Detection of single fluorescent microtubules and methods for determining their dynamics in living cells (original) (raw)
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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...
Cell biology international reports, 1981
Indirect immunofluorescence with rhodamine labelled antibodies and fluoresceinated colchicine (FC) are used to simultaneously localize microtubules and soluble tubulin in cultured ovarian granulosa cells. FC labelled tubulin is most concentrated in regions of the cell occupied by antitubulin stained microtubule bundles. Pretreatment of granulosa cells with colchicine results in a central accumulation of FC and antibody labelled tubulin that coincides with the disposition of 10-nm filament cables. In contrast, the microtubule disrupting agent nocodazole produces a diffuse tubulin distribution as detected with both FC and antibody probes. Taxol treatment, which enhances microtubule assembly, results in a striking concentration of microtubule bundles associated with the nucleus that avidly bind FC. These results suggest that disassembled tubulin is preferentially associated with cytoplasmic microtubules and possibly other formed elements of the cytoskeleton.
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.
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...
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.
Visualization of cell microtubules in their native state
Biology of the Cell, 2007
Background information. Over the past decades, cryo-electron microscopy of vitrified specimens has yielded a detailed understanding of the tubulin and microtubule structures of samples reassembled in vitro from purified components. However, our knowledge of microtubule structure in vivo remains limited by the chemical treatments commonly used to observe cellular architecture using electron microscopy.
Biophysical Journal, 2014
The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with~9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of~20 mm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells.