Cep70 promotes microtubule assembly in vitro by increasing microtubule elongation

Microtubules are dynamic cytoskeletal polymers present in all eukaryotic cells. In animal cells, they are organized by the centrosome, the major microtubule-organizing center. Many centrosomal proteins act coordinately to modulate microtubule assembly and organization. Our previous work has shown that Cep70, a novel centrosomal protein regulates microtubule assembly and organization in mammalian cells. However, the molecular details remain to be investigated. In this study, we investigated the molecular mechanism of how Cep70 regulates microtubule assembly using purified proteins. Our data showed that Cep70 increased the microtubule length without affecting the microtubule number in the purified system. These results demonstrate that Cep70 could directly regulate microtubule assembly by promoting microtubule elongation instead of microtubule nucleation.     

GTP hydrolysis during microtubule assembly

The GTP cap model of dynamic instability [Mitchison, T., & Kirschner, M.W. (1984) Nature (London) 312, 237] postulates that a GTP cap at the end of most microtubules stabilizes the polymer and allows continuing assembly of GTP-tubulin subunits while microtubules without a cap rapidly disassemble. This attractive explanation for observed microtubule behavior is based on the suggestion that hydrolysis of GTP is not coupled to assembly but rather takes place as a first-order reaction after a subunit is assembled onto a polymer end. Carlier and Pantaloni [Carlier, M., & Pantaloni, D. (1981) Biochemistry 20, 1918] reported a lag of hydrolysis behind microtubule assembly and a first-order rate constant for hydrolysis (kh) of 0.25/min. A lag has not been demonstrated by other investigators, and a kh value that specifies such a slow rate of hydrolysis is difficult to reconcile with reported steady-state microtubule growth rates and frequencies of disassembly. We have looked for a lag using tubulin free of microtubule-associated protein at concentrations of 18.5-74 microM, assembly with and without glycerol, and two independent assays of GTP hydrolysis. No lag was observed under any of the conditions employed, with initial rates of hydrolysis increasing in proportion to rates of assembly. If hydrolysis is uncoupled from assembly, we estimate that kh must be at least 2.5/min and could be much greater, a result that we argue may be advantageous to the GTP cap model. We also describe a preliminary model of assembly coupled to hydrolysis that specifies formation and loss of a GTP cap, thus allowing dynamic instability.     

Nucleotide specificity in microtubule assembly in vitro

A procedure is described for removing most of the GDP bound at the exchangeable GTP binding site (E site) of tubulin. Microtubule protein containing substoichiometric amounts of GDP at the E site is found to polymerize in response to: (a) two nonhydrolyzable ATP analogues, adenylyl imidodiphosphate (AMP-PNP) and adenylyl beta, gamma-methylenediphosphonate (AMP-PCP); and (b) substoichiometric levels of GTP or dGTP. The results are interpreted as suggesting that: (1) when GDP is removed from tubulin, the E site shows broad specificity for nucleoside triphosphates: (2) microtubule assembly can be induced by the binding of substoichiometric amounts of nucleoside triphosphate to the E site.     

Arginyl residues involvement in the microtubule assembly

Modification of pig brain tubulin with 2,3-butanedione, an arginine-specific reagent, resulted in a decrease of its microtubule formation capacity, with apparent first-order kinetics. However, microtubules already assembled were not affected by the reagent. The relation between the polymerization inhibition rate constant and the butanedione concentration followed a saturation curve whereas the colchicine binding activity remained unchanged over that concentration range. GTP partially prevented the decrease of tubulin polymerization induced by the butanedione treatment. This protective effect of GTP was increased by glycerol. The butanedione inhibition of tubulin polymerization appears to be related to the modification of no more than three arginyl residues. These data suggest that at least one of the arginyl residues plays an essential role in tubulin polymerization, probably through its interaction with the negatively charged phosphate moiety of the nucleotide.     

