Microtubule Assembly Dynamics Research Articles

Decreasing oncoprotein 18/stathmin levels reduces microtubule catastrophes and increases microtubule polymer in vivo.

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein which destabilizes microtubules. To characterize the function of Op18 in living cells, we used microinjection of anti-Op18 antibodies or antisense oligonucleotides to block either Op18 activity or expression in interphase newt lung cells. Anti-tubulin staining of cells microinjected with anti-Op18 and fixed 1-2 hours after injection showed an increase in total microtubule polymer. In contrast, microinjection of either non-immune IgG or anti-Op18 preincubated with bacterially-expressed Op18 had little effect on microtubule polymer level. Cells treated with Op18 antisense oligonucleotides for 4 days had (greater than or equal to)50% reduced levels of Op18 with no change in the soluble tubulin level. Measurement of MT polymer level in untreated, antisense or nonsense oligonucleotide treated cells demonstrated that reduced Op18 levels resulted in a 2.5-fold increase in microtubule polymer. Next, the assembly dynamics of individual microtubules at the peripheral regions of living cells were examined using video-enhanced contrast DIC microscopy. Microinjection of antibodies against oncoprotein 18 resulted in a 2.2-fold reduction in catastrophe frequency and a slight reduction in plus end elongation velocity compared to uninjected cells or cells microinjected with non-immune IgG. Preincubation of anti-Op18 antibody with recombinant Op18 greatly diminished the effects of the antibody. Similarly, treatment of cells with antisense oligonucleotides reduced catastrophes 2.5- to 3-fold compared to nonsense oligonucleotide treated or untreated cells. The other parameters of dynamic instability were unchanged after reducing Op18 with antisense oligonucleotides. These studies are consistent with Op18 functioning to regulate microtubule catastrophes during interphase in vivo.

Stage-specific Expression of a Schistosoma mansoniPolypeptide Similar to the Vertebrate Regulatory Protein Stathmin

The ubiquitous vertebrate protein stathmin is expressed and phosphorylated in response to a variety of external and internal signals. Stathmin, in turn, controls cell growth and differentiation through its capacity to regulate microtubule assembly dynamics. This is the first report on the molecular cloning and characterization of a stathmin-like protein (SmSLP) in an invertebrate, the human blood fluke Schistosoma mansoni. SmSLP is first synthesized at high levels in the intermediate molluscan host and completely disappears 48 h after penetration into the mammalian host. The protein is preferentially iodinated in intact immature parasites using the Bolton-Hunter reagent, can be quantitatively extracted in high salt buffers, and remains soluble after boiling. Native SmSLP was partially sequenced, and its complete structure was derived from the cloning and sequencing of its cDNA. The sequence is up to 26% identical to vertebrate stathmin sequences and contains two potential phosphorylation sites. Native SmSLP is indeed phosphorylated because phosphatase digestion shifts its mobility in electrofocusing gels. SmSLP associates with tubulin, as suggested by immune co-precipitation results. In vitro experiments demonstrated that SmSLP inhibits tubulin assembly and causes the depolymerization of preassembled microtubules, thus probably fulfilling regulatory roles in critical steps of schistosome development.

Phosphorylation by CDK1 regulates XMAP215 function in vitro.

XMAP215, a microtubule-associated protein isolated from Xenopus eggs, promotes microtubule assembly dynamics in an end-specific manner: addition of XMAP215 to purified porcine tubulin increases both elongation and shortening rates at microtubule plus ends, with minimal effects at minus ends. Previous results indicated that XMAP215 is phosphorylated during M phase, suggesting that its activity may be regulated by cell cycle phosphorylation. To test this hypothesis, we used video-enhanced DIC microscopy to examine the effects of XMAP215 phosphorylated by CDK1 on the assembly of purified tubulin. XMAP215 incubated with ATP at 30 degrees C for 10- 20 min in the absence of CDK1 exhibited a 4.1-fold increase in plus end elongation rate compared to purified tubulin. Elongation was promoted to a lesser degree (2.4-fold) by phosphorylated XMAP215. In contrast, XMAP215 phosphorylation did not alter the approximately 3-fold increase in shortening rate. XMAP215 binding to taxol microtubules was also not changed by phosphorylation. To further investigate mechanisms responsible for the faster microtubule shortening rate observed with XMAP215, we made microtubules with segments assembled prior to XMAP215 addition (proximal segments) and segments assembled in the presence of XMAP215 (distal segments). In 9 of 10 microtubules, the distal segment shortened faster (distal = 60.7 microm/min; proximal = 37.5 microm/min), suggesting that MTs assembled in the presence of XMAP215 have an altered lattice that results in subsequent faster shortening.

