Release of C-terminal tyrosine from tubulin and microtubules at steady state.

Microtubule protein preparations purified by cycles of assembly-disassembly contain the enzyme tubulinyltyrosine carboxypeptidase (TTCPase). Using these preparations, containing tubulinyl[14C]tyrosine, we studied the release of [14C]tyrosine from assembled and non-assembled tubulin under steady-state conditions. It was found that both states of aggregation were detyrosinated at similar rates by the action of the endogenous TTCPase. However, practically no release of [14C]tyrosine from the non-assembled tubulin pool was found when microtubules were previously eliminated from the incubation mixture. These results indicated that non-assembled tubulin requires to interact with microtubules to be detyrosinated. This interaction seems to occur through the incorporation of dimers into microtubules, since when the capability of tubulin to incorporate into microtubules was diminished by binding of colchicine a concomitant decrease in the rate of release of tyrosine was observed. When detyrosination was accelerated by increasing the concentration of TTCPase relative to the microtubule protein concentration, microtubules were found to be detyrosinated faster than was non-assembled tubulin. Using exogenous TTCPase in an incubation system in which the formation of microtubules was not allowed, tubulinyl[14C]tyrosine and tubulinyl[14C]tyrosine-colchicine complex were shown to have similar capabilities to act as substrates for this enzyme. Free colchicine was shown not to affect the activity of TTCPase.     

Alterations in number of protofilaments in microtubules assembled in vitro

Tubulin from bovine brain was polymerized in vitro using a variety of assembly conditions. Many of the formed microtubules were shown to contain 14 wall protofilaments. The number of microtubules containing 14 protofilaments increased with consecutive repetitions of cold-dissociation followed by reassembly in vitro.     

The Type II Arabidopsis Formin14 Interacts with Microtubules and Microfilaments to Regulate Cell Division

Formins have long been known to regulate microfilaments but have also recently been shown to associate with microtubules. In this study, Arabidopsis thaliana FORMIN14 (AFH14), a type II formin, was found to regulate both microtubule and microfilament arrays. AFH14 expressed in BY-2 cells was shown to decorate preprophase bands, spindles, and phragmoplasts and to induce coalignment of microtubules with microfilaments. These effects perturbed the process of cell division. Localization of AFH14 to microtubule-based structures was confirmed in Arabidopsis suspension cells. Knockdown of AFH14 in mitotic cells altered interactions between microtubules and microfilaments, resulting in the formation of an abnormal mitotic apparatus. In Arabidopsis afh14 T-DNA insertion mutants, microtubule arrays displayed abnormalities during the meiosis-associated process of microspore formation, which corresponded to altered phenotypes during tetrad formation. In vitro biochemical experiments showed that AFH14 bound directly to either microtubules or microfilaments and that the FH2 domain was essential for cytoskeleton binding and bundling. However, in the presence of both microtubules and microfilaments, AFH14 promoted interactions between microtubules and microfilaments. These results demonstrate that AFH14 is a unique plant formin that functions as a linking protein between microtubules and microfilaments and thus plays important roles in the process of plant cell division.     

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [¹⁴C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[¹⁴C]Tyrosine was released from assembled and non-assembled [¹⁴C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[¹⁴C]tyrosine preparation in the presence of CaCl₂ at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [¹⁴C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[³H]Phenylalanine was released from a preparation of tubulinyl-[³H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[¹⁴C]tyrosine and tubulinyl-[³H]phenylalanine was partially assembled, the ratio of ¹⁴C/³H found in the microtubules was the same as in the non-assembled tubulin fraction.     

Characterization of microtubule protofilament numbers. How does the surface lattice accommodate?

