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S A Endow

Publications and source records attributed to S A Endow.

At least 37 records · Page 2Linked to original sources
PubMed1996

Binding sites on microtubules of kinesin motors of the same or opposite polarity.

The kinesin motor proteins translocate toward either the plus or minus end of microtubules (MTs). Competitive microtubule binding assays were carried out with monomeric motor domains of the minus-end-directed nonclaret disjunctional (Ncd) and Kar3 and the plus-end-directed kinesin heavy chain (KHC) to determine whether motors of the same or opposite polarity compete for binding sites on MTs and to test the idea that motor polarity is determined by differences in binding sites on MTs of the motors. The stoichiometries of binding were approximately 1 motor:1 tubulin heterodimer for all three motors. Ncd and Kar3, both minus-end motors, severely inhibited the binding of one another to MTs, as predicted theoretically for binding of the two motors to the same site on MTs, indicating that the binding sites on MTs of Ncd and Kar3 are the same or overlap extensively. Motors of opposite polarity, KHC and Ncd or KHC and Kar3, showed partial or complete inhibition of binding to MTs under different experimental protocols. The differences in binding behavior could be due to experimental conditions or be inherent in the nature of motor binding to MTs. Alternatively, differences in KHC and Ncd or Kar3 binding sites on MTs may exist such that the motors bind to partially overlapping but nonidentical sites on MTs. These differences in binding sites may be related to the opposite polarity of translocation on MTs of the motors.

Animals↗
PubMed1996

A point mutation in the microtubule binding region of the Ncd motor protein reduces motor velocity.

Non-claret disjunctional (Ncd) is a kinesin-related microtubule motor protein in Drosophila that functions in meiotic spindle assembly in oocytes and spindle pole maintenance in early embryos. The partial loss-of-function mutant ncdD retains mitotic, but not meiotic, function. The predicted NcdD mutant protein contains a V556-->F mutation in the putative microtubule binding region of the Ncd motor domain. Here we report an analysis of the properties of recombinant Ncd and NcdD proteins. A GST-NcdD fusion protein translocated microtubules approximately 10-fold more slowly than the corresponding wild-type protein in gliding assays. The maximum microtubule-stimulated ATPase activity of an NcdD motor domain protein was reduced approximately 3-fold and an approximately 3-fold greater concentration of microtubules was required for half-maximal stimulation of ATPase activity, compared with the corresponding wild-type protein. The Km for ATP and basal rate of ATP turnover were, in contrast, similar for the NcdD mutant and wild-type Ncd motor domain proteins. Pelleting assays demonstrated that the binding of the mutant NcdD motor protein to microtubules was reduced in the absence of nucleotide, relative to wild-type. The reduced velocity of NcdD translocation on microtubules is therefore correlated with reductions in microtubule-stimulated ATPase activity and affinity of the mutant motor for microtubules. The characteristics of the NcdD motor explain its meiotic loss of function, and are consistent with partial motor activity of Ncd being sufficient for its mitotic, but not its meiotic, role.

Adenosine Triphosphatases↗
PubMed1996

Kinesin proteins: a phylum of motors for microtubule-based motility.

The cellular processes of transport, division and, possibly, early development all involve microtubule-based motors. Recent work shows that, unexpectedly, many of these cellular functions are carried out by different types of kinesin and kinesin-related motor proteins. The kinesin proteins are a large and rapidly growing family of microtubule-motor proteins that share a 340-amino-acid motor domain. Phylogenetic analysis of the conserved motor domains groups the kinesin proteins into a number of subfamilies, the members of which exhibit a common molecular organization and related functions. The kinesin proteins that belong to different subfamilies differ in their rates and polarity of movement along microtubules, and probably in the particles/organelles that they transport. The kinesins arose early in eukaryotic evolution and gene duplication has allowed functional specialization to occur, resulting in a surprisingly large number of different classes of these proteins adapted for intracellular transport of vesicles and organelles, and for assembly and force generation in the meiotic and mitotic spindles.

