Microtubule Treadmilling Research Articles

Collective Effects of Xmap215, EB1, CLASP2, and MCAK Lead to Robust Microtubule Treadmilling

Microtubule network remodeling is essential for fundamental cellular processes including cell division, differentiation, and motility. Microtubules are active biological polymers whose ends stochastically and independently switch between phases of growth and shrinkage. Microtubule treadmilling, in which the microtubule plus end grows while the minus end shrinks, is observed in cells; however, the underlying mechanisms are not known. Here, we use a combination of computational and in vitro reconstitution approaches to determine the conditions leading to robust microtubule treadmilling. We find that microtubules polymerized from tubulin alone can treadmill, albeit with opposite directionality and order-of-magnitude slower rates than observed in cells. We then employ computational simulations to predict that the combinatory effects of four microtubule-associated proteins (MAPs), namely EB1, XMAP215, CLASP2, and MCAK, can promote fast and sustained plus-end-leading treadmilling. Finally, we experimentally confirm the predictions of our computational model using a multi-MAP, in vitro microtubule dynamics assay to reconstitute robust plus-end-leading treadmilling, consistent with observations in cells. Our results demonstrate how microtubule dynamics can be modulated to achieve a dynamic balance between assembly and disassembly at opposite polymer ends, resulting in treadmilling over long periods of time. Overall, we show how the collective effects of multiple components give rise to complex microtubule behavior that may be used for global network remodeling in cells.

Collective effects of XMAP215, EB1, CLASP2, and MCAK lead to robust microtubule treadmilling

Microtubule network remodeling is essential for fundamental cellular processes including cell division, differentiation, and motility. Microtubules are active biological polymers whose ends stochastically and independently switch between phases of growth and shrinkage. Microtubule treadmilling, in which the microtubule plus end grows while the minus end shrinks, is observed in cells; however, the underlying mechanisms are not known. Here, we use a combination of computational and in vitro reconstitution approaches to determine the conditions leading to robust microtubule treadmilling. We find that microtubules polymerized from tubulin alone can treadmill, albeit with opposite directionality and order-of-magnitude slower rates than observed in cells. We then employ computational simulations to predict that the combinatory effects of four microtubule-associated proteins (MAPs), namely EB1, XMAP215, CLASP2, and MCAK, can promote fast and sustained plus-end-leading treadmilling. Finally, we experimentally confirm the predictions of our computational model using a multi-MAP, in vitro microtubule dynamics assay to reconstitute robust plus-end-leading treadmilling, consistent with observations in cells. Our results demonstrate how microtubule dynamics can be modulated to achieve a dynamic balance between assembly and disassembly at opposite polymer ends, resulting in treadmilling over long periods of time. Overall, we show how the collective effects of multiple components give rise to complex microtubule behavior that may be used for global network remodeling in cells.

Microtubule Treadmilling Reconstituted with a Minimal-component in vitro System

Microtubule Treadmilling Reconstituted with a Minimal-component in vitro System

Dynamic Instability and Treadmilling Coexist for In Vitro Microtubules

Microtubules are cytoskeletal polymers that play essential roles during multiple cellular processes. Dynamic nature of these polymers allows them to remodel the microtubule network spatially and temporally to fulfill their specific duties. Dynamic instability is a well known microtubule behavior in which both polymer ends independently switch between phases of growth and shrinkage. Earlier studies observed that microtubules can exhibit another mode of dynamics, a phenomenon called treadmilling, in which the growth rate at one end is comparable to the shrinkage rate of the other end. While microtubule dynamic instability has been widely studied, the conditions that lead to microtubule treadmilling are not well understood, and the relations between these two modes of microtubule dynamics are not well characterized. Here, we show that the dynamic instability and treadmilling of individual polymers coexist in vitro around 6uM tubulin in solution in the absence of microtubule associated proteins. We investigate different time scales to characterize the dynamic behavior of microtubules by calculating the net growth/shrinkage rate within the given time window and find that the microtubules exhibit treadmilling episodes in either direction, as well as growth or shrinkage at both ends. The treadmilling behavior arises due to the bias in the dynamic instability for a given time window, resulting in net shrinkage at one end and net growth at the other end. We find that, with tubulin alone, treadmilling towards the minus ends is more likely than towards the plus ends within time segments varying from 45 seconds to 10 minutes. By focusing on the microtubule dynamics at different time scales, this study provides a deeper understanding of the relations between dynamic instability and microtubule treadmilling.