Submembraneous microtubule cytoskeleton: regulation of microtubule assembly by heterotrimeric Gproteins

Heterotrimeric Gproteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. Gproteins also interact with microtubules and participate in microtubule-dependent centrosome/chromosome movement during cell division, as well as neuronal differentiation. In recent years, significant progress has been made in our understanding of the biochemical/functional interactions between Gprotein subunits (alpha and betagamma) and microtubules, and the molecular details emerging from these studies suggest that alpha and betagamma subunits of Gproteins interact with tubulin/microtubules to regulate the assembly/dynamics of microtubules, providing a novel mechanism for hormone- or neurotransmitter-induced rapid remodeling of cytoskeleton, regulation of the mitotic spindle for centrosome/chromosome movements in cell division, and neuronal differentiation in which structural plasticity mediated by microtubules is important for appropriate synaptic connections and signal transmission.     

Effect of gamma-irradiation on microtubule assembly in vitro

Microtubular protein was exposed to gamma-radiation from 500 to 1000 Gy, Within that dose range its polymerization ability was decreased by 20-60 per cent when samples were irradiated in assembled state, and by 40-75 per cent when irradiated in unassembled state. Microtubules assembled from irradiated subunits were shorter and of more uniform lengths than control microtubules. For the dose of 1000 Gy the mean length and its standard deviation were reduced to about one-half of the values of the control.     

Regulation of microtubule assembly and disassembly by nucleotides

The role of nucleotides in microtubular assembly and disassembly has been reviewed. Two possible functions of GTP hydrolysis during assembly are discussed: (1) hydrolysis renders sensitivity to factor(s) regulating microtubule depolymerization; (2) the energy of GTP hydrolysis is utilized for the subunit flow from one end of the microtubule to the other. In the second part of the review, experiments are considered showing that microtubular disassembly takes place in the cells only in the presence of ATP, and, therefore, this process is regulated via some ATP-dependent mechanism (most probably, phosphorylation of microtubule-associated proteins).     

Effects of purinenucleotide analogues on microtubule assembly

This minireview summarizes the syntheses of various purinenucleotide analogues and their effects on microtubule (Mt) assembly. 27 analogues were so far synthesized and, together with 3 analogues commercially available (ITP, XTP and dGTP), their effects on Microtubule assembly were investigated. The positions C2, C6, C8, and ribose moiety of purine nucleotides were modified or substituted. It was found that the microenvironments of the purine base and ribose moiety are important for the nucleotides to support Mt assembly. Introduction of amino group into position C2 of ATP, formation of 2-amino ATP, caused Mt assembly substantially. 2-Amino deoxy ATP and deoxy GTP are more potent than GTP in supporting assembly. The introduction of reactive thiol group into C6 (6-SH-GTP) largely reduces the activity of the analogue to support assembly. However, sequestering reactivity of the thiol group by association with methyl group largely recovers the ability of the analogue to promote assembly. Free rotation of the glycosidic linkage was found to be also innevitable in promoting assembly, as the introduction of sulfur atom between C8 of the purine base and C2' of the ribose moiety (formation of 8,2'-S-cyclo purine nucleotides) caused total inhibition. Purinenucleoside triphosphate supports assembly better than GTP but the deoxy-type analogues are totally inhibitory. 2-Amino-8-hydroxy ATP and other analogues support assembly much better than does GTP. However, their diphosphate analogues are totally incapable of supporting assembly. Introduction of a bulky fluorescent probes into C3' can be made to visualize the fluorescent signal in assembled Mts. Together with the suggestions proposed from electron chrystallography of zinc-induced tubulin sheets, interactions of the purine base and ribose moieties with surrounding amino acid residues are discussed.     

Double and single exponential kinetics of microtubule assembly in vitro

The kinetics of the microtubule protein assembly were studied in Mes buffer, pH 6.6, at 28 degrees C. The assembly under above conditions follow a kinetic expression containing two exponential terms. The observed two rate constants depend on protein concentration, and are on the order of 10(-2) sec-1 and 10(-3) sec-1. When CaCl2 is added to the system in low concentration, the kinetic expression becomes single exponential. The observed rate constant is independent of protein concentration and its value is 5 X 10(-3) sec-1. It is concluded that the double exponential kinetics correspond to favorable assembly conditions, probably to a high extent of nucleation, whereas the single exponential kinetics correspond to favorable assembly conditions, probably to a high extent of nucleation, whereas the single exponential kinetics is a slower process which occur under hindered assembly conditions.     

