共查询到20条相似文献,搜索用时 31 毫秒
1.
R. Wu J.R. Guzman-Sepulveda A.P. Kalra J.A. Tuszynski A. Dogariu 《Biochemistry and Biophysics Reports》2022
The assembly/disassembly of biological macromolecules plays an important role in their biological functionalities. Although the dynamics of tubulin polymers and their super-assembly into microtubule structures is critical for many cellular processes, details of their cyclical polymerization/depolymerization are not fully understood. Here, we use a specially designed light scattering technique to continuously examine the effects of temperature cycling on the process of microtubule assembly/disassembly. We observe a thermal hysteresis loop during tubulin assembly/disassembly, consistently with earlier reports on the coexistence of tubulin and microtubules as a phase transition. In a cyclical process, the structural hysteresis has a kinetic component that depends on the rate of temperature change but also an intrinsic thermodynamic component that depends on the protein topology, possibly related to irreversible processes. Analyzing the evolution of such thermal hysteresis loops over successive cycles, we found that the assembly/disassembly ceases after some time, which is indicative of protein aging leading to its inability to self-assemble after a finite number of temperature cycles. The emergence of assembly-incompetent tubulin could have major consequences for human pathologies related to microtubules, including aging, neurodegenerative diseases and cancer. 相似文献
2.
Tubulin oligomers and microtubule oscillations. Antagonistic role of microtubule stabilizers and destabilizers 总被引:2,自引:0,他引:2
Several types of non-equilibrium phenomena have been observed in microtubule polymerization, including dynamic instability, assembly overshoot and oscillations. They can be interpreted in terms of interactions between tubulin subunits (= alpha, beta heterodimers), microtubules, and a third state, oligomers, which represent intermediates between microtubule disassembly and the regeneration of assembly-competent subunits by GTP. Here we examine the role of oligomers by varying conditions that stabilize or destabilize microtubules and/or oligomers. By varying their ratio one can drive tubulin assembly either into steady-state microtubules or oligomers. These regimens of assembly conditions are separated by a region where microtubules oscillate. The oscillations can be simulated by computer modelling, based on a reaction scheme involving the three states of tubulin and nucleotide exchange on tubulin subunits, but not on microtubules or oligomers. 相似文献
3.
Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy 总被引:21,自引:14,他引:7
We describe here the continuous observations of the polymerization of individual microtubules in vitro by darkfield microscopy. In homogeneous preparations we verify that polymerization can occur onto both ends of microtubules. The assembly of microtubules is polar, with one end growing at three times the rate of the other. The differential rate of elongation can be used to determine the polarity of growth off cellular nucleating centers. We show that the microtubules grow off the proximal end of ciliary axonemes at a growth rate equal to that of the slow growing end of free microtubules, while growth off the distal end proceeds at the same rate as the fast growing end. Applying this technique to microtubule growth from metaphase chromosomes isolated from HeLa and CHO cells, we demonstrate that chromosomes initiate polymerization with the fast growing end facing away from the chromosome nucleation site. The opposite ends of free microtubules show different sensitivities to microtubule depolymerizing agents such as low temperature, Ca++ or colchicine as measured directly by darkfield microscopy. The differing rates of assembly and disassembly of each end of a microtubule suggest that a difference in polarity of growth off nucleating sites could serve as one basis for regulating the polymerization of different groups of microtubules in the same cell. 相似文献
4.
