Adopted from: http://micro.magnet.fsu.edu/cells/microtubules/microtubules.html
The Microtubule Dynamics and Motility Tools are used by researchers who try to understand the mechanisms of growing and shortening dynamics of microtubules (MTs) and related regulatory mechanisms associated with MTs functioning. MTs represent major structural components of the cytoskeleton that are involved in many cellular functions, especially in mitosis, cytokinesis (cell motility) and vesicular transport. They are assembled from heterodimers made of a GTP-bound forms of a-tubulin and b-tubulin proteins (Fig. 1). Uni-directional orientation of tubulin heterodimers in MTs results in polarized nature of MTs with a-tubulin exposed ends (called (-)-ends) being relatively stable ones and b-tubulin exposed end (called (+)-ends) being more unstable. Upon MT assembly, most of tubulin-bound GTP (with exception of subunits at the very ends of MTs) is hydrolyzed into GDP. The energy released by the hydrolysis of GTP to GDP results in decreased stability of MTs and leads to growing and shortening phase transitions at the microtubule ends (called dynamic instability). In addition, at the polymer mass steady state the growing and shortening transitions at (+)-end of a microtubule give rise to net growing, while the growing and shortening transitions at the opposite (-)-end of the microtubule gives rise to net shortening. This directional growth of MTs called treadmilling or flux. MTs also have a number of microtubule-associated proteins (MAPs) associated with their surfaces and ends. MAPs main functions are to regulate the polymerization dynamics of the microtubules and to mediate the functional interactions of microtubules with other cell components. Another class of proteins associated with MTs are motor proteins (kinesins and dyneins) responsible for directional transport of cargo vesicles. Proper dynamics of MTs required for normal cell functioning while aberrant behavior of MTs can lead to cellular dysfunction and/or death. Correspondingly, dynamics of MTs as well as associated with MTs cell signaling pathways are important targets for novel anti-cancer and anti- neurodegenerative therapies.
MCF7 GFP-Tub Cells
Tubulin stained green, Pericentrin (centrosome marker) stained red and DNA stained blue.
Image acquired on Olympus DSU Confocal Microscope
Dynamics of MTs as Anti-cancer therapy target:The research is concerned with the mechanism and regulation of microtubule polymerization and the role of microtubule polymerization dynamics in cell function. Our major interest is to understand the mechanism and the regulation of microtubule polymerization dynamics in relation to chromosome movements during mitosis. We study the molecular mechanisms of action of a number of antimitotic antitumor drugs (taxol, vinblastine etc), which act by modulating the dynamics of tubulin addition and loss at microtubule ends, and use these drugs as research tools to elucidate the roles of microtubule polymerization dynamics in mitosis. We are also interested in understanding of how certain MAPs (Statmin etc) modulate and perhaps regulate microtubule polymerization dynamics.
MTs dynamics are studied in vitro at the biochemical and the cellular levels. Time-Lapse Fluorescence microscopy (TLF) used to study MTs dynamics in living cancer cells in vitro. MTs are visualized either by injection of fluorophore-labeled tubulin (MAP-free total tubulin fraction from bovine brain) or by transfection of cells with vector encoding EGFP-a-tubulin recombinant protein. Stably transfected MCF7 cell line is available and routinely used as a model for the latter type of experiments. Video-Enhanced Differential Interference Contrast microscopy (VE-DIC) is used to study the dynamics of MTs assembled of purified tubulin heterodimers in vitro.
Image of MCF7 GFP Tub cells acquired by epifluorescent microscopy on Nikon Eclipse E800, x100.
Effects of mis-regulation of Tau MAP isoforms phosphorylation on Dynamics of MTs:
It is well established that neural specific microtubule associated protein tau is crucial for the proper development and maintenance of the nervous system. Mechanistically, tau binds directly to microtubules, promotes microtubule assembly and regulates microtubule dynamics, which are crucial for cell function and viability. Abnormal tau function has been associated with numerous neurodegenerative diseases. Tau can form insoluble intracellular fibers known as neuro-fibrillary tangles (NFT), which are one of the major hallmarks of several neurodegenerative diseases, including Alzheimer’s disease.