BKM-120 (Buparlisib): A Phosphatidyl-Inositol-3 Kinase Inhibitor with Anti-Invasive Properties in Glioblastoma.

Glioblastoma is an aggressive, invasive tumor of the central nervous system (CNS). There is a widely acknowledged need for anti-invasive therapeutics to limit glioblastoma invasion. BKM-120 is a CNS-penetrant pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for solid tumors, including glioblastoma. We observed that BKM-120 has potent anti-invasive effects in glioblastoma cell lines and patient-derived glioma cells in vitro. These anti-migratory effects were clearly distinguishable from cytostatic and cytotoxic effects at higher drug concentrations and longer durations of drug exposure. The effects were reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenografts. GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. BKM-120 reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition contribute to the anti-invasive effects of BKM-120. Our data suggest the CNS-penetrant PI3K inhibitor BKM-120 may have anti-invasive properties in glioblastoma.

Abstract LB-B19: BKM-120: a phosphatidyl-inositol-3 kinase inhibitor with anti-migratory properties in glioblastoma

Abstract Glioblastoma (GBM) is the most common glial brain tumor and is also one of the most lethal human cancers: patients have a median survival of 15 months with a five-year survival rate of only 3%. The current standard-of-care has remained the same for the last decade and consists of maximal safe surgical resection followed by radio- and chemotherapy. GBM cells are highly infiltrative, leading to invasion of normal brain tissue by tumor cells. These invasive tumor cells render GBM a surgically incurable disease and tumor recurrence is almost inevitable, with 90% of patients developing new lesions within 2-3 cm of the original site or at distant sites in the brain. Moreover, invasion may increase further during anti-angiogenic therapy with bevacizumab and no anti-invasive approaches are yet available clinically. The phosphatidyl-inositol-3 kinase (PI3K) pathway is frequently deregulated in cancer and is activated in the majority of GBM cases due to constitutive receptor tyrosine kinase activation as well as inactivating mutations/deletions of PTEN (33%) or activating PI3K mutations (17%). In addition PI3K plays a role in cell migration in some cell types and a number of small molecule PI3K inhibitors are under investigation in oncology clinical trials. BKM-120 (Buparlisib), is a CNS-penetrant selective pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for several types of solid tumor, including GBM. We initially observed that BKM-120 is a potent anti-invasive molecule in GBM cell lines and patient-derived glioma stem-like cells in vitro. The anti-migratory effects of BKM-120 were clearly distinguishable from cytostatic and cytotoxic effects that occurred at higher drug concentrations and after a longer duration of drug exposure. The blockade of migration was reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenograft model. Mechanistically we found that GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. However, BKM-120 substantially reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition may contribute to the anti-invasive effects of BKM-120. Overall, our data suggest that the CNS-penetrant PI3K inhibitor BKM-120 may be a useful anti-migratory drug for the treatment of highly invasive tumors such as glioblastoma. Citation Format: Maria Carmela Speranza, Michal O. Nowicki, Choi-Fong Cho, E. Antonio Chiocca, Sean E. Lawler. BKM-120: a phosphatidyl-inositol-3 kinase inhibitor with anti-migratory properties in glioblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B19.

Non-signalling energy use in the brain.

Energy use limits the information processing power of the brain. However, apart from the ATP used to power electrical signalling, a significant fraction of the brain's energy consumption is not directly related to information processing. The brain spends just under half of its energy on non-signalling processes, but it remains poorly understood which tasks are so energetically costly for the brain. We review existing experimental data on subcellular processes that may contribute to this non-signalling energy use, and provide modelling estimates, to try to assess the magnitude of their ATP consumption and consider how their changes in pathology may compromise neuronal function. As a main result, surprisingly little consensus exists on the energetic cost of actin treadmilling, with estimates ranging from < 1% of the brain's global energy budget up to one-half of neuronal energy use. Microtubule treadmilling and protein synthesis have been estimated to account for very small fractions of the brain's energy budget, whereas there is stronger evidence that lipid synthesis and mitochondrial proton leak are energetically expensive. Substantial further research is necessary to close these gaps in knowledge about the brain's energy-expensive non-signalling tasks.