Robert V. Zackroff Richard C. Weisenberg William J. Deery 《Journal of molecular biology》1980,139(4):641-677
In this paper we expand upon a previously reported observation of the effects of GDP on microtubule assembly. A ratio of GDP to GTP of ten (1 mm-GDP and 0.1 mm-GTP) is generally sufficient to completely block microtubule assembly, but only limited depolymerization is induced if GDP is added after assembly has reached a plateau in the presence of GTP. When added during polymerization, GDP arrests further elongation, and greater steady-state levels of assembly are obtained the later the time of addition of GDP. To explain this behavior we examined the rates of assembly and disassembly and the apparent critical concentration (C0) of tubulin in the presence of GDP. GDP-tubulin polymerizes very slowly as compared to GTP-tubulin, while depolymerization rates, as determined by dilution, are nearly identical in GTP and GDP. The C0 value calculated from the assembly and disassembly rates in GTP is within experimental error of the C0 value at steady-state determined directly. In the presence of GDP, however, the C0 value calculated from rate measurements is at least 60 times greater than that determined by equilibrium analysis. Our results indicate that the net assembly rate in GDP is not a valid measure of the reaction occurring at steady-state. A limited amount of depolymerization may occur upon addition of GDP to microtubules, and this appears to be due to a decrease in the fraction of protein able to participate in the polymerization reaction. The amount of tubulin “inactivated” by GDP is increased by the removal of microtubule-associated proteins. GDP-tubulin will stabilize existing microtubules, even when its polymerization cannot be demonstrated. These results are inconsistent with present models of microtubule assembly, and a new model involving co-operative interaction of microtubule-associated protein-tubulin oligomers at microtubule ends is proposed. 相似文献
5.
The polymerization of microtubule protein from beef brain is inefficient under the same conditions which are optimal for the assembly of microtubules isolated from hog brain (0.1 m piperazine-N,N′-bis(2-ethanesulfonic acid) buffer at pH 6.94). In examining the conditions required for microtubule polymerization in both beef brain extract and purified microtuble protein, it was determined that the pH optimum was pH 6.62 or 0.3 pH unit lower than the reported optimum for hog. Other assembly requirements (ionic strength, Mg2+ and nucleotide concentration, temperature) remained essentially the same as for hog. By separating and recombining fractions of tubulin and nontubulin components prepared from beef and hog microtubule protein, the requirement for the reduction in pH was found to be due to the tubulin and not to the microtubule-associated proteins. It was also determined that the efficiency of beef tubulin assembly, as measured by the yield of microtubule polymer, decreased rapidly after slaughter with a half-time of 19 min. Furthermore, when the overall efficiency of polymerization was reduced, the extent of assembly at each cycle of purification by disassembly and assembly was also observed to be depressed. The variations in the requirements for neuronal tubulin assembly in two closely related mammals suggest that the conditions required for assembly of microtubule protein in other tissues and cell types may also be different. 相似文献
6.
Current models of microtubule assembly from pure tubulin involve a nucleation phase followed by microtubule elongation at a constant polymer number. Both the rate of microtubule nucleation and elongation are thought to be tightly influenced by the free GTP-tubulin concentration, in a law of mass action-dependent manner. However, these basic hypotheses have remained largely untested due to a lack of data reporting actual measurements of the microtubule length and number concentration during microtubule assembly.Here, we performed simultaneous measurements of the polymeric tubulin concentration, of the free GTP-tubulin concentration, and of the microtubule length and number concentration in both polymerizing and depolymerizing conditions. In agreement with previous work we find that the microtubule nucleation rate is strongly dependent on the initial GTP-tubulin concentration. But we find that microtubule nucleation persists during microtubule elongation. At any given initial tubulin-GTP concentration, the microtubule nucleation rate remains constant during polymer assembly, despite the wide variation in free GTP-tubulin concentration. We also find a remarkable constancy of the rate of microtubule elongation during assembly. Apparently, the rate of microtubule elongation is intrinsic to the polymers, insensitive to large variations of the free GTP-tubulin concentration. Finally we observe that when, following assembly, microtubules depolymerize below the free GTP-tubulin critical concentration, the rate-limiting factor for disassembly is the frequency of microtubule catastrophe. At all time-points during disassembly, the microtubule catastrophe frequency is independent of the free GTP-tubulin concentration but, as the microtubule nucleation rate, is strongly dependent on the initial free GTP-tubulin concentration. We conclude that the dynamics of both microtubule assembly and disassembly depend largely on factors other than the free GTP-tubulin concentration. We propose that intrinsic structural factors and endogenous regulators, whose concentration varies with the initial conditions, are also major determinants of these dynamics. 相似文献
7.
