Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

A growing body of evidence points to an association of prion protein (PrP) with microtubular cytoskeleton. Recently, direct binding of PrP to tubulin has also been found. In this work, using standard light scattering measurements, sedimentation experiments, and electron microscopy, we show for the first time the effect of a direct interaction between these proteins on tubulin polymerization. We demonstrate that full-length recombinant PrP induces a rapid increase in the turbidity of tubulin diluted below the critical concentration for microtubule assembly. This effect requires magnesium ions and is weakened by NaCl. Moreover, the PrP-induced light scattering structures of tubulin are cold-stable. In preparations of diluted tubulin incubated with PrP, electron microscopy revealed the presence of approximately 50 nm disc-shaped structures not reported so far. These unique tubulin oligomers may form large aggregates. The effect of PrP is more pronounced under the conditions promoting microtubule formation. In these tubulin samples, PrP induces formation of the above oligomers associated with short protofilaments and sheets of protofilaments into aggregates. Noticeably, this is accompanied by a significant reduction of the number and length of microtubules. Hence, we postulate that prion protein may act as an inhibitor of microtubule assembly by inducing formation of stable tubulin oligomers.     

Phosphofructokinase from Dictyostelium discoideum is a potent inhibitor of tubulin polymerization

We identified the nonallosteric phosphofructokinase from the slime mold Dictyostelium discoideum as a potent protein factor that inhibits the rate of polymerization of tubulin at a molar ratio of 1 molecule to about 300 tubulin dimers for half-maximal action (IC50 = 32 nM). This effect was (i) assessed by turbidity measurements, pelleting of microtubules, and electron microscopy, (ii) observed when tubulin assembly was induced by taxol as well as by GTP in the presence of microtubule-associated proteins or glutamate, and (iii) specific as it was not produced by the phosphofructokinase from rabbit muscle. Also in contrast to the latter, neither tubulin nor microtubules modified the catalytic activity of the slime mold isozyme. Immunoelectron microscopy provided further evidence that D. discoideumphosphofructokinase physically interacts with tubulin, leading to the formation of aggregates. The process seems to be reversible since microtubules eventually formed in the presence of the inhibitor with concomitant reduction of tubulin aggregates. Limited proteolysis by subtilisin showed that the hypervariable C-termini of tubulin is not involved in the interaction with the enzyme. The possible physiological relevance of this novel function of D. discoideum phosphofructokinase different from its glycolytic action is discussed.     

Native tubulin-folding cofactor E purified from baculovirus-infected Sf9 cells dissociates tubulin dimers

Tubulin-folding cofactor E (TBCE) is an alpha-tubulin-binding protein involved in the formation of the tubulin dimer and in microtubule dynamics, through the regulation of tubulin heterodimer dissociation. TBCE has also been implicated in two important related human disorders, the Kenny-Caffey and Sanjad-Sakati syndromes. The expression of TBCE as a recombinant protein in bacteria results in the formation of insoluble inclusion bodies in the absence of denaturing agents. Although the active protein can be obtained from mammalian tissues, biochemical studies of TBCE present a special challenge. To express and purify native TBCE, a recombinant baculovirus expression system was used. Native wild-type TBCE purified from Sf9 extracts was sequentially purified chromatographically through cation exchange, hydrophobic interaction, and high-resolution gel-filtration columns. Mass spectrometric analysis identified 30% of the sequence of human TBCE. A stoichiometric excess of purified TBCE dissociated tubulin heterodimers. This reaction produced a highly unstable TBCE-alpha-tubulin complex, which formed aggregates. To distinguish between the aggregation of tubulin dimers induced by TBCE and tubulin dissociation, TBCE and tubulin were incubated with tubulin-folding cofactor A (TBCA). This cofactor captures the beta-tubulin released from the heterodimer with a stoichiometry of 1:1, as previously demonstrated. The beta-tubulin polypeptide was recovered as TBCA-beta-tubulin complexes, as demonstrated by non-denaturing gel electrophoresis and specific antibodies directed against beta-tubulin and TBCA.     

