MCPH1, mutated in primary microcephaly, is required for efficient chromosome alignment during mitosis.

MCPH1 gene, mutated in primary microcephaly, regulates cell progression into mitosis. While this role has been extensively investigated in the context of DNA damage, its function during unperturbed cell cycles has been given less attention. Here we have analyzed the dynamics of chromosome condensation and cell cycle progression in MCPH1 deficient cells under undamaging conditions. Our study demonstrates that chromosome condensation is uncoupled from cell cycle progression when MCPH1 function is lacking, resulting in cells that prematurely condense their chromosomes during mid G2-phase and delay decondensation at the completion of mitosis. However, mitosis onset occurs on schedule in MCPH1 deficient cells. We also revealed active Cdk1 to be mandatory for the premature onset of chromosome condensation during G2 and the maintenance of the condensed state thereafter. Interestingly, a novel cellular phenotype was observed while monitoring cell cycle progression in cells lacking MCPH1 function. Specifically, completion of chromosome alignment at the metaphase plate was significantly delayed. This deficiency reveals that MCPH1 is required for efficient chromosome biorientation during mitosis.

A nuclear matrix-associated factor, SAF-B, interacts with specific isoforms of AUF1/hnRNP D.

One class of heterogeneous nuclear ribonucleoproteins (hnRNPs), AUF1/hnRNP D, consists of four isoform proteins (p45, p42, p40, and p37) which are generated by alternative splicing. The present study was therefore undertaken to clarify any isoform-specific differences in terms of their functions and nucleocytoplasmic localization. All isoforms primarily localized in the nucleus. However, heterokaryon analysis and a study using RNA polymerase II inhibitor revealed that p40/p37 exhibited a continuous shuttling between the nucleus and cytoplasm. Constant nuclear retention activity was mapped to the p45/p42-specific sequence at the C-terminal region, which is retained by alternative splicing. Using this domain as a probe, we performed a yeast two-hybrid screening and we found that scaffold attachment factor B (SAF-B), a nuclear matrix-associated protein, exhibits protein-protein interaction to this region. Colocalization of p45/p42 and SAF-B was observed as a speckle in the nucleus. Interestingly, p45/p42 isoforms appeared to act as a negative regulator in gene expression by forming a complex with SAF-B. Thus, the present study revealed that the isoform-specific functions of AUF1/hnRNP D are defined by intracellular shuttling capacity.

Depletion of a microtubule-associated motor protein induces the loss of dendritic identity.

Dendrites are short stout tapering processes that are rich in ribosomes and Golgi elements, whereas axons are long thin processes of uniform diameter that are deficient in these organelles. It has been hypothesized that the unique morphological and compositional features of axons and dendrites result from their distinct patterns of microtubule polarity orientation. The microtubules within axons are uniformly oriented with their plus ends distal to the cell body, whereas microtubules within dendrites are nonuniformly oriented. The minus-end-distal microtubules are thought to arise via their specific transport into dendrites by the motor protein known as CHO1/MKLP1. According to this model, CHO1/MKLP1 transports microtubules with their minus ends leading into dendrites by generating forces against the plus-end-distal microtubules, thus creating drag on the plus-end-distal microtubules. Here we show that depletion of CHO1/MKLP1 from cultured neurons causes a rapid redistribution of microtubules within dendrites such that minus-end-distal microtubules are chased back to the cell body while plus-end-distal microtubules are redistributed forward. The dendrite grows significantly longer and thinner, loses its taper, and acquires a progressively more axon-like organelle composition. These results suggest that the forces generated by CHO1/MKLP1 are necessary for maintaining the minus-end-distal microtubules in the dendrite, for antagonizing the anterograde transport of the plus-end-distal microtubules, and for sustaining a pattern of microtubule organization necessary for the maintenance of dendritic morphology and composition. Thus, we would conclude that dendritic identity is dependent on forces generated by CHO1/MKLP1.

Filamentous polymers induced by overexpression of a novel centrosomal protein, Cep135.

A novel 135 kDa centrosomal component (Cep135) was identified by immunoscreening of a mammalian expression library with monoclonal antibodies raised against clam centrosomes. It is predicted to be a highly coiled-coil protein with an extensive alpha-helix, suggesting that Cep135 is a structural component of the centrosome. To evaluate how the protein is arranged in the centrosomal structure, we overexpressed Cep135 polypeptides in CHO cells by transient transfection. HA- or GFP-tagged full (amino acids 1-1144) as well as truncated (#10, 29-1144; Delta3, 29-812) polypeptides become localized at the centrosome and induce cytoplasmic dots of various size and number in CHO cells. Centrosomes are associated with massive approximately 7 nm filaments and dense particles organized in a whorl-like arrangement in which parallel-oriented dense lines appear with a regular approximately 7 nm periodicity. The same filamentous aggregates are also detected in cytoplasmic dots, indicating that overexpressed Cep135 can assemble into elaborate higher-ordered structures in and outside the centrosome. Sf9 cells infected with baculovirus containing Cep135 sequences induce filamentous polymers which are distinctive from the whorl seen in CHO cells; #10 forms highly packed spheroids, but the Delta3-containing structure looks loose. Both structures show an internal repeating unit of dense and less dense stripes. Although the distance between the outer end of two adjacent dense lines is similar between two types of polymers ( approximately 120 nm), the dense stripe of Delta3 polymers ( approximately 40 nm) is wider than #10 ( approximately 30 nm). The light band of Delta3 ( approximately 40 nm) is thus narrower than #10 ( approximately 60 nm). Since thin fibers are frequently seen to extend from one dense line to the next, the coiled-coil rod of Cep135 may span the light band. These results suggest that overexpressed Cep135 assemble into distinctive polymers in a domain-specific manner.

Function of a minus-end-directed kinesin-like motor protein in mammalian cells.

CHO2 is a mammalian minus-end-directed kinesin-like motor protein present in interphase centrosomes/nuclei and mitotic spindle fibers/poles. Expression of HA- or GFP-tagged subfragments in transfected CHO cells revealed the presence of the nuclear localization site at the N-terminal tail. This domain becomes associated with spindle fibers during mitosis, indicating that the tail is capable of interaction with microtubules in vivo. While the central stalk diffusely distributes in the entire cytoplasm of cells, the motor domain co-localizes with microtubules throughout the cell cycle, which is eliminated by mutation of the ATP-binding consensus motif from GKT to AAA. Overexpression of the full-length CHO2 causes mitotic arrest and spindle abnormality. The effect of protein expression was first seen around the polar region where microtubule tended to be bundled together. A higher level of protein expression induces more elongated spindles which eventually become disorganized by loosing the structural integrity between microtubule bundles. Live cell observation demonstrated that GFP-labeled microtubule bundles underwent continuous changes in their relative position to one another through repeated attachment and detachment at one end; this results in the formation of irregular number of microtubule focal points in mitotic arrested cells. Thus the primary action of CHO2 appears to cross-link microtubules and move toward the minus-end direction to maintain association of the microtubule end at the pole. In contrast to the full-length of CHO2, overexpression of neither truncated nor mutant polypeptides resulted in significant effects on mitosis and mitotic spindles, suggesting that the function of CHO2 in mammalian cells may be redundant with other motor molecules during cell division.

Arf proteins bind to mitotic kinesin-like protein 1 (MKLP1) in a GTP-dependent fashion.

Arf proteins comprise a family of 21-kDa GTP-binding proteins with many proposed functions in mammalian cells, including the regulation of several steps of membrane transport, maintenance of organelle integrity, and activation of phospholipase D. We performed a yeast two-hybrid screen of human cDNA libraries using a dominant activating allele, [Q71L], of human Arf3 as bait. Eleven independent isolates contained plasmids encoding the C-terminal tail of mitotic kinesin-like protein-1 (MKLP1). Further deletion mapping allowed the identification of an 88 amino acid Arf3 binding domain in the C-terminus of MKLP1. This domain has no clear homology to other Arf binding proteins or to other proteins in the protein databases. The C-terminal domain of MKLP1 was expressed and purified from bacteria as a GST fusion protein and shown to bind Arf3 in a GTP-dependent fashion. A screen for mutations in Arf3 that specifically lost the ability to bind MKLP1 identified 10 of 14 point mutations in the GTP-sensitive switch I or switch II regions of Arf3. Two-hybrid assays of the C-terminal domain of MKLP1 with each of the human Arf isoforms revealed strong interaction with each. Taken together, these data are all supportive of the conclusion that activated Arf proteins bind to the C-terminal "tail" domain of MKLP1.

Absence of limiting amino acids in calves fed a corn and soybean meal diet past three months of age.

We conducted three nitrogen balance trials using Holstein bull calves older than 16 wk (Trial 1; n = 8), 13 wk (Trial 2; n = 6), and 15 wk of age (Trial 3; n = 9) in a 4 x 4 (Trial 1) or 3 x 3 Latin square design (Trials 2 and 3) to identify limiting amino acids for a corn and soybean meal diet. All calves were trained to maintain reflex closure of the reticular groove after weaning at 5 wk of age. The basal diet was fed daily at 20 or 27 g/kg BW (Trial 1) and at 20 g/kg BW (Trials 2 and 3). The lower feeding level resulted in reduced urinary excretion of purine derivatives, suggesting reduced synthesis of ruminal microbial protein (Trial 1). In Trials 1 and 2, administration of DL-methionine plus L-lysine monohydrochloride through the reticular groove did not increase N retention compared with the supplement of isonitrogenous L-glutamine at either level of intake. In Trial 3, administration of either casein or isonitrogenous monosodium glutamate increased N retention to a similar extent above that observed with a N-free supplement. Results suggested that no specific amino acids were limiting for the corn-soybean meal diet. Administration of methionine plus lysine resulted in a remarkable increase in plasma methionine (Trials 1 and 2), especially at the lower intake level (Trial 1), and a decrease in plasma branched-chain amino acids at either intake level. Glutamine supplementation did not increase plasma branched-chain amino acids compared with the supplementation of diammonium citrate (Trial 2).

Nonuniform microtubular polarity established by CHO1/MKLP1 motor protein is necessary for process formation of podocytes.

Podocytes are unique cells that are decisively involved in glomerular filtration. They are equipped with a complex process system consisting of major processes and foot processes whose function is insufficiently understood (Mundel, P., and W. Kriz. 1995. Anat. Embryol. 192:385-397). The major processes of podocytes contain a microtubular cytoskeleton. Taking advantage of a recently established cell culture system for podocytes with preserved ability to form processes (Mundel, P., J. Reiser, A. Zúñiga Mejía Borja, H. Pavenstädt, G.R. Davidson, W. Kriz, and R. Zeller. 1997b. Exp. Cell Res. 36:248-258), we studied the functional significance of the microtubular system in major processes. The following data were obtained: (a) Microtubules (MTs) in podocytes show a nonuniform polarity as revealed by hook-decoration. (b) CHO1/ MKLP1, a kinesin-like motor protein, is associated with MTs in podocytes. (c) Treatment of differentiating podocytes with CHO1/MKLP1 antisense oligonucleotides abolished the formation of processes and the nonuniform polarity of MTs. (d) During the recovery from taxol treatment, taxol-stabilized (nocodazole- resistant) MT fragments were distributed in the cell periphery along newly assembled nocodazole-sensitive MTs. A similar distribution pattern of CHO1/MKLP1 was found under these circumstances, indicating its association with MTs. (e) In the recovery phase after complete depolymerization, MTs reassembled exclusively at centrosomes. Taken together, these findings lead to the conclusion that the nonuniform MT polarity in podocytes established by CHO1/MKLP1 is necessary for process formation.

Expression of the mitotic motor protein CHO1/MKLP1 in postmitotic neurons.

The kinesin-related motor protein CHO1/MKLP1 was initially thought to be expressed only in mitotic cells, where it presumably transports oppositely oriented microtubules relative to one another in the spindle mid-zone. We have recently shown that CHO1/MKLP1 is also expressed in cultured neuronal cells, where it is enriched in developing dendrites [Sharp et al. (1997a) J. Cell Biol., 138, 833-843]. The putative function of CHO1/MKLP1 in these postmitotic cells is to intercalate minus-end-distal microtubules among oppositely oriented microtubules within developing dendrites, thereby establishing their non-uniform microtubule polarity pattern. Here we used in situ hybridization to determine whether CHO1/MKLP1 is expressed in a variety of rodent neurons both in vivo and in vitro. These analyses revealed that CHO1/MKLP1 is expressed within various neuronal populations of the brain including those in the cerebral cortex, hippocampus, olfactory bulb and cerebellum. The messenger ribonucleic acid (mRNA) levels are high within these neurons well after the completion of their terminal mitotic division and throughout the development of their dendrites. After this, the levels decrease and are relatively low within the adult brain. Parallel analyses on developing hippocampal neurons in culture indicate that the levels of expression increase dramatically just prior to dendritic development, and then decrease somewhat after the dendrites have differentiated. Dorsal root ganglion neurons, which generate axons but not dendrites, express significantly lower levels of mRNA for CHO1/MKLP1 than hippocampal or sympathetic neurons. These results are consistent with the proposed role of CHO1/MKLP1 in establishing the dendritic microtubule array.

Serum substances that interfere with thyroid hormone assays in patients with chronic renal failure.

OBJECTIVE: Serum thyroid hormone concentrations in patients with chronic renal failure are usually low, despite normal serum TSH levels. We investigated the effect on thyroid hormone assays of serum dialysable organic acids that are elevated in uraemic patients. PATIENTS: Serum samples from 42 patients with chronic renal failure who were receiving haemodialysis and 37 sex- and age-matched healthy subjects were examined. DESIGN AND MEASUREMENTS: Serum thyroid hormone concentrations were measured with an analogue radioimmunoassay (RIA), a labelled antibody assay, and an equilibrium dialysis/RIA method. Serum concentrations of organic acids were determined with high performance liquid chromatography. RESULTS: Serum thyroid hormone levels determined by an analogue RIA and a labelled antibody assay in uraemic patients increased, and serum concentrations of organic acids decreased following haemodialysis. A significant association existed between serum free T3 (FT3) levels determined by an analogue RIA and serum concentrations of indoxyl sulphate (IS) prior to dialysis. There was also a significant association between serum free T4 (FT4) levels determined by an analogue RIA and serum concentration of IS and hippuric acid (HA) prior to dialysis. There was a significant association between the changes of serum concentrations of indole acetic acid (IAA) and FT4 concentrations prior to and following haemodialysis when determined by an analogue RIA. Serum FT3 and FT4 levels significantly decreased after the addition of IS to serum from healthy subjects when determined by an analogue RIA but not by a labelled antibody assay. Serum FT4 levels, but not FT3 levels, decreased after addition of IAA when determined by an analogue RIA. Serum FT4 concentrations determined by an equilibrium dialysis/RIA were significantly higher than those determined by the other two methods. The addition of IS, IAA, and HA to serum samples from healthy subjects significantly increased FT4 concentrations when determined by an equilibrium dialysis/RIA method. CONCLUSIONS: Increased serum levels of indoxyl sulphate, indole acetic acid and hippuric acid in sera of uraemic patients may interfere with thyroid hormone measurements when an analogue radioimmunoassay is used. In contrast, there was little Interference with a labelled antibody assay. Dialysable organic acids may also interfere with thyroid hormone assays determined by an equilibrium dialysis/radioimmunoassay method.

The nuclear/mitotic apparatus protein NuMA is a component of the somatodendritic microtubule arrays of the neuron.

Neurons are terminally post-mitotic cells that utilize their microtubule arrays for the growth and maintenance of axons and dendrites rather than for the formation of mitotic spindles. Recent studies from our laboratory suggest that the mechanisms that organize the axonal and dendritic microtubule arrays may be variations on the same mechanisms that organize the mitotic spindle in dividing cells. In particular, we have identified molecular motor proteins that serve analogous functions in the establishment of these seemingly very different microtubule arrays. In the present study, we have sought to determine whether a non-motor protein termed NuMA is also a component of both systems. NuMA is a approximately 230 kDa structural protein that is present exclusively in the nucleus during interphase. During mitosis, NuMA forms aggregates that interact with microtubules and certain motor proteins. As a result of these interactions, NuMA is thought to draw together the minus-ends of microtubules, thereby helping to organize them into a bipolar spindle. In contrast to mitotic cells, post-mitotic neurons display NuMA both in the nucleus and in the cytoplasm. NuMA appears as multiple small particles within the somatodendritic compartment of the neuron, where its levels increase during early dendritic differentiation. A partial but not complete colocalization with minus-ends of microtubules is suggested by the distribution of the particles during development and during drug treatments that alter the microtubule array. These observations provide an initial set of clues regarding a potentially important function of NuMA in the organization of microtubules within the somatodendritic compartment of the neuron.

Interaction of an Overexpressed gamma-Tubulin with Microtubules In Vivo and In Vitro.

gamma-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for gamma-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus gamma-tubulin. When CHO cells were transiently transfected with the gamma-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such gamma-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that gamma-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas gamma-tubulin purified from insect Sf9 cells (), interaction between gamma-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with gamma-tubulin monomers in vitro were associated with more gold particles conjugated with gamma-tubulin than in controls where no exogenous gamma-tubulin was added. However, binding of gamma-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although gamma-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (; ), which might be ascribed to the difference in the level of protein expression in transfected cells.

Expression of a minus-end-directed motor protein induces Sf9 cells to form axon-like processes with uniform microtubule polarity orientation.

Neurons extend two types of processes with distinct morphologies and patterns of microtubule polarity orientation. Axons are thin cylindrical processes containing microtubules that are uniformly oriented with their plus-ends-distal to the cell body while dendrites are stout tapering processes that contain nonuniformly oriented microtubules. We have proposed that these distinct microtubule patterns are established by molecular motors that transport microtubules into each type of process with the appropriate orientation. To test the feasibility of this proposal, we have embarked on a series of studies involving the expression of vertebrate motors in insect Sf9 cells. We previously focused on a kinesin-related protein termed CHO1/MKLP1, which localizes to the midzone of the mitotic spindle, and which has been shown to have the appropriate properties to transport microtubules of opposite orientation relative to one another. Expression of a fragment of CHO1/MKLP1 containing its motor domain induces Sf9 cells to extend processes with a stout tapering morphology and a nonuniform microtubule polarity pattern similar to dendrites. Here we focus on a minus-end-directed kinesin-related motor protein termed CHO2, which localizes to the non-overlapping regions of the mitotic spindle, and which has been shown to have the appropriate properties to transport microtubules with plus-ends-leading. Sf9 cells induced to express a fragment of CHO2 containing its motor domain extend processes with a long cylindrical morphology and a uniformly plus-end-distal microtubule polarity pattern similar to axons. These results show that motor proteins have the capacity to organize distinct patterns of microtubule polarity orientation during process outgrowth, and that these patterns are intimately related to the unique morphological characteristics of the processes. Moreover, mutation of three amino acids corresponding to the ATP binding site necessary for motor function suppresses the capacity of the CHO2 fragment to induce process formation and microtubule reorganization, indicating that at least in the case of CHO2, the transport properties of the motor are essential for it to elicit these effects.

Identification of a microtubule-associated motor protein essential for dendritic differentiation.

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.

Inhibition of a mitotic motor compromises the formation of dendrite-like processes from neuroblastoma cells.

Microtubules in the axon are uniformly oriented, while microtubules in the dendrite are nonuniformly oriented. We have proposed that these distinct microtubule polarity patterns may arise from a redistribution of molecular motor proteins previously used for mitosis of the developing neuroblast. To address this issue, we performed studies on neuroblastoma cells that undergo mitosis but also generate short processes during interphase. Some of these processes are similar to axons with regard to their morphology and microtubule polarity pattern, while others are similar to dendrites. Treatment with cAMP or retinoic acid inhibits cell division, with the former promoting the development of the axon-like processes and the latter promoting the development of the dendrite-like processes. During mitosis, the kinesin-related motor termed CHO1/MKLP1 is localized within the spindle midzone where it is thought to transport microtubules of opposite orientation relative to one another. During process formation, CHO1/ MKLP1 becomes concentrated within the dendrite-like processes but is excluded from the axon-like processes. The levels of CHO1/MKLP1 increase in the presence of retinoic acid but decrease in the presence of cAMP, consistent with a role for the protein in dendritic differentiation. Moreover, treatment of the cultures with antisense oligonucleotides to CHO1/MKLP1 compromises the formation of the dendrite-like processes. We speculate that a redistribution of CHO1/MKLP1 is required for the formation of dendrite-like processes, presumably by establishing their characteristic nonuniform microtubule polarity pattern.

TKRP125, a kinesin-related protein involved in the centrosome-independent organization of the cytokinetic apparatus in tobacco BY-2 cells.

Analysis of a cDNA for a 125 kDa polypeptide, previously isolated from phragmoplasts of tobacco BY-2 cells as a candidate for a plus end-directed microtubule motor, revealed this polypeptide to be a novel member of the kinesin superfamily. We named this protein TKRP125 (tobacco kinesin-related polypeptide of 125 kDa). The strong similarity between TKRP125 and members of the bimC subfamily in terms of the amino acid sequence of the amino-terminal motor domain indicated that TKRP125 belonged to the bimC subfamily. An antibody against a short peptide from the motor domain of TKRP125 inhibited the GTP- or ATP-dependent translocation of phragmoplast microtubules in membrane-permeabilized BY-2 cells, suggesting a role for TKRP125 in microtubule translocation, which is considered to be involved in the formation and/or maintenance of the bipolar structure of the phragmoplast. The expression of TKRP125 was found to be cell cycle-dependent. TKRP125 was not present in cells at the G1 phase. It began to appear at the S phase and accumulated during the G2 phase. The distribution of TKRP125 changed as the arrangement of microtubules changed with the progression of the cell cycle. TKRP125 was distributed along cortical microtubules during the S phase and along microtubules in the preprophase band and perinuclear microtubules in premitotic cells. It was also present in the nucleus in premitotic cells. In cells in M phase, TKRP125 was distributed along spindle microtubules. It accumulated at the equatorial plane of the spindle as the spindle elongated. In cytokinetic cells, TKRP125 was colocalized with phragmoplast microtubules. These observations suggest the possible involvement of TKRP125 in the cell cycle-dependent changes in arrays of microtubules, including the organization of the phragmoplast, and in the movement of chromosomes in anaphase cells.

Expression of a kinesin-related motor protein induces Sf9 cells to form dendrite-like processes with nonuniform microtubule polarity orientation.

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.