Direction determination in the minus-end-directed kinesin motor ncd

Motor proteins of the kinesin superfamily transport intracellular cargo along microtubules. Although different kinesin proteins share 30-50% amino-acid identity in their motor catalytic cores, some move to the plus end of microtubules whereas others travel in the opposite direction. Crystal structures of the catalytic cores of conventional kinesin (a plus-end-directed motor involved in organelle transport) and ncd (a minus-end-directed motor involved in chromosome segregation) are nearly identical; therefore, the structural basis for their opposite directions of movement is unknown. Here we show that the ncd 'neck' made up of 13 class-specific residues next to the superfamily-conserved catalytic core, is essential for minus-end-directed motility, as mutagenesis of these neck residues reverses the direction of ncd motion. By solving the 2.5 A structure of a functional ncd dimer, we show that the ncd neck (a coiled-coil) differs from the corresponding region in the kinesin neck (an interrupted beta-strand), although both necks interact with similar elements in the catalytic cores. The distinct neck architectures also confer different symmetries to the ncd and kinesin dimers and position these motors with appropriate directional bias on the microtubule.     

The directional preference of kinesin motors is specified by an element outside of the motor catalytic domain

Members of the kinesin superfamily share a similar motor catalytic domain yet move either toward the plus end (e.g., conventional kinesin) or the minus end (e.g., Ncd) of microtubules. The structural features that determine the polarity of movement have remained enigmatic. Here, we show that kinesin's catalytic domain (316 residues) in a dimeric construct (560 residues) can be replaced with the catalytic domain of Ncd and that the resultant motor moves in the kinesin direction. We also demonstrate that this chimera does not move processively over many tubulin subunits, which is similar to Ncd but differs from the highly processive motion of conventional kinesin. These findings reveal that the catalytic domain contributes to motor processivity but does not control the polarity of movement. We propose that a region adjacent to the catalytic domain serves as a mechanical transducer that determines directionality.     

Phosphotransferases associated with the regulation of kinesin motor activity

Kinesin, a plus-end-directed microtubule motor protein, functions in concert with accessory factors that have been shown to regulate enzyme activity and may also provide cargo specificity. This report identifies teh 79-kDa kinesin-associated phosphoprotein as a phosphoisoform of kinesin light chain. Increased phosphorylation of this light chain isoform is sufficient to account for the increase in kinesin-mediated microtubule-gliding activity. Additionally, it was found that the degree of phosphorylation of this isoform is regulated by a 100-kDa kinase and 150-kDa type 1 phosphatase. Both the kinesin light chain kinase and phosphatase co-purify with the kinesin heavy chain, suggesting that kinesin exists in a large complex capable of self-regulation.     

Identification and classification of 16 new kinesin superfamily (KIF) proteins in mouse genome

KIF (kinesin superfamily) proteins are microtubule-dependent molecular motors that play important roles in intracellular transport and cell division. The extent to which KIFs are involved in various transporting phenomena, as well as their regulation mechanism, are unknown. The identification of 16 new KIFs in this report doubles the existing number of KIFs known in the mouse. Conserved nucleotide sequences in the motor domain were amplified by PCR using cDNAs of mouse nervous tissue, kidney, and small intestine as templates. The new KIFs were studied with respect to their expression patterns in different tissues, chromosomal location, and molecular evolution. Our results suggest that (i) there is no apparent tendency among related subclasses of KIFs of cosegregation in chromosomal mapping, and (ii) according to their tissue distribution patterns, KIFs can be divided into two classes-i.e., ubiquitous and specific tissue-dominant. Further characterization of KIFs may elucidate unknown fundamental phenomena underlying intracellular transport. Finally, we propose a straightforward nomenclature system for the members of the mouse kinesin superfamily.     