Microtubules and the tax payer

Plant microtubules have evolved into a versatile tool to link environmental signals into flexible morphogenesis. Cortical microtubules define the axiality of cell expansion by control of cellulose orientation. Plant-specific microtubule structures such as preprophase band and phragmoplast determine symmetry and axiality of cell divisions. In addition, microtubules act as sensors and integrators for stimuli such as mechanic load, gravity, but also osmotic stress, cold and pathogen attack. Many of these functions are specific for plants and involve specific proteins or the recruitment of proteins to new functions. The review aims to ventilate the potential of microtubule-based strategies for biotechnological application by highlighting representative case studies. These include reorientation of cortical microtubules to increase lodging resistance, control of microtubule dynamics to alter the gravity-dependent orientation of leaves, the use of microtubules as sensitive thermometers to improve adaptive cold tolerance of chilling and freezing sensitive plants, the reduction of microtubule treadmilling to inhibit cell-to-cell transport of plant viruses, or the modulation of plant defence genes by pharmacological manipulation of microtubules. The specificity of these responses is controlled by a great variety of specific associated proteins opening a wide field for biotechnological manipulation of plant architecture and stress tolerance.

Patronin Regulates the Microtubule Network by Protecting Microtubule Minus Ends

Tubulin assembles into microtubule polymers that have distinct plus and minus ends. Most microtubule plus ends in living cells are dynamic; the transitions between growth and shrinkage are regulated by assembly-promoting and destabilizing proteins. In contrast, minus ends are generally not dynamic, suggesting their stabilization by some unknown protein. Here, we have identified Patronin (also known as ssp4) as a protein that stabilizes microtubule minus ends in Drosophila S2 cells. In the absence of Patronin, minus ends lose subunits through the actions of the Kinesin-13 microtubule depolymerase, leading to a sparse interphase microtubule array and short, disorganized mitotic spindles. In vitro, the selective binding of purified Patronin to microtubule minus ends is sufficient to protect them against Kinesin-13-induced depolymerization. We propose that Patronin caps and stabilizes microtubule minus ends, an activity that serves a critical role in the organization of the microtubule cytoskeleton.

Bacterial Cytoskeleton: Not Your Run-of-the-Mill Tubulin

Large plasmids of some Bacillus species encode a distinct tubulin homolog, TubZ, implicated in maintenance of the host plasmid. A recent study has shown that TubZ polymers exhibit treadmilling behavior in vivo, suggesting that they are involved in mitotic activity.

Dynamic interaction of NtMAP65-1a with microtubules in vivo

Plant microtubules are intrinsically more dynamic than those from animals. We know little about the dynamics of the interaction of plant microtubule-associated proteins (MAPs) with microtubules. Here, we have used tobacco and Arabidopsis MAPs with relative molecular mass 65 kDa (NtMAP65-1a and AtMAP65-1), to study their interaction with microtubules in vivo. Using fluorescence recovery after photobleaching we report that the turnover of both NtMAP65-1a and AtMAP65-1 bound to microtubules is four- to fivefold faster than microtubule treadmilling (13 seconds compared with 56 seconds, respectively) and that the replacement of NtMAP65-1a on microtubules is by random association rather than by translocation along microtubules. MAP65 will only bind polymerised microtubules and not its component tubulin dimers. The turnover of NtMAP65-1a and AtMAP65-1 on microtubules is similar in the interphase cortical array, the preprophase band and the phragmoplast, strongly suggesting that their role in these arrays is the same. NtMAP65-1a and AtMAP65-1 are not observed to bind microtubules in the metaphase spindle and their rate of recovery is consistent with their cytoplasmic localisation. In addition, the dramatic reappearance of NtMAP65-1a on microtubules at the spindle midzone in anaphase B suggests that NtMAP65-1a is controlled post-translationally. We conclude that the dynamic properties of these MAPs in vivo taken together with the fact that they have been shown not to effect microtubule polymerisation in vitro, makes them ideally suited to a role in crossbridging microtubules that need to retain spatial organisation in rapidly reorganising microtubule arrays.

