Perturbation Of Microtubules Research Articles

Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint.

Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1Δ or mad2Δ strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation.

Single‐molecule imaging reveals Tau trapping at nanometer‐sized dynamic hot spots near the plasma membrane that persists after microtubule perturbation and cholesterol depletion

Accumulation of aggregates of the microtubule‐binding protein Tau is a pathological hallmark of Alzheimer's disease. While Tau is thought to primarily associate with microtubules, it also interacts with and localizes to the plasma membrane. However, little is known about how Tau behaves and organizes at the plasma membrane of live cells. Using quantitative, single‐molecule imaging, we show that Tau exhibits spatial and kinetic heterogeneity near the plasma membrane of live cells, resulting in the formation of nanometer‐sized hot spots. The hot spots lasted tens of seconds, much longer than the short dwell time (∼ 40 ms) of Tau on microtubules. Pharmacological and biochemical disruption of Tau/microtubule interactions did not prevent hot spot formation, suggesting that these are different from the reported Tau condensation on microtubules. Although cholesterol removal has been shown to reduce Tau pathology, its acute depletion did not affect Tau hot spot dynamics. Our study identifies an intrinsic dynamic property of Tau near the plasma membrane that may facilitate the formation of assembly sites for Tau to assume its physiological and pathological functions.

Factor quinolinone inhibitors disrupt spindles and multiple LSF (TFCP2)-protein interactions in mitosis, including with microtubule-associated proteins.

Factor quinolinone inhibitors (FQIs), a first-in-class set of small molecule inhibitors targeted to the transcription factor LSF (TFCP2), exhibit promising cancer chemotherapeutic properties. FQI1, the initial lead compound identified, unexpectedly induced a concentration-dependent delay in mitotic progression. Here, we show that FQI1 can rapidly and reversibly lead to mitotic arrest, even when added directly to mitotic cells, implying that FQI1-mediated mitotic defects are not transcriptionally based. Furthermore, treatment with FQIs resulted in a striking, concentration-dependent diminishment of spindle microtubules, accompanied by a concentration-dependent increase in multi-aster formation. Aberrant γ-tubulin localization was also observed. These phenotypes suggest that perturbation of spindle microtubules is the primary event leading to the mitotic delays upon FQI1 treatment. Previously, FQIs were shown to specifically inhibit not only LSF DNA-binding activity, which requires LSF oligomerization to tetramers, but also other specific LSF-protein interactions. Other transcription factors participate in mitosis through non-transcriptional means, and we recently reported that LSF directly binds α-tubulin and is present in purified cellular tubulin preparations. Consistent with a microtubule role for LSF, here we show that LSF enhanced the rate of tubulin polymerization in vitro, and FQI1 inhibited such polymerization. To probe whether the FQI1-mediated spindle abnormalities could result from inhibition of mitotic LSF-protein interactions, mass spectrometry was performed using as bait an inducible, tagged form of LSF that is biotinylated by endogenous enzymes. The global proteomics analysis yielded expected associations for a transcription factor, notably with RNA processing machinery, but also to nontranscriptional components. In particular, and consistent with spindle disruption due to FQI treatment, mitotic, FQI1-sensitive interactions were identified between the biotinylated LSF and microtubule-associated proteins that regulate spindle assembly, positioning, and dynamics, as well as centrosome-associated proteins. Probing the mitotic LSF interactome using small molecule inhibitors therefore supported a non-transcriptional role for LSF in mediating progression through mitosis.

P301L tau mutation leads to alterations of cell cycle, DNA damage response and apoptosis: Evidence for a role of tau in cancer

