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Microtubule-based transport along axons, dendrites and axonemes
Abstract
MTs in cytoplasmic extensions including axons, dendrites and axonemes serve as polarized tracks for vectorial intracellular transport driven by MT-based motor proteins. Although axons and axonemes serve very different functions, increasing evidence suggests that the transport events, MT organization and the motors involved in their formation and function are conserved. Thus, there are obvious similarities in the mechanisms of axonal transport and IFT. The MT arrays of axons and axonemes are parallel, whereas those of dendrites are anti-parallel, but the functional significance of this difference and its consequences for mechanisms of transport along these processes are unclear. MT-based motor proteins of the dynein and kinesin superfamilies transport a variety of cargos including membrane-bound vesicles and macromolecular complexes along MTs of axons, dendrites and axonemes, and thus contribute to the formation, maintenance and function of these cytoplasmic extensions. Chemosensory neurons in the nematode C. elegans represent an appealing system for studying transport events along dendrites and axonemes that occur sequentially in a single cell.
... (5) The linear and parallel tracks followed by VP16-GFP-labelled particles in the squid axon are reminiscent of the interwoven microtubule/ actin bundles observed there by electron microscopy and confocal immunofluorescence imaging (Bearer & Reese, 1999). Such filaments are known to serve as tracks for endogenous transport (Schnapp et al., 1985;Vale, 1985;Signor & Scholey, 2000). ...
Anterograde transport of herpes simplex virus (HSV) from its site of synthesis in the neuronal cell body out the neuronal process to the mucosal membrane is crucial for transmission of the virus from one person to another, yet the molecular mechanism is not known. By injecting GFP-labeled HSV into the giant axon of the squid, we reconstitute fast anterograde transport of human HSV and use this as an assay to uncover the underlying molecular mechanism. HSV travels by fast axonal transport at velocities four-fold faster (0.9 µm/sec average, 1.2 µm/sec maximal) than that of mitochondria moving in the same axon (0.2 µm/sec) and ten-fold faster than negatively charged beads (0.08 µm/sec). Transport of HSV utilizes cellular transport mechanisms because it appears to be driven from inside cellular membranes as revealed by negative stain electron microscopy and by the association of TGN46, a component of the cellular secretory pathway, with GFP-labeled viral particles. Finally, we show that amyloid precursor protein (APP), a putative receptor for the microtubule motor, kinesin, is a major component of viral particles, at least as abundant as any viral encoded protein, while another putative motor receptor, JIP 1/2, is not detected. Conventional kinesin is also associated with viral particles. This work links fast anterograde transport of the common pathogen, HSV, with the neurodegenerative Alzheimer"s disease. This novel connection should prompt new ideas for treatment and prevention strategies.
... Microtubules (MT) are key determinants of neuronal polarity [10][11][12][13] and form the transport highways for cargo trafficking in axons and dendrites in neurons [6]. MT are formed from the association of dimers of α-tubulin and β-tubulin into protofilaments. ...
Gene products such as organelles, proteins and RNAs are actively transported to synaptic terminals for the remodeling of pre-existing neuronal connections and formation of new ones. Proteins described as molecular motors mediate this transport and utilize specialized cytoskeletal proteins that function as molecular tracks for the motor based transport of cargos. Molecular motors such as kinesins and dynein's move along microtubule tracks formed by tubulins whereas myosin motors utilize tracks formed by actin. Deficits in active transport of gene products have been implicated in a number of neurological disorders. We describe such disorders collectively as "transportopathies". Here we review current knowledge of critical components of active transport and their relevance to neurodegenerative diseases.
... Nonetheless, JNK plays pivotal roles in MT organization and stability in other cell types. For example, during neurogenesis, JNK regulates the bundling of MTs into parallel arrays to stabilize neurite structure and elongate axons (Horton and Ehlers, 2003;Maccioni and Cambiazo, 1995;Signor and Scholey, 2000). ...
Tissue elongation is a fundamental morphogenetic process that generates the proper anatomical topology of the body plan and vital organs. In many elongating embryonic structures, tissue lengthening is driven by Rho family GTPase-mediated cell rearrangement. During this dynamic process, the mechanisms that modulate intercellular adhesion to allow individual cells to change position without compromising structural integrity are not well understood. In vertebrates, Jun N-terminal kinase (JNK) is also required for tissue elongation, but the precise cellular role of JNK in this context has remained elusive. Here, we show that JNK activity is indispensable for the rearrangement of endoderm cells that underlies the elongation of the Xenopus gut tube. Whereas Rho kinase is necessary to induce cell intercalation and remodel adhesive contacts, we have found that JNK is required to maintain cell-cell adhesion and establish parallel microtubule arrays; without JNK activity, the reorganizing endoderm dissociates. Depleting polymerized microtubules phenocopies this effect of JNK inhibition on endoderm morphogenesis, consistent with a model in which JNK regulates microtubule architecture to preserve adhesive contacts between rearranging gut cells. Thus, in contrast to Rho kinase, which generates actomyosin-based tension and cell movement, JNK signaling is required to establish microtubule stability and maintain tissue cohesion; both factors are required to achieve proper cell rearrangement and gut extension. This model of gut elongation has implications not only for the etiology of digestive tract defects, but sheds new light on the means by which intra- and intercellular forces are balanced to promote topological change, while preserving structural integrity, in numerous morphogenetic contexts.
