Microtubules, motors, and mRNA localization mechanisms: watching fluorescent messages move.

Proper spatial and temporal localization of specific mRNAs is pivotal in the early stages of development. To dissect the mechanisms of localization, several groups are employing advanced fluorescence microscopy to track RNA movements in live oocytes and embryos.

Control of Drosophila perineurial glial growth by interacting neurotransmitter-mediated signaling pathways.

Drosophila peripheral nerves, similar structurally to the peripheral nerves of mammals, comprise a layer of axons and inner glia, surrounded by an outer perineurial glial layer. Although it is well established that intercellular communication occurs among cells within peripheral nerves, the signaling pathways used and the effects of this signaling on nerve structure and function remain incompletely understood. Here we demonstrate with genetic methods that the Drosophila peripheral nerve is a favorable system for the study of intercellular signaling. We show that growth of the perineurial glia is controlled by interactions among five genes: ine, which encodes a putative neurotransmitter transporter; eag, which encodes a potassium channel; push, which encodes a large, Zn(2+)-finger-containing protein; amn, which encodes a putative neuropeptide related to the pituitary adenylate cyclase activator peptide; and NF1, the Drosophila ortholog of the human gene responsible for type 1 neurofibromatosis. In other Drosophila systems, push and NF1 are required for signaling pathways mediated by Amn or the pituitary adenylate cyclase activator peptide. Our results support a model in which the Amn neuropeptide, acting through Push and NF1, inhibits perineurial glial growth, whereas the substrate neurotransmitter of Ine promotes perineurial glial growth. Defective intercellular signaling within peripheral nerves might underlie the formation of neurofibromas, the hallmark of neurofibromatosis.

A function for kinesin I in the posterior transport of oskar mRNA and Staufen protein.

The asymmetric localization of messenger RNA (mRNA) and protein determinants plays an important role in the establishment of complex body plans. In Drosophila oocytes, the anterior localization of bicoid mRNA and the posterior localization of oskar mRNA are key events in establishing the anterior-posterior axis. Although the mechanisms that drive bicoid and oskar localization have been elusive, oocyte microtubules are known to be essential. Here we report that the plus end-directed microtubule motor kinesin I is required for the posterior localization of oskar mRNA and an associated protein, Staufen, but not for the anterior-posterior localization of other asymmetric factors. Thus, a complex containing oskar mRNA and Staufen may be transported along microtubules to the posterior pole by kinesin I.

A kinesin mutation that uncouples motor domains and desensitizes the gamma-phosphate sensor.

Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr(291) of Drosophila kinesin heavy chain is identical in all superfamily members and is located in alpha-helix 5 on the microtubule-binding surface of the catalytic motor domain. Substitution of methionine at Thr(291) results in complete loss of function in vivo. In vitro, the T291M mutation disrupts the ATPase cross-bridge cycle of a kinesin motor/neck construct, K401-4 (Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514). The pre-steady-state kinetic analysis presented here shows that ATP binding is weakened significantly, and the rate of ATP hydrolysis is increased. The mutant motor also fails to distinguish ATP from ADP, suggesting that the contacts important for sensing the gamma-phosphate have been altered. The results indicate that there is a signaling defect between the motor domains of the T291M dimer. The ATPase cycles of the two motor domains appear to become kinetically uncoupled, causing them to work more independently rather than in the strict, coordinated fashion that is typical of kinesin.

Clonal tests of conventional kinesin function during cell proliferation and differentiation.

Null mutations in the Drosophila Kinesin heavy chain gene (Khc), which are lethal during the second larval instar, have shown that conventional kinesin is critical for fast axonal transport in neurons, but its functions elsewhere are uncertain. To test other tissues, single imaginal cells in young larvae were rendered null for Khc by mitotic recombination. Surprisingly, the null cells produced large clones of adult tissue. The rates of cell proliferation were not reduced, indicating that conventional kinesin is not essential for cell growth or division. This suggests that in undifferentiated cells vesicle transport from the Golgi to either the endoplasmic reticulum or the plasma membrane can proceed at normal rates without conventional kinesin. In adult eye clones produced by null founder cells, there were some defects in differentiation that caused mild ultrastructural changes, but they were not consistent with serious problems in the positioning or transport of endoplasmic reticulum, mitochondria, or vesicles. In contrast, defective cuticle deposition by highly elongated Khc null bristle shafts suggests that conventional kinesin is critical for proper secretory vesicle transport in some cell types, particularly ones that must build and maintain long cytoplasmic extensions. The ubiquity and evolutionary conservation of kinesin heavy chain argue for functions in all cells. We suggest interphase organelle movements away from the cell center are driven by multilayered transport mechanisms; that is, individual organelles can use kinesin-related proteins and myosins, as well as conventional kinesin, to move toward the cell periphery. In this case, other motors can compensate for the loss of conventional kinesin except in cells that have extremely long transport tracks.

Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport.

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.

Lethal kinesin mutations reveal amino acids important for ATPase activation and structural coupling.

To study the relationship between conventional kinesin's structure and function, we identified 13 lethal mutations in the Drosophila kinesin heavy chain motor domain and tested a subset for effects on mechanochemistry. S246F is a moderate mutation that occurs in loop 11 between the ATP- and microtubule-binding sites. While ATP and microtubule binding appear normal, there is a 3-fold decrease in the rate of ATP turnover. This is consistent with the hypothesis that loop 11 provides a structural link that is important for the activation of ATP turnover by microtubule binding. T291M is a severe mutation that occurs in alpha-helix 5 near the center of the microtubule-binding surface. It impairs the microtubule-kinesin interaction and directly effects the ATP-binding pocket, allowing an increase in ATP turnover in the absence of microtubules. The T291M mutation may mimic the structure of a microtubule-bound, partially activated state. E164K is a moderate mutation that occurs at the beta-sheet 5a/loop 8b junction, remote from the ATP pocket. Surprisingly, it causes both tighter ATP-binding and a 2-fold decrease in ATP turnover. We propose that E164 forms an ionic bridge with alpha-helix 5 and speculate that it helps coordinate the alternating site catalysis of dimerized kinesin heavy chain motor domains.

Intracellular motility: A special delivery service.

Recent studies have identified a delivery service that operates in specialised cell appendages: two motor proteins and a novel protein organelle use axonemal microtubules as tracks to shuttle essential components to the tips of flagella and the dendrites of sensory neurons.

Kinesins in the nervous system.

Both the development and the maintenance of neurons require a great deal of active cytoplasmic transport. Much of this transport is driven by microtubule motor proteins. Membranous organelles and other macromolecular assemblies bind motor proteins that then use cycles of adenosine 5'-triphosphate hydrolysis to move these 'cargoes' along microtubules. Different sets of cargoes are transported to distinct locations in the cell. The resulting differential distribution of materials almost certainly plays an important part in generating polarized neuronal morphologies and in maintaining their vectorial signalling activities. A number of different microtubule motor proteins function in neurons; presumably they are specialized for accomplishing different transport tasks. Questions about specific motor functions and the functional relationships between different motors present a great challenge. The answers will provide a much deeper understanding of fundamental transport mechanisms, as well as how these mechanisms are used to generate and sustain cellular asymmetries.

Kinesin mutations cause motor neuron disease phenotypes by disrupting fast axonal transport in Drosophila.

Previous work has shown that mutation of the gene that encodes the microtubule motor subunit kinesin heavy chain (Khc) in Drosophila inhibits neuronal sodium channel activity, action potentials and neurotransmitter secretion. These physiological defects cause progressive distal paralysis in larvae. To identify the cellular defects that cause these phenotypes, larval nerves were studied by light and electron microscopy. The axons of Khc mutants develop dramatic focal swellings along their lengths. The swellings are packed with fast axonal transport cargoes including vesicles, synaptic membrane proteins, mitochondria and prelysosomal organelles, but not with slow axonal transport cargoes such as cytoskeletal elements. Khc mutations also impair the development of larval motor axon terminals, causing dystrophic morphology and marked reductions in synaptic bouton numbers. These observations suggest that as the concentration of maternally provided wild-type KHC decreases, axonal organelles transported by kinesin periodically stall. This causes organelle jams that disrupt retrograde as well as anterograde fast axonal transport, leading to defective action potentials, dystrophic terminals, reduced transmitter secretion and progressive distal paralysis. These phenotypes parallel the pathologies of some vertebrate motor neuron diseases, including some forms of amyotrophic lateral sclerosis (ALS), and suggest that impaired fast axonal transport is a key element in these diseases.

