence similarity 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.

The nod gene is required for the distributive segregation of nonexchange chromosomes during meiosis in D. melanogaster. Loss-of-function nod mutations cause nondisjunction and loss of nonrecombinant chromosomes both at meiosis I and during subsequent mitotic divisions. We have cloned the nod locus, examined its expression patterns, and determined its coding sequence. In adults the nod transcript is only present in females, consistent with the observation that males do not use the distributive segregation system. However, the nod locus is also transcribed in the embryonic, larval, and pupal stages of development, and possibly in all dividing cells. Finally, the N-terminal domain of the predicted nod protein has amino acid similarity to the mechanochemical domain of kinesin heavy chain; however, the C-terminal domain is unlike that of kinesin heavy chain or of any previously reported protein. Thus, the nod protein is a member of the kinesin superfamily and may be a microtubule motor.

We identified and sequenced a cDNA clone encoding a kinesin-like protein from Drosophila. The predicted product of this cDNA has a carboxy-terminal domain that is substantially similar to the motor domain of kinesin heavy chain. The amino-terminal domain is unlike that found in previously identified kinesins or kinesin-like proteins. Analyses of this new sequence suggest that the maximal motor unit in the kinesin superfamily may be as little as 350 amino acids, and that the existence of both kinesin and kinesin-like molecules must be an evolutionarily ancient feature of eukaryotes. We also tested some of the biochemical properties of the protein encoded by this cDNA and found them to be similar to those of kinesin. Finally, the clone we isolated appears to correspond to the non-claret disjunctional (ncd) gene, which when mutant causes defects in meiotic and early embryonic mitotic chromosome segregation, and whose recently determined sequence predicts a kinesin-like domain.

The structure and function of kinesin heavy chain from D. melanogaster have been studied using DNA sequence analysis and analysis of the properties of truncated kinesin heavy chain synthesized in vitro. Analysis of the sequence suggests the existence of a 50 kd globular amino-terminal domain that contains an ATP binding consensus sequence, followed by another 50-60 kd domain that has sequence characteristics consistent with the ability to fold into an alpha helical coiled coil. The properties of amino- and carboxy-terminally truncated kinesin heavy chains synthesized in vitro reveal that a 60 kd amino-terminal fragment has the nucleotide-dependent microtubule binding activities of the intact kinesin heavy chain, and hence is likely to be a "motor" domain. Finally, the sequence data indicate the presence of a small carboxy-terminal domain. Because it is located at the end of the molecule away from the putative "motor" domain, we propose that this domain is responsible for interactions with other proteins, vesicles, or organelles. These data suggest that kinesin has an organization very similar to that of myosin even though there are no obvious sequence similarities between the two molecules.

An electron microscopic investigation of kinetochore structure during the first meiotic division in male Drosophila melanogaster is presented. The data suggest that the structure that is responsible for initial microtubule attachment and chromosome orientation is a single, bilaminar hemisphere on each half-bivalent. Following the initial attachment this structure undergoes morphogenesis to a double-disc structure that reflects the underlying duality of sister chromatids in the half-bivalent. Thus these data support Darlington's idea that sister chromatids disjoin to the same spindle pole because they share a single kinetochore. Additionally, these data suggest that the meiotic mutations ord and mel-S332 sometimes cause premature "doubling" of the kinetochore region though, as discussed, possibly for a trivial reason.