Animals↗
PubMed1996

Centrosome and spindle function of the Drosophila Ncd microtubule motor visualized in live embryos using Ncd-GFP fusion proteins.

The Ncd microtubule motor protein is required for meiotic and early mitotic chromosome distribution in Drosophila. Null mutant females expressing the Ncd motor fused to the Aequorea victoria green fluorescent protein (GFP), regulated by the wild-type ncd promoter, are rescued for chromosome segregation and embryo viability. Analysis of mitosis in live embryos shows cell cycle-dependent localization of Ncd-GFP to centrosomes and spindles. The distribution of Ncd-GFP in spindles during metaphase differs strikingly from that of tubulin: the tubulin staining is excluded by the chromosomes at the metaphase plate; in contrast, Ncd-GFP forms filaments along the spindle microtubules that extend across the chromosomes. The existence of Ncd-GFP fibers that cross the metaphase plate suggests that Ncd interacts functionally with chromosomes in metaphase. Differences are no longer observed in anaphase when the chromosomes have moved off the metaphase plate. A mutant form of Ncd fused to GFP also localizes to spindles in live embryos. Mutant embryos show frequent centrosome and spindle abnormalities, including free centrosomes that dissociate from interphase nuclei, precociously split centrosomes, and spindles with microtubule spurs or bridges to nearby spindles. The precociously split and free centrosomes indicate that the Ncd motor acts in cleavage stage embryos to maintain centrosome integrity and attachment to nuclei. The frequent spindle spurs of mutant embryos are associated with mis-segregating chromosomes that partially detach from the spindle in metaphase, but can be recaptured in early anaphase. This implies that the Ncd motor functions to prevent chromosome loss by maintaining chromosome attachment to the spindle in metaphase, consistent with the Ncd-GFP fibers that across the metaphase plate.

Adenosine Triphosphatases↗
PubMed1995

Haploidy and androgenesis in Drosophila.

Adrogenesis, development from paternal but not maternal chromosomes, can be induced to occur in some organisms, including vertebrates, but has only been reported to occur naturally in interspecific hybrids of the Sicilian stick insect. Androgenesis has not been described previously in Drosophila. We now report the recovery of androgenetic offspring from Drosophila melanogaster females mutant for a gene that affects an oocyte- and embryo-specific alpha-tubulin. The androgenetic exceptions are X,X diploid females that develop from haploid embryos and express paternal markers on all 4 chromosomes. The exceptional females arise by fusion of haploid cleavage nuclei or failure of newly replicated haploid chromosomes to segregate, rather than fusion of two inseminating sperm. The frequency of androgenetic offspring is greatly enhanced by a partial loss-of-function mutant of the NCD (nonclaret disjunctional) microtubule motor protein, suggesting that wild-type NCD functions is pronuclear fusion. Diploidization of haploid paternal chromosome complements results in complete genetic homozygosity, which could facilitate studies of gene variation and mutational load in populations.

Animals↗
PubMed1995

Determinants of motor polarity in the kinesin proteins.

Many of the proteins that are members of the kinesin family of microtubule motor proteins are plus-end motors; however, a few of the kinesin proteins have now been found to be minus-end microtubule motors. Overall structural features of the proteins can be used to identify further kinesins that are likely to be minus-end motors. Structural or biochemical differences that may serve as the basis of the "reversed" polarity of a unique subset of the kinesin proteins are discussed.

Adenosine Triphosphate↗
PubMed1995

Motor proteins 1: kinesins.

Progress regarding the kinesins is now being made at a rapid and accelerating rate. The in vivo-functions, and biophysical and enzymatic properties of kinesin itself are being explored at ever increasing levels of detail. The kinesin-related proteins now number several dozen, and although more is known about primary structure than function for most of the proteins, this trend is already reversing. For example, knowledge about the kinesin-related protein, ncd, is expanding rapidly, and more is already known about its three-dimensional structure than is known for kinesin heavy chain. This volume presents a comprehensive review of the major published works on kinesin and kinesin-related proteins. Hopefully, this manuscript will complement other recent review articles [17, 20, 25, 37, 60-62, 67, 69, 75, 85-88, 231, 233, 238, 244, 269-271, 281, 282, 292] or books [49, 227, 293] that have focused on more selective aspects of the kinesin family, or have been aimed more generally at MT motor proteins. In line with the stated purpose of the Protein Profile series, annual updates of the review on the kinesins are planned for at least the next few years.