... The highly specialized rod photoreceptors are located in the outermost layer of the vertebrate retina and mediate vision in dim light. While there is a diversity in the size and shape between different species (47), all rods are highly polarized with distinct compartments: an OS containing light-sensitive visual pigment, a narrow connecting cilium connecting the OS to the IS (35,44,(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58), and an IS containing the nucleus and synaptic terminus (Fig. 1 A). The cylindrical OS has an exceptional structure which contains hundreds of lipid bilayer membrane disks in a stack surrounded by, but not contiguous with, a plasma membrane (59). ...
In vertebrate eyes, the rod photoreceptor has a modified cilium with an extended cylindrical structure specialized for phototransduction called the outer segment (OS). The OS has numerous stacked membrane disks and can bend or break when subjected to mechanical forces. The OS exhibits axial structural variation, with extended bands composed of a few hundred membrane disks whose thickness is diurnally modulated. Using high-resolution confocal microscopy, we have observed OS flexing and disruption in live transgenic Xenopus rods. Based on the experimental observations, we introduce a coarse-grained model of OS mechanical rigidity using elasticity theory, representing the axial OS banding explicitly via a spring-bead model. We calculate a bending stiffness of ∼10(5) nN⋅μm(2), which is seven orders-of-magnitude larger than that of typical cilia and flagella. This bending stiffness has a quadratic relation to OS radius, so that thinner OS have lower fragility. Furthermore, we find that increasing the spatial frequency of axial OS banding decreases OS rigidity, reducing its fragility. Moreover, the model predicts a tendency for OS to break in bands with higher spring number density, analogous to the experimental observation that transgenic rods tended to break preferentially in bands of high fluorescence. We discuss how pathological alterations of disk membrane properties by mutant proteins may lead to increased OS rigidity and thus increased breakage, ultimately contributing to retinal degeneration.
... Microtubules are cytoskeletal polymers of a/b tubulin heterodimers. They contribute to the formation, main- tenance, and function of axons and dendrites (review in Signor and Scholey, 2000). Expression of regulators of microtubule dynamics in rat striatum was found to be modified by chronic morphine treatment. ...
It has been previously suggested that morphine can regulate the expression and function of some proteins of the cytoskeleton. In the present study, we used real-time quantitative polymerase chain reaction to examine the effects of chronic morphine administration, in rat striatum, on 14 proteins involved in microtubule polymerization and stabilization, intracellular trafficking, and serving as markers of neuronal growth and degeneration. Chronic morphine treatment led to modulation of the mRNA level of seven of the 14 genes tested. Glial fibrillary acidic protein (Gfap) and activity-regulated cytoskeleton-associated protein (Arc) mRNA were upregulated, while growth associated protein (Gap43), clathrin heavy chain (Cltc), alpha-tubulin, Tau, and stathmin were downregulated. In order to determine if the regulation of an mRNA correlates with a modulation of the expression of the corresponding protein, immunoblot analyses were performed. With the exception of Gap43, the levels of Cltc, Gfap, Tau, stathmin, and alpha-tubulin proteins were found to be in good agreement with those from mRNA quantification. These results demonstrate that neuroadaptation to chronic morphine administration in rat striatum implies modifications of the expression pattern of several genes and proteins of the cytoskeleton and cytoskeleton-associated components.
... The MT arrays within these processes are necessary to maintain the processes and may indirectly contribute to synaptic activity. They are believed to provide structural support for each process as well as tracks for the delivery of membrane vesicles (Horton and Ehlers, 2003;Overly et al., 1996;Signor and Scholey, 2000). ...
In most proliferating and migrating animal cells, the centrosome is the main site for microtubule (MT) nucleation and anchoring, leading to the formation of radial MT arrays in which MT minus ends are anchored at the centrosomes and plus ends extend to the cell periphery. By contrast, in most differentiated animal cell types, including muscle, epithelial and neuronal cells, as well as most fungi and vascular plant cells, MTs are arranged in noncentrosomal arrays that are non-radial. Recent studies suggest that these noncentrosomal MT arrays are generated by a three step process. The initial step involves formation of noncentrosomal MTs by distinct mechanisms depending on cell type: release from the centrosome, catalyzed nucleation at noncentrosomal sites or breakage of pre-existing MTs. The second step involves transport by MT motor proteins or treadmilling to sites of assembly. In the final step, the noncentrosomal MTs are rearranged into cell-type-specific arrays by bundling and/or capture at cortical sites, during which MTs acquire stability. Despite their relative stability, the final noncentrosomal MT arrays may still exhibit dynamic properties and in many cases can be remodeled.
Cells constantly need to move molecules and organelles throughout their interior. RNA produced in the nucleus must be exported to then be translated into proteins. Some of these proteins must then be imported back into the nucleus. Others are packaged into membrane vesicles, which in turn must be trafficked to the plasma membrane and exocytosed from the cell. The list of such examples has hundreds of entries [1]. An energetically ‘cheap’ way to transport microscopic particles is through diffusion [2]. The trick is done by Brownian movements: all particles in cytoplasm are bombarded by countless water molecules, and if these particles are smaller than a micron in size, momentous imbalances in hits by these smaller molecules results in random movement. This paradigm is surprisingly effective when particles only must spread over short distances. However, there are three major caveats that in many cases make diffusion ineffective. (1) Diffusion is direction-less, so if the cell need transport from a source to a well-defined target (e.g. from ER to Golgi), diffusion alone does not work. (2) Distance travelled by diffusion increases as a square root of time, while directional movement with constant speed generates displacement that grows linearly with time. Therefore, long-distance diffusive movements are slow. (3) Objects that are larger typically diffuse slower, so membrane vesicles, for example, diffuse far too slowly for cellular functions.