This report describes the effects of 10 mM procaine on microtubule assembly and on DNA synthesis, as followed by [3H]colchicine binding assays and [3H]thymidine incorporation respectively, in fertilized Paracentrotus lividus eggs. In the absence of microtubule assembly inhibitors, about 25% of the total egg tubulin is submitted to two cycles of polymerization prior to the first cell division, this polymerization process precedes DNA synthesis. If the zygotes are treated with 10 mM procaine in the course of the cell cycle, tubulin polymerization is inhibited or microtubules are disassembled. DNA synthesis is inhibited when procaine treatment is performed 10 min, before the initiation of the S-period. However, when the drug is applied in the course of this synthetic period, the process is normally accomplished, but the next S-period becomes inhibited. Moreover, procaine treatment increases the cytoplasmic pH of the fertilized eggs by about 0.6 to 0.8 pH units. This pH increase precedes microtubule disassembly and inhibition of DNA synthesis. Washing out the drug induces a decrease of the intracellular pH which returns to about the same value as that of the fertilized egg controls. This pH change is then followed by the reinitiation of microtubule assembly, DNA synthesis and cell division. Our results show that the inhibition of both tubulin polymerization and DNA synthesis in fertilized eggs treated with 10 mM procaine, appears to be related to the drug-induced increase in cytoplasmic pH. 相似文献
8.
Microtubule assembly in Saccharomyces cerevisiae is initiated from sites within spindle pole bodies (SPBs) in the nuclear envelope. Microtubule plus ends are thought to be organized distal to the SPBs, while minus ends are proximal. Several hypotheses for the function of microtubule motor proteins in force generation and regulation of microtubule assembly propose that assembly and disassembly occur at minus ends as well as at plus ends. Here we analyse microtubule assembly relative to the SPBs in haploid yeast cells expressing green fluorescent protein fused to alpha-tubulin, a microtubule subunit. Throughout the cell cycle, analysis of fluorescent speckle marks on cytoplasmic astral microtubules reveals that there is no detectable assembly or disassembly at minus ends. After laser-photobleaching, metaphase spindles recover about 63% of the bleached fluorescence, with a half-life of about 1 minute. After anaphase onset, photobleached marks in the interpolar spindle are persistent and do not move relative to the SPBs. In late anaphase, the elongated spindles disassemble at the microtubule plus ends. These results show for astral and anaphase interpolar spindle microtubules, and possibly for metaphase spindle microtubules, that microtubule assembly and disassembly occur at plus, and not minus, ends. 相似文献
9.
《The Journal of cell biology》1976,71(3):749-767
Methods have been developed for differentially inhibiting microtubule nucleation and elongation in vitro. By use of polyanions, assembly- competent tubulin solutions of several milligrams/milliliter can be prepared which do not exhibit appreciable spontaneous assembly during the time-course of an experiment. Microtubule elongation can be initiated by the addition of known numbers of microtubule fragments. A detailed analysis of the resulting process demonstrates that: (a) rings are not obligatory intermediates in the nucleation sequence, and neither rings nor protofilament sheets are obligatory intermediates in the elongation reaction. (b) The end of an elongating microtubule often has a short region of open protofilament sheet or "C-microtubule" similar to that observed in vivo. (c) The development of turbidity follows a simple exponential approach to an equilibrium value. (d) The final equilibrium values are independent of the number of added nucleating fragments, while the initial growth rates and half-times to reach equilibrium are dependent on the number of added nuclei. (e) The final lengths of the microtubules at equilibrium are inversely proportional to the number of added fragments. (f) The equilibrium constants are independent of microtubule length. (g) The number of assembly and disassembly sites per microtubule is not a function of microtubule length. (h) The forward rate constants, the final polymer concentrations, and growth rates of microtubules are dependent upon the concentration of polyanion present. These results are strongly supportive of the idea that microtubule assembly is a "condensation- polymerization" and provide basic information on the kinetics and length distributions of the elongation in vitro. 相似文献
10.