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BioTechniquesVol. 37, No. 1 WebWatchOpen AccessWebWatchKevin AhernKevin AhernSearch for more papers by this authorPublished Online:6 Jun 2018https://doi.org/10.2144/04371WW01AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Bug ZooWonder what that six-legged, skinny green life form crawling across your window is that looks like it came from another planet? Perhaps you're a nerdy entomologist who appreciates digital images of bugs? In either event, there's a treat in store for you at the MCZ Entomology Primary Type Specimen Database hosted at Harvard University. With a jaw-dropping 28,000 records and a growing number of digital images, scientists and entomological voyeurs alike will have enough to keep themselves busy here for hours. The site provides easy access to both of these groups, with a search engine that provides researchers a means to extract specific records and the Greatest Hits section that gives casual browsers a simple way to view dozens of the site's best digital photographs.http://mcz-28168.oeb.harvard.edu/mcztypedb.htmCulteral DiversitySpeaking of bugs, one of the best places to find the microscopic variety is at The World Data Centre for Microorganisms (WDCM). Sponsored by the World Federation for Culture Collections and seven other international groups, WDCM provides access to a catalog of catalogs—searchable indexes of more than 475 culture collections in 62 countries. A quick scroll through the collections reveals common culture sources, such as the ATCC, as well as relatively obscure ones, like the Quinone Database of Eubacteria. A thorough collection of links to genome sequencing projects also provides handy access to additional information about some of the species listed.http://wdcm.nig.ac.jp/Project OrientedIn the middle of bioinformatics is the word “inform,” and the designers of the Human Cancer Genetics (HCG) Bioinformatics site at Ohio State University appear to have made it the theme of the HCG site. With not one or even two, but rather three significant bioinformatics databases under the hood of the HCG site, these folks have been busy. From HemoPDB (transcriptional regulation in hematopoiesis and leukemia) to GenomeWide (discovery and analysis of alternative promoters) to FEAnnotations (annotation of promoters and first exons in the human genome), HCG covers a lot of territory and, if past performance is any indicator, more is likely on the way.http://bioinformatics.med.ohio-state.edu/Geek AlertWebWatch readers should beware that reading further about this site devoted to molecular biology programs created for the Linux operating system may start you on a path to geekdom. Linux, of course, is the open source programming project that is popular for high-end computing. The Molecular Linux page hosted at Bioinformatics.org provides one-click download access to a vast suite of software tools of interest to molecular biologists. These programs provide common functions for making sequence alignments, predicting PCR primers, performing phylogenetic analysis of sequence families, and displaying molecular structures in PDB files. True geeks will choose to download source code (programming instructions that can be compiled to run) that they can tweak and make perform as they choose. Executable code (already compiled and ready to run) is available for several programs as well.http://bioinformatics.org/software/index.php3Seeing GreenA quick scan through the Carnegie Cell Imaging page at Stanford University may leave you wondering if there is anything left in cells that hasn't been tagged, photographed, or captured on video by researchers using jellyfish green fluorescent protein (GFP). From 3-D imaging to cytokinesis, GFP has proven a godsend for imaging the action and movements of proteins in cellular structures. Curious about cytoskeletal dynamics? Then you'll appreciate the QuickTime-formatted movies depicting microtubule treadmilling in Arabidopsis. Are cellular organelles your thing? Then watch the time-lapse movies of changing organelle morphology and motility in root epidermal cells. A collection of protocols summarizes how the images were made and allows visitors to duplicate the results in their own laboratories.http://deepgreen.stanford.edu/FiguresReferencesRelatedDetails Vol. 37, No. 1 Follow us on social media for the latest updates Metrics Downloaded 78 times History Published online 6 June 2018 Published in print July 2004 Information© 2004 Author(s)PDF download

767. Intact Microtubules Are Required for Ad-Vector Induction of Chemokine Genes in Epithelial Cells

Upon entry in epithelial cells, adenovirus vectors (Ad-vectors) activate a cascade of signaling pathways including p38 MAPK (p38) and extracellular-signal regulated kinase (ERK). These signaling events occur within minutes and are triggered directly by the viral capsid. The activation of signaling by Ad-vectors leads to the expression of chemokine inflammatory genes such as IP-10. Shortly following internalization, Ad-vectors interact with microtubules that are required to mediate viral translocation to the nucleus. To better understand the role of microtubules in Ad vector induced signaling and chemokine gene expression, epithelial cells (REC) were treated with the microtubule depolymerizing reagent nocodazole (20mM). Nocodazole substantially reduced transduction of REC cells by a first generation Ad-vector, Adβgal, as determined by β-galactosidase expression 24 hours post-transduction. Vector internalization into REC however was unaffected as measured by flow cytometry confirming that microtubules were required to mediate nuclear translocation of Ad-vectors. To determine the effect of microtubule depolymerization on the induction of chemokine mRNA expression, RANTES and IP-10 gene expression was determined at 6 hours following transduction with Adβgal. Compared to untreated cells, nocodazole blocked the Adβgal induction of RANTES and IP-10 expression. Phosphorylated ERK or p38 was increased by nocodazole in the absence of Ad-vector transduction, a response that was not changed following the addition of Adβgal. Although indirect, this finding suggests that p38 and ERK signaling is linked to microtubules and is required but not sufficient to induce the expression of IP-10 and RANTES. In contrast, experiments with the microtubule-stabilizing agent, paclitaxel, did not affect Adβgal transduction of REC cells. Furthermore, Adβgal induction of p38/ERK phosphorylation and IP-10/RANTES gene expression was unchanged following paclitaxel suggesting that microtubule treadmilling or dynamic instability was not required for Ad-vector induction of host immune responses. These preliminary data suggest that intact microtubules are required for the Ad-vector induction of host inflammatory genes in epithelial cells. Understanding the biology of Ad-vector induced intracellular signaling will contribute significantly to the development of novel recombinant vectors for gene therapy.

