Genetic engineering of a Ca(2+) dependent chemical switch into the linear biomotor kinesin

Kinesin is a linear motor protein driven by energy released by ATP hydrolysis. In the present work, we genetically installed an M13 peptide sequence into Loop 12 of kinesin, which is one of the major microtubule binding regions of the protein. Because the M13 sequence has high affinity for Ca(2+)-calmodulin, the association of the engineered kinesin with microtubules showed a steep Ca(2+)-dependency in ATPase activity at Ca(2+) concentrations of pCa 6.5-8. The calmodulin-binding domain of plant kinesin-like calmodulin-binding protein is also known to confer Ca(2+)-calmodulin regulation to kinesins. Unlike this plant kinesin, however, our novel engineered kinesin achieves this regulation while maintaining the interaction between kinesin and microtubules. The engineered kinesin is switched on/off reversibly by an external signal (i.e., Ca(2+)-calmodulin) and, thus, can be used as a model system for a bio/nano-actuator.     

Microtubule‐binding protein doublecortin‐like kinase 1 (DCLK1) guides kinesin‐3‐mediated cargo transport to dendrites

In neurons, the polarized distribution of vesicles and other cellular materials is established through molecular motors that steer selective transport between axons and dendrites. It is currently unclear whether interactions between kinesin motors and microtubule‐binding proteins can steer polarized transport. By screening all 45 kinesin family members, we systematically addressed which kinesin motors can translocate cargo in living cells and drive polarized transport in hippocampal neurons. While the majority of kinesin motors transport cargo selectively into axons, we identified five members of the kinesin‐3 (KIF1) and kinesin‐4 (KIF21) subfamily that can also target dendrites. We found that microtubule‐binding protein doublecortin‐like kinase 1 (DCLK1) labels a subset of dendritic microtubules and is required for KIF1‐dependent dense‐core vesicles (DCVs) trafficking into dendrites and dendrite development. Our study demonstrates that microtubule‐binding proteins can provide local signals for specific kinesin motors to drive polarized cargo transport.     

鼠脑驱动蛋白的表达、纯化和结晶

驱动蛋白是一类利用水解ATP为ADP和磷酸的过程中释放的能量沿微管系统运动的蛋白。为了研究ATP中储存的化学能是如何转化为驱动蛋白的机械动能,鼠脑驱动蛋白的相关N-端区域在BL21-Codon Plus(DE3)-RP感受态大肠杆菌细胞中大量地表达。通过SP-强阳离子交换色谱和分子筛色谱的两步骤纯化,蛋白最终产量高达10 mg/L细胞培养液,蛋白纯度可以达到95%以上。纯化的蛋白具有水解ATP酶的活力,并与驱动蛋白抗体有特异性的反应。驱动蛋白可以在如下条件结晶:1.7 mol/L(NH4)2SO4,500 mmol/L NaCl,20%glycerol。晶体衍射的分辨率可以达到2.0。     

Highly processive motility is not a general feature of the kinesins

Evidence is presented that the kinesin-related ncd protein is not as processive as kinesin. In low surface density motility experiments, a dimeric ncd fusion protein behaved mechanistically more similar to non-processive myosins than to the highly processive kinesin. First, there was a critical microtubule length for motility; only microtubules longer than this critical length moved in low density ncd surfaces, which suggested that multiple ncd proteins must cooperate to move microtubules in the surface assay. Under similar conditions, native kinesin demonstrated no critical microtubule length, consistent with the behavior of a highly processive motor. Second, addition of methylcellulose to decrease microtubule diffusion decreased the critical microtubule length for motility. Also, the rates of microtubule motility were microtubule length dependent in methylcellulose; short microtubules, that interacted with fewer ncd proteins, moved more slowly than long microtubules that interacted with more ncd proteins. In contrast, short microtubules, that interacted with one or a few kinesin proteins, moved on average slightly faster than long microtubules that interacted with multiple kinesins. We conclude that a degree of processivity as high as that of kinesin, where a single dimer can move over distances on the order of one micrometer, may not be a general mechanistic feature of the kinesin superfamily. Received: 16 September 1997 / Accepted: 4 November 1997     

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 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.     