A bipolar kinesin.

Chromosome segregation during mitosis depends on the action of the mitotic spindle, a self-organizing, bipolar protein machine which uses microtubules (MTs) and their associated motors. Members of the BimC subfamily of kinesin-related MT-motor proteins are believed to be essential for the formation and functioning of a normal bipolar spindle. Here we report that KRP130, a homotetrameric BimC-related kinesin purified from Drosophila melanogaster embryos, has an unusual ultrastructure. It consists of four kinesin-related polypeptides assembled into a bipolar aggregate with motor domains at opposite ends, analogous to a miniature myosin filament. Such a bipolar 'minifilament' could crosslink spindle MTs and slide them relative to one another. We do not know of any other MT motors that have a bipolar structure.

Mutation of the axonal transport motor kinesin enhances paralytic and suppresses Shaker in Drosophila.

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.

A "slow" homotetrameric kinesin-related motor protein purified from Drosophila embryos.

Pan-kinesin peptide antibodies (Cole, D. G., Cande, W. Z., Baskin, R. J., Skoufias, D. A., Hogan, C. J., and Scholey, J. M. (1992) J. Cell Sci. 101, 291-301; Sawin, K. E., Mitchinson, T. J., and Wordeman, L. G. (1992) J. Cell Sci. 101, 303-313) were used to identify and isolate kinesin-related proteins (KRPs) from Drosophila melanogaster embryonic cytosol. These KRPs cosedimented with microtubules (MTs) polymerized from cytosol treated with AMP-PNP (adenyl-5'-yl imidodiphosphate), and one of them, KRP130, was further purified from ATP eluates of the embryonic MTs. Purified KRP130 behaves as a homotetrameric complex composed of four 130-kDa polypeptide subunits which displays a "slow" plus-end directed motor activity capable of moving single MTs at 0.04 +/- 0.01 microns/s. The 130-kDa subunit of KRP130 was tested for reactivity with monoclonal and polyclonal antibodies that are specific for various members of the kinesin superfamily. Results indicate that the KRP130 subunit is related to Xenopus Eg5 (Sawin, K. E., Le Guellec, K. L., Philippe, M., Mitchinson, T. J. (1992) Nature 359, 540-543), a member of the BimC subfamily of kinesins. Therefore, KRP130 appears to be the first Drosophila KRP, and the first member of the BimC subfamily in any organism, to be purified from native tissue as a multimeric motor complex.

Isolation and analysis of microtubule motor proteins.

Isolation of microtubule motor proteins is needed both for the discovery of new motors and for characterization of the products of motor-related genes. The sequences of motor-related genes cannot yet be used to predict the mechanochemical properties of the gene products. This was illustrated by the first kinesin-related gene product to be characterized. Protein expressed from the ncd gene moved toward the minus ends of microtubules (Walker et al., 1990; McDonald et al., 1990), while kinesin itself moves toward the plus ends. Until the relationship between mechanochemical function and amino acid sequence is more thoroughly understood, biochemical isolation and characterization of microtubule motor proteins will remain essential. Two approaches for getting useful quantities of microtubule motor proteins have been used: isolation from cytosol as described under Section II above and isolation from bacteria carrying cloned motor protein genes in expression vectors. Bacterial expression of functional microtubule motors has been successful to date in only a few cases (Yang et al., 1990; Walker et al., 1990, McDonald et al., 1990). Additional progress is expected with the expression of cloned genes from viral vectors in cultured eukaryotic cells, but broad success has not yet been reported. Biochemical isolation of motors from their natural cytosol has some distinct advantages. One can have confidence that a given motor will be folded properly and have normal post-translational modifications. In addition, if it exists in vivo as a heteromultimer, a microtubule motor isolated from its native cytosol will carry with it a normal complement of associated proteins. Studies of such associated proteins will be important in learning how motors accomplish their tasks in vivo. Drosophila cytosol should be a rich source of microtubule motors. Drosophila carry at least 11 and perhaps as many as 30 genes that are related to kinesin (Stewart et al., 1991; Endow and Hatsumi, 1991). The work of Tom Hays' lab indicates that Drosophila carry more than nine dynein related genes (Rasmussen et al., 1994). Relatively little effort to isolate the products of these genes from cytosol has been made. The only work that I am aware of has produced a kinesin-like microtubule motor (D.G. Cole, K.B. Sheehan, W.M. Saxton, and J.M. Scholey, in progress) that may be the Drosophila homolog of Xenopus eg5 (Sawin et al., 1992). This isolation was straightforward, and efforts to identify additional motors are almost assured of success.