Tau is a microtubule-associated protein, coded by the MAPT gene, which regulates microtubule (MT) polymerization and dynamics. Due to its key role in neurons, it is a major player in neurodegenerative diseases known as “tauopathies“. Since tau has emerged as a multitasking protein with a role in genome stability, it may act both in neurodegeneration and cancer. After demonstrating that tau can be considered as a risk factor for cancer, here we explored the mechanisms linking mutated tau to dysregulation of cancer-relevant processes, by employing lymphoblastoid cell lines (LCL) from patients affected by genetic tauopathy carrying the MAPT P301L mutation and healthy controls (wild-type, wt). In mutated LCL, we found reduced sensitivity to MT perturbation, along with decreased G2/M accumulation and cyclin B1 levels. Furthermore, mutated LCL displayed lower levels of phospho-Chk1 and phospho-Chk2 following hydrogen peroxide-induced oxidative stress, indicating a poorly effective DNA damage checkpoint, as well as reduced basal levels of p53. Such cells also exhibited lower levels of Bax and cleaved caspase-3, and increased levels of Cdc25A, upon oxidative stress, accounting for diminished apoptosis.Overall, these findings point to tau as a key player in biological pathways relevant for cancer, as evidenced by the differential response of mutated and wt cells to MT and DNA perturbation. The modulation of p53 is intriguing given its function as guardian of the genome.

Primary acute lymphoblastic leukemia cells are susceptible to microtubule depolymerization in G1 and M phases through distinct cell death pathways

Microtubule targeting agents (MTAs) are widely used cancer chemotherapeutics which conventionally exert their effects during mitosis, leading to mitotic or postmitotic death. However, accumulating evidence suggests that MTAs can also generate death signals during interphase, which may represent a key mechanism in the clinical setting. We reported previously that vincristine and other microtubule destabilizers induce death not only in M phase but also in G1 phase in primary acute lymphoblastic leukemia cells. Here, we sought to investigate and compare the pathways responsible for phase-specific cell death. Primary acute lymphoblastic leukemia cells were subjected to centrifugal elutriation, and cell populations enriched in G1 phase (97%) or G2/M phases (80%) were obtained and treated with vincristine. We found death of M phase cells was associated with established features of mitochondrial-mediated apoptosis, including Bax activation, loss of mitochondrial transmembrane potential, caspase-3 activation, and nucleosomal DNA fragmentation. In contrast, death of G1 phase cells was not associated with pronounced Bax or caspase-3 activation but was associated with loss of mitochondrial transmembrane potential, parylation, nuclear translocation of apoptosis-inducing factor and endonuclease G, and supra-nucleosomal DNA fragmentation, which was enhanced by inhibition of autophagy. The results indicate that microtubule depolymerization induces distinct cell death pathways depending on during which phase of the cell cycle microtubule perturbation occurs. The observation that a specific type of drug can enter a single cell type and induce two different modes of death is novel and intriguing. These findings provide a basis for advancing knowledge of clinical mechanisms of MTAs.

BP-M345, a New Diarylpentanoid with Promising Antimitotic Activity.

Previously, we reported the in vitro growth inhibitory effect of diarylpentanoid BP-M345 on human cancer cells. Nevertheless, at that time, the cellular mechanism through which BP-M345 exerts its growth inhibitory effect remained to be explored. In the present work, we report its mechanism of action on cancer cells. The compound exhibits a potent tumor growth inhibitory activity with high selectivity index. Mechanistically, it induces perturbation of the spindles through microtubule instability. As a consequence, treated cells exhibit irreversible defects in chromosome congression during mitosis, which induce a prolonged spindle assembly checkpoint-dependent mitotic arrest, followed by massive apoptosis, as revealed by live cell imaging. Collectively, the results indicate that the diarylpentanoid BP-M345 exerts its antiproliferative activity by inhibiting mitosis through microtubule perturbation and causing cancer cell death, thereby highlighting its potential as antitumor agent.

Tubulin mRNA stability is sensitive to change in microtubule dynamics caused by multiple physiological and toxic cues.

