Analysis of microtubule polymerization dynamics in live cells (original) (raw)
Related papers
Cell Motility and The Cytoskeleton, 1988
The ability to tag biological molecules fluorescently and to detect their distribution in living cells has promoted the study of cytoplasmic organization in general and microtubule dynamics in particular. The techniques that we have selected and developed allowed the determination of spatial and temporal changes of the microtubule network in living fibroblasts at the level of individual microtubules. We have employed two general approaches for determining pattern changes: direct video microscopy and photobleaching and subsequent observation. Direct observation of fluorescent microtubules by high-definition video microscopy provided good spatial resolution at several time points, but was limited to the less congested and thinner periphery of the cell. This approach was made possible by a relatively bright, photostable reporter, xrhodamine-tubulin, and showed that microtubules underwent rounds of assembly and disassembly from their ends. Bleaching and subsequent observation of lysed cells improved the signal to noise ratio by extracting soluble chromophore and permitted observations in congested areas, but was limited to a single time interval. This approach demonstrated that microtubule domains were replaced one by one and that turnover was most rapid at the cell periphery. Antibodies specific for nonbleached chromophore can be used to enhance the signal to noise ratio further or to extend spatial resolution by the use of immunoelectron microscopy. Direct video microscopy and photo-bleaching are two approaches to the study of dynamics that have complementary strengths and wide application to the biology of living cells.
Microtubule Dynamics Reconstituted In Vitro and Imaged by Single-Molecule Fluorescence Microscopy
Methods in Cell Biology, 2010
In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of microtubule-associated proteins (MAPs) in regulating the growth, shrinkage, and catastrophe of microtubules. The use of total internal reflection fluorescence microscopy with fluorescently labeled tubulin and MAPs has allowed us to study microtubule dynamics at the resolution of single molecules. In this chapter we present a practical overview of how these assays are performed in our laboratory: fluorescent labeling methods, strategies to prolong the time to photo-bleaching, preparation of stabilized microtubules, flow-cells, microtubule immobilization, and finally an overview of the workflow that we follow when performing the experiments. At all stages, we focus on practical tips and highlight potential stumbling blocks.
Localized alteration of microtubule polymerization in response to guidance cues
Journal of Neuroscience Research, 2010
Inhibition of microtubule dynamic instability prevents growth cone turning in response to guidance cues, yet specific changes in microtubule polymerization as growth cones encounter boundaries have not been investigated. In this study, we examined the rate and direction of microtubule polymerization in response to soluble nerve growth factor (NGF) and immobilized chondroitin sulfate proteoglycans (CSPGs) by expressing enhanced GFPEB3 in rat pheochromocytoma (PC12) cells. GFP-EB3 comets were monitored in live cells using time-lapse epifluorescent microscopy. Using an automated tracking system, the rate of microtubule polymerization was calculated as the frame-to-frame displacement of EB3 comets. Our results demonstrate that the rate of microtubule polymerization is increased following NGF treatment, while contact with CSPGs decreases microtubule polymerization rates. This reduction in microtubule polymerization rates was specifically localized to neurites in direct contact with CSPGs, and not at non-contacting neurites. Additionally, we found an increase in the percentage of microtubules polymerizing in the retrograde direction in neurites at CSPG boundaries with a concomitant decrease in the rate of retrograde microtubule polymerization. These results implicate localized changes in microtubule dynamics as an important component of the growth cone response to guidance cues.
Modern methods to interrogate microtubule dynamics
Integrative Biology, 2013
Microtubules are essential protein filaments required to organize and rearrange the interior of the cell. They must be stiff with mechanical integrity to support the structure of the cell. Yet, they must also be dynamic to enable rearrangements of the cell during cell division and development. This dynamic nature is inherent to microtubules and comes about through the hydrolysis of chemical energy stored in guanosine triphosphate (GTP). Dynamic instability has been studied with a number of microscopy techniques both in cells and in reconstituted systems. In this article, we review the techniques used to examine microtubule dynamic instability and highlight future avenues and still open questions about this vital and fascinating activity.