The book describes the history of the centrosome study and presents
the current state of knowledge about this cell organelle in the morphological,
biochemical and functional aspects. In addition to the classical function of the centrosome as a center of nucleation, anchoring and organization of MT,
the ideas of centrosome as a cellular regulatory center and as a structural
part of the mechanism controlling the dynamic morphology of the cells
are discussed in the book.
Key words: centrosome, centriole, basal body, primary cilium, microtubules.
Now the book being translated into English.
We are looking for funding and Publisher for the publication of this book in English.
We report here the development of a three-dimensional (3D) magnetic force microscope for applying forces to and measuring responses of biological systems and materials. This instrument combines a conventional optical microscope with a free-floating or specifically bound magnetic bead used as a mechanical probe. Forces can be applied by the bead to microscopic structures of interest (specimens), while the reaction displacement of the bead is measured. This enables 3D mechanical manipulations and measurements to be performed on specimens in fluids. Force is generated by the magnetically permeable bead in reaction to fields produced by external electromagnets. The displacement is measured by interferometry using forward light scattered by the bead from a focused laser beam. The far-field interference pattern is imaged on a quadrant photodetector from which the 3D displacement can be computed over a limited range about the focal point. The bead and specimen are mounted on a 3D translation stage and feedback techniques are used to keep the bead within this limited range. We demonstrate the system with application to beads attached to cilia in human lung cell cultures.
It has been a decade since a novel form of microtubule (MT)-based motility, i.e., intraflagellar transport (IFT), was discovered in Chlamydomonas flagella. Subsequent research has supported the hypothesis that IFT is required for the assembly and maintenance of all cilia and flagella and that its underlying mechanism involves the transport of nonmembrane-bound macromolecular protein complexes (IFT particles) along axonemal MTs beneath the ciliary membrane. IFT requires the action of the anterograde kinesin-II motors and the retrograde IFT-dynein motors to transport IFT particles in opposite directions along the MT polymer lattice from the basal body to the tip of the axoneme and back again. A rich diversity of biological processes has been shown to depend upon IFT, including flagellar length control, cell swimming, mating and feeding, photoreception, animal development, sensory perception, chemosensory behavior, and lifespan control. These processes reflect the varied roles of cilia and flagella in motility and sensory signaling.
The spatial organization of microtubules is crucial for different cellular processes. It is traditionally supposed that fibroblasts
have radial microtubule arrays consisting of long microtubules that run from the centrosome. However, a detailed analysis
of the microtubule array in the internal cytoplasm has never been performed. In the current study, we used laser photobleaching
to analyze the spatial organization of microtubules in the internal cytoplasm of cultured 3T3 fibroblasts. Cells were injected
with Cy-3-labeled tubulin, after which the growth of microtubules in the centrosome region and peripheral parts of cytoplasm
was assayed in the bleached zone. In most cases, microtubule growth in the bleached zone occurred rectilinearly; at distances
of up to 5 μm, microtubules seldom bend more than 10°–15°. We considered a growing fragment of the microtubule as a vector
with the beginning at the point of occurrence and the end at the point where the growth terminated (or the end point after
30 s if microtubule persistent growth proceeded for longer). We defined the direction of microtubule growth in different parts
of the cell using these vectors and measured the angle of their deviation from the vector of comparison. In the area of the
centrosome, we directed a comparison vector inside the bleached zone from the centrosome to the beginning of the growing microtubule
segment; in the lamella and trailing part of the fibroblast, we used the vector of comparison directed along the long axis
of the cell from its geometrical center to periphery. The microtubules growing straight away from the centrosome grew along
the cell radius. However, at a distance of 10 μm from the centrosome, radially growing microtubules comprised 40% of the overall
number, while at a distance of 20 μm, they made up only 25%. The rest of the microtubules grew in different directions, with
the preferred angle between their growth direction and cell radius equaling around 90 °. In the lamella and trailing part
of the fibroblast, 80% of all microtubules grew along the long axis of the cell or at an angle of no more than 20 °; 10–15%
of microtubules grew along axis of the cell but towards the centrosome. Thus, in 3T3 fibroblasts, the radial system of microtubules
is perturbed starting at a distance of several microns from the centrosome. In the internal cytoplasm, the microtubule system
is completely disordered and, in the stretched parts of the polarized cell (lamella, trailing edge), the microtubule system
again becomes well organized; microtubules are preferentially oriented along the long axis of the cell. From the results obtained,
we conclude that the orderliness of microtubules at the periphery of the fibroblast is not a consequence of their growth from
the centrosome; rather, their orientation is preset by local factors.
Besides the ventral tegmental area and the nucleus accumbens as the most investigated brain reward structures, several reports about the relation between volume and activity of the amygdala and drug-seeking behavior have emphasized the central role of the amygdala in the etiology of addiction. Considering its proposed important role and the limited number of human protein expression studies with amygdala in drug addiction, we performed a human postmortem proteomic analysis of amygdala tissue obtained from 8 opiate addicts and 7 control individuals. Results were validated by Western blot in an independent postmortem replication sample from 12 opiate addicts compared to 12 controls and 12 suicide victims, as a second "control sample". Applying 2D-electrophoresis and MALDI-TOF-MS analysis, we detected alterations of beta-tubulin expression and decreased levels of the heat-shock protein HSP60 in drug addicts. Western blot analysis in the additional sample demonstrated significantly increased alpha- and beta-tubulin concentrations in the amygdala of drug abusers versus controls (P = 0.021, 0.029) and to suicide victims (P = 0.006, 0.002). Our results suggest that cytoskeletal alterations in the amygdala determined by tubulin seem to be involved in the pathophysiology of drug addiction, probably via a relation to neurotransmission and cellular signaling. Moreover, the loss of neuroprotection against stressors by chaperons as HSP60 might also contribute to structural alteration in the brain of drug addicts. Although further studies have to confirm our results, this might be a possible pathway that may increase our understanding of drug addiction.