Endoplasmic reticulum membrane tubules are distributed by microtubules in living cells using three distinct mechanisms.

The microtubule-dependent motility of endoplasmic reticulum (ER) tubules is fundamental to the structure and function of the ER. From in vitro assays, three mechanisms for ER tubule motility have arisen: the 'membrane sliding mechanism' in which ER tubules slide along microtubules using microtubule motor activity; the 'microtubule movement mechanism' in which ER attaches to moving microtubules; and the 'tip attachment complex (TAC) mechanism' in which ER tubules attach to growing plus ends of microtubules. We have used multi-wavelength time-lapse epifluorescence microscopy to image the dynamic interactions between microtubules (by microinjection of X-rhodamine-labeled tubulin) and ER (by DiOC6(3) staining) in living cells to determine which mechanism contributes to the formation and motility of ER tubules in migrating cells in vivo. Newly forming ER tubules extended only in a microtubule plus-end direction towards the cell periphery: 31.4% by TACs and 68.6% by the membrane sliding mechanism. ER tubules, statically attached to microtubules, moved towards the cell center with microtubules through actomyosin-based retrograde flow. TACs did not change microtubule growth and shortening velocities, but reduced transitions between these states. Treatment of cells with 100 nM nocodazole to inhibit plus-end microtubule dynamics demonstrated that TAC motility required microtubule assembly dynamics, whereas membrane sliding and retrograde-flow-driven ER motility did not. Both plus-end-directed membrane sliding and TAC mechanisms make significant contributions to the motility of ER towards the periphery of living cells, whereas ER removal from the lamella is powered by actomyosin-based retrograde flow of microtubules with ER attached as cargo. TACs in the ER modulate plus-end microtubule dynamics.

Chapter 9 High-Resolution Video-Enhanced Differential Interference Contrast (VE-DIC) Light Microscopy

Publisher Summary Differential interference contrast (DIC) light microscopy was an immediate success after its introduction in the 1960s because it could produce high-contrast optical images of the edges of objects and fine-structural detail within transparent specimens. DIC contrast depends on gradients in optical path and not on absolute values of optical path (OP). As a result, DIC methods can produce clear optical sections of relatively thick transparent specimens. Resolution in DIC microscopy is also superior to phase contrast. VE–DIC has been important in many aspects of cell biology, including measuring the assembly dynamics of individual microtubules and forced production by microtubule assembly/disassembly. The quality of images in VE–DIC microscopy depends critically on both the quality of the image projected onto the camera detector by the microscope as well as on the electronic contrast enhancement capabilities of the camera. The chapter describes the basic concepts of DIC image formation and analog and digital contrast enhancement, the practical aspects of component selection, alignment and specimen chambers for VE–DIC, and several test specimens for measuring microscope and video performance.

Chapter 15 The Use and Action of Drugs in Analyzing Mitosis

Publisher Summary This chapter describes the use of drugs in studies of mitosis and their mode of action at a biochemical/mechanistic level and present general guidelines for using the drugs as tools. The chapter presents a brief overview of microtubule assembly dynamics and summarizes the current understanding of (i) the binding of vinblastine, colchicine, nocodazole, and taxol to tubulin and microtubules; (ii) the effects of the drugs on microtubule polymerization and dynamics in vitro and in cells; (iii) the levels and kinetics of cellular accumulation and loss of the compounds; (iv) the drug concentration dependence for effects on mitotic spindle organization; and (v) the advantages and disadvantages of using a particular drug for the study of microtubule assembly and dynamics. The chapter also describes the methods for measuring drug uptake into cultured cells, and summarizes some practical guidelines for drug use. First of all, one has to determine the effects of the drug on spindle microtubule organization and mitosis over a range of concentrations in the cells of choice. One should also ensure that the drug is completely solubilized and should determine the effects of varying the duration of drug incubation.