Frozen-hydrated specimens of microtubules assembled in vitro were observed by cryoelectron microscopy. Specimens were of both pure tubulin, and of microtubule protein isolated by three cycles of assembly and disassembly. It is shown that the characteristic image contrast of individual microtubules allows the microtubule protofilament number to be determined unambiguously. Microtubules with 13, 14 and 15 protofilaments are observed to coexist in specimens prepared under various assembly conditions. Confirmation of these results is obtained by observations of thin sections of pelleted samples fixed and stained using the glutaraldehyde/tannic acid technique. Images of individual microtubules show both characteristic contrast profiles across their width and typical variations of these profiles along their length. The profiles across the images indicate the protofilament number of the microtubule. The lengthwise variations indicate how the protofilaments are aligned with respect to the microtubule axis giving what has previously been called a supertwist. In 13 protofilament microtubules the protofilaments are paraxial. In 14 and 15 protofilament microtubules, the protofilaments are skewed with respect to the microtubule axis. The skew is greater for the 15 protofilament case than for 14 protofilaments. The skew allows the extra protofilaments to be accommodated by the surface lattice. These results should also be relevant to situations in vivo.     

Lattice defects in microtubules: protofilament numbers vary within individual microtubules

We have used cryo-electron microscopy of vitrified specimens to study microtubules assembled both from three cycle purified tubulin (3x-tubulin) and in cell free extracts of Xenopus eggs. In vitro assembled 3x-tubulin samples have a majority of microtubules with 14 protofilaments whereas in cell extracts most microtubules have 13 protofilaments. Microtubule polymorphism was observed in both cases. The number of protofilaments can change abruptly along individual microtubules usually by single increments but double increments also occur. For 3x-tubulin, increasing the magnesium concentration decreases the proportion of 14 protofilament microtubules and decreases the average separation between transitions in these microtubules. Protofilament discontinuities may correspond to dislocation-like defects in the microtubule surface lattice.     

Packing volume of sedimented microtubules: regulation and potential relationship to an intracellular matrix

To determine the contribution of microtubules to a hypothetical intracellular matrix, we have analyzed the space occupied by microtubules in vitro. Taxol-stabilized microtubules assembled from purified (three-times-cycled) bovine brain microtubule protein were pelleted by centrifugation under standardized conditions. The specific volume of the pellet, defined as the microliter volume per milligram protein, was 22.4. As suggested by others, this volume was strongly dependent on microtubule-associated proteins (MAPs), as shown by quantitation of the effects of purified MAP supplementation on specific volume. The specific volumes of microtubule pellets stripped of MAPs by high salt or chymotryptic digestion approached the mathematically optimal (least occupied space) and increased 14-fold with the highest MAP concentrations employed. Packing was also dependent on pH. Specific volumes comparable to those of MAP-depleted microtubules were attainable at pH's from 5.5 to 6.0, and specific volumes more than doubled at pH 7.5. MAP content was unaffected by pH. We present a theoretical analysis that suggests that as microtubules are centrifuged the mixture behaves as a liquid crystal. With packing, the mixture undergoes an isotropic-nematic phase transition in which the microtubules become oriented principally as parallel rods, mimicking their orientation in vivo. From the known concentration of microtubules in vivo, it can be inferred from our measurements that in some cells a large fraction, perhaps 40-50% of the cytosolic volume, is occupied by microtubules that form a mechanically irreducible space. Further theoretical analysis employing Ogston's formulation of the penetrability of fibrous networks suggests that the space between microtubules (in contrast to the extracellular matrix) imposes little barrier to the diffusion of macromolecules. A microtubule array thus achieves mechanical stability without affecting transport by diffusion. The space can accommodate other fibrous networks that could then affect transport, and, as we show, the space itself may be regulated by MAP content and intracellular pH.     

Ultrastructure of the photosynthetic ciliate Mesodinium rubrum

New cellular structures, bifurcated oral tentacles, were observed in many specimens of the photosynthetic ciliate Mesodinium rubrum from the northern Baltic Sea. Cross-sections of tentacles revealed rings (cylinders) of 14 microtubules with spokes. The number of microtubules per ring decreased from 14 to 12 or 11 inside the cell but no true kinetosomes were detected. These "micro-rings" were often associated with extrusomes and the tentacle tips consisted of extrusomes. A nucleus of a symbiotic alga was present, surrounded by algal cytoplasm containing plastids and delimited from the ciliate cytoplasm by two membranes. Each plastid was bounded by four membranes and was associated with one nucleomorph, suggesting a symbiotic origin as a cryptophyte. The unique symbiotic organization and the organelles of 14 microtubules make Mesodinium rubrum an organism of unusual evolutionary interest.     