Processivity of the motor protein kinesin requires two heads

A single kinesin molecule can move for hundreds of steps along a microtubule without dissociating. One hypothesis to account for this processive movement is that the binding of kinesin's two heads is coordinated so that at least one head is always bound to the microtubule. To test this hypothesis, the motility of a full-length single-headed kinesin heterodimer was examined in the in vitro microtubule gliding assay. As the surface density of single-headed kinesin was lowered, there was a steep fall both in the rate at which microtubules landed and moved over the surface, and in the distance that microtubules moved, indicating that individual single-headed kinesin motors are not processive and that some four to six single-headed kinesin molecules are necessary and sufficient to move a microtubule continuously. At high ATP concentration, individual single-headed kinesin molecules detached from microtubules very slowly (at a rate less than one per second), 100-fold slower than the detachment during two-headed motility. This slow detachment directly supports a coordinated, hand-over-hand model in which the rapid detachment of one head in the dimer is contingent on the binding of the second head.     

KIF2beta, a new kinesin superfamily protein in non-neuronal cells, is associated with lysosomes and may be implicated in their centrifugal translocation

Lysosomes concentrate juxtanuclearly in the region around the microtubule-organizing center by interaction with microtubules. Different experimental and physiological conditions can induce these organelles to move to the cell periphery by a mechanism implying a plus-end-directed microtubule-motor protein (a kinesin-like motor). The responsible kinesin-superfamily protein, however, is unknown. We have identified a new mouse isoform of the kinesin superfamily, KIF2beta, an alternatively spliced isoform of the known, neuronal kinesin, KIF2. Developmental expression pattern and cell-type analysis in vivo and in vitro reveal that KIF2beta is abundant at early developmental stages of the hippocampus but is then downregulated in differentiated neuronal cells, and it is mainly or uniquely expressed in non-neuronal cells while KIF2 remains exclusively neuronal. Electron microscopy of mouse fibroblasts and immunofluorescence of KIF2beta-transiently-transfected fibroblasts show KIF2 and KIF2beta primarily associated with lysosomes, and this association can be disrupted by detergent treatment. In KIF2beta-overexpressing cells, lysosomes (labeled with anti-lysosome-associated membrane protein-1) become abnormally large and peripherally located at some distance from their usual perinuclear positions. Overexpression of KIF2 or KIF2beta does not change the size or distribution of early, late and recycling endosomes nor does overexpression of different kinesin superfamily proteins result in changes in lysosome size or positioning. These results implicate KIF2beta as a motor responsible for the peripheral translocation of lysosomes.     

Crystal structure of the kinesin motor domain reveals a structural similarity to myosin

Kinesin is the founding member of a superfamily of microtubule based motor proteins that perform force-generating tasks such as organelle transport and chromosome segregation. It has two identical approximately 960-amino-acid chains containing an amino-terminal globular motor domain, a central alpha-helical region that enables dimer formation through a coiled-coil, and a carboxy-terminal tail domain that binds light chains and possibly an organelle receptor. The kinesin motor domain of approximately 340 amino acids, which can produce movement in vitro, is much smaller than that of myosin (approximately 850 amino acids) and dynein (1,000 amino acids), and is the smallest known molecular motor. Here, we report the crystal structure of the human kinesin motor domain with bound ADP determined to 1.8-A resolution by X-ray crystallography. The motor consists primarily of a single alpha/beta arrowhead-shaped domain with dimensions of 70 x 45 x 45 A. Unexpectedly, it has a striking structural similarity to the core of the catalytic domain of the actin-based motor myosin. Although kinesin and myosin have virtually no amino-acid sequence++ identity, and exhibit distinct enzymatic and motile properties, our results suggest that these two classes of mechanochemical enzymes evolved from a common ancestor and share a similar force-generating strategy.     

Nucleotide-dependent movements of the kinesin motor domain predicted by simulated annealing

A frailty model for multivariate correlated life times is considered. The model both extends, in a rather straight-forward way, ordinary survival analysis with its emphasis on hazard modeling and incorporates well-known variance components models to account for the dependence between events of related individuals. Different approaches to estimation and inference are considered. An example from an ongoing study of genetic and environmental influences on premature death in adults serves to motivate and illustrate the model. Multivariate frailty models offer a conceptually simple and promising framework for analysis of correlated event times data, even if current knowledge is too sparse for such models to be tested critically.     

vab-8 is a key regulator of posteriorly directed migrations in C. elegans and encodes a novel protein with kinesin motor similarity