Anterograde transport of herpes simplex virus (HSV) from its site of synthesis in the neuronal cell body out the neuronal process to the mucosal membrane is crucial for transmission of the virus from one person to another, yet the molecular mechanism is not known. By injecting GFP-labeled HSV into the giant axon of the squid, we reconstitute fast anterograde transport of human HSV and use this as an assay to uncover the underlying molecular mechanism. HSV travels by fast axonal transport at velocities four-fold faster (0.9 microm/sec average, 1.2 microm/sec maximal) than that of mitochondria moving in the same axon (0.2 microm/sec) and ten-fold faster than negatively charged beads (0.08 microm/sec). Transport of HSV utilizes cellular transport mechanisms because it appears to be driven from inside cellular membranes as revealed by negative stain electron microscopy and by the association of TGN46, a component of the cellular secretory pathway, with GFP-labeled viral particles. Finally, we show that amyloid precursor protein (APP), a putative receptor for the microtubule motor, kinesin, is a major component of viral particles, at least as abundant as any viral encoded protein, while another putative motor receptor, JIP 1/2, is not detected. Conventional kinesin is also associated with viral particles. This work links fast anterograde transport of the common pathogen, HSV, with the neurodegenerative Alzheimer's disease. This novel connection should prompt new ideas for treatment and prevention strategies.
Characterization of the behavior and regulation of pioneer microtubules at the larval Drosophila neuromuscular junction
Movement of fragmented DNA in dendrites of retinal neurons during the apoptotic cell death was investigated. The time-course of the movement of fragmented DNA in dendrites of retinal neurons undergoing apoptotic cell death induced by intravitreal N-methyl-d-aspartate (NMDA) injection were examined by in situ terminal dUTP-biotin nick end labeling of DNA fragments (TUNEL) method and fluorescence DNA detection technique by 4',6-diamidino-2-phenylindole (DAPI). The inhibitory effect of axoplasmic transport inhibitor, vincristine was also tested on the NMDA-induced fragmented DNA transport. The movement of fragmented DNA from apoptotic nuclei toward peripheral ends of the dendrites of the retinal neurons was clearly demonstrated. The transport of fragmented DNA, but not fragmentation per se, was completely inhibited by the co-administration of vincristine.
Intracellular membrane trafficking plays an essential role in the structural and functional organization of oligodendrocytes, which synthesize a large amount of membrane to form myelin. Rab proteins are key components in intracellular vesicular transport. We cloned a novel Rab protein from an oligodendrocyte cDNA library, designating it Rab40c because of its homology with Rab40a and Rab40b. The DNA sequence of Rab40c shows an 843-base pair open reading frame. The deduced amino acid sequence is a protein with 281 amino acids, with a molecular weight of 31,466 Da and an isoelectric point of 9.83. Rab40c presents a number of distinct structural features including a carboxyl terminal extension and amino acid substitutions in the consensus sequence of the GTP-binding motifs. The carboxyl terminal region contains motifs that permit isoprenylation and palmitoylation. Binding studies indicate that Rab40c binds guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S) with a K(d) of 21 microM and has a higher affinity for guanosine triphosphate (GTP) than for guanosine diphosphate (GDP). Rab40c is localized in the perinuclear recycling compartment, suggesting its involvement in endocytic events such as receptor recycling. The importance of this recycling in myelin formation is suggested by the increase in both Rab40c mRNA and Rab40c protein as oligodendrocytes differentiate.
Defects in cilia are associated with several human disorders, including Kartagener syndrome, polycystic kidney disease, nephronophthisis and hydrocephalus. We proposed that the pleiotropic phenotype of Bardet-Biedl syndrome (BBS), which encompasses retinal degeneration, truncal obesity, renal and limb malformations and developmental delay, is due to dysfunction of basal bodies and cilia. Here we show that individuals with BBS have partial or complete anosmia. To test whether this phenotype is caused by ciliary defects of olfactory sensory neurons, we examined mice with deletions of Bbs1 or Bbs4. Loss of function of either BBS protein affected the olfactory, but not the respiratory, epithelium, causing severe reduction of the ciliated border, disorganization of the dendritic microtubule network and trapping of olfactory ciliary proteins in dendrites and cell bodies. Our data indicate that BBS proteins have a role in the microtubule organization of mammalian ciliated cells and that anosmia might be a useful determinant of other pleiotropic disorders with a suspected ciliary involvement.
Temporal analysis in gene expression during differentiation of neural stem cells (NSCs) was performed by using in-house microarrays composed of 10,368 genes. The changes in mRNA level were measured during differentiation day 1, 2, 3, 6, 12, and 15. Out of 10,368 genes analyzed, 259 genes were up-regulated or down-regulated by 2-fold or more at least at one time-point during differentiation, and were classified into six clusters based on their expression patterns by K-means clustering. Clusters characterized by gradual increase have large numbers of genes involved in transport and cell adhesion; those which showed gradual decrease have much of genes in nucleic acid metabolism, cell cycle, transcription factor, and RNA processing. In situ hybridization (ISH) validated microarray data and it also showed that Fox M1, cyclin D2, and CDK4 were highly expressed in CNS germinal zones and ectonucleotide pyrophosphatase/phosphodiesterase 2 (Enpp2) was highly expressed in choroid plexus where stem/progenitor cells are possibly located. Together, this clustering analysis of expression patterns of functionally classified genes may give insight into understanding of CNS development and mechanisms of NSCs proliferation and differentiation.