Comparative study of the precipitation of low density lipoproteins by aortic proteodermatan sulphate and heparin

The aortic proteoglycans and heparin were shown to form insoluble complexes with human low density lipoproteins (LDL). The effect of temperature, polyethylene glycol and ionic strength on the formation of complexes between porcine aortic proteodermatan sulphate (PDS) and LDL has been studied by laser nephelometry and comparisons made with heparin LDL complexes. Turbidity was a nonlinear function of the quantity of LDL precipitated by PDS. The turbidity of aggregates was constant at temperatures between 2 degrees C and 30 degrees C but increased with temperature above 30 degrees C up to 50 degrees C. The formation of insoluble complexes decreased rapidly with increasing NaCl concentration. Polyethylene glycol enhanced the turbidity at 20 degrees C but not at 37 degrees C. It also increased the resistance of complexes to dissociation by increasing ionic strength. The turbidity of heparin--LDL complexes was linearly correlated with the quantity of precipitated LDL. The heparin-LDL aggregates were less sensitive to modification of temperature and ionic strength than the PDS-LDL aggregates. These results suggest that ionic interactions are weaker in PDS-LDL complexes than in the heparin-LDL complexes. Non-coulombic interactions and/or temperature dependent conformational changes may be involved in the stabilization of supramolecular PDS-LDL aggregates. No such interactions or changes appear to be involved in complex formation between heparin and LDL.     

Some features of the vinblastine-induced assembly of porcine tubulin.

Porcine tubulin precipitated by 10^?3, m vinblastine (VLB) contains approximately 0.50 molecule of VLB bound per 110,000-molecular-weight tubulin dimer. The amount of precipitate, followed by turbidity, is a linear function of the initial tubulin concentration. The rate of precipitation is roughly first order in protein concentration. Vindoline and velbanamine halves of VLB are ineffective separately or together in producing the tubular aggregates observed for VLB precipitates by electron microscopy. At 10^?3, m concentrations no turbidity is observed nor is there any competition with VLB-induced turbidity. Removal of GTP from tubulin by dialysis or incubation of tubulin in the absence of added GTP blocks VLB-induced assembly. Readdition of GTP at room temperature or above restores sensitivity to VLB precipitation. The β,γ methylene analog of GTP cannot substitute for GTP in this process. About 0.7 mol of added GTP is found bound per mole of tubulin dimer. During the course of VLB-induced assembly, roughly half of this GTP is displaced. These results show interesting similarities and differences in the VLB-induced assembly of tubulin and the normal in vitro assembly of microtubules. Further comparisons between both assembly processes should be useful.     

C-terminal cleavage of tubulin by subtilisin enhances ring formation

Following cleavage of alpha- and beta-tubulin C termini, under mild conditions we observed that microtubule-related polymers were formed, and also that ring aggregates were abundant. These ring aggregates were clearly detected by turbidity and electron microscope studies under standard assembly conditions. It was found that removal of the C-terminal fragments of tubulin (phosphocellulose-tubulin or Weisenberg tubulin) markedly favored Mg2(+)-induced ring formation. Binding of GDP to the exchangeable nucleotide site of cleaved tubulin further enhanced ring formation. The cleaved tubulin-GDP ring aggregates could be classified into three types: aggregates without apparent order, bidimensionally ordered ring aggregates, and stacks of rings. Temperature had little effect on the formation of these ring aggregates; however, they were very sensitive to ionic strength.     

Evidence for the spontaneous formation of disulfide crosslinked aggregates of tubulin during nondenaturing electrophoresis

