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Methods in Cell Biology 2010The spatiotemporal regulation of intracellular microtubule polymerization dynamics, by numerous microtubule-associated proteins and other mechanisms, is central to many... (Review)
Review
The spatiotemporal regulation of intracellular microtubule polymerization dynamics, by numerous microtubule-associated proteins and other mechanisms, is central to many cell processes. Here, we give an overview and practical guide on how to acquire and analyze time-lapse sequences of dynamic microtubules in live cells by either fluorescently labeling entire microtubules or by utilizing proteins that specifically associate only with growing microtubule ends and summarize the strengths and weaknesses of different approaches. We give practical recommendations for imaging conditions, and discuss important limitations of such analysis that are dictated by the maximum achievable spatial and temporal sampling frequencies.
Topics: Adenoviridae; Animals; Cell Physiological Phenomena; Cell Proliferation; Clinical Laboratory Techniques; Humans; Microscopy, Fluorescence; Microtubules; Models, Biological; Protein Multimerization
PubMed: 20719263
DOI: 10.1016/S0091-679X(10)97002-7 -
Current Opinion in Cell Biology Feb 2008Although the dynamic self-assembly behavior of microtubule ends has been well characterized at the spatial resolution of light microscopy (~200 nm), the single-molecule... (Review)
Review
Although the dynamic self-assembly behavior of microtubule ends has been well characterized at the spatial resolution of light microscopy (~200 nm), the single-molecule events that lead to these dynamics are less clear. Recently, a number of in vitro studies used novel approaches combining laser tweezers, microfabricated chambers, and high-resolution tracking of microtubule-bound beads to characterize mechanochemical aspects of MT dynamics at nanometer scale resolution. In addition, computational modeling is providing a framework for integrating these experimental results into physically plausible models of molecular scale microtubule dynamics. These nanoscale studies are providing new fundamental insights about microtubule assembly, and will be important for advancing our understanding of how microtubule dynamic instability is regulated in vivo via microtubule-associated proteins, therapeutic agents, and mechanical forces.
Topics: Animals; Cell Polarity; Humans; Microtubules; Nanotechnology
PubMed: 18243676
DOI: 10.1016/j.ceb.2007.12.003 -
The EMBO Journal Aug 2022Microtubules tightly regulate various cellular activities. Our understanding of microtubules is largely based on experiments using microtubule-targeting agents, which,...
Microtubules tightly regulate various cellular activities. Our understanding of microtubules is largely based on experiments using microtubule-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific microtubule populations, due to their slow effects on the entire pool of microtubules. To overcome this technological limitation, we have used chemo and optogenetics to disassemble specific microtubule subtypes, including tyrosinated microtubules, primary cilia, mitotic spindles, and intercellular bridges, by rapidly recruiting engineered microtubule-cleaving enzymes onto target microtubules in a reversible manner. Using this approach, we show that acute microtubule disassembly swiftly halts vesicular trafficking and lysosomal dynamics. It also immediately triggers Golgi and ER reorganization and slows the fusion/fission of mitochondria without affecting mitochondrial membrane potential. In addition, cell rigidity is increased after microtubule disruption owing to increased contractile stress fibers. Microtubule disruption furthermore prevents cell division, but does not cause cell death during interphase. Overall, the reported tools facilitate detailed analysis of how microtubules precisely regulate cellular architecture and functions.
Topics: Interphase; Microtubules; Spindle Apparatus
PubMed: 35686621
DOI: 10.15252/embj.2021110472 -
Current Biology : CB Aug 2003Microtubules are intrinsically polar structures. A consequence of this polarity is that the two ends of the microtubule polymer exhibit different properties. The more... (Review)
Review
Microtubules are intrinsically polar structures. A consequence of this polarity is that the two ends of the microtubule polymer exhibit different properties. The more dynamic plus ends and the mechanisms that regulate their behavior have been the focus of much recent attention. Here, we concentrate on the dynamics and regulation of minus ends, which play distinct but equally critical roles in microtubule function. In the first part of this review, we compare the in vitro and in vivo behavior of microtubules from a minus end perspective. This comparison suggests that cells possess conserved mechanisms to specifically inhibit minus end polymerization, and perhaps also to actively promote depolymerization. In the second part, we focus on the spatial positioning of minus ends, which is achieved by localized microtubule nucleation, minus end capping and minus end anchoring as well as by motor-dependent sorting. These mechanisms are used in different biological contexts to generate the diversity of organized microtubule arrays in cells.
Topics: Biological Transport; Centrosome; Microtubules; Models, Biological; Molecular Motor Proteins; Tubulin
PubMed: 12906817
DOI: 10.1016/s0960-9822(03)00530-x -
The Journal of Cell Biology Jun 2017The primary goal of a dividing somatic cell is to accurately and equally segregate its genome into two new daughter cells. In eukaryotes, this process is performed by a... (Review)
Review
The primary goal of a dividing somatic cell is to accurately and equally segregate its genome into two new daughter cells. In eukaryotes, this process is performed by a self-organized structure called the mitotic spindle. It has long been appreciated that mechanical forces must be applied to chromosomes. At the same time, the network of microtubules in the spindle must be able to apply and sustain large forces to maintain spindle integrity. Here we consider recent efforts to measure forces generated within microtubule networks by ensembles of key proteins. New findings, such as length-dependent force generation, protein clustering by asymmetric friction, and entropic expansion forces will help advance models of force generation needed for spindle function and maintaining integrity.