Nervous system assembly requires the directed migrations of cells and axon growth cones along the dorsoventral and anteroposterior axes. Although guidance mechanisms for dorsoventral migrations are conserved from nematodes to mammals, mechanisms for anteroposterior migrations are unknown. In C. elegans, the gene vab-8, which specifically functions in posteriorly directed migrations, encodes two isoforms of a novel intracellular protein that act cell-autonomously in different migrations. VAB-8L, which contains a domain similar to kinesin-like motors, functions in all vab-8-dependent axon growth cone migrations. VAB-8S, which lacks this N-terminal domain, functions in a subset of vab-8-dependent cell migrations. Continuous expression of VAB-8L in the ALM mechanosensory neuron, which normally requires vab-8 early in its development for posteriorly directed cell migration, redirects its anteriorly projecting axon posteriorly. We propose that regulation of vab-8 activity is a mechanism for controlling the direction of cell and axon growth cone migrations.     

Delta-1 is a regulator of neurogenesis in the vertebrate retina

Controversy exists about the effect of tissue edema on cerebral microcirculation. High spatial resolution is required for observation of extravasation and microcirculation during focal vasogenic edema formation. To study the relationship between tissue edema and perfusion, we developed a technique for simultaneous visualization of extravasation and microvessel perfusion in rats. Focal intracortical microvascular injury was generated with a 1-sec Nd-YAG laser pulse. Evans blue albumin (EBA) was infused 30 min before decapitation to study extravasation and FITC-dextran was injected 30 sec prior to decapitation to examine microvessel perfusion. Computerized scanning laser-excited fluorescence microscopy followed by high resolution image analysis permitted quantitative assessment of both parameters on single fresh-frozen brain sections. Studied at 30 min (3.66 +/- 0.15 mm), 2 hr (4.14 +/- 0.08 mm, P < .05), and 8 hr (4.69 +/- 0.18 mm, P < .01) after injury, the diameter of the circular, sharply demarcated zone of EBA-extravasation increased progressively. At 30 min, microvessels at a zone surrounding the area of EBA-extravasation contained 69 +/- 14% (P < .05) more fluorescent FITC-filling than in the control hemisphere, but the density of perfused microvessels was unchanged. At 2 hr, secondary tissue changes had already occurred in a zone surrounding the initial laser lesion. While severe reduction in the density (-76 +/- 13%, P < .05) of perfused microvessels was observed within 400 to 240 microm inside the border of EBA extravasation, perfusion indexes were normal despite the presence of extravasated plasma constituents within 0-80 microm from the border. In a narrow zone (80 microm) outside the border of extravasation, individual microvessels contained 34 +/- 9% (P < .01) less FITC-fluorescence than those in a homologous area of the uninjured contralateral hemisphere. This report demonstrates the feasibility of simultaneous measurement and high-resolution mapping of indices of microvascular perfusion (density, filling) and extravasated plasma constituents in damaged and intact brain areas. In this model, the presence of extravasated plasma constituents the size of proteins did not immediately influence indices of cortical microcirculation. However, microvascular perfusion may be perturbed surrounding such an area of advancing vasogenic edema formation.     

Functional role of plateau potentials in vertebrate motor neurons

The expression of plateau potentials in spinal motor neurons is regulated by neuromodulatory substances. Recent experiments have shed new light on this regulation at the cellular level. It is now possible to evaluate the existence of plateau potentials in intact organisms, including humans, and to address the functional role of plateau potentials in motor control, as well as in information transfer in the brain.     