Phosphocellulose-purified tubulin has been shown to form a characteristic "ladder" of nonmicrotubular aggregates during nondenaturing gel electrophoresis (J. J. Correia and R. C. Williams, Jr. (1985) Arch. Biochem. Biophys. 239, 120-129). In this paper we describe evidence that the intersubunit bonds responsible for formation of these oligomeric particles are disulfides. Two-dimensional nondenaturing-denaturing gel electrophoresis demonstrates that each aggregate zone is composed of alpha- and beta-subunits of tubulin. Omission of beta-mercaptoethanol during the sodium dodecyl sulfate (SDS)-electrophoresis step causes a pattern of aggregates to appear and implicates disulfide linkages in their stabilization. Molecular weights, estimated from mobilities in the second (SDS) dimension of two-dimensional gels, suggest that the aggregates are crosslinked in units of monomers, not heterodimers. Consistent with this conclusion, alpha- or beta-subunits alone (isolated by isoelectric focusing) will form the same ladder of aggregates. The disulfide crosslinking of tubulin is also achievable in solution. It is favored by high concentrations of alcohol, the presence of oxidizing agents, high pH, and high temperature, conditions that denature tubulin and cause rapid noncovalent aggregation or precipitation. When aggregate formation was monitored as a function of time by SDS-gel electrophoresis in the absence of beta-mercaptoethanol and by quantitative sulfhydryl and disulfide titrations, the most effective conditions for the crosslinking reaction included greater than 75% alcohol, excess H2O2, or excess iodine. These results suggest that proximity of a hydrophobic gel matrix, high pH, the presence of oxidizing agents, high protein concentration, tubulin's propensity to aggregate nonspecifically, and the availability of as many as 20 sulfhydryls in alpha beta-tubulin contribute, during nondenaturing gel electrophoresis, to the spontaneous formation of disulfide-crosslinked tubulin aggregates.     

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.     

Assembly of tubulin by classic myelin basic protein isoforms and regulation by post-translational modification

Myelin basic protein (MBP), a highly cationic protein that maintains the structure of the myelin sheath, associates with tubulin in vivo. The in vitro assembly of tubulin by MBP was examined here using several assays. The unmodified C1 component of 18.5 kDa bovine MBP (bC1) assembled tubulin into microtubules in a dose-dependent manner via filamentous intermediates, and was able simultaneously to promote the formation of microtubule bundles. The critical tubulin concentration in the presence of bC1 was 0.69 +/- 0.05 microM. The effects of post-translational modifications (such as deamidation and phosphorylation) were assayed by comparing the bC1-bC6 components of 18.5 kDa bovine MBP; an increasing level of modification enhanced the ability of MBP to assemble tubulin. The effects of charge reduction via deimination were examined using recombinant murine isoforms emulating the unmodified C1 and deiminated C8 isoforms of 18.5 kDa MBP; both rmC1 and rmC8 exhibited a comparable ability to assemble tubulin. The effects of alternate exon recombination of the classic MBP variants were tested using the recombinant murine 21.5, 17.22, and 14 kDa isoforms. The isoforms containing regions derived from exon II of the classic MBP gene, 21.5 and 17.22 kDa MBP, showed no substantial difference in the extent of tubulin polymerization and bundling when compared to those of 18.5 kDa MBP. The 14 kDa isoform and two terminal deletion mutants of rmC1 were able to induce microtubule polymerization, but not bundling, to the same degree as the longer proteins. Finally, bC1 was shown to disrupt and aggregate planar sheets of crystalline tubulin stabilized by paclitaxel, establishing that these structures are not suitable substrates for the formation of MBP cocrystals.     

Mechanism of microtubule assembly. Changes in polymer structure and organization during assembly of sea urchin egg tubulin

Assembly of tubulin, purified from eggs of the sea urchin Stronglyocentrotus purpuratus, was examined at physiological (18 degrees C) and nonphysiological (37 degrees C) temperatures. Critical concentrations for assembly were 0.71 mg/ml at 18 degrees C and 0.21 mg/ml at 37 degrees C. At tubulin concentrations above 1.2 mg/ml at 18 degrees C and 0.5 mg/ml at 37 degrees C, a concentration-dependent "overshoot" in turbidity and in small-angle light scattering was observed; turbidity and scattering increased rapidly to a peak, then decreased asymptotically toward a steady-state value. Quantitative sedimentation analysis revealed that the mass of assembled polymer reached and maintained a constant level during overshoot of turbidity. Changes in the wavelength dependence of turbidity were consistent with the initial formation of sheets of tubulin, followed by conversion of the sheets to microtubules, both at 18 and 37 degrees C. Examination by negative-stain electron microscopy showed that sheetlike structures predominated during the early stages of overshoot assembly, while complete microtubules were present at steady state. Furthermore, measurements of average polymer length revealed that the overshoots in turbidity and in light scattering are unlikely to be caused by polymer length redistribution. Qualitative observations of solution birefringence suggested that the polymer became progressively more aligned during assembly. These results suggest that the turbidity/light-scattering overshoots reflect changes in the form or in the organization of the assembling polymer, or both.     

