Microtubule Assembly In Cells Research Articles

The active Hsc70/tau complex can be exploited to enhance tau turnover without damaging microtubule dynamics.

The pathological accumulation of abnormally hyperphosphorylated and aggregated tau, a neuronal microtubule (MT)-associated protein that functions to maintain MT stability, is implicated in a number of hereditary and sporadic neurodegenerative diseases including frontotemporal dementia and Alzheimer's disease. Targeting tau for the treatment of these diseases is an area of intense interest and toward that end, modulation of cellular molecular chaperones is a potential therapeutic target. In particular, the constitutive Hsp70 isoform, Hsc70, seems highly interconnected with tau, preserving tau protein levels and synergizing with it to assemble MTs. But the relationship between tau and Hsc70, as well as the impact of this interaction in neurons and its therapeutic implications remain unknown. Using a human dominant negative Hsc70 that resembles isoform selective inhibition of this important chaperone, we found for the first time that Hsc70 activity is required to stimulate MT assembly in cells and brain. However, surprisingly, active Hsc70 also requires active tau to regulate MT assembly in vivo, suggesting that tau acts in some ways as a co-chaperone for Hsc70 to coordinate MT assembly. This was despite tau binding to Hsc70 as substrate, as determined biochemically. Moreover, we show that while chronic Hsc70 inhibition damaged MT dynamics, intermittent treatment with a small molecule Hsp70 inhibitor lowered tau in brain tissue without disrupting MT integrity. Thus, in tauopathies, where MT injury would be detrimental to neurons, the unique relationship of tau with the Hsc70 machinery can be exploited to deplete tau levels without damaging MT networks.

Molecular Dynamics Study of the Effect of Polyamine on Microtubule Conformations

Microtubules (MTs) play important roles in cell division, intracellular protein transport, and cell structure and movement in eukaryotic cells. MTs are hollow supramolecular polymers made of alpha and beta tubulin heterodimers. Microtubule assembly in cells require a proper orientation of tubulin molecules to the MTs end. Experiments have shown that polyamines such as spermine can facilitate the diffusion of tubulin dimers in the process of nuclei growth or MT growth. The mechanism of this aggregation is the bridging interaction of polyamines with highly negative C-terminal tail of tubulin surface which helps tubulin together. Recent experiments also show that with a high concentration of spermine, bundles of taxol-stabilized MTs undergo a shape transformation to bundles of inverted tubulin tubules. However, the molecular mechanism underlying the transformation is unclear. We performed all atom molecular dynamics simulation of tubulin clusters in presence of spermine in an explicit water solvent to study the structural dynamics of alpha/beta tubulin and microtubule protofilament. We discuss our results on the interaction between spermine and tubulin proteins and the effect of spermine on conformational dynamics of MT protofilaments.

Visualization and Analysis of Microtubule Dynamics Using Dual Color-Coded Display of Plus-End Labels

Investigating spatial and temporal control of microtubule dynamics in live cells is critical to understanding cell morphogenesis in development and disease. Tracking fluorescently labeled plus-end-tracking proteins over time has become a widely used method to study microtubule assembly. Here, we report a complementary approach that uses only two images of these labels to visualize and analyze microtubule dynamics at any given time. Using a simple color-coding scheme, labeled plus-ends from two sequential images are pseudocolored with different colors and then merged to display color-coded ends. Based on object recognition algorithms, these colored ends can be identified and segregated into dynamic groups corresponding to four events, including growth, rescue, catastrophe, and pause. Further analysis yields not only their spatial distribution throughout the cell but also provides measurements such as growth rate and direction for each labeled end. We have validated the method by comparing our results with ground-truth data derived from manual analysis as well as with data obtained using the tracking method. In addition, we have confirmed color-coded representation of different dynamic events by analyzing their history and fate. Finally, we have demonstrated the use of the method to investigate microtubule assembly in cells and provided guidance in selecting optimal image acquisition conditions. Thus, this simple computer vision method offers a unique and quantitative approach to study spatial regulation of microtubule dynamics in cells.