Microtubule Treadmilling in Vitro Investigated by Fluorescence Speckle and Confocal Microscopy

Whether polarized treadmilling is an intrinsic property of microtubules assembled from pure tubulin has been controversial. We have tested this possibility by imaging the polymerization dynamics of individual microtubules in samples assembled to steady-state in vitro from porcine brain tubulin, using a 2% glycerol buffer to reduce dynamic instability. Fluorescence speckled microtubules were bound to the cover-glass surface by kinesin motors, and the assembly dynamics of plus and minus ends were recorded with a spinning-disk confocal fluorescence microscopy system. At steady-state assembly, 19% of the observed microtubules ( n = 89) achieved treadmilling in a plus-to-minus direction, 34% in a minus-to-plus direction, 37% grew at both ends, and 10% just shortened. For the population of measured microtubules, the distribution of lengths remained unchanged while a 20% loss of original and 27% gain of new polymer occurred over the 20-min period of observation. The lack of polarity in the observed treadmilling indicates that stochastic differences in dynamic instability between plus and minus ends are responsible for polymer turnover at steady-state assembly, not unidirectional treadmilling. A Monte Carlo simulation of plus and minus end dynamics using measured dynamic instability parameters reproduces our experimental results and the amount of steady-state polymer turnover reported by previous biochemical assays.

Microtubule Treadmilling in Vitro Investigated by Fluorescence Speckle and Confocal Microscopy

Microtubule Treadmilling in Vitro Investigated by Fluorescence Speckle and Confocal Microscopy

Microtubule treadmilling: what goes around comes around.

"I'll see it when I believe it" Daniel Mazia Microtubules are centrally involved in many essential cell functions, including mitosis, vesicle motility, and the control of morphogenesis. Further, they appear to be involved in the control of cell cycle progression. To carry out these tasks properly, microtubules assume a protean array of different stability states and degrees of organization and they respond rapidly to requirements of the cell by modification of their organization and stability. In the typical fibroblast cell in culture, microtubules rapidly exchange their subunits with tubulin in the cytoplasmic pool, and control of this rapid turnover appears to be essential to their intrinsic capacity to perform such tasks as the separation of chromosomes in mitosis. Microtubules are not simple equilibrium polymers, but rather, they are capable of unusual nonequilibrium dynamic behaviors. One such behavior, termed treadmilling, involving the intrinsic flow of subunits from one polymer end to the other, is created by differences in the critical subunit concentrations at the opposite microtubule ends. Treadmilling was considered by many to be an in vitro dynamic behavior that did not play an important role in microtubule function in cells. However, recent evidence has established that treadmilling is a major in vivo mechanism underlying the dynamics of microtubule arrays.

Microtubule treadmilling in vivo.

In vivo, cytoplasmic microtubules are nucleated and anchored by their minus ends at the centrosome and are believed to turn over by a mechanism termed dynamic instability: depolymerization and repolymerization at their plus ends. In cytoplasmic fragments of fish melanophores, microtubules were shown to detach from their nucleation site and depolymerize from their minus ends. Free microtubules moved toward the periphery by treadmilling-growth at one end and shortening from the opposite end. Frequent release from nucleation sites may be a general property of centrosomes and permit a minus-end mechanism of microtubule turnover and treadmilling.

Direct observation of microtubule treadmilling by electron microscopy.