Effects of vanadate on the assembly and disassembly of purified tubulin

Sodium-orthovanadate (100-700 microM) added to purified pig brain microtubule protein (molar ratios 13-90 moles vanadate/mole tubulin) inhibits to a considerable extent the assembly (up to 65%) and the disassembly rates (up to 60%) of microtubules, as determined by turbidimetry. Vanadate added to preformed microtubules did not appreciably alter the turbidity level of the samples, however, the disassembly rates were decreased in the same manner as when vanadate was added prior to polymerization. Microtubule protein kept on ice for 3-6 hours became more susceptible to vanadate than freshly prepared protein. The effect of vanadate was independent of the GTP concentration at which the polymerization assays were performed (0.025 to 1 mM GTP). In the presence of taxol, which increases the rate and extent of microtubule formation, vanadate had no effect on assembly rates. Disassembly was inhibited, however, much less than in the presence of vanadate alone. Electron microscopy and polyacrylamide gel electrophoresis did not reveal differences between microtubules prepared in the presence or in the absence of vanadate. This is consistent with the notion that vanadate does not interfere with the interaction between tubulin and the high-molecular weight microtubule-associated proteins. Apparently vanadate brings about an allosteric change of the microtubule protein(s) resulting in the abnormal polymerization kinetics of tubulin found in our study. The above results may be relevant for studies where the effects of vanadate on intracellular motility are interpreted as being solely due to a specific inhibition of ATPases.     

Interactions of the sponge-derived antimitotic tripeptide hemiasterlin with tubulin: comparison with dolastatin 10 and cryptophycin 1

The sponge-derived antimitotic tripeptide hemiasterlin was previously shown to inhibit tubulin polymerization. We have now demonstrated that hemiasterlin resembles most other antimitotic peptides in noncompetitively inhibiting the binding of vinblastine to tubulin (apparent K(i) value, 7.0 microM), competitively inhibiting the binding of dolastatin 10 to tubulin (apparent K(i) value, 2.0 microM), stabilizing the colchicine binding activity of tubulin, inhibiting nucleotide exchange on beta-tubulin, and inducing the formation of tubulin oligomers that are stable to gel filtration in the absence of free drug, even at low drug concentrations. The tubulin oligomerization reaction induced by hemiasterlin was compared to the reactions induced by dolastatin 10 and cryptophycin 1. Like dolastatin 10, hemiasterlin induced formation of a tubulin aggregate that had the morphological appearance primarily of ring-like structures with a diameter of about 40 nm, while the morphology of the cryptophycin 1 aggregate consisted primarily of smaller rings (diameter about 30 nm). However, the hemiasterlin aggregate differed from the dolastatin 10 aggregate in that its formation was not associated with turbidity development, and the morphology of the hemiasterlin aggregate (as opposed to the dolastatin 10 aggregate) did not change greatly when microtubule-associated proteins were present (tight coils and pinwheels are observed with dolastatin 10 but not with hemiasterlin or cryptophycin 1). Opacification of tubulin-dolastatin 10 mixtures was inhibited by hemiasterlin at 22 degrees C and stimulated at 0 degrees C, while cryptophycin 1 was inhibitory at both reaction temperatures.     

Strongylocentrotus purpuratus spindle tubulin. I. Characteristics of its polymerization and depolymerization in vitro

Tubulin was extracted from spindles isolated from embryos of the sea urchin Strongylocentrotus purpuratus, repolymerized in vitro, and purified through three cycles of temperature-dependent assembly and disassembly. In addition to the tubulin, these preparations contain a protein of 80 kdaltons and a small but variable amount of actin. At 37 degrees C, the tubulin polymerizes with a critical concentration of 0.15-0.2 mg/ml into smooth-walled polymers which contain predominantly 14 protofilaments. Removal of the 80 kdalton protein and the actin by DEAE-chromatography does not change the critical concentration for polymerization. At 15 degrees C, which is within the range of physiological temperatures for S. purpuratus embryos, the spindle tubulin will self-assemble, but the rate of total polymer formation is very slow, requiring hours in the test tube. This rate can be increased by shearing the polymerizing microtubules, creating more ends for assembly, indicating that the slow rate of polymer formation is due to a slow rate of self-initiation. If spindle tubulin is polymerized at 37 degrees C and then lowered to 15 degrees C, some polymer will be retained, the percentage of which depends on the protein concentration. These results demonstrate that spindle tubulin from S. purpuratus will assemble at 37 degrees C with a low critical concentration for polymerization in the absence of detectable MAPs and will self-assemble and maintain steady state levels of polymer at physiological temperatures.     

