Sharyn A. Endow

A standardized kinesin nomenclature

In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered.

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)

A new kinesin tree

Dagenbach, E. M. and Endow, S. A. (2004). A new kinesin tree. J. Cell Sci. 117 , [3-7][1]. The neck analysis entries in Table 1 were not aligned correctly in both the print and online versions of this paper. The corrected [Table 1][2] is shown below. We apologise for any inconvenience caused.

A structural pathway for activation of the kinesin motor ATPase.

Molecular motors move along actin or microtubules by rapidly hydrolyzing ATP and undergoing changes in filament‐binding affinity with steps of the nucleotide hydrolysis cycle. It is generally accepted that motor binding to its filament greatly increases the rate of ATP hydrolysis, but the structural changes in the motor associated with ATPase activation are not known. To identify the conformational changes underlying motor movement on its filament, we solved the crystal structures of three kinesin mutants that decouple nucleotide and microtubule binding by the motor, and block microtubule‐activated, but not basal, ATPase activity. Conformational changes in the structures include a disordered loop and helices in the switch I region and a visible switch II loop, which is disordered in wild‐type structures. Switch I moved closer to the bound nucleotide in two mutant structures, perturbing water‐mediated interactions with the Mg2+. This could weaken Mg2+ binding and accelerate ADP release to activate the motor ATPase. The structural changes we observe define a signaling pathway within the motor for ATPase activation that is likely to be essential for motor movement on microtubules.

Differential replication of ribosomal gene repeats in polytene nuclei of drosophila

The genes coding for the 18S and 28S rRNAs in D. melanogaster were examined using Southern transfers of DNA from diploid or polytene tissue. A ribosomal gene repeat 12 kb in length is present in DNA from diploid tissue of males and is the major repeat on the Y chromosome. This repeat is present in low amounts on the X chromosome, which contains major repeats of 17 and 11.5 kb. In polytene nuclei of males, the 12 kb band is disproportionately replicated, and only a very low amount of the 11.5 kb repeat and no 17 kb repeat are detected. Polytene nuclei of females contain reduced amounts of the 17 kb repeat relative to the 11.5 kb repeat. This disproportionate replication of specific ribosomal gene repeats suggests that polytenization of the rDNA may involve an extrachromosomal mechanism. Evidence that genes from only one nucleolus organizer are replicated during polytenization in X/Y and X/X flies is discussed. A method for analyzing DNA from tissue of individual larvae was developed to test for population heterogeneity in ribosomal gene structure. Heterogeneity was observed in the ribosomal genes of three Ore R lines, four other D. melanogaster strains and between males and females of the same strain.

Determinants of molecular motor directionality

Work over the past two years has led to a breakthrough in our understanding of the molecular basis of the directionality of the kinesin motor proteins. This breakthrough has come first from the reversal of directionality of the kinesin-related motor Ncd, followed closely by the reversal of kinesin’s directionality and the finding that the Ncd ‘neck’ can convert Ncd or kinesin, which are intrinsically plus-end-directed microtubule motors, into a minus-end motor. These findings raise several outstanding questions, foremost, how does the neck function in motor directionality?

Rotation of the stalk/neck and one head in a new crystal structure of the kinesin motor protein, Ncd

Molecular motors undergo conformational changes to produce force and move along cytoskeletal filaments. Structural changes have been detected in kinesin motors; however, further changes are expected because previous crystal structures are in the same or closely related conformations. We report here a 2.5 Å crystal structure of the minus‐end kinesin, Ncd, with the coiled‐coil stalk/neck and one head rotated by ∼75° relative to the other head. The two heads are asymmetrically positioned with respect to the stalk and show asymmetry of nucleotide state: one head is fully occupied, but the other is unstably bound to ADP. Unlike previous structures, our new atomic model can be fit into cryoelectron microscopy density maps of the motor attached to microtubules, where it appears to resemble a one‐head‐bound motor with the stalk rotated towards the minus end. Interactions between neck and motor core residues, observed in the head that moves with the stalk, are disrupted in the other head, permitting rotation of the stalk/neck. The rotation could represent a force‐producing stroke that directs the motor to the minus end.

Differential replication of satellite DNA in polyploid tissues of Drosophila virilis

Satellite DNA amounts were examined in adult tissues of Drosophila virilis, a species whose DNA contains three prominent satellites. Satellite amounts in DNA from six of the seven tissues were lower than in DNA from diploid (adult brain) tissue. Satellite amounts in adult ovary DNA, however, were equivalent to or greater than diploid levels. When DNA from pupal ovaries was examined, a 30% increase in satellite amounts over diploid levels was found. An RNA-DNA hybridization experiment showed that the ribosomal RNA genes in pupal ovary DNA were under-replicated relative to diploid DNA levels.

Satellite DNA sequences of Drosophila melanogaster

Abstract Three of the four major satellite DNAs of Drosophila melanogaster were isolated by centrifugation in Ag + Cs 2 SO 4 gradients. The satellites were characterized by buoyant density analysis in neutral and alkaline CsCl and by thermal melt analysis, and were judged to be pure by these criteria. Complementary RNA was synthesized using Escherichia coli RNA polymerase, [α-32P]NTPs, and the separated satellite DNA strands as template. The complementary RNA was analyzed by RNA sequencing techniques. The sequences determined for the satellites are simple in composition and are related to one another. Satellite I is composed of A-A-T and A-T in the molar ratio of 2A-A-T:3A-T. Satellite II consists of the ten-nucleotide repeating sequence: A-A-T-A-A-C-A-T-A-G. Satellite IV is composed of A-A-G, A-G and G in the molar ratios of 3A-A-G: 4A-G: IG. One sequence present in long regions of satellite IV is A-A-G A-G. The sequences of the three satellites may be represented by the general expression (AAN)m(AN)n. The similarity of the sequences provides evidence for theories concerning the origin of families of satellite sequences.

Assembly pathway of the anastral Drosophila oocyte meiosis I spindle

Oocyte meiotic spindles of many species are anastral and lack centrosomes to nucleate microtubules. Assembly of anastral spindles occurs by a pathway that differs from that of most mitotic spindles. Here we analyze assembly of the Drosophila oocyte meiosis I spindle and the role of the Nonclaret disjunctional (Ncd) motor in spindle assembly using wild-type and mutant Ncd fused to GFP. Unexpectedly, we observe motor-associated asters at germinal vesicle breakdown that migrate towards the condensed chromosomes, where they nucleate microtubules at the chromosomes. Newly nucleated microtubules are randomly oriented, then become organized around the bivalent chromosomes. We show that the meiotic spindle forms by lateral associations of microtubule-coated chromosomes into a bipolar spindle. Lateral interactions between microtubule-associated bivalent chromosomes may be mediated by microtubule crosslinking by the Ncd motor, based on analysis of fixed oocytes. We report here that spindle assembly occurs in an ncd mutant defective for microtubule motility, but lateral interactions between microtubule-coated chromosomes are unstable, indicating that Ncd movement along microtubules is needed to stabilize interactions between chromosomes. A more severe ncd mutant that probably lacks ATPase activity prevents formation of lateral interactions between chromosomes and causes defective microtubule elongation. Anastral Drosophila oocyte meiosis I spindle assembly thus involves motor-associated asters to nucleate microtubules and Ncd motor activity to form and stabilize interactions between microtubule-associated chromosomes during the assembly process. This is the first complete account of assembly of an anastral spindle and the specific steps that require Ncd motor activity, revealing new and unexpected features of the process.