Animals↗
PubMed1994

Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules preferentially at the minus ends.

Mutants of the yeast Kar3 protein are defective in nuclear fusion, or karyogamy, during mating and show slow mitotic growth, indicating a requirement for the protein both during mating and in mitosis. DNA sequence analysis predicts that Kar3 is a microtubule motor protein related to kinesin, but with the motor domain at the C-terminus of the protein rather than the N-terminus as in kinesin heavy chain. We have expressed Kar3 as a fusion protein with glutathione S-transferase (GST) and determined the in vitro motility properties of the bacterially expressed protein. The GST-Kar3 fusion protein bound to a coverslip translocates microtubules in gliding assays with a velocity of 1-2 microns/min and moves towards microtubule minus ends, unlike kinesin but like kinesin-related Drosophila ncd. Taxol-stabilized microtubules bound to GST-Kar3 on a coverslip shorten as they glide, resulting in faster lagging end, than leading end, velocities. Comparison of lagging and leading end velocities with velocities of asymmetrical axoneme-microtubule complexes indicates that microtubules shorten preferentially from the lagging or minus ends. The minus end-directed translocation and microtubule bundling of GST-Kar3 is consistent with models in which the Kar3 protein crosslinks internuclear microtubules and mediates nuclear fusion by moving towards microtubule minus ends, pulling the two nuclei together. In mitotic cells, the minus end motility of Kar3 could move chromosomes polewards, either by attaching to kinetochores and moving them polewards along microtubules, or by attaching to kinetochore microtubules and pulling them polewards along other polar microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

Mutants of the Drosophila ncd microtubule motor protein cause centrosomal and spindle pole defects in mitosis.

Nonclaret disjunctional (ncd) is a kinesin-related microtubule motor protein required for meiotic and early mitotic chromosome distribution in Drosophila. ncd translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends, and is associated with spindles in chromosome/spindle preparations. Here we report a new mutant of ncd caused by partial deletion of the predicted coiled-coil central stalk. The mutant protein exhibits a velocity of translocation and ability to generate torque in motility assays comparable to near full-length ncd, but only partially rescues a null mutant for chromosome mis-segregation. Antibody staining experiments show that the partial loss-of-function and null mutants cause centrosomal and spindle pole defects, including centrosome splitting and loss of centrosomes from spindle poles, and localize ncd to centrosomes as well as spindles of wild-type embryos. Association of ncd with spindles and centrosomes is microtubule- and cell cycle-dependent: inhibition of microtubule assembly with colchicine abolishes ncd staining and centrosomal staining is observed in prometaphase, metaphase and anaphase, but diminishes in late anaphase/telophase. The cell cycle dependence of centrosomal staining and the defects of mutants provide clear evidence for activity of the ncd motor protein near or at the spindle poles in mitosis. The ncd motor may interact with centrosomal microtubules and spindle fibers to attach centrosomes to spindle poles, and mediate poleward translocation (flux) of kinetochore fibers, a process that may underlie poleward movement of chromosomes in mitosis. Together with previous work, our findings indicate that ncd is important in maintaining spindle poles in mitosis as well as in meiosis.

Animals↗
PubMed1994

Molecular phylogeny of the kinesin family of microtubule motor proteins.

The rapidly expanding kinesin family of microtubule motor proteins includes proteins that are involved in diverse microtubule-based functions in the cell. Phylogenetic analysis of the motor regions of the kinesin proteins reveals at least five clearly defined groups that are likely to identify kinesins with different roles in basic cellular processes. Two of the groups are consistent with overall sequence similarity, while two groups contain proteins that are related in overall structure or function but show no significant sequence similarity outside the motor domain. One of these groups consists only of kinesin proteins with predicted C-terminal motor domains; another includes only kinesins required for mitotic spindle bipolarity. Drosophila Nod, presently an ungrouped protein, may represent a class of kinesins that, like the myosin I proteins, function as monomers. The analysis indicates that many types of kinesin proteins exist in eukaryotic organisms. At least two of the five groups identified in this analysis are expected to be present in most, or all, eukaryotes.