Radiosensitizing effect of epothilone B on human epithelial cancer cells

A combined modality treatment employing radiation and chemotherapy plays a central role in the management of solid tumors. In our study, we examined the cytotoxic and radiosensitive effect of the microtubule stabilizer epothiloneB on two human epithelial tumor cell lines in vitro and its influence on the microtubule assembly. Cancer cells were treated with epothiloneB in proliferation assays and in combination with radiation in colony-forming assays. For the analysis of ionizing radiation-induced DNA damage and the influence of the drug on its repair a γH2AX foci assay was used. To determine the effect of epothiloneB on the microtubule assembly in cells and on purified tubulin, immunofluorescence staining and tubulin polymerization assay, respectively, were conducted. EpothiloneB induced a concentration- and application-dependent antiproliferative effect on the cells, with IC(50)values in the low nanomolar range. Colony forming assays showed a synergistic radiosensitive effect on both cell lines which was dependent on incubation time and applied concentration of epothiloneB. The γH2AX assays demonstrated that ionizing radiation combined with the drug resulted in a concentration-dependent increase in the number of double-strand breaks and suggested a reduction in DNA repair capacity. EpothiloneB produced enhanced microtubule bundling and abnormal spindle formation as revealed by immunofluorescence microscopy and caused microtubule formation from purified tubulin. The results of this study showed that epothiloneB displays cytotoxic antitumor activity at low nanomolar concentrations and also enhances the radiation response in the tumor cells tested; this may be induced by a reduced DNA repair capacity triggered by epothiloneB. It was also demonstrated that epothiloneB in fact targets microtubules in a more effective manner than paclitaxel.

A central role for polyamines in microtubule assembly in cells

Owing to preferential electrostatic adsorption of multivalent cations on highly anionic surfaces, natural multivalent polyamines and especially quadrivalent spermine can be considered as potential regulators of the complex dynamical properties of anionic MTs (microtubules). Indeed, the C-terminal tails of tubulin display many negative residues in a row which should enable the formation of a correlated liquid-like phase of multivalent counterions on its surface. Although it is known that polyamine counterions promote MT assembly in vitro, little is known about the relevance of this interaction in vivo. In the present study, we have explored the relationship between polyamine levels and MT assembly in HeLa and epithelial NRK (normal rat kidney) cells using DFMO (alpha-difluoromethylornithine), an irreversible inhibitor of ornithine decarboxylase, and APCHA [N-(3-aminopropyl)-N-cyclohexylamine], a spermine synthase inhibitor. Under conditions of intracellular polyamine depletion, the MT network is clearly disrupted and the MT mass decreases. Addition of spermine to polyamine-depleted cells reverses this phenotype and rapidly promotes the extensions of the MT network. Finally, we show that polyamine levels modulate the coating of MTs with MAP4 (MT-associated protein 4), an MT-stabilizing protein, and the spatial distribution of EB1 (end-binding protein 1), an MT plus-end-binding protein. In addition, polyamines favour the formation of gap junctions in NRK cells, a process which requires MT extensions at the cell periphery. The present study provides a basis for a better understanding of the role played by polyamines in MT assembly and establishes polyamine metabolism as a potential cellular target for modulating MT functions.

Doublecortin-like Kinase Controls Neurogenesis by Regulating Mitotic Spindles and M Phase Progression

The mechanisms controlling neurogenesis during brain development remain relatively unknown. Through a differential protein screen with developmental versus mature neural tissues, we identified a group of developmentally enriched microtubule-associated proteins (MAPs) including doublecortin-like kinase (DCLK), a protein that shares high homology with doublecortin (DCX). DCLK, but not DCX, is highly expressed in regions of active neurogenesis in the neocortex and cerebellum. Through a dynein-dependent mechanism, DCLK regulates the formation of bipolar mitotic spindles and the proper transition from prometaphase to metaphase during mitosis. In cultured cortical neural progenitors, DCLK RNAi Lentivirus disrupts the structure of mitotic spindles and the progression of M phase, causing an increase of cell-cycle exit index and an ectopic commitment to a neuronal fate. Furthermore, both DCLK gain and loss of function in vivo specifically promote a neuronal identity in neural progenitors. These data provide evidence that DCLK controls mitotic division by regulating spindle formation and also determines the fate of neural progenitors during cortical neurogenesis.

Effects of docetaxel on antigen presentation-related functions of human monocyte-derived dendritic cells

Docetaxel (TXT) is a unique chemotherapeutic agent that has been approved for treating various types of malignancies. TXT stabilizes microtubule assembly in cells and causes various dysfunctions of microtubule-dependent cellular events. Patients with advanced malignancies are beginning to receive TXT in combination with immunotherapy; however, the influence of TXT at clinically achievable serum concentrations (less than 10(-6) M) on antigen presentation-related functions of human monocyte-derived dendritic cells (Mo-DCs) remains unclear. Immature Mo-DCs (imMo-DCs) were generated from peripheral blood monocytes with interleukin-4 and granulocyte-macrophage colony-stimulating factor in vitro. Mature Mo-DCs (mMo-DCs) were induced from imMo-DCs with tumor necrosis factor-alpha and prostaglandin E(2). TXT at concentrations lower than 10(-7) M did not significantly affect cellular viability, phagocytosis, or expression of antigen presentation-related molecules of Mo-DCs. In contrast, TXT at concentrations lower than 10(-9) M significantly suppressed directional motility of imMo-DCs toward MIP-1alpha and of mMo-DCs toward MIP-3beta. However, TXT had no effect on either CCR1 expression by imMo-DCs or CCR7 expression by mMo-DCs. No gross changes in the microtubule skeleton were evident by immunofluorescence microscopy after treatment with TXT at less than 10(-8) M. However, reduced numbers of imMo-DCs with podosomes localized primarily in one cell region were observed. The present results indicate that different concentrations of TXT influence antigen presentation-related functions differently. In particular, TXT at relatively low therapeutic doses disrupts chemotactic motility of Mo-DCs.