The localization, mass, and dynamics of microtubules are important in many processes. Cells may actively monitor the state of their microtubules and respond to perturbation, but how this occurs outside mitosis is poorly understood. We used gene-expression analysis in quiescent cells to analyze responses to subtle and strong perturbation of microtubules. Genes encoding α-, β, and γ-tubulins (TUBAs, TUBBs, and TUBGs), but not δ- or ε-tubulins (TUBDs or TUBEs), exhibited the strongest differential expression response to microtubule-stabilizing versus destabilizing drugs. Quantitative PCR of exon versus intron sequences confirmed that these changes were caused by regulation of tubulin mRNA stability and not transcription. Using tubulin mRNA stability as a signature to query the Gene Expression Omnibus (GEO) database, we find that tubulin genes respond to toxins known to damage microtubules. Importantly, we find many other experimental perturbations, including multiple signaling and metabolic inputs that trigger tubulin differential expression, suggesting their novel, to our knowledge, role in the regulation of the microtubule cytoskeleton. Mechanistic follow-up confirms that one important physiological signal, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activity, indeed regulates tubulin mRNA stability via changes in microtubule dynamics. We propose that tubulin gene expression is regulated as part of many coordinated biological responses, with wide implications in physiology and toxicology. Furthermore, we present a new way to discover microtubule regulation using transcriptomics.

Single‐cell profiling screen identifies microtubule‐dependent reduction of variability in signaling

Populations of isogenic cells often respond coherently to signals, despite differences in protein abundance and cell state. Previously, we uncovered processes in the Saccharomyces cerevisiae pheromone response system (PRS) that reduced cell‐to‐cell variability in signal strength and cellular response. Here, we screened 1,141 non‐essential genes to identify 50 “variability genes”. Most had distinct, separable effects on strength and variability of the PRS, defining these quantities as genetically distinct “axes” of system behavior. Three genes affected cytoplasmic microtubule function: BIM1, GIM2, and GIM4. We used genetic and chemical perturbations to show that, without microtubules, PRS output is reduced but variability is unaffected, while, when microtubules are present but their function is perturbed, output is sometimes lowered, but its variability is always high. The increased variability caused by microtubule perturbations required the PRS MAP kinase Fus3 and a process at or upstream of Ste5, the membrane‐localized scaffold to which Fus3 must bind to be activated. Visualization of Ste5 localization dynamics demonstrated that perturbing microtubules destabilized Ste5 at the membrane signaling site. The fact that such microtubule perturbations cause aberrant fate and polarity decisions in mammals suggests that microtubule‐dependent signal stabilization might also operate throughout metazoans.

Antiproliferative and antimetastatic action of quercetin on A549 non-small cell lung cancer cells through its effect on the cytoskeleton

To our knowledge, this study is the first to investigate the effect of the dietary flavonoid quercetin on the main cytoskeletal elements, namely microfilaments, microtubules and vimentin intermediate filaments, as well as cytoskeleton-driven processes in A549 non-small cell lung cancer cells. The methyl-thiazol-diphenyl-tetrazolium assay, annexin V/propidium iodide test, electron microscopic examination, cell cycle analysis based on DNA content, real-time PCR assays, in vitro scratch wound-healing assay, fluorescence staining of F-actin, β-tubulin and vimentin were performed to assess the effects of quercetin on A549 cells. Our results showed that quercetin triggered BCL2/BAX-mediated apoptosis, as well as necrosis and mitotic catastrophe, and inhibited the migratory potential of A549 cells. The disassembling effect of quercetin on microfilaments, microtubules and vimentin filaments along with its inhibitory impact on vimentin and N-cadherin expression might account for the decreased migration of A549 cells in response to quercetin treatment. We also suggest that the possible mechanism underlying quercetin-induced mitotic catastrophe involves the perturbation of mitotic microtubules leading to monopolar spindle formation, and, consequently, to the failure of cytokinesis. We further propose that cytokinesis failure could also be a result of the depletion of actin filaments by quercetin. These findings are important to our further understanding of the detailed mechanism of the antitumor activity of quercetin and render this flavonoid a potentially useful candidate for combination therapy with conventional antimicrotubule drugs, nucleic acid-directed agents or novel cytoskeletal-directed agents.

Independent variability of microtubule perturbations associated with dystrophinopathy.