Topics: Animals; Cell Division; Humans; Mechanotransduction, Cellular; Microtubule Proteins; Microtubules; Spindle Apparatus; Stress, Mechanical
PubMed: 28490474
DOI: 10.1083/jcb.201612064 -
Biomolecules Jun 2023Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such...
Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well-developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite the development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes, and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that are solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in , with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.
Topics: Saccharomyces cerevisiae; Microscopy, Fluorescence; Microtubules; Software
PubMed: 37371519
DOI: 10.3390/biom13060939 -
TheScientificWorldJournal Jun 2008Originally characterized as regulators of cytokinesis, septins were later implicated in other cellular processes. Recent studies show that septins have a broader role in... (Review)
Review
Originally characterized as regulators of cytokinesis, septins were later implicated in other cellular processes. Recent studies show that septins have a broader role in microtubule-dependent processes, such as karyokinesis, exocytosis, and maintenance of cell shape. Many members of the septin family have been shown to colocalize or interact with the microtubule cytoskeleton, suggesting that these might be general properties of septins. Septins could play an important role in regulating microtubule dynamics by interacting with microtubule-associated proteins (MAPs) that modulate microtubule stability. Being able to associate with both microtubules and actin, septins can play an important role as adaptors between the two cytoskeletons and as regulators of processes in which both actin and microtubules are involved. As septins are associated with various neurodegenerative diseases and cancer, a better understanding of the biology of septins and their interactions with microtubules is important in order to develop possible therapeutic strategies for these diseases.
Topics: Animals; Cytoskeletal Proteins; Humans; Microtubules
PubMed: 18604445
DOI: 10.1100/tsw.2008.87 -
Current Biology : CB Dec 2009Mitosis depends on the mitotic spindle, a subcellular protein machine that uses dynamic microtubules and mitotic motors to assemble itself and to coordinate chromosome... (Review)
Review
Mitosis depends on the mitotic spindle, a subcellular protein machine that uses dynamic microtubules and mitotic motors to assemble itself and to coordinate chromosome movements. Spindle function depends critically on the interplay of microtubule polymer dynamics and the motor proteins and non-motor microtubule-associated proteins (MAPs) that crosslink adjacent microtubules. These microtubule crosslinkers can organize microtubules into bundles with specific polarity patterns and some of them can slide adjacent microtubules in relation to one another. Here, we discuss the functions and mechanisms of action of three such crosslinkers: the motors kinesin-5 and kinesin-14, and the non-motor MAPs of the Ase1p family.
Topics: Animals; Microtubules; Mitosis; Molecular Motor Proteins; Protein Transport
PubMed: 20064413
DOI: 10.1016/j.cub.2009.10.047 -
Seminars in Cell & Developmental Biology Jan 2015Microtubules are essential cellular polymers assembled from tubulin heterodimers. The tubulin dimer consists of a compact folded globular core and intrinsically... (Review)
Review
Microtubules are essential cellular polymers assembled from tubulin heterodimers. The tubulin dimer consists of a compact folded globular core and intrinsically disordered C-terminal tails. The tubulin tails form a lawn of densely grafted, negatively charged, flexible peptides on the exterior of the microtubule, potentially akin to brush polymers in the field of synthetic materials. These tails are hotspots for conserved, chemically complex posttranslational modifications that have the potential to act in a combinatorial fashion to regulate microtubule polymer dynamics and interactions with microtubule effectors, giving rise to a "tubulin code". In this review, I summarize our current knowledge of the enzymes that generate the astonishing tubulin chemical diversity observed in cells and describe recent advances in deciphering the roles of tubulin C-terminal tails and their posttranslational modifications in regulating the activity of molecular motors and microtubule associated proteins. Lastly, I outline the promises, challenges and potential pitfalls of deciphering the tubulin code.
Topics: Animals; Humans; Intrinsically Disordered Proteins; Microtubules; Protein Processing, Post-Translational; Tubulin
PubMed: 25307498
DOI: 10.1016/j.semcdb.2014.09.026 -
Nature Communications Jul 2020Microtubules are dynamic tubulin polymers responsible for many cellular processes, including the capture and segregation of chromosomes during mitosis. In contrast to...
Microtubules are dynamic tubulin polymers responsible for many cellular processes, including the capture and segregation of chromosomes during mitosis. In contrast to textbook models of tubulin self-assembly, we have recently demonstrated that microtubules elongate by addition of bent guanosine triphosphate tubulin to the tips of curving protofilaments. Here we explore this mechanism of microtubule growth using Brownian dynamics modeling and electron cryotomography. The previously described flaring shapes of growing microtubule tips are remarkably consistent under various assembly conditions, including different tubulin concentrations, the presence or absence of a polymerization catalyst or tubulin-binding drugs. Simulations indicate that development of substantial forces during microtubule growth and shortening requires a high activation energy barrier in lateral tubulin-tubulin interactions. Modeling offers a mechanism to explain kinetochore coupling to growing microtubule tips under assisting force, and it predicts a load-dependent acceleration of microtubule assembly, providing a role for the flared morphology of growing microtubule ends.
Topics: Animals; Cryoelectron Microscopy; Electron Microscope Tomography; Microtubules; Models, Biological; Molecular Dynamics Simulation; Polymerization; Swine; Tubulin; Tubulin Modulators
PubMed: 32724196
DOI: 10.1038/s41467-020-17553-2