Unlike most organelles, which are surrounded by cytoplasm, the flagellum protrudes from the cell surface extending tens or even hundreds of microns into the external medium. This elongated organelle must import all the macromolecules required for its assembly, maintenance, and function including >
In neuronal axons, various kinds of membranous components are transported along microtubules bidirectionally. However, only two kinds of mechanochemical motor proteins, kinesin and brain dynein, had been identified as transporters of membranous organelles in mammalian neurons. Recently, a series of genes that encode proteins closely related to kinesin heavy chain were identified in several organisms including Schizosaccharomyces pombe, Aspergillus niddulans, Saccharomyces cerevisiae, Caenorhabditus elegans, and Drosophila. Most of these members of the kinesin family are implicated in mechanisms of mitosis or meiosis. To address the mechanism of intracellular organelle transport at a molecular level, we have cloned and characterized five different members (KIF1-5), that encode the microtubule-associated motor domain homologous to kinesin heavy chain, in murine brain tissue. Homology analysis of amino acid sequence indicated that KIF1 and KIF5 are murine counterparts of unc104 and kinesin heavy chain, respectively, while KIF2, KIF3, and KIF4 are as yet unidentified new species. Complete amino acid sequence of KIF3 revealed that KIF3 consists of NH2-terminal motor domain, central alpha-helical rod domain, and COOH-terminal globular domain. Complete amino acid sequence of KIF2 revealed that KIF2 consists of NH2-terminal globular domain, central motor domain, and COOH-terminal alpha-helical rod domain. This is the first identification of the kinesin-related protein which has its motor domain at the central part in its primary structure. Northern blot analysis revealed that KIF1, KIF3, and KIF5 are expressed almost exclusively in murine brain, whereas KIF2 and KIF4 are expressed in brain as well as in other tissues. All these members of the kinesin family are expressed in the same type of neurons, and thus each one of them may transport its specific organelle in the murine central nervous system.
To understand the roles of kinesin and its relatives in cell division, it is necessary to identify and characterize multiple members of the kinesin superfamily from mitotic cells. To this end we have raised antisera to peptides corresponding to highly conserved regions of the motor domains of several known members of the kinesin superfamily. These peptide antibodies react specifically with the motor domains of kinesin and ncd protein, as expected, and they also react with several polypeptides (including kinesin heavy chain) that cosediment with microtubules (MTs) precipitated from AMPPNP-treated sea urchin egg cytosol. Subsequent fractionation of ATP eluates of these MTs yields a protein of relative molecular mass 330 x 10(3) that behaves as a complex of three polypeptides that are distinct from conventional kinesin subunits or fragments thereof. This complex contains 85 kDa and 95 kDa polypeptides, which react with our peptide antibodies, and a 115 kDa polypeptide, which does not. This triplet of polypeptides, which we refer to as KRP(85/95), binds to purified sea urchin egg tubulin in an AMPPNP-enhanced, ATP-sensitive manner and induces the formation of microtubule bundles. We therefore propose that the triplet corresponds to a novel sea urchin egg kinesin-related protein.
Recent evidence has suggested that the principal polypeptide component of the microtubule motor protein kinesin may be a member of an extended superfamily of related motor proteins. To gain insight into how large the kinesin superfamily might be and to begin determining the potential functions in which various superfamily members might participate, we identified and partially characterized six additional members of the Drosophila kinesin superfamily. Genes encoding these proteins were identified by using the polymerase chain reaction with degenerate primers corresponding to highly conserved regions of the kinesin heavy-chain motor domain. Partial sequencing of the six genes revealed that they encode proteins that are 40-60% identical to the motor domain of the kinesin heavy-chain sequence. The cytogenetic locations as well as the developmental and tissue-specific expression patterns have been determined. The data suggest that each of these six kinesin-like proteins may have functions in a wide variety of cell types and tissues.
The distribution of membrane-bound organelles was studied in cultured hippocampal neurons after antisense oligonucleotide suppression of the kinesin-heavy chain (KHC). We observed reduced 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) fluorescent staining in neurites and growth cones. In astrocytes, KHC suppression results in the disappearance of the DiOC6(3)-positive reticular network from the cell periphery, and a parallel accumulation of label within the cell center. On the other hand, mitochondria microtubules and microfilaments display a distribution that closely resembles that observed in control cells. KHC suppression of neurons and astrocytes completely inhibited the Brefeldin A-induced spreading and tubulation of the Golgi-associated structure enriched in mannose-6-phosphate receptors. In addition, KHC suppression prevents the low pH-induced anterograde redistribution of late endocytic structures. Taken collectively, these observations suggest that in living neurons, kinesin mediates the anterograde transport of tubulovesicular structures originated in the central vacuolar system (e.g., the endoplasmic reticulum) and that the regulation of kinesin-membrane interactions may be of key importance for determining the intracellular distribution of selected organelles.
The Chlamydomonas FLA10 gene was shown to encode a flagellar kinesin-like protein (Walther, Z., M. Vashishtha, and J.L. Hall. 1994. J. Cell Biol. 126:175-188). By using a temperature-sensitive allele of FLA10, we have determined that the FLA10 protein is necessary for both the bidirectional movement of polystyrene beads on the flagellar membrane and intraflagellar transport (IFT), the bidirectional movement of granule-like particles beneath the flagellar membrane (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. (USA). 90:5519-5523). In addition, we have correlated the presence and position of the IFT particles visualized by light microscopy with that of the electron dense complexes (rafts) observed beneath the flagellar membrane by electron microscopy. A role for FLA10 in submembranous or flagellar surface motility is also strongly supported by the immunolocalization of FLA10 to the region between the axonemal outer doublet microtubules and the flagellar membrane.