Mechanism for oscillatory assembly of microtubules 总被引:1,自引:0,他引:1
Dampened oscillations of microtubule assembly can accompany polymerization at high tubulin subunit concentrations. This presumably results from a synchronization of dynamic instability behavior, which generates a large population of rapidly disassembling microtubules, that liberate tubulin-GDP oligomers. Subunits in oligomers cannot assemble until they dissociate, to allow GDP-GTP exchange. To determine whether rapidly disassembling microtubules generate oligomers directly, we measured the rate of dilution-induced disassembly of tubulin-GDP microtubules and the rate of dissociation of GDP from the so-formed tubulin-GDP subunits. The rate of GDP dissociation from liberated subunits was found to correspond to that of tubulin-GDP subunits (t1/2 = 5 s), rather than tubulin-GDP oligomers. This indicates that tubulin-GDP subunits are released from microtubules undergoing rapid disassembly. Oligomers apparently form in a side reaction from the high concentration of tubulin-GDP subunits liberated from the synchronously disassembling microtubule population. The rate of subunit dissociation is 0.11 s-1 with oligomers formed by concentrating tubulin-GDP subunits and 0.045 s-1 with oligomers formed by cold-induced microtubule disassembly. This difference provides evidence that the conformation of tubulin-GDP subunits released from rapidly disassembling microtubules differs from tubulin-GDP subunits that were not recently in the microtubule lattice. 相似文献
11.
The dynamic assembly and disassembly of microtubules and the mechanical properties of these polymers are essential for many key cellular processes. Mathematical and computational modeling, especially coupled mechanochemical modeling, has contributed significantly to our understanding of microtubule dynamics. However, critical discrepancies exist between experimental observations and modeling results that need to be resolved before further progress toward a complete model can be made. Open sheet structures ranging in length from several hundred nanometers to one micron have often been observed at the growing ends of microtubules in in vitro studies. Existing modeling studies predict these sheet structures to be short and rare intermediates of microtubule disassembly rather than important components of the assembly process. Atomic force microscopy (AFM) studies also reveal interesting step-like gaps of the force-indentation curve that cannot yet be explained by existing theoretical models. We have carried out computational studies to compare the mechanical properties of two alternative models: a more conventional model where tubulin dimers are added directly into a microtubule lattice, and one that considers an additional type of tubulin lateral interaction proposed to exist in intermediate sheet structures during the microtubule assembly process. The first model involves a single type of lateral interactions between tubulin subunits, whereas the latter considers a second type that can convert to the canonical lateral contact during microtubule closure into a cylinder. Our analysis shows that only the second model can reproduce the AFM results over a broad parameter range. We propose additional studies using different sizes of AFM tips that would allow to unambiguously distinguish the relative validity of the two models. 相似文献
12.
Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau. 总被引:36,自引:14,他引:36
下载免费PDF全文
![点击此处可从《Molecular biology of the cell》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Microtubule-associated proteins (MAP), such as tau, modulate the extent and rate of microtubule assembly and play an essential role in morphogenetic processes, such as axonal growth. We have examined the mechanism by which tau affects microtubule polymerization by examining the kinetics of microtubule assembly and disassembly through direct observation of microtubules using dark-field microscopy. Tau increases the rate of polymerization, decreases the rate of transit into the shrinking phase (catastrophe), and inhibits the rate of depolymerization. Tau strongly suppresses the catastrophe rate, and its ability to do so is independent of its ability to increase the elongation rate. Thus, tau generates a partially stable but still dynamic state in microtubules. This state is perturbed by phosphorylation by MAP2 kinase, which affects all three activities by lowering the affinity of tau for the microtubule lattice. 相似文献
13.
