New features of microtubule behaviour observed in vivo

The microtubule cytoskeleton is thought to be intimately involved in generating and maintaining cell polarity and can generate many different morphological structures from a few structural elements. The mechanism by which these structures are generated has been partially elucidated from studies of microtubule polymerization both in vitro and in vivo. Microtubules in vitro exist in growing (polymerizing) and shrinking (depolymerizing) populations that interconvert infrequently. This behaviour, termed dynamic instability, permits microtubules in the cell rapidly to explore different arrangements and allows selective stabilization of specific morphologies. To investigate the regulation of these processes, we have implemented techniques for direct observation of fluorescently labelled microtubules and developed them to observe the dynamic behaviour of individual microtubules in single living cells. Sammak and Borisy recently used this technique to show that the dynamics of microtubules in fibroblasts is explained by dynamic instability. Although we also conclude here that dynamic instability explains much of microtubule behaviour in vivo, we find significant deviations from the properties of tubulin in vitro. These results suggest that local cytoplasmic factors strongly influence microtubule dynamics; such control has important implications for cellular morphogenesis.     

Stabilization of microtubule dynamics at anaphase onset promotes chromosome segregation

Microtubules of the mitotic spindle form the structural basis for chromosome segregation. In metaphase, microtubules show high dynamic instability, which is thought to aid the 'search and capture' of chromosomes for bipolar alignment on the spindle. Microtubules suddenly become more stable at the onset of anaphase, but how this change in microtubule behaviour is regulated and how important it is for the ensuing chromosome segregation are unknown. Here we show that in the budding yeast Saccharomyces cerevisiae, activation of the phosphatase Cdc14 at anaphase onset is both necessary and sufficient for silencing microtubule dynamics. Cdc14 is activated by separase, the protease that triggers sister chromatid separation, linking the onset of anaphase to microtubule stabilization. If sister chromatids separate in the absence of Cdc14 activity, microtubules maintain high dynamic instability; this correlates with defects in both the movement of chromosomes to the spindle poles (anaphase A) and the elongation of the anaphase spindle (anaphase B). Cdc14 promotes localization of microtubule-stabilizing proteins to the anaphase spindle, and dephosphorylation of the kinetochore component Ask1 contributes to both the silencing of microtubule turnover and successful anaphase A.     

Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain

Microtubules are cytoskeletal polymers of tubulin involved in many cellular functions. Their dynamic instability is controlled by numerous compounds and proteins, including colchicine and stathmin family proteins. The way in which microtubule instability is regulated at the molecular level has remained elusive, mainly because of the lack of appropriate structural data. Here, we present the structure, at 3.5 A resolution, of tubulin in complex with colchicine and with the stathmin-like domain (SLD) of RB3. It shows the interaction of RB3-SLD with two tubulin heterodimers in a curved complex capped by the SLD amino-terminal domain, which prevents the incorporation of the complexed tubulin into microtubules. A comparison with the structure of tubulin in protofilaments shows changes in the subunits of tubulin as it switches from its straight conformation to a curved one. These changes correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability. Moreover, the tubulin-colchicine complex sheds light on the mechanism of colchicine's activity: we show that colchicine binds at a location where it prevents curved tubulin from adopting a straight structure, which inhibits assembly.     

Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs

Microtubules are involved in the transport of vesicles in interphase and of the chromosomes during mitosis. Their arrangement and orientation in the cell are therefore of prime importance and specific patterns are believed to be generated by modulations of the intrinsic dynamic instability of microtubules. Here it is shown that the interphase-metaphase transition of microtubule arrays is under the control of the cdc2 kinase that precisely regulates the dynamics and steady-state length of microtubules.     