The microtubules (MTs) within neuronal processes are highly organized with regard to their polarity and yet are not attached to any detectable nucleating structure. Axonal MTs are uniformly oriented with their plus ends distal to the cell body, whereas dendritic MTs are of both orientations. Here, we sought to test the capacity of motor-driven MT transport to organize distinct MT patterns during process outgrowth. We focused on CHO1/MKLP1, a kinesin-related protein present in the midzonal region of the mitotic spindle where MTs of opposite orientation overlap. Insect ovarian Sf9 cells induced to express the N-terminal portion of the molecule form MT-rich processes with a morphology similar to that of neuronal dendrites (Kuriyama et al., 1994). Nascent processes contain uniformly plus-end-distal MTs, but these are joined by minus-end-distal MTs as the processes continue to develop. Thus, this CHO1/MKLP1 fragment establishes a nonuniform MT polarity pattern and does so by a similar sequence of events as occurs with the dendrite, the antecedent of which is a short process with a uniform MT polarity orientation. Two lines of evidence suggest that these results are elicited by motor-driven MT transport. First, there is a depletion of MTs from the cell body during process outgrowth. Second, the same polarity pattern is obtained when net MT assembly is suppressed pharmacologically during process formation. Collectively, these findings provide precedent for the idea that motor-driven transport can organize MTs into distinct patterns of polarity orientation during process outgrowth.
The quintessential feature of the dendritic microtubule array is its nonuniform pattern of polarity orientation. During the development of the dendrite, a population of plus end-distal microtubules first appears, and these microtubules are subsequently joined by a population of oppositely oriented microtubules. Studies from our laboratory indicate that the latter microtubules are intercalated within the microtubule array by their specific transport from the cell body of the neuron during a critical stage in development (Sharp, D.J., W. Yu, and P.W. Baas. 1995. J. Cell Biol. 130:93- 104). In addition, we have established that the mitotic motor protein termed CHO1/MKLP1 has the appropriate properties to transport microtubules in this manner (Sharp, D.J., R. Kuriyama, and P.W. Baas. 1996. J. Neurosci. 16:4370-4375). In the present study we have sought to determine whether CHO1/MKLP1 continues to be expressed in terminally postmitotic neurons and whether it is required for the establishment of the dendritic microtubule array. In situ hybridization analyses reveal that CHO1/MKLP1 is expressed in postmitotic cultured rat sympathetic and hippocampal neurons. Immunofluorescence analyses indicate that the motor is absent from axons but is enriched in developing dendrites, where it appears as discrete patches associated with the microtubule array. Treatment of the neurons with antisense oligonucleotides to CHO1/MKLP1 suppresses dendritic differentiation, presumably by inhibiting the establishment of their nonuniform microtubule polarity pattern. We conclude that CHO1/MKLP1 transports microtubules from the cell body into the developing dendrite with their minus ends leading, thereby establishing the nonuniform microtubule polarity pattern of the dendrite.
Heterotrimeric kinesin-II is a plus end- directed microtubule (MT) motor protein consisting of distinct heterodimerized motor subunits associated with an accessory subunit. To probe the intracellular transport functions of kinesin-II, we microinjected fertilized sea urchin eggs with an anti-kinesin-II monoclonal antibody, and we observed a dramatic inhibition of ciliogenesis at the blastula stage characterized by the assembly of short, paralyzed, 9+0 ciliary axonemes that lack central pair MTs. Control embryos show no such defect and form swimming blastulae with normal, motile, 9+2 cilia that contain kinesin-II as detected by Western blotting. Injection of anti-kinesin-II into one blastomere of a two-cell embryo leads to the development of chimeric blastulae covered on one side with short, paralyzed cilia, and on the other with normal, beating cilia. We observed a unimodal length distribution of short cilia on anti-kinesin-II-injected embryos corresponding to the first mode of the trimodal distribution of ciliary lengths observed for control embryos. This short mode may represent a default ciliary assembly intermediate. We hypothesize that kinesin-II functions during ciliogenesis to deliver ciliary components that are required for elongation of the assembly intermediate and for formation of stable central pair MTs. Thus, kinesin-II plays a critical role in embryonic development by supporting the maturation of nascent cilia to generate long motile organelles capable of producing the propulsive forces required for swimming and feeding.
Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca2+-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca2+/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin- dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca2+-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain.
We previously described a kinesin-dependent movement of particles in the flagella of Chlamydomonas reinhardtii called intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519-5523). When IFT is inhibited by inactivation of a kinesin, FLA10, in the temperature-sensitive mutant, fla10, existing flagella resorb and new flagella cannot be assembled. We report here that: (a) the IFT-associated FLA10 protein is a subunit of a heterotrimeric kinesin; (b) IFT particles are composed of 15 polypeptides comprising two large complexes; (c) the FLA10 kinesin-II and IFT particle polypeptides, in addition to being found in flagella, are highly concentrated around the flagellar basal bodies; and, (d) mutations affecting homologs of two of the IFT particle polypeptides in Caenorhabditis elegans result in defects in the sensory cilia located on the dendritic processes of sensory neurons. In the accompanying report by Pazour, G.J., C.G. Wilkerson, and G.B. Witman (1998. J. Cell Biol. 141:979-992), a Chlamydomonas mutant (fla14) is described in which only the retrograde transport of IFT particles is disrupted, resulting in assembly-defective flagella filled with an excess of IFT particles. This microtubule- dependent transport process, IFT, defined by mutants in both the anterograde (fla10) and retrograde (fla14) transport of isolable particles, is probably essential for the maintenance and assembly of all eukaryotic motile flagella and nonmotile sensory cilia.
