Changes In Microtubule Dynamics Research Articles

Minimal Mechanisms of Microtubule Length Regulation in Living Cells.

The microtubule cytoskeleton is responsible for sustained, long-range intracellular transport of mRNAs, proteins, and organelles in neurons. Neuronal microtubules must be stable enough to ensure reliable transport, but they also undergo dynamic instability, as their plus and minus ends continuously switch between growth and shrinking. This process allows for continuous rebuilding of the cytoskeleton and for flexibility in injury settings. Motivated by in vivo experimental data on microtubule behavior in Drosophila neurons, we propose a mathematical model of dendritic microtubule dynamics, with a focus on understanding microtubule length, velocity, and state-duration distributions. We find that limitations on microtubule growth phases are needed for realistic dynamics, but the type of limiting mechanism leads to qualitatively different responses to plausible experimental perturbations. We therefore propose and investigate two minimally-complex length-limiting factors: limitation due to resource (tubulin) constraints and limitation due to catastrophe of large-length microtubules. We combine simulations of a detailed stochastic model with steady-state analysis of a mean-field ordinary differential equations model to map out qualitatively distinct parameter regimes. This provides a basis for predicting changes in microtubule dynamics, tubulin allocation, and the turnover rate of tubulin within microtubules in different experimental environments.

The Role of MAPRE2 and Microtubules in Maintaining Normal Ventricular Conduction.

Brugada syndrome is associated with loss-of-function SCN5A variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within MAPRE2, which encodes EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome. A mapre2 knockout zebrafish model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated protein 9) and validated by Western blot. Larval hearts at 5 days post-fertilization were isolated for voltage mapping and immunocytochemistry. Adult fish hearts were used for ECG, patch clamping, and immunocytochemistry. Morpholinos were injected into embryos at 1-cell stage for knockdown experiments. A transgenic zebrafish line with cdh2 tandem fluorescent timer was used to study adherens junctions. Microtubule plus-end tracking and patch clamping were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with MAPRE2 knockdown and knockout, respectively. Voltage mapping of mapre2 knockout hearts showed a decrease in ventricular maximum upstroke velocity of the action potential and conduction velocity, suggesting loss of cardiac voltage-gated sodium channel function. ECG showed QRS prolongation in adult knockout fish, and patch clamping showed decreased sodium current density in knockout ventricular myocytes and arrhythmias in knockout iPSC-CMs. Confocal imaging showed disorganized adherens junctions and mislocalization of mature Ncad (N-cadherin) with mapre2 loss of function, associated with a decrease of detyrosinated tubulin. MAPRE2 knockdown in iPSC-CMs led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of ttl encoding tubulin tyrosine ligase in mapre2 knockout larvae rescued tubulin detyrosination and ventricular maximum upstroke velocity of the action potential. Genetic ablation of mapre2 led to a decrease in voltage-gated sodium channel function, a hallmark of Brugada syndrome, associated with disruption of adherens junctions, decrease of detyrosinated tubulin as a marker of microtubule stability, and changes in microtubule dynamics. Restoration of the detyrosinated tubulin fraction with ttl knockdown led to rescue of voltage-gated sodium channel-related functional parameters in mapre2 knockout hearts. Taken together, our study implicates microtubule dynamics in the modulation of ventricular conduction.

The role of MAPRE2 and microtubules in Brugada syndrome pathogenesis

Abstract Background Brugada syndrome (BrS) is associated with loss-of-function variants in SCN5A (encoding NaV1.5), which are found in only ∼20% of patients. Recent genome-wide association studies identified a novel locus within an intron of MAPRE2 (encoding microtubule end-binding protein 2, EB2), implicating microtubule (MT) involvement in BrS. Purpose To understand the role of MAPRE2 and MT in BrS pathogenesis. Methods A mapre2 knock-out (KO) zebrafish model was generated using CRISPR/Cas9 and validated by western blot. Larval hearts at 5 day-post-fertilization (dpf) were isolated for voltage mapping and immunohistochemistry with confocal imaging. Morpholino targeting mapre2 or ttl (encoding tubulin tyrosine ligase) were injected into zebrafish embryos at the one-cell stage. A transgenic zebrafish line with cdh2 (encoding N-cadherin) tandem fluorescent timer (tFT) was used to study adherens junction. MT plus-end tracking experiments were performed in iPSC-derived cardiomyocytes (CM) with MAPRE2 knockdown (KD) using siRNA. ECG was recorded in adult fish. Results Voltage mapping of larval hearts (14 WTs vs. 19 KOs) showed a significant decrease in ventricular conduction velocity (17.4±1.5 vs. 13.4±0.8 mm/s; p<0.05) and action potential upstroke velocity (Vmax; 86.1±3.6 vs. 73.9±3.2 1/s; p<0.05), suggesting loss of cardiac NaV function. Correspondingly, ECG of adult fish (11 WTs vs. 10 KOs) showed a significant increase in QRS duration (27.8±0.5 vs. 32.3±0.7 ms; p<0.0001). Confocal imaging of KO hearts showed disorganized adherens junction based on N-cadherin staining. Similarly, morpholino KD of mapre2 in the cdh2 tFT line showed ∼40% mislocalization of mature N-cadherin relative to ZO-1 staining in larval ventricles (8 KDs vs 9 control hearts; p<0.05), suggesting disruption of adherens junction. In terms of MT, immunohistochemistry showed a 23% decrease of detyrosinated tubulin relative to total alpha tubulin in KO hearts (16 vs. 8 WT hearts; p<0.01). MAPRE2 KD in iPSC-CM (337 control vs. 285 KD MT in 4 sets of cells) showed an increase in MT velocity (7.9±0.2 vs. 8.8±0.2 µm/min; p<0.001), distance (5.8±0.2 vs. 7.1±0.2 µm; p<0.0001), and duration (46.2±1.4 vs. 52.2±1.6 s; p<0.001), suggesting changes to MT dynamics. Finally, morpholino KD of ttl in mapre2 KO embryos restored the fraction of detyrosinated tubulin (46% increase; 16 controls vs. 16 KDs; p<0.01) as well as Vmax (74.3±3.0 vs. 88.3±2.1 1/s; 13 controls vs. 9 KDs; p<0.01) to WT levels. Conclusion Genetic ablation of mapre2 led to a decrease in cardiac NaV function, a hallmark of BrS. This is associated with disruption of adherens junction, a decrease of detyrosinated tubulin as a marker of MT stability, and changes in MT dynamics. Restoration of detyrosinated tubulin fraction with ttl KD led to a rescue of NaV function in zebrafish larval hearts. Taken together, MAPRE2 loss-of-function may contribute to BrS pathogenesis by disruption of adherens junction via changes in MT dynamics.