Animals↗
PubMed1993

Structural and functional domains of the Drosophila ncd microtubule motor protein.

Nonclaret disjunctional (ncd) is a kinesin-related microtubule motor protein that is required for proper chromosome distribution in Drosophila. Despite its sequence similarity to kinesin heavy chain, ncd translocates with the opposite polarity as kinesin, toward microtubule minus ends. We have expressed different regions of the protein in bacteria and analyzed the proteins for function. Results indicate that ncd consists of three domains: a basic, proline-rich N-terminal "tail," a central alpha-helical coiled-coil stalk, and a C-terminal motor domain. The ncd N terminus proteins bundle microtubules in motility assays and show ATP-independent binding to microtubules in solution. Truncated proteins, lacking the tail but containing the predicted motor domain and differing lengths of the stalk, did not support microtubule gliding in in vitro assays but showed microtubule-stimulated MgATPase activity in solution. Addition of a nonspecific N terminus to two of the truncated proteins restored directional gliding and rotation of microtubules in motility assays, demonstrating that these properties map to the predicted mechanochemical domain of ncd. Physical properties of the C terminus proteins indicate that the stalk region is important for dimerization and that the ncd protein probably exists and functions as a dimer.

Adenosine Triphosphatases↗
PubMed1993

Meiosis, mitosis and microtubule motors.

A framework for understanding the complex movements of mitosis and meiosis has been provided by the recent discovery of microtubule motor proteins, required for the proper distribution of chromosomes or the structural integrity of the mitotic or meiotic spindle. Although overall features of mitosis and meiosis are often assumed to be similar in mechanism, it is now clear that they differ in several important aspects. These include spindle structure and assembly, and timing of chromosome segregation to opposite poles. Here we review progress in the functional characterization of several newly identified microtubule motor proteins, emphasizing their possible roles in spindle structure and function.

Animals↗
PubMed1993

Chromosome distribution, molecular motors and the claret protein.

Recently, proteins have been identified that are required for proper distribution of chromosomes in meiosis and mitosis. Unexpectedly, several of these are microtubule motor proteins. This finding has prompted further investigation into the basis of meiotic and mitotic chromosome movement. The claret protein, or ncd, is one of the new motor proteins that may perform several functions in meiosis and early mitosis in Drosophila.

Animals↗
PubMed1993

Constitutive magnification by the Ybb- chromosome of Drosophila melanogaster.

Ybb- is an rDNA-deficient chromosome of Drosophila that has often been used in magnification experiments to induce high-frequency reversion of bobbed (bb) chromosomes. We observed previously that Ybb- causes ring chromosome loss even when the rings are bb+, suggesting that Ybb- induces magnifying sister chromatid exchanges in bb+ rings. Here we show that the Ybb- chromosome causes low levels of bb magnification in bb+ flies. We refer to the ability of Ybb- to bypass the rDNA deficiency requirement for magnification as 'constitutive' magnification. We have magnified the ribosomal genes on the Ybb- chromosome and analysed the revertant chromosomes using genetic and molecular methods. We find that magnified Ybb- chromosomes also exhibit constitutive magnifier activity. Molecular analysis shows that both type 1 and type 2 intron+ ribosomal gene repeats are associated with magnified Ybb- chromosomes. Type 2 introns have been described previously in the rDNA of both X and Y chromosomes. However, type 1 intervening sequences are thought to be present only in X, but not Y, ribosomal genes. Some of the Ybb- type 1 insertions differ from those present in the rDNA of X chromosomes in that they contain an EcoRI site, and some may be present in tandem arrays. The constitutive magnifier activity of Ybb- may reside either in the structurally unusual ribosomal gene intervening sequences associated with the chromosome, or in the locus on YL that is required for magnification to occur.

Animals↗