Several enzymes, including cytoplasmic and flagellar outer arm dynein, share an Mr 8,000 light chain termed LC8. The function of this chain is unknown, but it is highly conserved between a wide variety of organisms. We have identified deletion alleles of the gene (fla14) encoding this protein in Chlamydomonas reinhardtii. These mutants have short, immotile flagella with deficiencies in radial spokes, in the inner and outer arms, and in the beak-like projections in the B tubule of the outer doublet microtubules. Most dramatically, the space between the doublet microtubules and the flagellar membrane contains an unusually high number of rafts, the particles translocated by intraflagellar transport (IFT) (Kozminski, K.G., P.L. Beech, and J.L. Rosenbaum. 1995. J. Cell Biol. 131:1517-1527). IFT is a rapid bidirectional movement of rafts under the flagellar membrane along axonemal microtubules. Anterograde IFT is dependent on a kinesin whereas the motor for retrograde IFT is unknown. Anterograde IFT is normal in the LC8 mutants but retrograde IFT is absent; this undoubtedly accounts for the accumulation of rafts in the flagellum. This is the first mutation shown to specifically affect retrograde IFT; the fact that LC8 loss affects retrograde IFT strongly suggests that cytoplasmic dynein is the motor that drives this process. Concomitant with the accumulation of rafts, LC8 mutants accumulate proteins that are components of the 15-16S IFT complexes (Cole, D.G., D.R. Deiner, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993-1008), confirming that these complexes are subunits of the rafts. Polystyrene microbeads are still translocated on the surface of the flagella of LC8 mutants, indicating that the motor for flagellar surface motility is different than the motor for retrograde IFT.
Kinesin is believed to generate force for the movement of organelles in anterograde axonal transport. The identification of
genes that encode kinesin-like proteins suggests that other motors may provide anterograde force instead of or in addition
to kinesin. To gain insight into the specific functions of kinesin, the effects of mutations in the kinesin heavy chain gene
(khc) on the physiology and ultrastructure of Drosophila larval neurons were studied. Mutations in khc impair both action
potential propagation in axons and neurotransmitter release at nerve terminals but have no apparent effect on the concentration
of synaptic vesicles in nerve terminal cytoplasm. Thus kinesin is required in vivo for normal neuronal function and may be
active in the transport of ion channels and components of the synaptic release machinery to their appropriate cellular locations.
Kinesin appears not to be required for the anterograde transport of synaptic vesicles or their components.
unc-104 encodes a novel kinesin paralog that may act as a microtubule-based motor in the nervous system. Neuronal cell lineages and axonogenesis are normal in unc-104 null mutants, but axons have few synaptic vesicles and make only a few small synapses. By contrast, neuron cell bodies have surfeits of similar vesicles tethered together within the cytoplasm. Based on behavioral and cellular phenotypes, we suggest that UNC-104 is a neuron-specific motor used for anterograde translocation of synaptic vesicles along axonal microtubules. Other membrane-bounded organelles are transported normally.
Eight classes of chemosensory neurons in C. elegans fill with fluorescein when living animals are placed in a dye solution. Fluorescein enters the neurons through their exposed sensory cilia. Mutations in 14 genes prevent dye uptake and disrupt chemosensory behaviors. Each of these genes affects the ultrastructure of the chemosensory cilia or their accessory cells. In each case, the cilia are shorter or less exposed than normal, suggesting that dye contact is the principal factor under selection. Ten genes affect many or all of the sensory cilia in the head. The daf-19 (m86) mutation eliminates all cilia, leaving only occasional centrioles in the dendrites. The cilia in che-13 (e1805), osm-1 (p808), osm-5 (p813), and osm-6 (p811) mutants have normal transition zones and severely shortened axonemes. Doublet-microtubules, attached to the membrane by Y links, assemble ectopically proximal to the cilia in these mutants. The amphid cilia in che-11 (e1810) are irregular in diameter and contain dark ground material in the middle of the axonemes. Certain mechanocilia are also affected. The amphid cilia in che-10 (e1809) apparently degenerate, leaving dendrites with bulb-shaped endings filled with dark ground material. The mechanocilia lack striated rootlets. Cilia defects have also been found in che-2, che-3, and daf-10 mutants. The osm-3 (p802) mutation specifically eliminates the distal segment of the amphid cilia. Mutations in three genes affect sensillar support cells. The che-12 (e1812) mutation eliminates matrix material normally secreted by the amphid sheath cell. The che-14 (e1960) mutation disrupts the joining of the amphid sheath and socket cells to form the receptor channel. A similar defect has been observed in daf-6 mutants. Four additional genes affect specific classes of ciliated sensory neurons. The mec-1 and mec-8 (e398) mutations disrupt the fasciculation of the amphid cilia. The cat-6 (e1861) mutation disrupts the tubular bodies of the CEP mechanocilia. A cryophilic thermotaxis mutant, ttx-1 (p767), lacks fingers on the AFD dendrite, suggesting this neuron is thermosensory.
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.
Axoplasm from the squid giant axon contains a soluble protein translocator that induces movement of microtubules on glass, latex beads on microtubules, and axoplasmic organelles on microtubules. We now report the partial purification of a protein from squid giant axons and optic lobes that induces these microtubule-based movements and show that there is a homologous protein in bovine brain. The purification of the translocator protein depended primarily on its unusual property of forming a high affinity complex with microtubules in the presence of a nonhydrolyzable ATP analog, adenylyl imidodiphosphate. The protein, once released from microtubules with ATP, migrates on gel filtration columns with an apparent molecular weight of 600 kilodaltons and contains 110-120 and 60-70 kilodalton polypeptides. This protein is distinct in molecular weight and enzymatic behavior from myosin or dynein, which suggests that it belongs to a novel class of force-generating molecules, for which we propose the name kinesin.