Absence of the protein dystrophin causes Duchenne muscular dystrophy. Dystrophin directly binds to microtubules in vitro, and its absence in vivo correlates with disorganization of the subsarcolemmal microtubule lattice, increased detyrosination of α-tubulin, and altered redox signaling. We previously demonstrated that the dystrophin homologue utrophin neither binds microtubules in vitro nor rescues microtubule lattice organization when overexpressed in muscles of dystrophin-deficient mdx mice. Here, we fine-mapped the dystrophin domain necessary for microtubule binding to spectrin-like repeats 20–22. We show that transgenic mdx mice expressing a full-length dystrophin/utrophin chimera completely lacking microtubule binding activity are surprisingly rescued for all measured dystrophic phenotypes, including full restoration of microtubule lattice organization. Conversely, despite the presence of dystrophin at the sarcolemma, β-sarcoglycan-deficient skeletal muscle presents with a disorganized and densified microtubule lattice. Finally, we show that the levels of α-tubulin detyrosination remain significantly elevated to that of mdx levels in transgenic mdx mice expressing nearly full-length dystrophin. Our results demonstrate that the microtubule-associated perturbations of mdx muscle are distinct, separable, and can vary independently from other parameters previously ascribed to dystrophin deficiency.

Cell Cycle-Dependent Mechanisms Underlie Vincristine-Induced Death of Primary Acute Lymphoblastic Leukemia Cells.

Microtubule-targeting agents (MTA), such as the taxanes and vinca alkaloids, are used to treat a variety of cancers due to their ability to perturb microtubule dynamics. In cell culture, MTAs exert their anticancer effects primarily by causing mitotic arrest and cell death. However, accumulating indirect evidence suggests that MTAs may exert their cytotoxicity in human tumors by interfering with interphase microtubules. In this study, we sought to develop and characterize an experimental system in which to test the hypothesis that MTAs induce cell death during interphase. Primary adult acute lymphoblastic leukemia (ALL) cells treated with vincristine only weakly exhibited colocalization between mitotic and apoptotic markers and major characteristics of mitotic death, such as an increase in cells with 4N DNA content before the appearance of cells with <2N DNA content, suggesting a mixed response. Therefore, we separated ALL cells into distinct phases of the cell cycle by centrifugal elutriation, labeled cells with 5-ethynyl-2'-deoxyuridine (EdU), and then treated each population with vincristine. Cells isolated during G1 underwent cell death without evidence of EdU uptake, indicating that the cytotoxic effects of vincristine took place during G1 Conversely, cells isolated during S or G2-M phases underwent death following mitotic arrest. Thus, vincristine induces distinct death programs in primary ALL cells depending on cell-cycle phase, and cells in G1 are particularly susceptible to perturbation of interphase microtubules. Primary ALL cells may therefore provide a powerful model system in which to study the multimodal mechanisms underlying MTA-induced cell death. Cancer Res; 76(12); 3553-61. ©2016 AACR.

Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli.

Enteropathogenic Escherichia coli (EPEC) is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl(-)/HCO3(-) exchanger SLC26A3 [downregulated in adenoma (DRA)], contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. In the current study, Caco-2 cells were used to investigate DRA trafficking under basal conditions and in response to EPEC. Apical Cl(-)/HCO3(-) exchange activity was measured as DIDS-sensitive (125)I(-) uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 μM) increased apical Cl(-)/HCO3(-) exchange activity. Dynasore, a dynamin inhibitor, also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice treated with colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent on the EPEC virulence genes espG1 and espG2. Intriguingly, the EPEC-induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that the EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis.

Distinct Pathways of Vincristine Induced Cell Death in Primary Acute Lymphoblastic Leukemia Cells

Microtubule-targeting agents (MTAs), such as the taxanes and vinca alkaloids, are anti-cancer agents that perturb microtubule function. In cell culture, these drugs exert their cytotoxicity primarily by causing mitotic arrest and cell death. However, accumulated indirect evidence suggests that MTAs may exert their cytotoxicity in tumors by interfering with interphase microtubules. We therefore sought to develop and characterize a cell system in which MTAs induced interphase death. In primary adult acute lymphoblastic leukemia (ALL) cell cultures treated with vincristine, the major characteristics of mitotic death, which include an increase in cells with 4N DNA content prior to the appearance of cells with sub-G1 DNA content, as well as the co-existence of mitotic and apoptotic markers, were only weakly represented, suggesting a mixed response. The ALL cells were therefore separated into distinct phases of the cell cycle by centrifugal elutriation and labeled with 5-ethynyl-2'-deoxyuridine (EdU) prior to treatment with vincristine. A proportion of cells in G1 phase underwent vincristine-induced cell death without evidence of EdU staining, indicating that they had died without entering S phase and had thus died directly from G1 phase. In contrast, cells in S or G2/M phases underwent death following mitotic arrest. Thus, vincristine induces distinct, cell cycle-dictated pathways of death in primary ALL cells, with cells in G1 phase evidently susceptible to perturbation of interphase microtubules. Hence, primary ALL cells provide a powerful model system to study clinically relevant mechanisms of MTA-induced cell death. Supported by NIH CA109821