Various transgenic mouse models implicate mis-regulation of tau phosphorylation in neuronal cell death. In Alzheimer’s disease brain, tau is highly phosphorylated, thus abnormal tau phosphorylation has been proposed as one potential cause of the disease. The specific hypothesis we are testing is that single or combinatorial tau phosporylation events might cause dysfunction of microtubules and thereby cause tau-mediated neurodegenerative abnormalities. Ongoing studies of the ability of each phosphorylation event, individually and in combination, to affect the ability of tau to influence microtubule behavior should provide valuable insights into mechanisms of normal and pathological tau action.
Specific Aims:
1. Implement novel automated detection and tracking methods to assess the abilities of various phosphorylated tau molecules to regulate microtubule dynamics and bundling, both in vitro and in cultured cells.
2. Determine if tau phosphorylation effects tau binding to microtubules.
3. Assess the effects of phosphorylation events upon its aggregation behavior.
Microtubule dynamics in vitro, image acquired using darkfield microscopy
Regulatory role of the microtubule-associated protein tau upon kinesin processivity along MTs:
Despite a strong correlation of irregularities in tau protein with neurodegenerative disease, (mutation, isoform composition shift, hyper-phosphorylation, aggregation) and in vivo experimental data demonstrating drastic impairment of axonal transport following tau over-expression, the molecular mechanisms of tau-opathy remain elusive. Additionally, our understanding of the functional consequences associated with the prominent shift in naturally occurring tau isoforms through development, by alternative mRNA splicing, is also largely incomplete. Our recent work focuses on the potential regulatory role of the microtubule-associated protein tau upon kinesin processivity along microtubules in the contexts of both neurodegenerative disease and neuronal development. In the work presented here, we utilize a familiar reconstituted in vitro system, kinesin-driven microtubule gliding, to directly investigate the relationship between tau, microtubules and kinesin, in search of qualitative functional differences between the wild type and disease-implicated tau molecules. Of the six naturally occurring tau isoforms, the first molecules assayed were 3-Repeat and 4-Repeat tau, both of the “short” splice-variant class lacking both N-terminal exons, termed here “3R0N” and “4R0N”, respectively. Previous investigations of tau protein by in vitroin vitro at low tau:tubulin stoichiometry – presumably due to steric effects conferred by its large N-terminal domain. However, all previously reported in vitro studies of tau’s effect upon kinesin motility are limited in their physiological relevance by the use of a Taxol-directed and stabilized microtubule assembly regimen, which is then followed by tau decoration. Recent cryo-electron microscopy studies utilizing gold-nanoparticle-labeled tau have demonstrated that pre-assembly by Taxol causes tau to bind solely to the outside of MTs, whereas the Taxol-free assembly of MTs, instead directed by tau itself, results in tau binding to an internal site as well as to the MT surface. Recent biochemical studies have also shown that tau-binding can be characterized by two kinetic states- one with rapid dissociation from MTs and another high-affinity state that cannot be competed off of the MT following assembly. motility assay are limited to 4R-Long tau (4R2N), and demonstrate little effect of tau upon kinesin processivity within a physiologically relevant concentration range, as opposed to MAP-2c which severely inhibits kinesin binding and processivity
Our aim in these studies was to recreate a physiologically meaningful system where tau is free to interact with and guide the assembly of MTs as it would in vivo, allowing for the possibility that the two tau sites may possess different functional roles and that kinesin may act as a molecular reporter to bring these physiologically significant differences – as well as differences between wild-type isoforms, mutants, and modification states - to light.
Custom-designed, semi-automated tracking software was developed and used to locate entire MT-bodies through an image time series, acquired by epifluorescent microscopy, in order to simultaneously collect gliding velocity, trajectory, MT-body curvature and MT length data. By applying sophisticated feature-detection algorithms to track MTs through each image time-series, in a semi-automated manner, we were able to track movement of entire-MT-bodies while simultaneously monitoring accuracy – essentially managing a massive amount of spatiotemporal data in seconds, whereas this type of analysis would simply be impractical by any pre-existing manual technique. Simultaneous monitoring of MT length was of particular importance in the analysis gliding velocity because of the potential for MT dynamic instability in our drug-free, non-stabilized conditions, where length changes could have influenced velocity measurements.
Single Frame of Kinesin-Driven Gliding Assay:Microtubules Assembled with 3R0N Tau,Imaged by Epifluorescent Microscopy, 100x.
MT-Gliding Trajectory Plot:4R0N-Assembled versus 4R0N-Decorated MTs.Gliding paths through time series centered at origin.
Adopted from: http://www.cytochemistry.net/Cell-biology/microtubule_structure.htm
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