We have identified and characterized 95 mutations that reduce or abolish dye filling of amphid and phasmid neurons and that have little effect on viability, fertility or movement. Twenty-seven mutations occurred spontaneously in strains with a high frequency of transposon insertion. Sixty-eight were isolated after treatment with EMS. All of the mutations result in defects in one or more chemosensory responses, such as chemotaxis to ammonium chloride or formation of dauer larvae under conditions of starvation and overcrowding. Seventy-five of the mutations are alleles of 12 previously defined genes, mutations which were previously shown to lead to defects in amphid ultrastructure. We have assigned 20 mutations to 13 new genes, called dyf-1 through dyf-13. We expect that the genes represented by dye-filing defective mutants are important for the differentiation of amphid and phasmid chemosensilla.
Kinesin heavy chain and kinesin-related polypeptides (KRPs) comprise a family of motor proteins with diverse intracellular transport functions. Using pan-kinesin peptide antibodies that react with these proteins, we have previously purified from sea urchin eggs a trimeric microtubule-binding and bundling protein, KRP (85/95) (ref. 8) comprising subunits of M(r) 115,000 (115K), 95K and 85K. We report here that kinesin-related genes encode the 85K and 95K subunits, and that the protein can be immunoprecipitated from cytosol as a trimeric complex using an 85K monoclonal antibody. We also find that purified KRP(85/95) directs movements towards the 'plus' ends of microtubules. To our knowledge, this protein is the first kinesin-related motor to be purified from its natural host cell in a native multimeric state.
To investigate the possibility that kinesin transports vesicles bearing proteins essential for ion channel activity, the effects of kinesin (Khc) and ion channel mutations were compared in Drosophila using established tests. Our results show that Khc mutations produce defects and genetic interactions characteristic of paralytic (para) and maleless (mle) mutations that cause reduced expression or function of the alpha-subunit of voltage-gated sodium channels. Like para and mle mutations, Khc mutations cause temperature-sensitive (TS) paralysis. When combined with para or mle mutations, Khe mutations cause synthetic lethality and a synergistic enhancement of TS-paralysis. Furthermore, Khc: mutations suppress Shaker and ether-a-go-go mutations that disrupt potassium channel activity. In light of previous physiological tests that show that Khc mutations inhibit compound action potential propagation in segmental nerves, these data indicate that kinesin activity is required for normal inward sodium currents during neuronal action potentials. Tests for phenotypic similarities and genetic interactions between kinesin and sodium/potassium ATPse mutations suggest that impaired kinesin function does not affect the driving force on sodium ions. We hypothesize that a loss of kinesin function inhibits the anterograde axonal transport of vesicles bearing sodium channels.
Cells transport and sort proteins and lipids, after their synthesis, to various destinations at appropriate velocities in
membranous organelles and protein complexes. Intracellular transport is thus fundamental to cellular morphogenesis and functioning.
Microtubules serve as a rail on which motor proteins, such as kinesin and dynein superfamily proteins, convey their cargoes.
This review focuses on the molecular mechanism of organelle transport in cells and describes kinesin and dynein superfamily
proteins.
The kinesin superfamily comprises a large and structurally diverse group of microtubule-based motor proteins that produce a variety of force-generating activities within cells. This review addresses how the structures of kinesin proteins provide clues as to their biological functions and motile properties. We discuss structural features common to all kinesin motors, as well as specialized features that enable subfamilies of related motors to carry out specialized activities. We also discuss how the kinesin motor domain uses chemical energy from ATP hydrolysis to move along microtubules.
Chemosensation in the nervous system of the nematode Caenorhabditis elegans depends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia.
Intraflagellar transport (IFT) is important in the formation and maintenance of many cilia, such as the motile cilia that drive the swimming of cells and embryos, the nodal cilia that generate left-right asymmetry in vertebrate embryos, and the sensory cilia that detect sensory stimuli in some animals. The heterotrimeric kinesin-II motor protein drives the anterograde transport of macromolecular complexes, called rafts, along microtubule tracks from the base of the cilium to its distal tip, whereas cytoplasmic dynein moves the rafts back in the retrograde direction. We have used fluorescence microscopy to visualize for the first time the intracellular transport of a motor and its cargo in vivo. We observed the anterograde movement of green fluorescent protein (GFP)-labelled kinesin-II motors and IFT rafts within sensory cilia on chemosensory neurons in living Caenorhabditis elegans.
Members of the kinesin family of motor proteins are assembled from kinesin-related polypeptides that share conserved 'motor' domains linked to diverse 'tail' domains. Recent work suggests that tail diversity underlies the differences in quaternary structure observed among native kinesin holoenzymes.
Dyneins: molecular structure and cellular functions
- E Holzbauer
- R B Vallee
Holzbauer, E. & Vallee, R.B. (1994) Dyneins: molecular structure and cellular functions. Ann. Rev.
Cell Biol. 10, 339-372
Identification of a MT-associated motor protein essential for dendritic differentiation
- D J Sharp
- W Yu
- L Ferhat
- R Kuriyama
- D C Rueger
- P W Bass
Sharp, D.J., Yu, W., Ferhat, L., Kuriyama, R., Rueger, D.C. & Bass, P.W. (1997) Identification of a
MT-associated motor protein essential for dendritic differentiation. J. Cell Biol. 138, 833-843