Assembly dynamics of microtubules at molecular resolution

Microtubules are highly dynamic protein polymers that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers, information on the assembly dynamics of microtubules has been limited, both in vitro and in vivo, to measurements of average growth and shrinkage rates over several thousands of tubulin subunits. As a result there is a lack of information on the sequence of molecular events that leads to the growth and shrinkage of microtubule ends. Here we use optical tweezers to observe the assembly dynamics of individual microtubules at molecular resolution. We find that microtubules can increase their overall length almost instantaneously by amounts exceeding the size of individual dimers (8 nm). When the microtubule-associated protein XMAP215 (ref. 6) is added, this effect is markedly enhanced and fast increases in length of about 40-60 nm are observed. These observations suggest that small tubulin oligomers are able to add directly to growing microtubules and that XMAP215 speeds up microtubule growth by facilitating the addition of long oligomers. The achievement of molecular resolution on the microtubule assembly process opens the way to direct studies of the molecular mechanism by which the many recently discovered microtubule end-binding proteins regulate microtubule dynamics in living cells.     

Inhibition of neurite polarity by tau antisense oligonucleotides in primary cerebellar neurons

Neurons in culture can have fundamentally distinct morphologies which permit their cytological identification and the recognition of their neurites as axons or dendrites. Microtubules may have a role in determining morphology by the selective stabilization of spatially distinct microtubule subsets. The plasticity of a neurite correlates inversely with the stability of its component microtubules: microtubules in growth cones are very dynamic, and in initial neurites there is continuous incorporation of labelled subunits, whereas in mature neurites, microtubules are highly stabilized. The binding of microtubule-associated proteins to the microtubules very probably contributes to this stability. Cerebellar neurons in dissociated culture initially extend exploratory neurites and, after a relatively constant interval, become polarized. Polarity becomes evident when a single neurite exceeds the others in length. These stable neurites cease to undergo the retractions and extensions characteristic of initial neurites and assume many features of axons and dendrites. We have now studied the role of the neuronal microtubule-associate protein tau in neurite polarization by selectively inhibiting tau expression by the addition of antisense oligonucleotides to the culture media. Although the extension of initial exploratory neurites occurred normally, neurite asymmetry was inhibited by the failure to elaborate an axon.     

Assembly of microtubules at the tip of growing axons

The growth of axons in the developing nervous system depends on the elongation of the microtubules that form their principal longitudinal structural element. It is not known whether individual microtubules in the axon elongate at their proximal ends, close to the cell body, and then move forward into the lengthening axon, or whether tubulin subunits are transported to the tip of the axon and assembled there onto the free ends of microtubules. The former possibility is supported by studies of slow axonal transport in mature nerves from which it has been deduced that microtubule assembly occurs principally at the neuronal cell body. By contrast, the polarity of microtubules in axons, which have their 'plus' or 'fast-growing' ends distal to the cell body, suggests that assembly occurs at the growing tip, or growth cone, of the axon. We have addressed this question by topically applying Colcemid (N-desacetyl-N-methylcolchicine), and other drugs which alter microtubule stability, to different regions of isolated nerve cells growing in tissue culture. We find that the sensitivity to these drugs is greatest at the growth cone by at least two orders of magnitude, suggesting that this is a major site of microtubule assembly during axonal growth.     

Actin microfilament dynamics in locomoting cells

The dynamic behaviour of actin filaments has been directly observed in living, motile cells using fluorescence photoactivation. In goldfish epithelial keratocytes, the actin microfilaments in the lamellipodium remain approximately fixed relative to the substrate as the cell moves over them, regardless of cell speed. The rate of turnover of actin subunits in the lamellipodium is remarkably rapid. Cell movement is directly and tightly coupled to the formation of new actin filaments at the leading edge.     

Dynamic instability of microtubule growth

We report here that microtubules in vitro coexist in growing and shrinking populations which interconvert rather infrequently. This dynamic instability is a general property of microtubules and may be fundamental in explaining cellular microtubule organization.     