Interaction of tyrosine hydroxylase with tubulin

Bovine adrenal medulla tyrosine hydroxylase associates with microtubules during tubulin assembly. Limited proteolytic digestion of tyrosine hydroxylase does not affect the enzymatic activity but prevents its association with tubulin. A possible interpretation is that an ionic interaction occurs between microtubules and a negatively charged region of the enzyme which is removed by the protease treatment. Tyrosine hydroxylase is able to induce purified tubulin assembly as do the microtubule associated proteins; however, the association induced by tyrosine hydroxylase corresponds to the formation of aggregates or organized structures different from microtubules. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and electron microscopy of proteins obtained from bovine adrenal medulla show the presence of tubulin in this tissue.     

Interaction of the myelin basic protein with the anionic detergent sodium dodecyl sulphate.

The interaction of the myelin basic protein and two peptides derived from it with the anionic detergent SDS (sodium dodecyl sulphate) was studied. At molar ratios of detergent/protein of up to approx. 20:1 the transient increase in turbidity (as measured by increases in A230) is proportional to the ratio. Between ratios of 30:1 and 100:1 the effect of the detergent is constant and maximal. At molar ratios exceeding 100:1 the transient increase in turbidity decreases with increasing amounts of detergent. With increasing ionic strength the rapid development of turbidity is inhibited, whereas the slow decay of turbidity is not affected. Neither of the peptide fragments produced by cleavage of the myelin basic protein at the single tryptophan residue, nor both when mixed, produce measurable turbidity when mixed with SDS. Under similar conditions poly-L-lysine of similar molecular size to the basic protein shows the increase in turbidity but not the decay. The interaction between the protein and SDS is interpreted in molecular terms, which involve the initial ionic interaction of the detergent with protein resulting in aggregation and turbidity in the solution. Within the aggregated complexes molecules rearrange to maximize hydrophobic interactions.     

Direct photoaffinity labeling of tubulin with guanosine 5'-triphosphate

Irradiation of tubulin in the presence of [3H]GTP or [3H]GDP at 254 nm led to the covalent incorporation of nucleotide into the protein. The specific nature of the labeling was shown in the following manner: with tubulin depleted of exchangeable nucleotide, the amount of labeling increased to a plateau value as the [3H]GTP concentration was increased, with saturation being reached at a ratio of approximately 1.5; the same amount of labeling was obtained with GTP/tubulin ratios of 1 and 100; [3H]GMP was not incorporated into the dimer, nor did GMP inhibit the incorporation of [3H]GTP; [3H]ATP was not incorporated; [3H]GTP incorporation did not occur into denatured tubulin or into serum albumin. When [alpha-32P]GTP was used in the irradiation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the carboxymethylated protein demonstrated that the incorporated label was associated with the beta subunit. The radiation treatment did cause changes in the tubulin molecule resulting in a decrease in assembly competence and in sulfhydryl groups, but these effects were minimized when a large excess of GTP was present during irradiation. Labeling of tubulin in the assembled state was much less than that observed in the free state.     

Thermodynamics of reversible monomer-dimer association of tubulin

The equilibrium between the rat brain tubulin alpha beta dimer and the dissociated alpha and beta monomers has been studied by analytical ultracentrifugation with use of a new method employing short solution columns, allowing rapid equilibration and hence short runs, minimizing tubulin decay. Simultaneous analysis of the equilibrium concentration distributions of three different initial concentrations of tubulin provides clear evidence of a single equilibrium characterized by an association constant, Ka, of 4.9 X 10(6) M-1 (Kd = 2 X 10(-7) M) at 5 degrees, corresponding to a standard free energy change on association delta G degrees = -8.5 kcal mol-1. Colchicine and GDP both stabilize the dimer against dissociation, increasing the Ka values (at 4.5 degrees C) to 20 X 10(6) and 16 X 10(6) M-1, respectively. Temperature dependence of association was examined with multiple three-concentration runs at temperatures from 2 to 30 degrees C. The van't Hoff plot was linear, yielding positive values for the enthalpy and entropy changes on association, delta S degrees = 38.1 +/- 2.4 cal deg-1 mol-1 and delta H degrees = 2.1 +/- 0.7 kcal mol-1, and a small or zero value for the heat capacity change on association, delta C p degrees. The entropically driven association of tubulin monomers is discussed in terms of the suggested importance of hydrophobic interactions to the stability of the monomer association and is compared to the thermodynamics of dimer polymerization.     