Diethylstilbestrol induces metaphase arrest and inhibits microtubule assembly

Diethylstilbestrol produced a dose-dependent increase in the mitotic index of the human prostatic tumour cell line DU 145. This is the result of metaphase arrest which may be induced by the action of diethylstilbestrol on spindle microtubules. Evidence is presented to show that diethylstilbestrol affects microtubules. Diethylstilbestrol completely inhibited the assembly of isolated brain microtubules although only partial disassembly could be induced. In contrast to other microtubule poisons, the inhibitory effect of diethylstilbestrol on taxol-induced self-assembly could be reversed by the addition of GTP.     

Enthalpy changes in microtubule assembly from pure tubulin

The enthalpy changes that occur in the self-assembly of tubulin into microtubules were examined by adiabatic differential heat capacity microcalorimetry and by isothermal batch microcalorimetry. Tubulin solutions at concentrations between 7 and 17 mg/mL were heated from 0 to 40 degrees C at heating rates of 1 or 2 deg/min in pH 6.8 or 7.0 assembly buffers containing 20 mM MES, 100 mM glutamic acid, 5 mM MgCl2, 3.4 M glycerol, and either 0.5 mM GMP-PCP or 1 mM GTP. The assembly reaction in the presence of GTP was characterized by a complex heat-uptake pattern consisting of a broad endotherm with a sharper exotherm superimposed on it, similar to assembly in a GTP phosphate buffer [Hinz, H.-J., Gorbunoff, M.J., Price, B., & Timasheff, S.N. (1979) Biochemistry 18,3084]. Replacement of GTP by the nonhydrolyzable analogue resulted in a pattern typical for an endothermic reaction only. These results have permitted the assignment of the endothermic process to microtubule assembly and of the exothermic process to the resultant GTP hydrolysis. In these studies equilibration was found to be slow, several hours of cooling being required for the system to return to its original state. Turbidity scans also revealed hysteresis between consecutive scans and a displacement of the depolymerization transition midpoint to a lower temperature than that of assembly. The disassembly of microtubules was examined in batch calorimetry experiments in pH 7.0 phosphate, 1 mM GTP, 16 mM MgCl2, and 3.4 M glycerol, in which tubulin assembled into microtubules was diluted to below the critical concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sites of microtubule assembly and disassembly in the mitotic spindle

We have microinjected biotinylated tubulin into mitotic fibroblast cells to identify the sites in the spindle at which new subunits are incorporated into microtubules (MTs). Labeled subunits were visualized in the electron microscope using an antibody to biotin followed by a secondary antibody coupled to colloidal gold. Astral MTs incorporate labeled subunits very rapidly by elongation of existing MTs and by new nucleation from the centrosome. At a slower rate, kinetochore MTs incorporate subunits at the kinetochore progressively during metaphase, suggesting a slow poleward flux of subunits in the kinetochore fiber. When cells injected in metaphase were examined in anaphase, a significant fraction of kinetochore MTs was unlabeled, suggesting that depolymerization had occurred at the kinetochore concomitant with chromosome to pole movement. The existence of opposite fluxes at the kinetochore during metaphase and anaphase suggests that two separate forces are responsible for chromosome congression and anaphase movement.     

Levuglandin E2 inhibits mitosis and microtubule assembly

Levuglandin E2 (LGE2) is a gamma-keto aldehyde produced by rearrangement of the prostaglandin endoperoxide PGH2 under the aqueous conditions of its biosynthesis. We show that exogenous LGE2 enters cells and efficiently inhibits the first synchronous cell division of fertilized sea urchin eggs. We attribute this inhibition to covalent modification of tubulin and thereby to inhibition of microtubule assembly.     