Movement and segregation of kinetochores experimentally detached from mammalian chromosomes

The kinetochore is a specialized structure at the centromere of eukaryotic chromosomes that attaches chromosomes to the mitotic spindle. Recently, several lines of evidence have suggested that kinetochores may have more than a passive role in the movement of chromosomes during mitosis and meiosis. Kinetochores seem to attract and 'capture' microtubules that grow from the spindle poles and microtubules may lengthen or shorten by the addition or subtraction of tubulin subunits at their kinetochore-associated ends. An attractive hypothesis is that kinetochores function as 'self-contained engines running on a microtubule track'. Here, we show that kinetochores can be experimentally detached from chromosomes when caffeine is applied to Chinese hamster ovary cells that are arrested in the G1/S phase of the cell cycle. The detached kinetochore fragments can still interact with spindle microtubules and complete all the mitotic movements in the absence of other chromosomal components. As these cells enter mitosis before DNA synthesis is completed, chromosome replication need not be a prerequisite for the pairing, alignment and segregation of kinetochores.     

The microtubule aster formation and its role in nuclear envelope assembly around the sperm chromatin in<Emphasis Type="Italic">Xenopus</Emphasis> egg extracts

Nuclear envelope separates cell genome from cyto-plasm in eucaryotic cells and plays a pivotal role in the cell life. The nuclear envelope is composed of two jointed membranes, the inner membrane and the out membrane, embedded the nuclear pore complexes. The out membrane is continuous with the endoplasmic reticulum (ER). The ARTICLES inner membrane faces to and connects with the chromatin through the nuclear lamina, an intermediate filamentous network thought to play a structural role for…     

Structure and elasticity of microtubule-associated protein tau

Tau is one of the diverse group of microtubule-associated proteins that bind to microtubules and may thereby influence their structure and function. It occurs in the mammalian brain, mainly in axons, and is a component of the neurofibrillary tangles of Alzheimer's disease. Tau was recently sequenced, but there remains a short-age of structural data on the protein. We have now prepared paracrystals of tau suitable for electron microscopy and image processing. They show distinct transverse banding and polarity, indicating that the protein subunits are aligned with the same orientations. In contrast to other paracrystals, those of tau protein can stretch or contract continuously by more than three-fold; the axial repeats range from 22 to 68 nm. After scaling to a common period, the density distributions are closely superimposable. This suggests that tau is an elastic molecule.     

Dynamics and mechanics of the microtubule plus end

An important function of microtubules is to move cellular structures such as chromosomes, mitotic spindles and other organelles around inside cells. This is achieved by attaching the ends of microtubules to cellular structures; as the microtubules grow and shrink, the structures are pushed or pulled around the cell. How do the ends of microtubules couple to cellular structures, and how does this coupling regulate the stability and distribution of the microtubules? It is now clear that there are at least three properties of a microtubule end: it has alternate structures; it has a biochemical transition defined by GTP hydrolysis; and it forms a distinct target for the binding of specific proteins. These different properties can be unified by thinking of the microtubule as a molecular machine, which switches between growing and shrinking modes. Each mode is associated with a specific end structure on which end-binding proteins can assemble to modulate dynamics and couple the dynamic properties of microtubules to the movement of cellular structures.     

结构动力稳定性判定新准则

利用非线性弹簧模型分析了静力失稳与动力失稳的区别,指出拟静力刚度准则不能用于判定结构动力稳定.给出了结构动力系统特征能量的概念,发现结构动力失稳源于结构特征能量超过输入到结构中的总能量,由此提出了一种判定结构动力稳定性的新准则.实例分析表明,本文建议的准则可以准确分析并判定结构在任意动力荷载激励下的动力失稳.     

Adaptation at the output of the chemotaxis signalling pathway

In the bacterial chemotaxis network, receptor clusters process input, and flagellar motors generate output. Receptor and motor complexes are coupled by the diffusible protein CheY-P. Receptor output (the steady-state concentration of CheY-P) varies from cell to cell. However, the motor is ultrasensitive, with a narrow operating range of CheY-P concentrations. How the match between receptor output and motor input might be optimized is unclear. Here we show that the motor can shift its operating range by changing its composition. The number of FliM subunits in the C-ring increases in response to a decrement in the concentration of CheY-P, increasing motor sensitivity. This shift in sensitivity explains the slow partial adaptation observed in mutants that lack the receptor methyltransferase and methylesterase and why motors show signal-dependent FliM turnover. Adaptive remodelling is likely to be a common feature in the operation of many molecular machines.     