Kinectin, a major kinesin-binding protein on ER

Previous studies have shown that microtubule-based organelle transport requires a membrane receptor but no kinesin-binding membrane proteins have been isolated. Chick embryo brain microsomes have kinesin bound to their surface, and after detergent solubilization, a matrix with an antibody to the kinesin head domain (SUK-4) (Ingold et al., 1988) bound the solubilized kinesin and retained an equal amount of a microsome protein of 160-kD. Similarly, velocity sedimentation of solubilized membranes showed that kinesin and the 160-kD polypeptide cosedimented at 13S. After alkaline treatment to remove kinesin from the microsomes, the same 160-kD polypeptide doublet bound to a kinesin affinity resin and not to other proteins tested. Biochemical characterization localized this protein to the cytoplasmic face of brain microsomes and indicated that it was an integral membrane protein since it was resistant to alkaline washing. mAbs raised to chick 160-kD protein demonstrated that it was absent in the supernatant and concentrated in the dense microsome fraction. The dense microsome fraction also had the greatest amount of microtubule-dependent motility. With immunofluorescence, the antibodies labeled the ER in chick embryo fibroblasts (similar to the pattern of bound kinesin staining in the same cells) (Hollenbeck, P. J. 1989. J. Cell Biol. 108:2335-2342), astroglia, Schwann cells and dorsal root ganglion cells but staining was much less in the Golgi regions of these cells. Because this protein is a major kinesin-binding protein of motile vesicles and would be expected to bind kinesin to the organelle membrane, we have chosen the name, kinectin, for this protein.     

Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells

To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOH-terminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesin-like protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with translational regulation in certain mRNAs. After transient expression in CV-1 cells, the kinesin heavy chain showed both a diffuse distribution and a filamentous staining pattern that coaligned with microtubules but not vimentin intermediate filaments. Altering the number and distribution of microtubules with taxol or nocodazole produced corresponding changes in the localization of the expressed kinesin heavy chain. The expressed NH2-terminal motor and the COOH-terminal tail domains, but not the alpha-helical coiled coil rod domain, also colocalized with microtubules. The finding that both the kinesin motor and tail domains can interact with cytoplasmic microtubules raises the possibility that kinesin could crossbridge and induce sliding between microtubules under certain circumstances.     

Identification and characterization of a gene encoding a kinesin-like protein in Drosophila

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.     

Rat coagulating gland secretion contains a kinesin heavy chain-like protein acting as a type IV transglutaminase substrate

By a proteomic approach, we demonstrated in rat coagulating gland secretion the presence of a 120 kDa protein which shares at least 80% identity at the amino acid level with the most closely related kinesin heavy chain codified by the kinesin superfamily protein Kif5c gene. In addition, we identified 30 and 66 kDa proteolytic fragments of such a kinesin heavy chain-like protein, corresponding to the 73-299 N-terminal and 300-860 C-terminal regions, respectively. Finally, we demonstrated the occurrence in coagulating gland secretion of a 200 kDa protein probably derived by cross-linking reaction of the kinesin heavy chain-like protein with type IV transglutaminase. In fact, kinesin heavy chain-like protein and its 66 kDa proteolytic fragment were also found to act as effective acyl donor substrates for the enzyme in vitro.     