Abstract 1158: Microtubule perturbation regulates remodeling of tumor microenvironment by modulating the composition of tumor cell secretome

Abstract Microtubules (MTs) represent one of the most effective targets in cancer chemotherapy. However, MT-targeting drugs, such as the taxanes, often fail in the metastatic setting and mechanisms underlying drug resistance are poorly elucidated. An important process during tumor development and metastasis involves the dynamic remodeling of the 3D microenvironment surrounding the tumor cells which enables them to successfully proliferate and metastasize. We hypothesized that tumor cell MTs may regulate the cellular signaling process that controls tumor-mediated remodeling of the microenvironment by paracrine activation of fibroblasts. To assess the role of MT dynamics in the secretion of factors from the tumor cells that mediate communication with the microenvironment, we collected conditioned medium (CM) from untreated or paclitaxel (PTX)-pre-treated metastatic breast cancer MDA-MB-231 cells and applied it to HMF3S human mammary fibroblasts. We observed that CM from untreated 231 cells resulted in fibroblast activation as evidenced by their increased directional 3D cell motility towards a serum gradient. CM from PTX-pretreated 231 cells, at concentrations that suppress MT dynamics, but not from the PTX-resistant β-tubulin mutation 231 clone, 231K20T cells, did not result in activation of fibroblasts' 3D motility, suggesting that MT-mediated secretion of soluble factor(s) (secretome) underlies fibroblast activation. Mass-spectrometry analysis of CM derived from untreated or PTX-pretreated 231 cells identified proteins whose secretion was diminished following suppression of MT dynamics and which are involved in cell motility, cell-cell communication, or ECM remodeling, such as TGFβ, CTGF, c-Met, fibronectin, and lysyl oxidase 2. Treatment of 231-derived CM with an anti-TGFβ blocking antibody diminished HMF3S fibroblast motility in 3D, suggesting that the presence of TGFβ in the tumor cell secretome mediated the activation of tumor associated fibroblasts. To further probe the MT-dependent regulation of TGFβ secretion we showed that PTX treatment prevented trafficking of intracellular TGFβ to the cell surface in 231 cells, likely underlying its reduced secretion. Confocal reflectance and two-photon microscopic imaging revealed that CM treatment of HMF3S fibroblasts induced significant collagen remodeling comparable to treatment with exogenous TGFβ alone. Our findings point to a novel role of interphase MTs in tumor cell secretome and raise the possibility that MTs may regulate secretion of distinct soluble factors in different types of tumor cells, which then mediate the communication between tumor cell compartment and the tumor microenvironment. These novel insights have significant implications for the mechanism of action and resistance to MT inhibitors. Citation Format: Katsuhiro Kita, Andy Tran, Lewis M. Brown, Duane C. Hassane, Shawn Carey, Alexandre Matov, Cynthia A. Reinhart-King, Paraskevi Giannakakou. Microtubule perturbation regulates remodeling of tumor microenvironment by modulating the composition of tumor cell secretome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1158. doi:10.1158/1538-7445.AM2014-1158

Abstract 923: Analysis of microtubule perturbations and androgen receptor localization in circulating tumor cells from castration resistant prostate cancer patients as predictive biomarkers of clinical response to docetaxel chemotherapy