22] Use of drugs to study role of microtubule assembly dynamics in living cells

22] Use of drugs to study role of microtubule assembly dynamics in living cells

Estimation of the diffusion-limited rate of microtubule assembly

Microtubule assembly is a complex process with individual microtubules alternating stochastically between extended periods of assembly and disassembly, a phenomenon known as dynamic instability. Since the discovery of dynamic instability, molecular models of assembly have generally assumed that tubulin incorporation into the microtubule lattice is primarily reaction-limited. Recently this assumption has been challenged and the importance of diffusion in microtubule assembly dynamics asserted on the basis of scaling arguments, with tubulin gradients predicted to extend over length scales exceeding a cell diameter, approximately 50 microns. To assess whether individual microtubules in vivo assemble at diffusion-limited rates and to predict the theoretical upper limit on the assembly rate, a steady-state mean-field model for the concentration of tubulin about a growing microtubule tip was developed. Using published parameter values for microtubule assembly in vivo (growth rate = 7 microns/min, diffusivity = 6 x 10(-12) m2/s, tubulin concentration = 10 microM), the model predicted that the tubulin concentration at the microtubule tip was approximately 89% of the concentration far from the tip, indicating that microtubule self-assembly is not diffusion-limited. Furthermore, the gradients extended less than approximately 50 nm (the equivalent of about two microtubule diameters) from the microtubule tip, a distance much less than a cell diameter. In addition, a general relation was developed to predict the diffusion-limited assembly rate from the diffusivity and bulk tubulin concentration. Using this relation, it was estimated that the maximum theoretical assembly rate is approximately 65 microns/min, above which tubulin can no longer diffuse rapidly enough to support faster growth.

The Role of Microtubule Assembly Dynamics in Mitotic Force Generation and Functional Organization of Living Cells

This article summarizes the author's presentation at the Baylor Medical School Symposium on the Biophysics of Microtubules, held April 12 to 14, 1996, in Houston, Texas. It presents a brief historical sketch and discusses the role that assembly/disassembly of microtubules is likely to be playing in force generation for chromosome movement and related organellar positioning in living cells. The article starts out with how polarized light microscopy of living cells had laid the foundation for this concept in the 1950s and 1960s, but was then eclipsed for some 2 decades following the discovery of force generation by microtubule sliding powered by an ATP-hydrolyzing motor protein, dynein. The intriguing recent discoveries: that microtubules undergo dynamic instability; that they both assemble and disassemble right at the kinetochore where they are attached to the chromsome; and that assembling and disassembling microtubules can of themselves push and pull reasonable loads in model experiments, even in the absence of hydrolyzable nucleotides, have refocused serious attention on the probable role played by assembly/disassembly of microtubules. This mode of force generation may well be intricately coupled, and interact, with force-generating and/or dynamic attachment roles played by “motor” proteins, especially at the kinetochore.

Microtubule assembly in clarifiedXenopus egg extracts

Crude cytoplasmic extracts made from Xenopus eggs have proven to be uniquely useful in the studies of the mechanism of spindle microtubule assembly dynamics and chromosome movement during progression through the cell cycle. We examined microtubule dynamic instability in the Xenopus system using video-enhanced differential interference contrast microscopy (VE-DIC), which required high-speed centrifugation in order to clarify crude Xenopus extracts of refractile particles. Surprisingly, the resultant clarified, undiluted extracts exhibited virtually no microtubule catastrophe, even in the presence of high MPF (cyclin B/p34cdc2 kinase) activity and mitogen-activated protein (MAP) kinase activity, a down-stream kinase also implicated in regulating microtubule dynamics. Microtubule elongation occurred at plus ends, and interphase microtubules grew at 17-30 microns/min while metaphase [meiotic, myelin basic protein kinase activity which is diagnostic for cytostatic factor (CSF)-arrested] microtubules grew at about 10 microns/min. Plus-end shortening rates for both interphase and metaphase extracts were > 50 microns/min. Addition of okadaic acid, a protein phosphatase inhibitor known to activate MAP kinase activity and cause an increase in microtubule turnover in extracts made from sea urchin eggs, had no effect on microtubule catastrophe in either interphase or metaphase Xenopus extracts. In addition, the microtubules assembled in interphase extracts were less sensitive to dilution than those in metaphase. This study is the first to describe the dynamic instability of microtubules in Xenopus extracts without the addition of exogenous tubulins or other buffer contaminants.