The shapes of the motor domains of two oppositely directed microtubule motors, ncd and kinesin: a neutron scattering study

The shapes of the motor domains of kinesin and ncd, which move in opposite directions along microtubules, have been investigated. Using proteins expressed in Escherichia coli, it was found that at high salt (> 200 mM) Drosophila ncd motor domain (R335-K700) and human kinesin motor domain (M1-E349) were both sufficiently monomeric to allow an accurate determination of their radii of gyration (Rg) and their molecular weights. The measured Rg values of the ncd and kinesin motor domains in D2O were 2.06 +/- 0.06 and 2.05 +/- 0.04 nm, respectively, and the molecular weights were consistent with those computed from the amino acid compositions. Fitting of the scattering curves to approximately 3.5 nm resolution showed that the ncd and kinesin motor domains can be described adequately by triaxial ellipsoids having half-axes of 1.42 +/- 0.38, 2.24 +/- 0.44, and 3.65 +/- 0.22 nm, and half-axes of 1.52 +/- 0.23, 2.00 +/- 0.25, and 3.73 +/- 0.10 nm, respectively. Both motor domains are described adequately as somewhat flattened prolate ellipsoids with a maximum dimension of approximately 7.5 nm. Thus, it appears that the overall shapes of these motor domains are not the major determinants of the directionality of their movement along microtubules.     

The coiled-coil helix in the neck of kinesin

Kinesin is a microtubule-dependent motor protein. We have recently determined the X-ray structure of monomeric and dimeric kinesin from rat brain. The dimer consists of two motor domains, held together by their alpha-helical neck domains forming a coiled coil. Here we analyze the nature of the interactions in the neck domain (residues 339-370). Overall, the neck helix shows a heptad repeat (abcdefg)n typical of coiled coils, with mostly nonpolar residues in positions a and d. However, the first segment (339-355) contains several nonclassical residues in the a and d positions which tend to weaken the hydrophobic interaction along the common interface. Instead, stabilization is achieved by a hydrophobic "coat" formed by the a and d residues and the long aliphatic moieties of lysines and glutamates, extending away from the coiled-coil core. By contrast, the second segment of the kinesin neck (356-370) shows a classical leucine zipper pattern in which most of the hydrophobic residues are buried at the highly symmetrical dimer interface. The end of the neck reveals the structure of a potential coiled-coil "trigger" sequence.     

Molecular cloning and expression of the KIF3A gene in the frog brain and testis

KIF3A is a member of the kinesin superfamily proteins (KIFs), but its gene has been cloned only in mouse and sea urchin. We have cloned a homolog of KIF3A from the frog, Rana rugosa (rrKIF3A). The sequence encoded a 699 amino acid protein that shares 93% similarity with mouse KIF3A (mKIF3A) and 69% with sea urchin kinesin-related protein (SpKRP85). The putative ATP-binding domain was completely identical to that of mKIF3A and SpKRP85. The level of rrKIF3A mRNA appeared to be high in the brain and testis of adult frogs, but low in the heart, lung and kidney. The results suggest that the rrKIF3A gene is expressed in the brain and testis more than other tissues of adult frogs examined, and that KIF3A is widely distributed in eukaryotic organisms.     

Direct glutamate-mediated presynaptic inhibition of sensory afferents by the postsynaptic motor neurons

An in vitro preparation of the crayfish central nervous system was used to study a negative feedback control exerted by the glutamatergic motor neurons (MNs) on to their presynaptic cholinergic sensory afferents. This negative control consists in small amplitude, slowly developing depolarizations of the primary afferents (sdPADs) strictly timed with MN bursts. They were not blocked by picrotoxin, but were sensitive to glutamate non-N-methyl-D-aspartate (NMDA) antagonists. Intracellular recordings were performed within thin branches of sensory terminals while electrical antidromic stimulation were applied to the motor nerves, or while glutamate (the MN neurotransmitter) was pressure-applied close to the recording site. Electrical motor nerve stimulations and glutamate pressure application had similar effects on to sensory terminals issued from the coxo-basipodite chordotonal organ (CBTs): like sdPADs, both stimulation-induced depolarizations were picrotoxin-resistant and were dramatically reduced by non-NMDA antagonist bath application. These results indicate that sdPADs are likely directly produced by MNs during locomotor activity. A functional scheme is proposed.     