Effects of kinesin mutations on neuronal functions.

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.

Kinesin heavy chain is essential for viability and neuromuscular functions in Drosophila, but mutants show no defects in mitosis.

The in vivo function of the microtubule motor protein kinesin was examined in Drosophila using genetics and immunolocalization. Kinesin heavy chain mutations (khc) cause abnormal behavior and lethality. Mutant larvae exhibit loss of mobility and tactile responsiveness in the most posterior segments, followed by general paralysis and death during larval or pupal development. Adults homozygous for a temperature-sensitive allele also exhibit a loss in mobility and sensory responses. The data indicate that kinesin function is essential and suggest that kinesin has an important role in the neuromuscular system, perhaps as a motor for axonal transport. The possibility of more general cellular functions remains open, but observation of embryogenesis and morphogenesis in khc mutants suggests that mitosis and the cell cycle can proceed in spite of impaired kinesin function. Immunolocalization suggests that kinesin may have some general cellular functions but that it is not a major component of mitotic spindles.

Evidence that the head of kinesin is sufficient for force generation and motility in vitro.

Kinesin is a mechanochemical protein that converts the chemical energy in adenosine triphosphate into mechanical force for movement of cellular components along microtubules. The regions of the kinesin molecule responsible for generating movement were determined by studying the heavy chain of Drosophila kinesin, and its truncated forms, expressed in Escherichia coli. The results demonstrate that (i) kinesin heavy chain alone, without the light chains and other eukaryotic factors, is able to induce microtubule movement in vitro, and (ii) a fragment likely to contain only the kinesin head is also capable of inducing microtubule motility. Thus, the amino-terminal 450 amino acids of kinesin contain all the basic elements needed to convert chemical energy into mechanical force.

Cell cycle-dependent changes in the dynamics of MAP 2 and MAP 4 in cultured cells.

To examine the behavior of microtubule-associated proteins (MAPs) in living cells, MAP 4 and MAP 2 have been derivatized with 6-iodoacetamido-fluorescein, and the distribution of microinjected MAP has been analyzed using a low light level video system and fluorescence redistribution after photobleaching. Within 1 min following microinjection of fluoresceinated MAP 4 or MAP 2, fluorescent microtubule arrays were visible in interphase or mitotic PtK1 cells. After cold treatment of fluorescent MAP 2-containing cells (3 h, 4 degrees C), microtubule fluorescence disappeared, and the only fluorescence above background was located at the centrosomes; microtubule patterns returned upon warming. Loss of microtubule immunofluorescence after nocodozole treatment was similar in MAP-injected and control cells, suggesting that injected fluorescein-labeled MAP 2 did not stabilize microtubules. The dynamics of the MAPs were examined further by FRAP. FRAP analysis of interphase cells demonstrated that MAP 2 redistributed with half-times slightly longer (60 +/- 25 s) than those for MAP 4 (44 +/- 20 s), but both types of MAPs bound to microtubules in vivo exchanged with soluble MAPs at rates exceeding the rate of tubulin turnover. These data imply that microtubules in interphase cells are assembled with constantly exchanging populations of MAP. Metaphase cells at 37 degrees C or 26 degrees C showed similar mean redistribution half-times for both MAP 2 and MAP 4; these were 3-4 fold faster than the interphase rates (MAP 2, t1/2 = 14 +/- 6 s; MAP 4, t1/2 = 17 +/- 5 s). The extent of recovery of spindle fluorescence in MAP-injected cells was to 84-94% at either 26 or 37 degrees C. Although most metaphase tubulin, like the MAPs, turns over rapidly and completely under physiologic conditions, published work shows either reduced rates or extents of turnover at 26 degrees C, suggesting that the fast mitotic MAP exchange is not simply because of fast tubulin turnover. Exchange of MAP 4 bound to telophase midbodies occurred with dynamics comparable to those seen in metaphase spindles (t1/2 = approximately 27 s) whereas midbody tubulin exchange was slow (greater than 300 s). These data demonstrate that the rate of MAP exchange on microtubules is a function of time in the cell cycle.