Minus-end-directed motor Ncd exhibits processive movement that is enhanced by microtubule bundling in vitro

Drosophila Ncd, a kinesin-14A family member, is essential for meiosis and mitosis. Ncd is a minus-end-directed motor protein that has an ATP-independent microtubule binding site in the tail region, which enables it to act as a dynamic crosslinker of microtubules to assemble and maintain the spindle. Although a tailless Ncd has been shown to be nonprocessive, the role of the Ncd tail in single-molecule motility is unknown. Here, we show that individual Ncd dimers containing the tail region can move processively along microtubules at very low ionic strength, which provides the first evidence of processivity for minus-end-directed kinesins. The movement of GFP-Ncd consists of both a unidirectional and a diffusive element, and it was sensitive to ionic strength. Motility of a truncation series of Ncd and removal of the tubulin tail suggested that the Ncd tail serves as an electrostatic tether to microtubules. Under higher ionic conditions, Ncd showed only a small bias in diffusion along "single" microtubules, whereas it exhibited processive movement along "bundled" microtubules. This property may allow Ncd to accumulate preferentially in the vicinity of focused microtubules and then to crosslink and slide microtubules, possibly contributing to dynamic spindle self-organization.     

Microtubule-binding domain of tau proteins

Limited chymotryptic digestion of whole tau proteins produced a fragment of Mr 14,000 (CT14), which was able to bind to microtubules reconstituted from tubulin alone in the presence of taxol. This fragment was also found to persist in microtubules when microtubules consisting of tau proteins and tubulin were digested by chymotrypsin. Analysis of amino acid composition revealed that CT14 was rich in lysine and proline residues, suggesting unique structure of microtubule-binding domain of tau proteins. Amino-terminal sequence of CT14 was determined to be Ser-Ser-Pro-Gly-Ser-Pro-Gly-Thr-Pro-Gly-Ser-Arg-Ser-Arg-X-Pro-Ser-Leu-Pr o. No heterogeneity was detected in this amino-terminal sequence of 19 residues. Five species of polypeptides consisting of tau proteins were separated from each other by gel electrophoresis and subjected to chymotryptic digestion. CT14 was produced from each of the tau polypeptides by chymotryptic digestion, indicating that all tau polypeptides have a common microtubule-binding domain.     

Cortical microtubule labeling: Insight of AFH14 in non-dividing cells

We recently reported that AFH14 participated in microtubule and actin filament interaction in cell division, and the AFH14 (FH1FH2) was important to the directly binding activity of microtubules and microfilaments. To preliminarily understand the function and localization of AFH14 in non-dividing cells, we overexpressed FH1FH2-RFP in onion epidermal cells, and found a fluorescence labeled filamentous network. The result of double labeling with different cytoskeleton reporter proteins indicated that FH1FH2-RFP co-localized with cortical microtubules. Treatment of cells expressing FH1FH2-RFP with cytoskeleton disrupting drugs confirms that FH1FH2-RFP binds to microtubules. Moreover, the binding of FH1FH2-RFP to microtubules were revealed to be dynamic by fluorescence recovery after photobleaching (FRAP) experiment. Time-lapse confocal microscopy showed that FH1FH2-RFP could display a dynamics similar to the microtubule dynamic instability. These data suggest that FH1FH2 domain may lead AFH14 function on cortical microtubules in non-dividing cells, and FH1FH2-RFP may be utilized as a microtubule reporter protein in living onion epidermal cells.Key words: cortical microtubule, AFH14, non-dividing cell, microtubule dynamics, FRAP     

Overexpression of 14-3-3ζ Promotes Tau Phosphorylation at Ser262 and Accelerates Proteosomal Degradation of Synaptophysin in Rat Primary Hippocampal Neurons

β-amyloid peptide accumulation, tau hyperphosphorylation, and synapse loss are characteristic neuropathological symptoms of Alzheimer’s disease (AD). Tau hyperphosphorylation is suggested to inhibit the association of tau with microtubules, making microtubules unstable and causing neurodegeneration. The mechanism of tau phosphorylation in AD brain, therefore, is of considerable significance. Although PHF-tau is phosphorylated at over 40 Ser/Thr sites, Ser²⁶² phosphorylation was shown to mediate β-amyloid neurotoxicity and formation of toxic tau lesions in the brain. In vitro, PKA is one of the kinases that phosphorylates tau at Ser²⁶², but the mechanism by which it phosphorylates tau in AD brain is not very clear. 14-3-3ζ is associated with neurofibrillary tangles and is upregulated in AD brain. In this study, we show that 14-3-3ζ promotes tau phosphorylation at Ser²⁶² by PKA in differentiating neurons. When overexpressed in rat hippocampal primary neurons, 14-3-3ζ causes an increase in Ser²⁶² phosphorylation, a decrease in the amount of microtubule-bound tau, a reduction in the amount of polymerized microtubules, as well as microtubule instability. More importantly, the level of pre-synaptic protein synaptophysin was significantly reduced. Downregulation of synaptophysin in 14-3-3ζ overexpressing neurons was mitigated by inhibiting the proteosome, indicating that 14-3-3ζ promotes proteosomal degradation of synaptophysin. When 14-3-3ζ overexpressing neurons were treated with the microtubule stabilizing drug taxol, tau Ser²⁶² phosphorylation decreased and synaptophysin level was restored. Our data demonstrate that overexpression of 14-3-3ζ accelerates proteosomal turnover of synaptophysin by promoting the destabilization of microtubules. Synaptophysin is involved in synapse formation and neurotransmitter release. Our results suggest that 14-3-3ζ may cause synaptic pathology by reducing synaptophysin levels in the brains of patients suffering from AD.     

The nuclear F‐actin interactome of Xenopus oocytes reveals an actin‐bundling kinesin that is essential for meiotic cytokinesis

Nuclei of Xenopus laevis oocytes grow 100 000‐fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F‐actin scaffold for mechanical stability. We now developed a method for mapping F‐actin interactomes and identified a comprehensive set of F‐actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin‐bundling Kinesin). NabKin not only binds microtubules but also F‐actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F‐actin is negatively regulated by Importin‐β and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F‐actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin‐bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F‐actin and that such link is essential for cytokinesis.     

Disruption of arabinogalactan proteins disorganizes cortical microtubules in the root of Arabidopsis thaliana

The cortical array of microtubules inside the cell and arabinogalactan proteins on the external surface of the cell are each implicated in plant morphogenesis. To determine whether the cortical array is influenced by arabinogalactan proteins, we first treated Arabidopsis roots with a Yariv reagent that binds arabinogalactan proteins. Cortical microtubules were markedly disorganized by 1 microM beta-D-glucosyl (active) Yariv but not by up to 10 microM beta-D-mannosyl (inactive) Yariv. This was observed for 24-h treatments in wild-type roots, fixed and stained with anti-tubulin antibodies, as well as in living roots expressing a green fluorescent protein (GFP) reporter for microtubules. Using the reporter line, microtubule disorganization was evident within 10 min of treatment with 5 microM active Yariv and extensive by 30 min. Active Yariv (5 microM) disorganized cortical microtubules after gadolinium pre-treatment, suggesting that this effect is independent of calcium influx across the plasma membrane. Similar effects on cortical microtubules, over a similar time scale, were induced by two anti-arabinogalactan-protein antibodies (JIM13 and JIM14) but not by antibodies recognizing pectin or xyloglucan epitopes. Active Yariv, JIM13, and JIM14 caused arabinogalactan proteins to aggregate rapidly, as assessed either in fixed wild-type roots or in the living cells of a line expressing a plasma membrane-anchored arabinogalactan protein from tomato fused to GFP. Finally, electron microscopy of roots prepared by high-pressure freezing showed that treatment with 5 microM active Yariv for 2 h significantly increased the distance between cortical microtubules and the plasma membrane. These findings demonstrate that cell surface arabinogalactan proteins influence the organization of cortical microtubules.     

Characterization of the ATP-sensitive binding of Tetrahymena 30 S dynein to bovine brain microtubules

Dynein was obtained by high salt extraction of Tetrahymena cilia and purified by DEAE-Sephacel chromatography. This fraction consisted of a mixture of 30 S dynein (80%) and the 14 S ATPase (15%). The column purification effectively removed tubulin and adenylate kinase. Sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that the 30 S dynein was composed of a major heavy chain (approximately 400 kD, three copies), three intermediate chains (70, 85, and 100 kD), and a group of light chains (approximately 20 kD). The binding of the column-purified dynein to bovine brain microtubules was characterized as follows. (i) Titration of the dynein with microtubules showed a linear increase in turbidity up to an equivalence point of 2.7 mg of dynein/mg of tubulin with apparently tight binding; (ii) the addition of ATP caused the turbidity of the solution of decrease to a level equal to the sum of free dynein plus microtubules; (iii) transmission electron microscopy indicated that microtubules were decorated with dynein arms spaced at a 24-nm longitudinal repeat and that the dynein decoration was removed upon addition of ATP; (iv) cross-section images of microtubules that were saturated with dynein showed six to seven dynein arms around a microtubule consisting of 14 protofilaments, corresponding to a molar ratio of one dynein/six tubulin dimers; (v) the dynein arms were bound primarily by their broader end which corresponds to the end normally bound to the B-subfiber in vivo. Experiments with purified 30 and 14 S dyneins indicated that the dynein-microtubule binding activity and the ATP-induced dissociation were the properties of the 30 S dynein alone. These studies demonstrate that the 30 S dynein under our conditions (50 mM PIPES, pH 6.96, 4 mM MgSO4) interacts with bovine brain microtubules through the ATP-sensitive site of the dynein arm.     

Effects of proteolysis of the extending parts of the high-molecular-weight microtubule-associated proteins on interactions between microtubules

Digestion of assembled microtubules with agarose-bound trypsin was performed to obtain microtubules which lack the extending projections, the non-tubulin-binding part of the high-molecular-weight microtubule-associated proteins. The assembly kinetics and the minimum protein concentration for assembly were the same for these trypsinated microtubules as for normal, untreated microtubules. Furthermore, the digested microtubules gave rise to the same change in turbidity per polymer mass as that found for normal microtubules. However, electron microscopy of pelleted microtubules revealed a closer packing after trypsin treatment. A substantially lower increase in specific viscosity was found upon assembly. At concentrations of above approx. 1.5 mg/ml, the viscosity of trypsin-treated microtubules was almost independent of the protein concentration, in contrast to the turbidity, which still increased. Both microtubules and the trypsin-digested microtubules were easily oriented by shear, although the flow linear dichroism signal for the microtubules after trypsin treatment was only half of that found for perfectly oriented normal microtubules. At higher shear force gradients, digested microtubules aggregated side by side as shown by electron microscopy. This was not found for normal microtubules. Even although the extending parts of the high-molecular-weight proteins are not needed for assembly, they were found to play an important role in microtubule orientation and interactions between microtubules, probably by acting as spacers between microtubules.     

Kinesin-14 Family Proteins HSET/XCTK2 Control Spindle Length by Cross-Linking and Sliding Microtubules

Kinesin-14 family proteins are minus-end directed motors that cross-link microtubules and play key roles during spindle assembly. We showed previously that the Xenopus Kinesin-14 XCTK2 is regulated by Ran via the association of a bipartite NLS in the tail of XCTK2 with importin α/β, which regulates its ability to cross-link microtubules during spindle formation. Here we show that mutation of the nuclear localization signal (NLS) of human Kinesin-14 HSET caused an accumulation of HSET in the cytoplasm, which resulted in strong microtubule bundling. HSET overexpression in HeLa cells resulted in longer spindles, similar to what was seen with NLS mutants of XCTK2 in extracts, suggesting that Kinesin-14 proteins play similar roles in extracts and in somatic cells. Conversely, HSET knockdown by RNAi resulted in shorter spindles but did not affect pole formation. The change in spindle length was not dependent on K-fibers, as elimination of the K-fiber by Nuf2 RNAi resulted in an increase in spindle length that was partially rescued by co-RNAi of HSET. However, these changes in spindle length did require microtubule sliding, as overexpression of an HSET mutant that had its sliding activity uncoupled from its ATPase activity resulted in cells with spindle lengths shorter than cells overexpressing wild-type HSET. Our results are consistent with a model in which Ran regulates the association of Kinesin-14s with importin α/β to prevent aberrant cross-linking and bundling of microtubules by sequestering Kinesin-14s in the nucleus during interphase. Kinesin-14s act during mitosis to cross-link and slide between parallel microtubules to regulate spindle length.     

Competitive Inhibition of High-Affinity Oryzalin Binding to Plant Tubulin by the Phosphoric Amide Herbicide Amiprophos-Methyl

Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in vitro polymerization of isolated plant tubulin (L.C. Morejohn, D.E. Fosket [1984] Science 224: 874-876), yet little other biochemical information exists concerning this compound. To characterize further the mechanism of action of APM, its interactions with tubulin and microtubules purified from cultured cells of tobacco (Nicotiana tabacum cv Bright Yellow-2) were investigated. Low micromolar concentrations of APM depolymerized preformed, taxol-stabilized tobacco microtubules. Remarkably, at the lowest APM concentration examined, many short microtubules were redistributed into fewer but 2.7-fold longer microtubules without a substantial decrease in total polymer mass, a result consistent with an end-to-end annealing of microtubules with enhanced kinetic properties. Quasi-equilibrium binding measurements showed that tobacco tubulin binds [14C]oryzalin with high affinity to produce a tubulin-oryzalin complex having a dissociation constant (Kd) = 117 nM (pH 6.9; 23[deg]C). Also, an estimated maximum molar binding stoichiometry of 0.32 indicates pharamacological heterogeneity of tobacco dimers and may be related to structural heterogeneity of tobacco tubulin subunits. APM inhibits competitively the binding of [14C]oryzalin to tubulin with an inhibition constant (Ki) = 5 [mu]M, indicating the formation of a moderate affinity tubulin-APM complex that may interact with the ends of microtubules. APM concentrations inhibiting tobacco cell growth were within the threshold range of APM concentrations that depolymerized cellular microtubules, indicating that growth inhibition is caused by microtubules depolymerization. APM had no apparent effect on microtubules in mouse 3T3 fibroblasts. Because cellular microtubules were depolymerized at APM and oryzalin concentrations below their respective Ki and Kd values, both herbicides are proposed to depolymerize microtubules by a substoichiometric endwise mechanism.     

Characterization of smooth muscle caldesmon as a microtubule-associated protein.

We have previously shown that nonmuscle caldesmon copurified with brain microtubules binds to microtubules in vitro [Ishikawa et al.: FEBS Lett. 299:54-56, 1992]. To explore the role of caldesmon in the functions of microtubules, further characterization was performed using smooth muscle caldesmon, whose molecular structure and function have been best-characterized in all caldesmon species. Smooth muscle caldesmon bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with the binding constant of 1.1 x 10(6) M-1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. Partial digestion of the caldesmon with alpha-chymotrypsin revealed that the binding site of the caldesmon for microtubules lay in the 34-kDa C-terminal domain. When the caldesmon was in the dimeric form in the absence of a reducing agent, the caldesmon cross-linked microtubules to form bundles. Further, the caldesmon potentiated the polymerization of tubulin, and inhibited the in vitro movement of microtubules on dynein. These results suggest that caldesmon may be involved in the regulation by Ca2+ of the functions of microtubules.     

Aggregation of melanosomes in melanophores is accompanied by the reorganization of microtubules and intermediate filaments

The structure of the cytoskeleton in cultured melanophores of the fish Gymnocorymbus ternetzi during aggregation of melanosomes was studied. It has been shown that the motion of pigment granules is accompanied by a reorganization of microtubules and intermediate filament systems. In melanophores with dispersed pigment granules, microtubules are wavy and form a loose network whilst intermediate filaments in such cells form a dense network around the dispersed melanosomes. During aggregation microtubules and intermediate filaments become radially oriented. It was also shown that the surface area of melanophores increased during aggregation.