Time series characterization of simulated microtubule dynamics in the nerve growth cone.

The process of neurite outgrowth is critically dependent on proper microtubule assembly. However, characterizing the dynamics of microtubule assembly and their quantitative relationship to neurite outgrowth is a difficult task. The difficulty can be reduced by using time series analysis which has broad application in characterizing the dynamics of stochastic, or "noisy," behaviors. Here we apply time series analysis to quantitatively compare simulated microtubule assembly and neurite outgrowth in vitro. Microtubule length life histories were simulated assuming constant growth and shrinkage rates coupled with random selection of growth and shrinkage times, a formulation based on the dynamic instability model of microtubule assembly. Net length displacements of simulated microtubules were calculated at discrete, evenly spaced times, and the resulting time series were characterized by both spectral and autocorrelation analysis. Depending on the sampling rate and the dynamic parameters, simulated microtubules exhibited significant autocorrelation and periodicity. To make a comparison to neurite outgrowth, we characterized the dynamic behavior of simulated microtubule populations and found it was not significantly different from that of single microtubules. The net displacements of rat superior cervical ganglion neurite tips were measured and characterized using time series methods. Their behavior was consistent with the microtubule dynamics for appropriate simulation parameters and sampling rates. Our results show that time series analysis can provide a useful tool for quantitative characterization of microtubule dynamics and neurite outgrowth and for assessing the relationship between them.

Microtubule-associated protein 2 alters the dynamic properties of microtubule assembly and disassembly

The influence of microtubule-associated protein 2 (MAP2) on the dynamics of microtubule assembly and disassembly from axonemal fragments was characterized in vitro in solutions of pure tubulin and varying concentrations of MAP2. A mechanistic description of interactions between MAP2 and individual microtubules was developed from analysis of recorded images obtained by video-enhanced differential-interference-contrast light microscopy. MAP2 decreased the rates and lengths of shortening events and decreased the frequency of transitions between growth and shortening over a wide range of concentrations, thereby producing the increases in net microtubule growth previously described by light-scattering techniques. Increases in rates and lengths of elongation phases, as well as rescue frequencies (i.e. transition from shortening to growth), were observed under conditions in which microtubules are expected to be saturated with MAP2. During early stages of nucleated assembly, MAP2 greatly increased the number of microtubules growing from a given axoneme and caused elongation of "curved" structures which may be sheet-like microtubules.

Newly assembled microtubules are concentrated in the proximal and distal regions of growing axons.

We have investigated the sites of microtubule (MT) assembly in neurons during axon growth by taking advantage of the relationship between the proportion of tyrosinated alpha-tubulin (tyr-tubulin) in MTs and their age. Specifically, young (newly assembled) MTs contain more tyr-tubulin than older (more long-lived) MTs. To quantify the relative proportion of tyr-tubulin in MTs, cultured rat sympathetic neurons were permeabilized under conditions that stabilize existing MTs and remove unassembled tubulin. The MTs were then double-stained with antibodies to tyr-tubulin (as a measure of the amount of tyr-tubulin in MTs) and to beta-tubulin (as a measure of total MT mass), using immunofluorescence procedures. Cells were imaged with a cooled charge-coupled device camera and the relative proportion of tyr-tubulin in the MTs was quantified by computing the ratio of the tyr-tubulin fluorescence to the beta-tubulin fluorescence using a novel application of digital image processing and analysis techniques. The amount of tyr-tubulin in the MTs was highest in the cell body and at the growth cone; peak ratios in these two regions were approximately 10-fold higher than for the axon shaft. Moving out from the cell body into the axon, the tyr-tubulin content declined over an average distance of 40 microns to reach a constant low value within the axon shaft and then rose again more distally, over an average distance of 110 microns, to reach a peak at the growth cone (average axon length = 358 microns). These observations indicate that newly assembled MTs are concentrated in the proximal and distal regions of growing axons and therefore that the cell body and growth cone are the most active sites of MT assembly dynamics in neurons that are actively extending axons.