Using an immunoelectron microscopic procedure, we directly observed the concurrent addition and loss of chicken brain tubulin subunits from the opposite ends of microtubules containing erythrocyte tubulin domains. The polarity of growth of the brain tubulin on the ends of erythrocyte microtubules was determined to be similar to growth off the ends of Chlamydomonas axonemes. The flux rate for brain tubulin subunits in vitro was low, approximately 0.9 micron/h. Tubulin subunit flux did not continue through the entire microtubule as expected, but ceased when erythrocyte tubulin domains became exposed, resulting in a metastable configuration that persisted for at least several hours. We attribute this to differences in the critical concentrations of erythrocyte and brain tubulin. The exchange of tubulin subunits into the walls of preformed microtubules other than at their ends was also determined to be insignificant, the exchange rate being less than the sensitivity of the assay, or less than 0.2%/h.

Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching.

The rate of exchange of tubulin that is incorporated into spindle microtubules with dimeric tubulin in the cytoplasm has been measured in sea urchin eggs by studying fluorescence redistribution after photobleaching (FRAP). Dichlorotriazinyl amino fluorescein (DTAF) has been used to label bovine brain tubulin. DTAF-tubulin has been injected into fertilized eggs of Lytechinus variegatus and allowed to equilibrate with the endogenous tubulin pool. Fluorescent spindles formed at the same time that spindles were seen in control eggs, and the injected embryos proceeded through many cycles of division on schedule, suggesting that DTAF-tubulin is a good analogue of tubulin in vivo. A microbeam of argon laser light has been used to bleach parts of the fluorescent spindles, and FRAP has been recorded with a sensitive video camera. Laser bleaching did not affect spindle structure, as seen with polarization optics, nor spindle function, as seen by rate of progress through mitosis, even when one spindle was bleached several times in a single cell cycle. Video image analysis has been used to measure the rate of FRAP and to obtain a low resolution view of the fluorescence redistribution process. The half-time for spindle FRAP is approximately 19 s, even when an entire half-spindle is bleached. Complete exchange of tubulin in nonkinetochore spindle and astral microtubules appeared to occur within 60-80 s at steady state. This rate is too fast to be explained by a simple microtubule end-dependent exchange of tubulin. Efficient microtubule treadmilling would be fast enough, but with current techniques we saw no evidence for movement of the bleached spot during recovery, which we would expect on the basis of Margolis and Wilson's model (Nature (Lond.)., 1981, 293:705)--fluorescence recovers uniformly. Microtubules may be depolymerizing and repolymerizing rapidly and asynchronously throughout the spindle and asters, but the FRAP data are most compatible with a rapid exchange of tubulin subunits all along the entire lengths of nonkinetochore spindle and astral microtubules.

A model for the microtubule organizing activity of the centrosomes and kinetochores in mammalian cells

In this report I review shortly recent evidence on the role of centrosomes and kinetochores in organized microtubule assembly in cells. I arrive at the conclusion that models for these organizing centres must provide an explanation for the following observations: 1. 1. Both centrosomes and kinetochores induce microtubule assembly in their immediate vicinity at a tubulin concentration below the cytoplasmic critical concentration. 2. 2. Initially, the newly assembled microtubules are not necessarily anchored to the MTOC's. 3. 3. The assembly initiating and microtubule stabilizing activity of the MTOC's is abrogated by lowering the critical tubulin concentration in the cytoplasm. 4. 4. Microtubules attach to the centrosomes with their minus end and to the kinetochores with their plus end. 5. 5. Interactions between centrosomes and kinetochores or microtubules derived from them are important in guiding microtubule elongation and stabilizing kinetically unfavored microtubule sets (kinetochore microtubules). Models that are based on the presence of seeds or templates in the MTOC's do not predict observations 2 and 3. Models which conceive MTOC's as sites where microtubules are anchored at their minus end which is consequently capped do not predict observations 3 and 4. We propose a model that explains all the observations summarized above. Both centrosomes and kinetochores induce assembly at low tubulin concentrations by being domains where the critical tubulin concentration is lower than elsewhere in the cytoplasm. Once formed, microtubules may become more or less securely fixed to the MTOC by their minus (centrosome) or plus end (kinetochore). Because this anchoring may occur through lateral bonds between the microtubule surface and a component of the MTOC the end can remain free to add or loose subunits. The model allows cells to build microtubule sets of different polarity and stability. Unlike the seed and minus end capping models it is compatible with mechanisms of intracellular motility based on microtubule treadmilling.