Abstract Although the recent availability of novel treatment for castration resistant prostate cancer (CRPC) has shown improvements in overall survival, CRPC is incurable and lethal disease. It has been recognized that tumor progression despite castrate levels of androgens is still associated with active signaling from the androgen receptor (AR) which affects gene transcription following its nuclear accumulation. Taxanes bind to and stabilize cellular microtubules (MTs) perturbing the fine and intricate organization of the microtubule network thus, impairing MT-based cell processes like cell division and intracellular trafficking. Our recent work showed that AR translocation from the cytoplasm to the nucleus is MT-dependent and that taxane treatment sequesters AR into the cytoplasm inhibiting the receptors transcriptional activity. While this novel mechanism of taxane activity may explain the clinical efficacy of taxanes in CRPC, clinically we still fail to understand the molecular basis of patient response or resistance to taxanes. We hypothesized that engagement of the MT-AR pathway by the taxanes can be used as read-out of effective drug-target engagement in cancer cells and can be used as biomarker predictive of taxane activity in CRPC patients. To assess the predictive role of these biomarkers, we are currently conducting a prospective clinical study in which we isolate circulating tumor cells (CTCs) from the peripheral blood of CRPC patient undergoing docetaxel treatment. We have enrolled 50 of the 80 total patients, from which we isolated CTCs before (at baseline) and during docetaxel treatment (at day 8 of first cycle of docetaxel and upon relapse) using two different approaches: the EpCAM-based immunomagnetic enrichment (CellSearch) and the ficolling technique to isolate unenriched PBMCs inclusive of CTCs. To avoid issues with leucocyte contamination we subject the EpCAM captured cells are to staining for DAPI , PSMA, CD-45, AR and Tubulin and image them by confocal microscopy. CTCs are defined as nucleated, PSMA+, CD-45- cells. The unenriched PBMC population is also subjected to the same multiplex confocal microscopy protocol. Microtubule network morphology and AR subcellular localization in then assessed in CTCs and each biomarker alone and in combination is correlated with clinical response to docetaxel treatment defined by PCWG2 recommendation. So far we have collected and analyzed baseline and cycle 1-Day 8 samples from all 50 patients and relapse samples from 31 patients. Determination of effective drug-target engagement on C1D8 may allow early detection of molecular response to treatment, or lack of molecular response, which will ultimately allow for chemotherapy customization for the individual patient. Citation Format: Shinsuke Tasaki, Matthew Sung, Alexandre Matov, Giuseppe Galletti, Elan Diamond, Neil Bander, Kathy Zhou, Scott Tagawa, David Nanus, Paraskevi Giannakakou. Analysis of microtubule perturbations and androgen receptor localization in circulating tumor cells from castration resistant prostate cancer patients as predictive biomarkers of clinical response to docetaxel chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 923. doi:10.1158/1538-7445.AM2014-923

Biochemical and Structural Insights into Microtubule Perturbation by CopN from Chlamydia pneumoniae

Although the actin network is commonly hijacked by pathogens, there are few reports of parasites targeting microtubules. The proposed member of the LcrE protein family from some Chlamydia species (e.g. pCopN from C. pneumoniae) binds tubulin and inhibits microtubule assembly in vitro. From the pCopN structure and its similarity with that of MxiC from Shigella, we definitively confirm CopN as the Chlamydia homolog of the LcrE family of bacterial proteins involved in the regulation of type III secretion. We have also investigated the molecular basis for the pCopN effect on microtubules. We show that pCopN delays microtubule nucleation and acts as a pure tubulin-sequestering protein at steady state. It targets the β subunit interface involved in the tubulin longitudinal self-association in a way that inhibits nucleotide exchange. pCopN contains three repetitions of a helical motif flanked by disordered N- and C-terminal extensions. We have identified the pCopN minimal tubulin-binding region within the second and third repeats. Together with the intriguing observation that C. trachomatis CopN does not bind tubulin, our data support the notion that, in addition to the shared function of type III secretion regulation, these proteins have evolved different functions in the host cytosol. Our results provide a mechanistic framework for understanding the C. pneumoniae CopN-specific inhibition of microtubule assembly.

Specific polar subpopulations of astral microtubules control spindle orientation and symmetric neural stem cell division.