Characterization of 20S component in tubulin from mammalian brain.

Tubulin from porcine brain, purified by at least two cycles of assembly and disassembly, was characerized at different pH values by sedimentation velocity analysis and turbidimetric measurements. At pH 6.4 the depolymerized material was composed of two major species sedimenting with so20,w values of 6 and 36 and a minor one of 20S. By raising the pH, the amount of the 20S component increased and that of the 36S decreased, whereas that of the 6S component was unaltered. At pH 7.6 the mixture contained 20S and 6S components but hardly any 36S. The 20S species can be separated from the 6S ones by gel filtration on agarose A-15m at pH 7.6. On electron microscopic examination this preparation contains far fewer double rings compared to the material at pH 6.4, but single rings could often be seen. Sodium dodecyl sulfate gel electrophoresis of the 20S and 36S components showed that they consist almost entirely of tubulin and some higher and lower molecular weight fractions. Turbidity measurements showed that the minimal protein concentration necessary for polymerization increases with increasing pH. The turbidity plateau reached at a given pH can be raised by decreasing the pH. From these results it is suggested that the 20S component is an intermediate of the 36S species. The results further indicate the existence of a pH-dependent equilibrium between the 20S species and the 36Soligomers.     

Mechanical properties of brain tubulin and microtubules

We measured the elasticity and viscosity of brain tubulin solutions under various conditions with a cone and plate rheometer using both oscillatory and steady shearing modes. Microtubules composed of purified tubulin, purified tubulin with taxol and 3x cycled microtubule protein from pig, cow, and chicken behaved as mechanically indistinguishable viscoelastic materials. Microtubules composed of pure tubulin and heat stable microtubule-associated proteins were also similar but did not recover their mechanical properties after shearing like other samples, even after 60 min. All of the other microtubule samples were more rigid after flow orientation, suggesting that the mechanical properties of anisotropic arrays of microtubules may be substantially greater than those of randomly arranged microtubules. These experiments confirm that MAPs do not cross link microtubules. Surprisingly, under conditions where microtubule assembly is strongly inhibited (either 5 degrees or at 37 degrees C with colchicine or Ca++) tubulin was mechanically indistinguishable from microtubules at 10-20 microM concentration. By electron microscopy and ultracentrifugation these samples were devoid of microtubules or other obvious structures. However, these mechanical data are strong evidence that tubulin will spontaneously assemble into alternate structures (aggregates) in nonpolymerizing conditions. Because unpolymerized tubulin is found in significant quantities in the cytoplasm, it may contribute significantly to the viscoelastic properties of cytoplasm, especially at low deformation rates.     

Enhancement of tubulin assembly as monitored by a rapid filtration assay

The early kinetics of microtubule formation from lamb brain tubulin isolated by affinity chromatography can be followed by a newly developed filter assay. The rapid collection of microtubules on glass fiber filters permits the calculation of the moles of tubulin polymerized. The filter assay gives both a rate and extent of polymerization that are identical to those obtained by turbidity or sedimentation analysis, respectively. The microtubules trapped by the filter are readily depolymerized by cold (t12= 3 min) and slowly by colchicine (t1/2= 32min). Tubulin purified by affinity chromatography requires a high protein concentration (>4 mg/ml) for polymerization. Although 5m glycerol allows polymerization to occur at tubulin concentrations below 2 mg/ml, the maximum amount of microtubule formation is observed at low tubulin concentration when microtubule-associated proteins are present. These proteins are not retained by the affinity resin; however, they can be eluted from diethylaminoethyl-Sephadex by solutions containing 0.3m KCl. Microtubule-associated proteins enhance both the rate of polymerization and the total amount of tubulin polymerized as assessed by the filter assay, suggesting that they are involved in both initiation and elongation of microtubules.