V I Gel'fand 《Tsitologiia》1984,26(4):362-370
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). 相似文献
14.
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. 相似文献
15.
Uematsu Y Kogo Y Ohishi I 《Biology of the cell / under the auspices of the European Cell Biology Organization》2007,99(3):141-150
BACKGROUND INFORMATION: C(2) toxin produced by Clostridium botulinum types C and D ADP-ribosylates actin monomers and inactivates their polymerization activities. The disassembly of actin filaments by C(2) toxin induces a polarization of cultured human leukaemia cell lines. RESULTS: The polarization induced by C(2) toxin was temperature dependent and was prevented by nocodazole, a microtubule-disrupting agent, whereas it was promoted by paclitaxel, a microtubule-stabilizing agent. The fluorescence staining of polarized cells indicated an increase in microtubule assembly accompanying disassembly of actin filaments. Furthermore, several actin-filament-disrupting agents, other than C(2) toxin, also induced microtubule assembly and cell polarization, irrespective of their different mechanisms of action. The effects induced by some of the agents, which have lower binding affinities for actin, were reversible in response to the re-assembly of actin filaments. CONCLUSIONS: Thus the disassembly of actin filaments by C(2) toxin and actin-filament-disrupting agents induces assembly of microtubules followed by polarization of human leukaemia cell lines, indicating that the assembly/disassembly equilibrium of actin filaments influences the dynamics of microtubules, which control cell morphology and, in turn, diverse cellular processes. 相似文献
16.
R. Manso-Martinez 《Molecular and cellular biochemistry》1982,45(1):3-11
Summary Depolymerization kinetics of microtubules assembled to steady-state by pod ophyllotoxin treatment show a dose-dependent effect of this mitotic poison on the net rate of microtubule disassembly. Pulse-chase experiments with microtubules at steady-state indicate that the depolymerization effect induced by superstoichiometric concentrations of podophyllotoxin relative to tubulin is polar and time-dependent, i.e. the rate of tubulin loss decreases along with the time of treatment in the presence of the drug. Under these conditions the rate of microtubule disassembly is much faster than one could expect from a unique effect of drug-tubulin complex on the microtubule assembly end. Podophyllotoxin-tubulin complex is not able to induce active depolymerization of microtubules, while free podophyllotoxin is. These results are consistent with the hypothesis that this drug acts on the microtubule assembly-disassembly process by two different mechanisms: 1) as a free drug, it actively promotes polar depolymerization of microtubules, and 2) as a drug-tubulin complex, it retards the addition of subunits into the microtubule ends. 相似文献
17.
The Mn4Ca complex bound to photosystem II (PSII) is the active site of photosynthetic water oxidation. Its assembly involves binding and light-driven oxidation of manganese, a process denoted as photoactivation. The disassembly of the Mn complex is a thermally activated process involving distinct intermediates. Starting from intermediate states of the disassembly, which was initiated by a temperature jump to 47 degrees C, we photoactivated PSII membrane particles and monitored the activity recovery by O2 polarography and delayed chlorophyll fluorescence measurements. Oxidation state and structural features of the formed intermediates of the Mn complex were assayed by X-ray absorption spectroscopy at the Mn K-edge. The photoactivation time courses, which exhibit a lag phase characteristic of intermediate formation only when starting with the apo-PSII, suggest that within approximately 5 min of photoactivation of apo-PSII, a binuclear Mn complex is formed. It is proposed that a MnIII2(di-mu-oxo) complex is a key intermediate both in the disassembly and in the assembly reaction paths. 相似文献
18.