Alterations in dynamics of microtubule assembly during axonal neuritogenesis in NB2a/d1 cells

During dibutyryl cyclic AMP (dbcAMP)-mediated differentiation, axonal neurites elaborated by mouse NB2a/d1 neuroblastoma cells are initially colchicine-labile but attain colchicine-stability after 7 days. To examine whether or not differences in tubulin subunit turnover could account for the development of colchicine-stability, anti-tubulin antibodies were delivered into NB2a/d1 cells at various times during dbcAMP-mediated neurite outgrowth. These antibodies prevented initial neurite elaboration, and induced neurite retraction in cells treated with dbcAMP for up to 3 days, but did not induce neurite retraction for cells treated for 7 days. We conclude that a less dynamic, more slowly-turning over population of microtubules develops within neurites of cells treated with dbcAMP for 7 days.

Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro.

The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a "GTP cap." A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of "stabilizing cap," possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.

Rat testis during 2,5-hexanedione intoxication and recovery: II. Dynamics of pyrrole reactivity, tubulin content, and microtubule assembly

Charles River CD rats (200 g) were intoxicated with 1 % 2,5-hexanedione (2,5-HD) in the drinking water for 5 weeks followed by a 17-week recovery period. Pyrrole reactivity of testis proteins increased early during intoxication and then returned toward normal during recovery. Testis tubulin content first increased as germ cells were lost and then fell over time while atrophy was maintained. Purified testis tubulin demonstrated a decreased nucleation time for microtubule assembly at 2 weeks, maintained this alteration throughout intoxication, and then returned to normal assembly kinetics during recovery. The assembly abnormality was accompanied by the presence of a unique crosslinked tubulin species. These findings support the hypothesis that alterations in Sertoli cell microtubules result in germ cell loss following 2,5-HD exposure.

Microtubule dynamics in the chromosomal spindle fiber: Analysis by fluorescence and high‐resolution polarization microscopy

We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24 degrees C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24 degrees C. In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into "rods" composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.

Several metabolic factors governing the dynamics of microtubule assembly and disassembly.

Several metabolic factors governing the dynamics of microtubule assembly and disassembly.

Microinjection of fluorescent tubulin into dividing sea urchin cells.

To follow the dynamics of microtubule (MT) assembly and disassembly during mitosis in living cells, tubulin has been covalently modified with the fluorochrome 5-(4,6-dichlorotriazin-2-yl)aminofluorescein and microinjected into fertilized eggs of the sea urchin Lytechinus variegatus. The changing distribution of the fluorescent protein probe is visualized in a fluorescence microscope coupled to an image intensification video system. Cells that have been injected with fluorescent tubulin show fluorescent linear polymers that assemble very rapidly and radiate from the spindle poles, coincident with the position of the astral fibers. No fluorescent polymer is apparent in other areas of the cytoplasm. When fluorescent tubulin is injected near the completion of anaphase, little incorporation of fluorescent tubulin into polymer is apparent, suggesting that new polymerization does not occur past a critical point in anaphase. These results demonstrate that MT polymerization is very rapid in vivo and that the assembly is both temporally and spatially regulated within the injected cells. Furthermore, the microinjected tubulin is stable within the sea urchin cytoplasm for at least 1 h since it can be reutilized in successive daughter cell spindles. Control experiments indicate that the observed fluorescence is dependent on MT assembly. The fluorescence is greatly diminished upon treatment of the cells with cold or colchicine agents known to cause the depolymerization of assembled MT. In addition, cells injected with fluorescent bovine serum albumin or assembly-incompetent fluorescent tubulin do not exhibit fluorescence localized in the spindle but rather appear diffusely fluorescent throughout the cytoplasm.