Fission yeast mod5p regulates polarized growth through anchoring of tea1p at cell tips

Microtubules have a central role in eukaryotic cell polarity, in part through interactions between microtubule end-binding proteins and the cell cortex. In the fission yeast Schizosaccharomyces pombe, microtubules and the polarity modulator tea1p maintain cylindrical cell shape and strictly antipodal cell growth. The tea1p protein is transported to cell tips by association with growing microtubule plus ends; once at cell tips, tea1p releases from microtubule ends and associates with the cell cortex, where it coordinates polarized growth. Here we describe a cortical protein, mod5p, that regulates the dynamic behaviour of tea1p. In mod5Delta cells, tea1p is efficiently transported on microtubules to cell tips but fails to anchor properly at the cortex and thus fails to accumulate to normal levels. mod5p contains a signal for carboxy-terminal prenylation and in wild-type cells is associated with the plasma membrane at cell tips. However, in tea1Delta cells, although mod5p remains localized to the plasma membrane, mod5p is no longer restricted to the cell tips. We propose that tea1p and mod5p act in a positive-feedback loop in the microtubule-mediated regulation of cell polarity.     

Organization of microtubules in dendrites and axons is determined by a short hydrophobic zipper in microtubule-associated proteins MAP2 and tau

Here we report that the microtubule-associated proteins MAP2 and tau share two separable functional domains. One is the microtubule-binding site which serves to nucleate microtubule assembly; the second is a short C-terminal alpha-helical sequence which can crosslink microtubules by means of a hydrophobic zipper interaction into dense stable parallel arrays characteristic of axons or dendrites. Thus, interactions between molecules of a single type are capable of drastically reorganizing microtubules and completely suppressing their dynamic properties.     

淬火介质对耐磨铸钢组织和性能的影响

分别研究了以水和机油作为淬火介质的42CrMo钢和双相耐磨钢淬火+回火后的显微组织和性能.确定了双相耐磨钢和42CrMo钢的最佳热处理工艺及性能.结果表明:双相耐磨钢经水淬和油淬回火后的显微组织均为板条马氏体+针状贝氏体+碳化物;42CrMo钢经水淬和油淬回火后的显微组织分别为回火马氏体+残余奥氏体和回火马氏体+铁素体+贝氏体.经过480h的低应力磨料磨损实验发现,水淬+280℃回火处理的42CrMo钢耐磨性能最好,其相对耐磨性可达高锰钢的1.558倍.     

点支式钢化和夹胶玻璃板的循环疲劳试验研究

为研究四点支承玻璃板在循环荷载作用下的力学性能,对采用浮头式驳接头点支承的8块钢化玻璃板和15块夹胶玻璃板进行了板中心集中力加载疲劳破坏试验,并观测了循环荷载作用下玻璃板中心点的挠度和关键点的动静态应力变化.试验表明:钢化玻璃在循环荷载作用下的循环荷载-板中心挠度曲线呈弱非线性,随着荷载循环次数的增加其抗弯刚度没有明显变化;夹胶玻璃在循环作用下的荷载-板中心挠度曲线与试验环境温度有关,在温度低于15℃时无滞回环,而温度在22～30℃范围内时,则有明显的滞回环;夹胶玻璃的等幅循环加载荷载-板中心挠度曲线在循环初期挠度随着循环次数的增加而逐渐增大,但循环次数超过5×104后,板中心挠度几乎不随循环次数的增加而变化.点支式钢化玻璃和夹胶玻璃经过5×105次循环加载后,卸载至零,再进行静载破坏试验,其静载抗弯承载力没有降低,表明钢化玻璃和夹胶玻璃抗疲劳性能很好.