Effects of taxol on human neutrophils.

Taxol, a plant alkaloid, promotes and stabilizes microtubule assembly in cells and cellfree systems. In the present study, the effects of taxol on various functional, morphologic, and biochemical phenomena in human peripheral blood PMN (Hypaque-Ficoll) were examined. Taxol (10(-7) M) inhibited PMN chemotaxis stimulated by N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) or endotoxin-activated serum by more than 60%. The inhibition was not readily reversed by washing, and taxol itself was not a chemoattractant, nor is it a secretagogue. Spontaneous nondirected migration, cell spreading on a glass surface, and orientation of cell organelles in response to a chemoattractant gradient were also inhibited by taxol. Taxol (10(-5) M) decreased killing of Staphylococcus aureus, but did not alter phagocytosis of heat-killed Candida or hexose monophosphate shunt activity in resting or stimulated PMN. Ultrastructural studies showed that PMN incubated in f-met-leu-phe, taxol, or both had increased (p less than 0.001) numbers of centrosome-associated microtubules, and the microtubules of cells incubated in taxol with or without f-met-leu-phe were organized into bundles. Taxol (10(-5) M) markedly inhibited post-translational tyrosinolation of alpha-chains of tubulin in both resting and f-met-leu-phe-stimulated PMN. The data indicate that taxol inhibits PMN locomotion and bacterial killing, supporting a role for microtubules in these processes. The ultrastructural and biochemical data also support the view that taxol mediates its effects on PMN by its effect on microtubules.

A model for the microtubule organizing activity of the centrosomes and kinetochores in mammalian cells

In this report I review shortly recent evidence on the role of centrosomes and kinetochores in organized microtubule assembly in cells. I arrive at the conclusion that models for these organizing centres must provide an explanation for the following observations: 1. 1. Both centrosomes and kinetochores induce microtubule assembly in their immediate vicinity at a tubulin concentration below the cytoplasmic critical concentration. 2. 2. Initially, the newly assembled microtubules are not necessarily anchored to the MTOC's. 3. 3. The assembly initiating and microtubule stabilizing activity of the MTOC's is abrogated by lowering the critical tubulin concentration in the cytoplasm. 4. 4. Microtubules attach to the centrosomes with their minus end and to the kinetochores with their plus end. 5. 5. Interactions between centrosomes and kinetochores or microtubules derived from them are important in guiding microtubule elongation and stabilizing kinetically unfavored microtubule sets (kinetochore microtubules). Models that are based on the presence of seeds or templates in the MTOC's do not predict observations 2 and 3. Models which conceive MTOC's as sites where microtubules are anchored at their minus end which is consequently capped do not predict observations 3 and 4. We propose a model that explains all the observations summarized above. Both centrosomes and kinetochores induce assembly at low tubulin concentrations by being domains where the critical tubulin concentration is lower than elsewhere in the cytoplasm. Once formed, microtubules may become more or less securely fixed to the MTOC by their minus (centrosome) or plus end (kinetochore). Because this anchoring may occur through lateral bonds between the microtubule surface and a component of the MTOC the end can remain free to add or loose subunits. The model allows cells to build microtubule sets of different polarity and stability. Unlike the seed and minus end capping models it is compatible with mechanisms of intracellular motility based on microtubule treadmilling.

Taxol induces the assembly of free microtubules in living cells and blocks the organizing capacity of the centrosomes and kinetochores.

Taxol, a potent promoter of microtubule polymerization in vitro, induces massive assembly of free microtubules in cultured cells as visualized by immunocytochemistry and electron microscopy. The centrosomes and kinetochores largely lost their capacity to organize microtubule assembly, as became evident by the disappearance of the cytoplasmic microtubule complex and the mitotic spindle. The taxol-induced microtubules were partially resistant to nocodazole, an inhibitor of tubulin polymerization. Moreover, taxol induced microtubule assembly in cells pretreated with nocodazole. Increasing the ratio of nocodazole to taxol restored the ability of the centrosomes and kinetochores to specifically induce microtubule assembly in their immediate vicinity. The data suggest that taxol lowers the critical tubulin concentration in vivo as well as in vitro and that the organizing capacity of the microtubule-organizing centers depends on the cytoplasmic polymerization threshold.