Erythrocyte microtubule assembly in vitro. Tubulin oligomers limit the rate of microtubule self-assembly

Chicken erythrocyte tubulin containing a unique beta tubulin variant polymerizes with greater efficiency (lower critical concentration) but at a slower rate than chicken brain tubulin. In a previous study we demonstrated that the low net rate of assembly is partly due to the presence of large oligomers and rings which reduce the initial rate of subunit elongation on microtubule seeds (Murphy, D.B., and Wallis, K.T. (1985) J. Biol. Chem. 260, 12293-12301). In this study we show that erythrocyte tubulin oligomers also retard the rate of microtubule nucleation and the net rate of self-assembly. The inhibitory effect is most likely to be due to the increased stability of erythrocyte tubulin oligomers, including a novel polymer of coiled rings that forms during the rapid phase of microtubule polymerization. The slow rate of dissociation of rings and coils into dimers and small oligomers appears to limit both the nucleation and elongation steps in the self-assembly of erythrocyte microtubules.     

Cytoplasmic microtubule assembly is altered in cytoplasts.

Nucleation of microtubule (MT) organization of the cytoplasmic microtubule complex (CMTC) from the microtubule organizing centres (MTOC) was studied in enucleated cytoplasts of human diploid fibroblast (MRC-5) and mouse peritoneal macrophages in culture. Cytoplasts of both cell types could not organize the complete CMTC. Aberrant MT patterns were seen in MRC-5 cells while mouse macrophages showed occurrence of few short MT. The studies suggest that nucleus may have a role in determining CMTC.     

PACSIN proteins bind tubulin and promote microtubule assembly

PACSINs are intracellular adapter proteins involved in vesicle transport, membrane dynamics and actin reorganisation. In this study, we report a novel role for PACSIN proteins as components of the centrosome involved in microtubule dynamics. Glutathione S-transferase (GST)-tagged PACSIN proteins interacted with protein complexes containing α- and γ-tubulin in brain homogenate. Analysis of cell lysates showed that all three endogenous PACSINs co-immunoprecipitated dynamin, α-tubulin and γ-tubulin. Furthermore, PACSINs bound only to unpolymerised tubulin, not to microtubules purified from brain. In agreement, the cellular localisation of endogenous PACSIN 2 was not affected by the microtubule depolymerising reagent nocodazole. By light microscopy, endogenous PACSIN 2 localised next to γ-tubulin at purified centrosomes from NIH 3T3 cells. Finally, reduction of PACSIN 2 protein levels with small-interfering RNA (siRNA) resulted in impaired microtubule nucleation from centrosomes, whereas microtubule centrosome splitting was not affected, suggesting a role for PACSIN 2 in the regulation of tubulin polymerisation. These findings suggest a novel function for PACSIN proteins in dynamic microtubuli nucleation.     

Nucleotide binding and phosphorylation in microtubule assembly in vitro.

Two non-hydrolyzable analogs of GTP, guanylyl-β,γ-methylene diphosphonate and guanylyl imidodiphosphate, have been found to induce rapid and efficient microtubule assembly in vitro by binding at the exchangeable site (E-site) on tubulin. Characterization of microtubule polymerization by several criteria, including polymerization kinetics, nucleotide binding to depolymerized and polymerized microtubules, and microtubule stability, reveals strong similarities between microtubule assembly induced by GTP and non-hydrolyzable GTP analogs. Nucleoside triphosphates which bind weakly or not at all to tubulin, such as ATP, UTP and CTP, are shown to induce microtubule assembly by means of a nucleoside diphosphate kinase (NDP-kinase, EC 2.7.4.6.) activity which is not intrinsic to tubulin. The NDP-kinase mediates microtubule polymerization by phosphorylating tubulin-bound GDP in situ at the E-site. Although hydrolysis of exchangeably bound GTP occurs, it is found to be uncoupled from the polymerization reaction. The non-exchangeable nucleotide binding site on tubulin (N-site) is not directly involved in microtubule assembly in vitro. The N-site is shown to contain almost exclusively GTP which is not hydrolyzed during microtubule assembly. A scheme is presented in which GTP acts as an allosteric effector at the E-site during microtubule assembly in vitro.