Mitotic spindle orientation is crucial for symmetric vs asymmetric cell division and depends on astral microtubules. Here, we show that distinct subpopulations of astral microtubules exist, which have differential functions in regulating spindle orientation and division symmetry. Specifically, in polarized stem cells of developing mouse neocortex, astral microtubules reaching the apical and basal cell cortex, but not those reaching the central cell cortex, are more abundant in symmetrically than asymmetrically dividing cells and reduce spindle orientation variability. This promotes symmetric divisions by maintaining an apico-basal cleavage plane. The greater abundance of apical/basal astrals depends on a higher concentration, at the basal cell cortex, of LGN, a known spindle-cell cortex linker. Furthermore, newly developed specific microtubule perturbations that selectively decrease apical/basal astrals recapitulate the symmetric-to-asymmetric division switch and suffice to increase neurogenesis in vivo. Thus, our study identifies a novel link between cell polarity, astral microtubules, and spindle orientation in morphogenesis.

The microtubule-associated protein 1A (MAP1A) is an early molecular target of soluble Aβ-peptide.

A progressive accumulation of amyloid β-protein (Aβ) is widely recognized as a pathological hallmark of Alzheimer's disease (AD). Substantial progress has been made toward understanding the neurodegenerative cascade initiated by small soluble species of Aβ and recent evidence supports the notion that microtubule rearrangements may be proximate to neuritic degeneration and deficits in episodic declarative memory. Here, we examined primary cortical neurons for changes in markers associated with synaptic function following exposure to sublethal concentrations of non-aggregated Aβ-peptide. This data show that soluble Aβ species at a sublethal concentration induce degradation of the microtubule-associated protein 1A (MAP1A) without concurrently affecting dendritic marker MAP2 and/or the pre-synaptic marker synaptophysin. In addition, MAP1A was found to highly co-localize with the postsynaptic density-95 (PSD-95) protein, proposing that microtubule perturbations might be central for the Aβ-induced neuronal dysfunctions as PSD-95 plays a key role in synaptic plasticity. In conclusion, this study suggests that disruption of MAP1A could be a very early manifestation of Aβ-mediated synaptic dysfunction-one that presages the clinical onset of AD by years. Moreover, our data support the notion of microtubule-stabilizing agents as effective AD drugs.

Interpolar microtubules are dispensable in fission yeast meiosis II

The mitotic spindle consists of two types of microtubules. Dynamic kinetochore microtubules capture kinetochores, whereas stable interpolar microtubules serve as the structural backbone that connects the two spindle poles. Both have been believed to be indispensable for cell division in eukaryotes. Here we demonstrate that interpolar microtubules are dispensable for the second division of meiosis in fission yeast. Even when interpolar microtubules are disrupted by a microtubule-depolymerizing drug, spindle poles separate and chromosomes segregate poleward in second division of meiosis in most zygotes, producing viable spores. The forespore membrane, which encapsulates the nucleus in second division of meiosis and is guided by septins and the leading-edge proteins, is responsible for carrying out meiotic events in the absence of interpolar microtubules. Furthermore, during physiological second division of meiosis without microtubule perturbation, the forespore membrane assembly contributes structurally to spindle pole separation and nuclear division, generating sufficient force for spindle pole separation and subsequent events independently of interpolar microtubules.

Disruption of Microtubule Network Rescues Aberrant Actin Comets in Dynamin2-Depleted Cells

A large GTPase dynamin, which is required for endocytic vesicle formation, regulates the actin cytoskeleton through its interaction with cortactin. Dynamin2 mutants impair the formation of actin comets, which are induced by Listeria monocytogenes or phosphatidylinositol-4-phosphate 5-kinase. However, the role of dynamin2 in the regulation of the actin comet is still unclear. Here we show that aberrant actin comets in dynamin2-depleted cells were rescued by disrupting of microtubule networks. Depletion of dynamin2, but not cortactin, significantly reduced the length and the speed of actin comets induced by Listeria. This implies that dynamin2 may regulate the actin comet in a cortactin-independent manner. As dynamin regulates microtubules, we investigated whether perturbation of microtubules would rescue actin comet formation in dynamin2-depleted cells. Treatment with taxol or colchicine created a microtubule-free space in the cytoplasm, and made no difference between control and dynamin2 siRNA cells. This suggests that the alteration of microtubules by dynamin2 depletion reduced the length and the speed of the actin comet.