BIOLOGICAL OVERVIEW

Kinesin is expressed in virtually all cells of both vertebrates and invertebrates. The majority of kinesin appears to be free in cytosol, but various studies have shown that it can associate with endoplasmic recticulum (ER), vesicles, mitochondria, and other organelles. Function disruption tests indicate that it is critical for fast organelle transport in axons, although the set of cargoes it carries is not clearly defined (see for example Goldstein, 2000). Other studies of non-neuronal cells or cell-free systems suggest that kinesin is important for the positioning of lysosomes, mitochondria, and ER. It is also thought to be important for vesicle transport from the Golgi to the plasma membrane, one of the late steps in the secretion pathway, and in Golgi-to-ER membrane recycling, which is an indirect but essential part of the early secretion pathway. For example, based on the effects of anti-kinesin heavy chain antibodies or a KHC tail fragment microinjected into sea urchin embryos, kinesin has been proposed to be important for outward transport, to the cortex, of a class of vesicles used for rapid membrane repair. Furthermore, based on immunolocalization and on the effects of anti-KHC antibodies on brefeldin A-induced Golgi-to-ER membrane transport in vertebrate cultured cells, kinesin has been proposed to be the motor for Golgi-to-ER membrane recycling (R. P. Brendza, 2000a and references therein).

Kinesin heavy chain (Khc) is the plus end directed microtubule motor protein of Drosophila. Conventional kinesin (often referred to simply as kinesin) is an abundant microtubule motor protein that functions in a number of important intracellular transport processes. Kinesin is a heterotetramer, comprised of 2 kinesin heavy chains and 2 light chains. The heavy chains dimerize to form an elongated stalk with 2 amino-terminal globular domains ('motor domains') at one end and the light chains at the other end. The light chains and stalk are expected to bind the cytoplasmic cargoes that conventional kinesin transports. Each motor domain couples a cycle of ATP turnover to conformational changes and a cycle of microtubule binding and release that generates displacement toward the microtubule plus end. A single motor domain is not processive. In contrast, dimerized motor domains are remarkably processive, making hundreds of steps before releasing from the microtubule. This feature is probably critical for the resolute transport of small organelles that can present only one or a few kinesin molecules to a microtubule (Brendza, 1999 and references therein).

The Kinesin heavy chain moves its cytoplasmic cargo toward the plus ends of microtubules. Microtubules are relatively long, straight polymers that course throughout the cytoplasm. In undifferentiated and in fibroblastoid cell types, the minus ends of microtubules are usually near the cell center, whereas the plus ends are usually near the periphery. This is consistent with the idea that most microtubule-based, outward vesicle movements (Golgi-to-ER or Golgi-to-plasma membrane) are driven by plus end-directed kinesins, and most inward movements are driven by cytoplasmic dyneins. In differentiated cells, a variety of microtubule orientations are seen. For instance, in the axons of neurons, almost all plus ends are away from the cell center and toward the terminal. In some but not all polarized epithelial cells, microtubules are oriented with their plus ends toward the basal pole and their minus ends near the apical pole. In such situations, in which the polarity is relatively uniform, models have been developed that invoke appropriate microtubule motors for various steps in vesicle or other organelle transport (R. P. Brendza, 2000a and references therein).

To address questions about kinesin function in motility processes, other than axonal transport, in an intact organism, an examination was carried out of the effects of recessive, Khc null mutations on various cell types in Drosophila. When the entire organism is homozygous for a null mutation, it becomes paralyzed and dies during the midlarval stage, preventing studies of Khc function during the remainder of development. To circumvent this lethality, a mitotic recombination strategy was used to generate chimeras with a few homozygous null cells in otherwise healthy heterozygous organisms. The fates of the descendants of such Khc null cells were then studied by light and electron microscopy. A focus was placed on two predictions of the hypothesis that kinesin is an important motor for vesicle transport in the late secretory and recycling pathways. (1) Because disruption of vesicle traffic should block membrane growth and thus cell proliferation, the proliferation of Khc null imaginal cells was assessed. Surprisingly, the null cells proliferate normally to produce large clones of adult cells. (2) Because disruption of vesicle traffic can cause striking changes in the organization of ER and Golgi as well as defective secretion, postmitotic cells that rely heavily on the secretory pathway were studied in detail. Defects consistent with a role for kinesin in the Golgi-to-ER recycling pathway are not seen. However, defects were seen consistent with a role for kinesin in long-distance late secretory vesicle transport (Golgi-to-plasma membrane) (R. P. Brendza, 2000a).