MTrack: Automated Detection, Tracking, and Analysis of Dynamic Microtubules
Scientific Reports
Microtubules are polar, dynamic filaments fundamental to many cellular processes. In vitro reconstitution approaches with purified tubulin are essential to elucidate different aspects of microtubule behavior. To date, deriving data from fluorescence microscopy images by manually creating and analyzing kymographs is still commonplace. Here, we present Mtrack, implemented as a plug-in for the open-source platform Fiji, which automatically identifies and tracks dynamic microtubules with sub-pixel resolution using advanced objection recognition. Mtrack provides automatic data interpretation yielding relevant parameters of microtubule dynamic instability together with population statistics. the application of our software produces unbiased and comparable quantitative datasets in a fully automated fashion. this helps the experimentalist to achieve higher reproducibility at higher throughput on a user-friendly platform. We use simulated data and real data to benchmark our algorithm and show that it reliably detects, tracks, and analyzes dynamic microtubules and achieves sub-pixel precision even at low signal-to-noise ratios. Microtubules are dynamic filaments essential for many cellular processes such as intracellular transport, cell motility and chromosome segregation. They assemble from dimeric αβ-tubulin subunits that polymerize in a head-to-tail fashion into polar filaments 1 (Fig. 1). Microtubules show a behavior termed dynamic instability, which can be empirically described by four parameters: (1) the polymerization velocity at which microtubules grow (vg), (2) the depolymerization velocity at which microtubules shrink (vs), (3) the catastrophe frequency at which microtubules switch from growth to shrinkage (fc), and (4) the rescue frequency at which microtubules switch from shrinkage to growth (fs) 2. This dynamic behavior is intrinsic to microtubules. In a cellular context, however, the dynamic properties of microtubules are modulated by motors and accessory proteins known as microtubule associated proteins (MAPs) 3-7. In most cases, the cellular context is too complex to study a single protein's contribution to microtubule dynamics. Therefore, biochemical activities of individual proteins have primarily been characterized in vitro using purified components and total-internal reflection fluorescence (TIRF) microscopy 8-17. Furthermore, microtubule dynamics are strongly affected by a set of drugs routinely used to treat diseases such as cancer 18 and malaria 19. Owing to their clinical relevance, it is a viable need to understand the exact regulation of microtubule dynamics by a given drug and thereby elucidate the underlying molecular mechanisms. Given the growing interest in biochemical reconstitution systems 3,4,20 , automation of data analysis will unveil the full potential of the experimental approaches as described above. Quantitatively deriving dynamic microtubule parameters from fluorescence microscopy images by manually creating and analyzing kymographs (spatial position over time) is still common practice 21. This limits the collection of statistically significant amounts of data. Moreover, manual analysis can bias data collection and introduce variability. Thus, methods have been developed that allow microtubule detection and/or tracking 22-27. However, to date, there is no fully automated workflow that provides detection and tracking of microtubules followed by automated data analysis and statistics collection. Here, we present the software MTrack, which detects, tracks, measures, and analyses the behavior of fluorescently labeled microtubules imaged by TIRF microscopy
Journal of Cell Biology, 1993
Recent experiments have demonstrated that the behavior of the interphase microtubule array is cell-type specific: microtubules in epithelial cells are less dynamic than microtubules in fibroblasts (Pepper-kok et al., 1990; Wadsworth and McGrail, 1990). To determine which parameters of microtubule dynamic instability behavior are responsible for this difference, we have examined the behavior of individual microtubules in both cell types after injection with rhodamine-labeled tubulin subunits. Individual microtubules in both cell types were observed to grow, shorten, and pause, as expected. The average amount of time microtubules remained within the lamellae of CHO fibroblasts, measured from images acquired at 10-s intervals, was significantly shorter than the average amount of time microtubules remained within lamellae of PtK1 epithelial cells. Further analysis of individual microtubule behavior from images acquired at 2-s intervals reveals that microtubules in PtK1 cells undergo mul...
Microtubule Motility Analysis based on Time-Lapse Fluorescence Microscopy
arXiv: Subcellular Processes, 2019
This paper describes an investigation into part of the mechanical mechanisms underlying the formation of mitotic spindle, the cellular machinery responsible for chromosomal separation during cell division. In normal eukaryotic cells, spindles are composed of microtubule filaments that radiate outward from two centrosomes. In many transformed cells, however, centrosome number is misregulated resulting in cells with more than two centrosomes. Addressing the question of how these cells accommodate these additional structures by coalescing supernumerary centrosomes to form normal spindles will provide a powerful insight toward understanding the proliferation of cancer cells and developing new therapeutics. The process of centrosome coalescence is thought to involve motor proteins that function to slide microtubules relative to one another. Here we use in vitro motility assays combined with fluorescence microscopy to visualize, characterize and quantify microtubule-microtubule interactio...
plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics
2011
Here we introduce plusTipTracker, a Matlab-based open source software package that combines automated tracking, data analysis, and visualization tools for movies of fluorescently-labeled microtubule (MT) plus end binding proteins (+TIPs). Although +TIPs mark only phases of MT growth, the plusTipTracker software allows inference of additional MT dynamics, including phases of pause and shrinkage, by linking collinear, sequential growth tracks. The algorithm underlying the reconstruction of full MT trajectories relies on the spatially and temporally global tracking framework described in (Jaqaman et al., 2008). Post-processing of track populations yields a wealth of quantitative phenotypic information about MT network architecture that can be explored using several visualization modalities and bioinformatics tools included in plusTipTracker. Graphical user interfaces enable novice Matlab users to track thousands of MTs in minutes. In this paper we describe the algorithms used by plusTipTracker and show how the package can be used to study regional differences in the relative proportion of MT subpopulations within a single cell. The strategy of grouping +TIP growth tracks for the analysis of MT dynamics has been introduced before (Matov et al., 2010). The numerical methods and analytical functionality incorporated in plusTipTracker substantially advance this previous work in terms of flexibility and robustness. To illustrate the enhanced performance of the new software we thus compare computer-assembled +TIP-marked trajectories to manually-traced MT trajectories from the same movie used in (Matov et al., 2010).
Peeping in on the cytoskeleton: light microscopy approaches to actin and microtubule organization
The actin and microtubule cytoskeletons consist of a dynamic network of polymer filaments that enable a variety of fundamental cellular processes, including cell shape, cell motility, chromosome separation and cytokinesis, synaptic plasticity, organelle distribution and intracellular transport and trafficking. Regional organization and remodelling of these filaments form the basis of their diverse critical functions and our ability to visualize these events with high spatiotemporal resolution is a powerful approach to uncover the cellular logic of cytoskeleton function. In recent years, a range of light microscopy techniques along with novel labelling and analytical tools has become available which allows unprecedented access to the cytoskeletal organization and dynamics. This article summarizes accessible technology platforms and strategies that allow one to peep into the inner life of these polymer systems.