Expression of Neuronal Kinesin Heavy Chain Is Developmentally Regulated in the Central Nervous System of the Rat

Abstract: The kinesin family of motor proteins comprises at least two isoforms of conventional kinesin encoded by different genes: ubiquitous kinesin, expressed in all cells and tissues, and neuronal kinesin, expressed exclusively in neuronal cells. In the present study, we have analyzed the expression of the two kinesin isoforms by immunochemistry at different stages of development of the rat CNS. We have found that the level of expression of neuronal kinesin is five to eight times higher in developing than in adult rat brains, whereas that of ubiquitous kinesin is only ∼2.5 times higher in maturing versus adult brains. Moreover, we have studied the distribution of neuronal kinesin by light microscopic immunocytochemistry in the rat brain at different postnatal ages and have found this protein not only to be more highly expressed in juvenile than in adult rat brains but also to show a different pattern of distribution. In particular, tracts of axonal fibers were clearly stained at early postnatal stages of development but were markedly unlabeled in adult rat brains. Our results indicate that the expression of at least one isoform of conventional neuron-specific kinesin is up-regulated in the developing rat CNS and suggest that this protein might play an important role in microtubule-based transport during the maturation of neuronal cells in vivo.     

Preparation and characterization of a novel rice plant-specific kinesin

Kinesin is an ATP-driven motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. The kinesin family is classified into subfamilies. Kinesin species derived from vertebrates have been well characterized. In contrast, plant kinesins have yet to be adequately characterized. In this study, we expressed the motor domain of a novel rice plant-specific kinesin, K16, in Escherichia coli, and then determined its enzymatic characteristics and compared them with those of kinesin 1. Our findings demonstrated that the rice kinesin motor domain has different enzymatic properties from those of well known kinesin 1.     

Subunits interactions in kinesin motors

Kinesins form a large and diverse superfamily of proteins involved in numerous important cellular processes. The majority of them are molecular motors moving along microtubules. Conversion of chemical energy into mechanical work is accomplished in a sequence of events involving both biochemical and conformational alternation of the motor structure called the mechanochemical cycle. Different members of the kinesin superfamily can either perform their function in large groups or act as single molecules. Conventional kinesin, a member of the kinesin-1 subfamily, exemplifies the second type of motor which requires tight coordination of the mechanochemical cycle in two identical subunits to accomplish processive movement toward the microtubule plus end. Recent results strongly support an asymmetric hand-over-hand model of "walking" for this protein. Conformational strain between two subunits at the stage of the cycle where both heads are attached to the microtubule seems to be a major factor in intersubunit coordination, although molecular and kinetic details of this phenomenon are not yet deciphered. We discuss also current knowledge concerning intersubunit coordination in other kinesin subfamilies. Members of the kinesin-3 class use at least three different mechanisms of movement and can translocate in monomeric or dimeric forms. It is not known to what extent intersubunit coordination takes place in Ncd, a dimeric member of the kinesin-14 subfamily which, unlike conventional kinesin, exercises a power-stroke toward the microtubule minus end. Eg5, a member of the kinesin-5 subfamily is a homotetrameric protein with two kinesin-1-like dimeric halves controlled by their relative orientation on two microtubules. It seems that diversity of subunit organization, quaternary structures and cellular functions in the kinesin superfamily are reflected also by the divergent extent and mechanism of intersubunit coordination during kinesin movement along microtubules.     

Inhibition of Kinesin Synthesis In Vivo Inhibits the Rapid Transport of Representative Proteins for Three Transport Vesicle Classes into the Axon

Abstract: We have previously demonstrated that the in vivo vitreal injection of an antisense oligonucleotide directed to the kinesin heavy chain inhibits retinal kinesin synthesis by 82% and concomitantly inhibits rapid transport of total protein into the optic nerve by 70%. These results establish a major role for kinesin in rapid axonal transport in vivo. Recently, the cloning of a family of kinesin-like molecules from the mammalian brain has been reported, and some of these proteins are also expressed in neurons. To assign a specific function to the kinesin heavy chain we inhibited the kinesin synthesis with an antisense kinesin oligonucleotide and assessed the axonal transport into the optic nerve of representative proteins from each of three vesicle classes that contain rapidly transported proteins. Marker proteins used were substance P for peptide-containing synaptic vesicles, the amyloid precursor protein for plasma membrane precursor vesicles, and several integral synaptic vesicle proteins. Our results indicate that the major anterograde motor protein for all three vesicle classes utilizes kinesin heavy chain, although we discuss alternative explanations.     