A mechanosensory bristle shaft forms as a fluted cylinder of cuticle around a long cytoplasmic extension that projects outward from a trichogen cell body. During bristle shaft differentiation, which occurs in pupae, the core of the extension contains many parallel microtubules running from the base toward the tip. Because the Golgi and nucleus of the trichogen are located in the cell body beneath the pupal epidermis, it is likely that secretion of the shaft cuticle components requires a great deal of vesicular traffic from the Golgi into and along the shaft-forming extension. The mechanosensory bristles in Khc^null wing clones are sometimes kinked. To examine possible cuticle secretion defects in more detail, Khc^null clones were examined throughout the adult epidermis. The longest null bristles had defects that were obvious even with a low-power light microscope. Some lay flat or twisted along the epidermal sheet rather than projecting outward from its surface. A number of those that did project outward were tested by direct mechanical manipulation. Beheaded flies will remain viable for several days in a moist chamber. They stand motionless but can respond to bristle deflections with reflex grooming behaviors (R. P. Brendza, 2000a).

Outside of test clones, the deflection of individual bristles with a tungsten needle caused normal pivoting at the base and elicited grooming reflexes. Inside test clones, bristles were so flaccid that attempted deflections usually caused a bend or kink rather than the pivoting needed to elicit a grooming reflex. Bristles from null and control clones were studied in detail by SEM. Khc^null bristles have a variety of structural defects. The longest test class bristles, the scutellar macrochaetae (300-400 µm), are usually ~20% shorter than the analogous wild-type bristles. This length defect is less evident in shorter macrochaetae and is not seen in microchaetae. The tips of test bristles are often contorted, and the contortions are most severe at the tips of long machrochaetae, which always exhibit flattened, flared, or twisted tips. Mutant microchaetae (65-70 µm) show less dramatic defects, such as bluntness or slight tip swelling. No defects are observed in the remainder of the integument, including the bristle sockets, the tiny (10-15 µm) nonsensory hairs of the epidermal cells, or the epidermal cuticle sheet. The severities of head, thoracic, and abdominal bristle defects were not detectably affected by clone size (R. P. Brendza, 2000a).

The evident weakness of Khc^null bristle shafts suggests that cuticle secretion from the shaft-forming extensions of trichogen cells is defective during differentiation. Consistent with this, SEM images showed defects in cuticle fluting. To study bristle cuticle pattern and thickness in more detail, serial cross-sections of wild-type and Khc^null scutellar bristles were compared by TEM. Overall, the cuticle layers of null bristles are quite thin. This effect is more pronounced at the tips of bristles than at their bases. These results suggest that KHC is critical for long-distance transport of secretory vesicles that bear cuticle precursors from the Golgi into and along the bristle shaft-forming extension. However, the fact that some cuticle secretion occurs even at the tips of the longest null bristles suggests that vesicle transport can continue at a low level despite the absence of KHC (R. P. Brendza, 2000a).