Tryptophan is present in glial cells and photoreceptors in the chicken retina

Tryptophan is a large neutral amino acid which is utilized in the biosynthesis of neuroactive substances such as serotonin and melatonin. However, it has been unclear where pools of tryptophan might be localized. Using a specific antiserum against tryptophan, we demonstrate that in the chicken retina tryptophan is present in radial glial cells and photoreceptors, but not in other neuronal elements. These data suggest that serotonergic neurones are probably dependent upon the transfer of tryptophan from the glial cells in order to manufacture serotonin and other tryptophan derivatives in the brain. If glia do supply tryptophan to neurones then this process will have significant practical implications for our basic understanding of and pharmacological manipulation of serotonergic systems.     

Complex formation of SMAP/KAP3, a KIF3A/B ATPase motor-associated protein, with a human chromosome-associated polypeptide

We have recently isolated SMAP (Smg GDS-associated protein; Smg GDS: small G protein GDP dissociation stimulator) as a novel Smg GDS-associated protein, which has Armadillo repeats and is phosphorylated by Src tyrosine kinase. SMAP is a human counterpart of mouse KAP3 (kinesin superfamily-associated protein) that is associated with mouse KIF3A/B (a kinesin superfamily protein), which functions as a microtubule-based ATPase motor for organelle transport. We isolated here a SMAP-interacting protein from a human brain cDNA library, identified it to be a human homolog of Xenopus XCAP-E (Xenopus chromosome-associated polypeptide), a subunit of condensins that regulate the assembly and structural maintenance of mitotic chromosomes, and named it HCAP (Human chromosome-associated polypeptide). Tissue and subcellular distribution analyses indicated that HCAP was ubiquitously expressed and highly concentrated in the nuclear fraction, where SMAP and KIF3B were also present. SMAP was extracted as a ternary complex with HCAP and KIF3B from the nuclear fraction in the presence of Mg-ATP. The results suggest that SMAP/KAP3 serves as a linker between HCAP and KIF3A/B in the nucleus, and that SMAP/KAP3 plays a role in the interaction of chromosomes with an ATPase motor protein.     

Polynucleotide phosphorylase is a component of a novel plant poly(A) polymerase

We have isolated cDNA clones encoding a novel RNA-binding protein that is a component of a multisubunit poly(A) polymerase from pea seedlings. The encoded protein bears a significant resemblance to polynucleotide phosphorylases (PNPases) from bacteria and chloroplasts. More significantly, this RNA-binding protein is able to degrade RNAs with the resultant production of nucleotide diphosphates, and it can add extended polyadenylate tracts to RNAs using ADP as a donor for adenylate moieties. These activities are characteristic of PNPase. Antibodies raised against the cloned protein simultaneously immunoprecipitate both poly(A) polymerase and PNPase activity. We conclude from these studies that PNPase is the RNA-binding cofactor for this poly(A) polymerase and is an integral player in the reaction catalyzed by this enzyme. The identification of this RNA-binding protein as PNPase, which is a chloroplast-localized enzyme known to be involved in mRNA 3'-end determination and turnover (Hayes, R., Kudla, J., Schuster, G., Gabay, L., Maliga, P., and Gruissem, W. (1996) EMBO J. 15, 1132-1141), raises interesting questions regarding the subcellular location of the poly(A) polymerase under study. We have reexamined this issue, and we find that this enzyme can be detected in chloroplast extracts. The involvement of PNPase in polyadenylation in vitro provides a biochemical rationale for the link between chloroplast RNA polyadenylation and RNA turnover which has been noted by others (Lisitsky, I., Klaff, P., and Schuster, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13398-13403).     

Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein

Microtubule-dependent motor, murine KIF3B, was disrupted by gene targeting. The null mutants did not survive beyond midgestation, exhibiting growth retardation, pericardial sac ballooning, and neural tube disorganization. Prominently, the left-right asymmetry was randomized in the heart loop and the direction of embryonic turning. lefty-2 expression was either bilateral or absent. Furthermore, the node lacked monocilia while the basal bodies were present. Immunocytochemistry revealed KIF3B localization in wild-type nodal cilia. Video microscopy showed that these cilia were motile and generated a leftward flow. These data suggest that KIF3B is essential for the left-right determination through intraciliary transportation of materials for ciliogenesis of motile primary cilia that could produce a gradient of putative morphogen along the left-right axis in the node.