Physiological evidence for involvement of a kinesin-related protein during anaphase spindle elongation in diatom central spindles

We have developed a new model system for studying spindle elongation in vitro using the pennate, marine diatom Cylindrotheca fusiformis. C. fusiformis can be grown in bulk to high densities while in log phase growth and synchronized by a simple light/dark regime. Isolated spindles can be attained in quantities sufficient for biochemical analysis and spindle tubulin is approximately 5% of the total protein present. The spindle isolation procedure results in a 10-fold enrichment of diatom tubulin and a calculated 40-fold increase in spindle protein. Isolated spindles or spindles in permeabilized cells can elongate in vitro by the same mechanism and with the same pharmacological sensitivities as described for other anaphase B models (Cande and McDonald, 1986; Masuda et al., 1990). Using this model, in vitro spindle elongation rate profiles were developed for a battery of nucleotide triphosphates and ATP analogs. The relative rates of spindle elongation produced by various nucleotide triphosphates parallel relative rates seen for kinesin-based motility in microtubule gliding assays. Likewise ATP analogs that allow discrimination between myosin-, dynein-, and kinesin-mediated motility produce relative spindle elongation rates characteristic of kinesin motility. Also, isolated spindle fractions are enriched for a kinesin related protein as identified by a peptide antibody against a conserved region of the kinesin superfamily. These data suggest that kinesin-like motility contributes to spindle elongation during anaphase B of mitosis.     

The overall conformation of conventional kinesins studied by small angle X-ray and neutron scattering

The quaternary structures of several monomeric and dimeric kinesin constructs from Homo sapiens and Drosophila melanogaster were analyzed using small angle x-ray and neutron scattering. The experimental scattering curves of these proteins were compared with simulated scattering curves calculated from available crystallographic coordinates. These comparisons indicate that the overall conformations of the solution structures of D. melanogaster and H. sapiens kinesin heavy chain dimers are compatible with the crystal structure of dimeric kinesin from Rattus norvegicus. This suggests that the unusual asymmetric conformation of dimeric kinesin in the microtubule-independent ADP state is likely to be a general feature of the kinesin heavy chain subfamily. An intermediate length Drosophila construct (365 residues) is mostly monomeric at low protein concentration whereas at higher concentrations it is dimeric with a tendency to form higher oligomers.     

Molecular basis of dystrobrevin interaction with kinesin heavy chain: structural determinants of their binding

Dystrobrevins are a family of widely expressed dystrophin-associated proteins that comprises alpha and beta isoforms and displays significant sequence homology with several protein-binding domains of the dystrophin C-terminal region. The complex distribution of the multiple dystrobrevin isoforms suggests that the variability of their composition may be important in mediating their function. We have recently identified kinesin as a novel dystrobrevin-interacting protein and localized the dystrobrevin-binding site on the cargo-binding domain of neuronal kinesin heavy chain (Kif5A). In the present study, we assessed the kinetics of the dystrobrevin-Kif5A interaction by quantitative pull-down assay and surface plasmon resonance (SPR) analysis and found that beta-dystrobrevin binds to kinesin with high affinity (K(D) approximately 40 nM). Comparison of the sensorgrams obtained with alpha and beta-dystrobrevin at the same concentration of analyte showed a lower affinity of alpha compared to that of beta-dystrobrevin, despite their functional domain homology and about 70% sequence identity. Analysis of the contribution of single dystrobrevin domains to the interaction revealed that the deletion of either the ZZ domain or the coiled-coil region decreased the kinetics of the interaction, suggesting that the tertiary structure of dystrobrevin may play a role in regulating the interaction of dystrobrevin with kinesin. In order to understand if structural changes induced by post-translational modifications could affect dystrobrevin affinity for kinesin, we phosphorylated beta-dystrobrevin in vitro and found that it showed reduced binding capacity towards kinesin. The interaction between the adaptor/scaffolding protein dystrobrevin and the motor protein kinesin may play a role in the transport and targeting of components of the dystrophin-associated protein complex to specific sites in the cell, with the differences in the binding properties of dystrobrevin isoforms reflecting their functional diversity within the same cell type. Phosphorylation events could have a regulatory role in this context.     

