Microtubules and microscopes: how the development of light microscopic imaging technologies has contributed to discoveries about microtubule dynamics in living cells.
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ABSTRACT:
SUBMITTER: Waterman-Storer CM
PROVIDER: S-EPMC25623 | biostudies-literature | 1998 Dec
REPOSITORIES: biostudies-literature
Publications
Microtubules and microscopes: how the development of light microscopic imaging technologies has contributed to discoveries about microtubule dynamics in living cells.
Molecular biology of the cell 19981201 12
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Project description:Microtubules (MTs) are cytoskeletal polymers composed of repeating subunits of tubulin that are ubiquitously expressed in eukaryotic cells. They undergo a stochastic process of polymerization and depolymerization from their plus ends termed dynamic instability. MT dynamics is an ongoing process in all cell types and has been the target for the development of several useful anticancer drugs, which compromise rapidly dividing cells. Recent studies also suggest that MT dynamics may be particularly important in neurons, which develop a highly polarized morphology, consisting of a single axon and multiple dendrites that persist throughout adulthood. MTs are especially dynamic in dendrites and have recently been shown to polymerize directly into dendritic spines, the postsynaptic compartment of excitatory neurons in the CNS. These transient polymerization events into dendritic spines have been demonstrated to play important roles in synaptic plasticity in cultured neurons. Recent studies also suggest that MT dynamics in the adult brain function in the essential process of learning and memory and may be compromised in degenerative diseases, such as Alzheimer's disease. This raises the possibility of targeting MT dynamics in the design of new therapeutic agents.
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Project description:The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTP-rhodamine-tubulin revealed tubulin incorporation at 7.9 (+/- 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (+/- 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP-expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states.
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Project description:Maytansine is a potent microtubule-targeted compound that induces mitotic arrest and kills tumor cells at subnanomolar concentrations. However, its side effects and lack of tumor specificity have prevented successful clinical use. Recently, antibody-conjugated maytansine derivatives have been developed to overcome these drawbacks. Several conjugates show promising early clinical results. We evaluated the effects on microtubule polymerization and dynamic instability of maytansine and two cellular metabolites (S-methyl-DM1 and S-methyl-DM4) of antibody-maytansinoid conjugates that are potent in cells at picomolar levels and that are active in tumor-bearing mice. Although S-methyl-DM1 and S-methyl-DM4 inhibited polymerization more weakly than maytansine, at 100 nmol/L they suppressed dynamic instability more strongly than maytansine (by 84% and 73%, respectively, compared with 45% for maytansine). However, unlike maytansine, S-methyl-DM1 and S-methyl-DM4 induced tubulin aggregates detectable by electron microscopy at concentrations ≥2 μmol/L, with S-methyl-DM4 showing more extensive aggregate formation than S-methyl-DM1. Both maytansine and S-methyl-DM1 bound to tubulin with similar K(D) values (0.86 ± 0.2 and 0.93 ± 0.2 μmol/L, respectively). Tritiated S-methyl-DM1 bound to 37 high-affinity sites per microtubule (K(D), 0.1 ± 0.05 μmol/L). Thus, S-methyl-DM1 binds to high-affinity sites on microtubules 20-fold more strongly than vinblastine. The high-affinity binding is likely at microtubule ends and is responsible for suppression of microtubule dynamic instability. Also, at higher concentrations, S-methyl-DM1 showed low-affinity binding either to a larger number of sites on microtubules or to sedimentable tubulin aggregates. Overall, the maytansine derivatives that result from cellular metabolism of the antibody conjugates are themselves potent microtubule poisons, interacting with microtubules as effectively as or more effectively than the parent molecule.
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Project description:Class VI β-tubulin (β6) is the most divergent tubulin produced in mammals and is found only in platelets and mature megakaryocytes. To determine how this unique tubulin isotype affects microtubule assembly and organization, we expressed the cDNA in tissue culture cells under the control of a tetracycline regulated promoter. The β6 coassembled with other endogenous β-tubulin isotypes into a normal microtubule array; but once the cells entered mitosis it caused extensive fragmentation of the microtubules, disrupted the formation of the spindle apparatus, and allowed entry into G1 phase without cytokinesis to produce large multinucleated cells. The microtubule fragments persisted into subsequent cell cycles and accumulated around the membrane in a marginal band-like appearance. The persistence of the fragments could be traced to a pronounced suppression of microtubule dynamic instability. Impairment of centrosomal nucleation also contributed to the loss of a normal microtubule cytoskeleton. Incorporation of β6 allowed microtubules to resist the effects of colcemid and maytansine, but not vinblastine or paclitaxel; however, cellular resistance to colcemid or maytansine did not occur because expression of β6 prevented cell division. The results indicate that many of the morphological features of megakaryocyte differentiation can be recapitulated in non-hematopoietic cells by β6 expression and they provide a mechanistic basis for understanding these changes.
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Project description:Although microtubules in plant cells have been extensively studied, the mechanisms that regulate the spatial organization of microtubules are poorly understood. We hypothesize that the interaction between microtubules and cytoplasmic flow plays an important role in the assembly and orientation of microtubules. To test this hypothesis, we developed a new computational modeling framework for microtubules based on theory and methods from the fluid-structure interaction. We employed the immersed boundary method to track the movement of microtubules in cytoplasmic flow. We also incorporated details of the encounter dynamics when two microtubules collide with each other. We verified our computational model through several numerical tests before applying it to the simulation of the microtubule-cytoplasm interaction in a growing plant cell. Our computational investigation demonstrated that microtubules are primarily oriented in the direction orthogonal to the axis of cell elongation. We validated the simulation results through a comparison with the measurement from laboratory experiments. We found that our computational model, with further calibration, was capable of generating microtubule orientation patterns that were qualitatively and quantitatively consistent with the experimental results. The computational model proposed in this study can be naturally extended to many other cellular systems that involve the interaction between microstructures and the intracellular fluid.
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Project description:Compounds that disrupt microtubule dynamics, such as colchicine, paclitaxel, or Vinca alkaloids, have been broadly used in biological studies and have found application in clinical anticancer medications. However, their main disadvantage is the lack of specificity towards cancerous cells, leading to severe side effects. In this paper, we report the first synthesis of 12 new visible light photoswitchable colchicine-based microtubule inhibitors AzoCols. Among the obtained compounds, two photoswitches showed light-dependent cytotoxicity in cancerous cell lines (HCT116 and MCF-7). The most promising compound displayed a nearly twofold increase in potency. Moreover, dissimilar inhibition of purified tubulin polymerisation in cell-free assay and light-dependent disruption of microtubule organisation visualised by immunofluorescence imaging sheds light on the mechanism of action as microtubule photoswitchable destabilisers. The presented results provide a foundation towards the synthesis and development of a novel class of photoswitchable colchicine-based microtubule polymerisation inhibitors.
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