Conventional kinesin has long been suspected of being a vesicle motor. Initially this stemmed from its discovery in axoplasm, which is rich in Golgi-derived transport vesicles, and its co-localization with vesicles in cultured cells. A number of studies have focused on the identification of specific types of vesicles that kinesin might carry, but the results have not provided a consistent answer. For example, in a study of vesicle/tubule transport in the recycling pathway, antibody inhibition of KHC blocked brefeldin A-induced movement of Golgi membranes into the ER in cultured NRK cells. Conversely, antisense oligonucleotide inhibition of KHC in cultured rat astrocytes and gene disruption in cultured mouse extraembryonic cells does not prevent brefeldin A-induced Golgi-to-ER membrane transfer. With regard to Golgi-to-plasma membrane vesicle transport, antisense oligonucleotide inhibition of KHC in cultured vertebrate neurons impairs delivery of vesicles containing certain synaptic proteins to axon terminals. In contrast, Khc mutations in Drosophila (Gho, 1992) and Caenorhabditis elegans do not prevent the accumulation of normal levels of synaptic vesicles at axon terminals (R. P. Brendza, 2000a and references therein).

It has also been proposed that conventional kinesin is a motor for other elements of the cytoplasm, including mitochondria, lysosomes, ER, and intermediate filaments (Yabe, 1999 and reviews by Goodson, 1997; Lane, 1998; Goldstein, 1999). However, function disruption tests have again yielded conflicting data. Antisense oligonucleotide inhibition of KHC in cultured rat astrocytes causes a retraction of the ER network. In contrast, antibody inhibition of KHC in sea urchin embryonic cells or gene disruption in mouse extraembryonic cells has no dramatic effects on ER organization. Antibody inhibition of KHC in human fibroblasts and gene disruption in mouse extraembryonic cells causes mitochondria, which are normally dispersed throughout the cytoplasm, to cluster near the cell center. However, no effect on mitochondrial distribution is seen in rat astrocytes injected with Khc antisense oligonucleotides or in mutant strains of C. elegans and fungi (R. P. Brendza, 2000a and references therein).

Overall, in the context of the cells and processes examined in Drosophila, the results suggest (1) that conventional kinesin is not essential for vesicle movement from the Golgi to either the ER or the plasma membrane in most cells; (2) that it is important for proper long-range vesicle transport in some elongated cells, and (3) that it is not required for the proper distribution of ER or mitochondria, at least in photoreceptor cells. Comparisons of control and Khc^null clones indicate that in the undifferentiated cells of imaginal discs, neither the rates nor the extents of cell proliferation are affected by a depletion of KHC. This should put to rest any lingering suspicion that conventional kinesin might have an essential role in mitosis. Furthermore, this comparision shows that conventional kinesin is not essential for any of the interphase motility processes required for imaginal cell growth. Whether the positioning of ER, lysosomes, or mitochondria is critical for cell growth is not clear. However, it is clear that vesicle transport in the secretory pathway is essential. For instance, in S. cereviseae, mutations that inhibit the Golgi-to-ER recycling pathway or the Golgi-to-plasma membrane 'late' secretory pathway cause a rapid halt to cell growth and division. More directly relevant to the results of this study, when mitotic recombination was used in Drosophila to create cells null for Rop, a homolog of the late secretory gene SEC1 of yeast, imaginal cells could not proliferate sufficiently to produce clones of adult cells. Mutations in syntaxin, which encode a t-SNARE thought to interact with ROP protein, also block imaginal cell proliferation. Thus, the proliferation and development of Drosophila imaginal cells is indeed sensitive to disruption of known elements of the secretion pathway. The fact that imaginal cell proliferation is not affected by a loss of KHC suggests that both the recycling pathway and the late secretion pathway can operate at normal rates with little or no conventional kinesin in small cells (R. P. Brendza, 2000a and references therein).

Defects in eye differentiation caused by a loss of KHC were mild and again are not consistent with major secretory pathway defects. Previous work has shown that disruption of the secretory pathway in photoreceptors should cause easily recognized changes in ultrastructure. For example, expression in the adult eye of a dominant negative form of a protein thought to function early in the secretory pathway, Rab1, causes hypertrophy and swelling of the ER, vesiculation or absence of the Golgi, and dramatic shrinkage of rhabdomeres. Shrunken rhabdomeres are also caused by mutations that block the transport of vesicles bearing rhodopsin from the Golgi to the plasma membrane. Although a loss of KHC did cause some mild structural defects in a few photoreceptor cell bodies, it did not cause shrunken rhabdomeres or any major changes in the organization of cytoplasmic organelles, including mitochondria, the nucleus, ER, and Golgi (R. P. Brendza, 2000a and references therein).

If conventional kinesin is dispensable for the secretion pathway in imaginal and retinal cells, how is vesicle movement away from the Golgi accomplished? Four alternative and nonexclusive force-generating systems come to mind: diffusion, minus end-directed microtubule motors, cytoplasmic myosins, and plus end-directed kinesin-related proteins. In some differentiated vertebrate epithelial cells, microtubule arrays have mixed polarity with some minus ends near the apical cortex and some near the cell center. Therefore, minus end-directed motors such as cytoplasmic dynein might be sufficient for vesicle transport in both directions. The involvement of plus end-directed kinesin-related motors is an attractive possibility. There are several different types of motors in the kinesin superfamily that might function in the recycling and late secretion pathways (reviewed by Goldstein, 1999). Whether these motors are expressed in imaginal cells is uncertain. However, it is reasonable to think that one or more of them is present and active in vesicle transport (R. P. Brendza, 2000a and references therein).

The elongated trichogen cells that create mechanosensory bristles (~1-2 × 100-400 µm) clearly do require conventional kinesin for normal secretion of cuticle precursors. Previous studies of another elongated Drosophila cell type, the larval motor neuron (~0.3 × 300-2000 µm), have shown that conventional kinesin is important in axons for membrane excitability, terminal growth, neurotransmitter secretion, and fast organelle transport (Gho, 1992; Hurd, 1996a; Hurd, 1996b; Gindhart, 1998). In the shaft-forming extensions of trichogen cells, as in axons, parallel arrays of microtubules are prominent. In both motor axons and bristles, the loss of KHC has a graded effect; distal regions are most strongly affected, and the defects become more severe as cell length increases (Gho, 1992; Hurd, 1996a; R. P. Brendza, 2000a). These observations suggest that conventional kinesin function is especially important for long-range vesicle movements, processes that are likely to demand efficient, highly processive transport machinery (R. P. Brendza, 2000a).

It is possible that conventional kinesin is a 'specialty motor,' a major contributor only to long-distance transport in specialized cells, whereas more ordinary motility is accomplished by other motors, such as cytoplasmic myosins and kinesin-related proteins. The evolutionary conservation of KHC in metazoans and its ubiquitous expression in both undifferentiated and differentiated cell types argue against this hypothesis, but it remains possible. Alternatively, conventional kinesin could be a more general motor, combining with kinesin-related proteins and myosins as a contributor to both short- and long-range movement of a variety of organelles. In this case, the other organelle/vesicle motors could compensate for the absence of kinesin sufficiently to prevent dramatic defects in cells with transport tracks of normal length. However, as track length and the requirement for transport efficiency increases, the lack of kinesin would cause progressively more severe defects. That is consistent with what has been seen in the R. P. Brendza (2000a) study. Some of the conflicting results seen in KHC function disruption tests (Goodson, 1997; Lane, 1998) could then be due to differences in cell geometry and to variations in the sets of myosins and kinesin-related motors that are expressed in the different systems studied; that is, kinesin gets more or less support from other motors depending on cell size, cell type, and perhaps culture conditions. In either case, as has been found for mitotic chromosome movements, it is probable that interphase organelle/vesicle movements are driven by the cooperative activities of multiple types of motors and that monogamous motor-cargo relationships in common transport processes are rare. For both chromosomes and interphase organelles, a clear view of such cooperative or multilayered transport systems will require rigorous definitions of specific motor-cargo relationships, including linkage mechanisms and regulatory controls (R. P. Brendza, 2000a).

GENE STRUCTURE

cDNA clone length - 3547

Bases in 5' UTR - 320

Exons - 4

Bases in 3' UTR - 299

PROTEIN STRUCTURE

Amino Acids - 975

Structural Domains

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, it is proposed 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 (Yang, 1989).

date revised: 12 April 2001

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