The transport of Staufen2-containing ribonucleoprotein complexes involves kinesin motor protein and is modulated by mitogen-activated protein kinase pathway

There is increasing evidence showing that mRNA is transported to the neuronal dendrites in ribonucleoprotein (RNP) complexes or RNA granules, which are aggregates of mRNA, rRNA, ribosomal proteins, and RNA-binding proteins. In these RNP complexes, Staufen, a double-stranded RNA-binding protein, is believed to be a core component that plays a key role in the dendritic mRNA transport. This study investigated the molecular mechanisms of the dendritic mRNA transport using green fluorescent protein-tagged Staufen2 produced employing a Sindbis viral expression system. The kinesin heavy chain was found to be associated with Staufen2. The inhibition of kinesin resulted in a significant decrease in the level of dendritic transport of the Staufen2-containing RNP complexes in neurons under non-stimulating or stimulating conditions. This suggests that the dendritic transport of the Staufen2-containing RNP complexes use kinesin as a motor protein. A mitogen-activated protein kinase inhibitor, PD98059, inhibited the activity-induced increase in the amount of both the Staufen2-containing RNP complexes and Ca(2+)/calmodulin-dependent protein kinase II alpha-subunit mRNA in the distal dendrites of cultured hippocampal neurons. Overall, these results suggest that dendritic mRNA transport is mediated via the Staufen2 and kinesin motor proteins and might be modulated by the neuronal activity and mitogen-activated protein kinase pathway.     

Nucleotide-dependent interaction of the N-terminal domain of MukB with microtubules

The MukB protein from Escherichia coli has a domain structure that is reminiscent of the eukaryotic motor proteins kinesin and myosin: N-terminal globular domains, a region of coiled-coil, and a specialised C-terminal domain. Sequence alignment of the N-terminal domain of MukB with the kinesin motor domain indicated an approximately 22% sequence identity. These observations raised the possibility that MukB might be a prokaryotic motor protein and, due to the sequence homology shared with kinesin, might bind to microtubules (Mts). We found that a construct encoding the first 342 residues of MukB (Muk342) binds specifically to Mts and shares a number of properties with the motor domain of kinesin. Visualisation of the Muk342 decorated Mt complexes using negative stain electron microscopy indicated that the Muk342 smoothly decorates the outside of Mts. Biochemical data demonstrate that Muk342 decorates Mts with a binding stoichiometry of one Muk342 monomer per tubulin monomer. These findings strongly suggest that MukB has a role in force generation and that it is a prokaryotic homologue of kinesin and myosin.     

Subcellular localization and sequence of sea urchin kinesin heavy chain: evidence for its association with membranes in the mitotic apparatus and interphase cytoplasm [published erratum appears in J Cell Biol 1991 Aug;114(4):following 863]

Kinesin was previously immunolocalized to mitotic apparatuses (MAs) of early sea urchin blastomeres (Scholey, J.M., M.E. Porter, P.M. Grissom, and J.R. McIntosh. 1985. Nature [Lond.]. 318:483-486). Here we report evidence that this MA-associated motor protein is a conventional membrane-bound kinesin, rather than a kinesin-like protein. Our evidence includes the observation that the deduced amino acid sequence of this sea urchin kinesin heavy chain is characteristic of a conventional kinesin. In addition, immunolocalizations using antibodies that distinguish kinesin from kinesin-like proteins confirm that conventional kinesin is concentrated in MAs. Finally, our immunocytochemical data further suggest that conventional kinesin is associated with membranes which accumulate in MAs and interphase asters of early sea urchin embryos, and with vesicles that are distributed in the perinuclear region of coelomocytes. Thus kinesin may function as a microtubule-based vesicle motor in some MAs, as well as in the interphase cytoplasm.