Although it is now apparent that the intracellular pH may rise considerably above neutrality under physiological conditions, information on the effect of alkaline pH on microtubule assembly and disassembly is still quite fragmentay. We have studied the assembly/disassembly of bovine brain microtubule protein at alkaline pH in vitro. When microtubules are assembled to a new steady state at pH less than 7 and pH is then made more alkaline, they undergo a rapid disassembly to a new steady state. This disassembly is reversed by acidification. The degree of disassembly is determined largely by the pH- dependence of the critical concentration, which increases five to eight times, from pH 7 to 8. A fraction of assembly-incompetent tubulin is identified that increases with pH, but its incompetency is largely reversed with acidification. Measurements of microtubule lengths are used to indicate that disassembly occurs by uniform shortening of microtubules. A comparison of shortening by alkalinization with dilution suggests that the intrinsic rate of disassembly is accelerated by increasing pH. The capacity for initiating assembly is progressively lost with incubation at alkaline pH (although some protection is afforded by sulfhydryl-reducing agents). However, direct assembly from depolymerized mixtures is possible at least up to pH 8.3, and the steady state achieved at these alkaline pH values is stable. Such preparations are readily disassembled by cold and podophyllotoxin (PLN). Disassembly induced by PLN is also markedly enhanced at alkaline pH, suggesting a corresponding enhancement of “treadmilling.” The implications of physiological events leading to alkaline shifts of pH for microtubule assembly/disassembly are discussed, particularly in the light of recent hypotheses regarding treadmilling and its role in controlling the distribution of microtubules in vivo. 相似文献
19.
BACKGROUND: CLIP-170 is a microtubule binding protein specifically located at microtubule plus ends, where it modulates their dynamic properties and their interactions with intracellular organelles. The mechanism by which CLIP-170 is targeted to microtubule ends remains unclear today, as well as its precise effect on microtubule dynamics. RESULTS: We used the N-terminal part of CLIP-170 (named H2), which contains the microtubule binding domains, to investigate how it modulates in vitro microtubule dynamics and structure. We found that H2 primarily promoted rescues (transitions from shrinkage to growth) of microtubules nucleated from pure tubulin and isolated centrosomes, and stimulated microtubule nucleation. Electron cryomicroscopy revealed that H2 induced the formation of tubulin rings in solution and curved oligomers at the extremities of microtubules in assembly conditions. CONCLUSIONS: These results suggest that CLIP-170 targets specifically at microtubule plus ends by copolymerizing with tubulin and modulates microtubule nucleation, polymerization, and rescues by the same basic mechanism with tubulin oligomers as intermediates. 相似文献
20.
The phosphorylation of p25/TPPP by LIM kinase 1 inhibits its ability to assemble microtubules 总被引:1,自引:0,他引:1
Acevedo K Li R Soo P Suryadinata R Sarcevic B Valova VA Graham ME Robinson PJ Bernard O 《Experimental cell research》2007,313(20):4091-4106
LIM kinase 1 (LIMK1) is a key regulator of actin dynamics as it phosphorylates and inactivates cofilin, an actin-depolymerizing factor. LIMK1 activity is also required for microtubule disassembly in endothelial cells. A search for LIMK1-interacting proteins identified p25alpha, a phosphoprotein that promotes tubulin polymerization. We found that p25 is phosphorylated by LIMK1 on serine residues in vitro and in cells. Immunoblotting analysis revealed that p25 is not a brain specific protein as previously reported, but is expressed in all mouse tissues. Immunofluorescence analysis demonstrated that endogenous p25 is co-localized with microtubules and is also found in the nucleus. Down-regulation of p25 by siRNA decreased microtubule levels while its overexpression in stable NIH-3T3 cell lines increased cell size and levels of stable tubulin. Bacterially expressed unphosphorylated p25 promotes microtubule assembly in vitro; however, when phosphorylated in cells, p25 lost its ability to assemble microtubule. Our results represent a surprising connection between the tubulin and the actin cytoskeleton mediated by LIMK1. We propose that the LIMK1 phosphorylation of p25 blocks p25 activity, thus promoting microtubule disassembly. 相似文献