Plant Defence Induction by Meyerozyma guilliermondii in Vitis vinifera L.

This article emphasizes the crucial importance of yeast Meyerozyma guilliermondii (Patent CECT13190) as a biological control agent (BCA) in eliciting defensive responses in vine plants, and is supported by comprehensive physiological, proteomic, and transcriptomic analyses. The results demonstrate that the BCA M. guilliermondii can induce enhanced defensive responses, as reflected in the regulation of key proteins. Notably, the upregulated expression of calmodulin and pathogenesis-related protein 10 (PR-10) are indicative of a complex interplay between calcium signalling, salicylic acid accumulation, and the elicitation of plant defence responses against pathogens. Furthermore, changes in microtubule dynamics and proteins related to protein synthesis and folding are observed, confirming the elicitation of defence responses. The correspondence between proteomic and transcriptomic analyses for genes codifying pathogenesis-related proteins, such as Vcgns1, VviTL1, and Vcchit1b, reinforces the empirical robustness of our findings. Collectively, our research explores the modulation of plant defences by the BCA, opening promising avenues for innovative agricultural strategies that enhance crop resilience and productivity.

PO-02-153 THE ROLE OF MAPRE2 AND MICROTUBULES IN CARDIAC ELECTROPHYSIOLOGY AND BRUGADA SYNDROME

Brugada syndrome (BrS) is associated with loss-of-function variants in SCN5A (encoding NaV1.5), yet these are only found in ∼20% of probands. Recent genome-wide association studies identified a novel locus within an intron of MAPRE2 (encoding microtubule end-binding protein 2, EB2), which implicates microtubule (MT) involvement in BrS. To understand the role of MAPRE2 and MT in cardiac electrophysiology and BrS pathogenesis. Using CRISPR/Cas9, a mapre2 knock-out (KO) mutant was generated in zebrafish and validated by western blot. Morpholino targeting ttl (encoding tubulin tyrosine ligase) was injected into zebrafish embryos at the one-cell stage. Larval hearts at 5 day-post-fertilization (dpf) were isolated for voltage mapping and immunohistochemistry. MT plus-end tracking experiments were performed in iPSC-derived cardiomyocytes with MAPRE2 knockdown (KD) using siRNA. ECG was recorded in adult fish. Voltage mapping of larval hearts (14 WTs vs. 19 KOs) showed a significant decrease in ventricular conduction velocity (17.4±1.5 vs. 13.4±0.8 mm/s; p<0.05) and action potential upstroke velocity (Vmax; 86.1±3.6 vs. 73.9±3.2 1/s; p<0.05), suggesting loss of cardiac NaV function. Correspondingly, ECG of adult fish (11 WTs vs. 10 KOs) showed a significant increase in QRS duration (27.8±0.5 vs. 32.3±0.7 ms; p<0.0001). Immunohistochemistry showed a 23% decrease of detyrosinated (glu) tubulin relative to total alpha tubulin in KO hearts (16 vs. 8 WT hearts; p<0.01). MT plus-end tracking experiments (337 control vs. 285 KD MT in 4 sets of cells) showed an increase in MT velocity (7.9±0.2 vs. 8.8±0.2 μm/min; p<0.001), distance (5.8±0.2 vs. 7.1±0.2 μm; p<0.0001), and duration (46.2±1.4 vs. 52.2±1.6 s; p<0.001) with MAPRE2 KD, suggesting changes to MT dynamics. Finally, morpholino KD of ttl in mapre2 KO embryos restored the fraction of glu-tubulin (46% increase; 16 controls vs. 16 ttl KDs; p<0.01) and Vmax (74.3±3.0 vs. 88.3±2.1 1/s; 13 controls vs. 9 KDs; p<0.01) to WT levels. Genetic ablation of mapre2 led to a decrease in NaV function, a hallmark of BrS. This is associated with a decrease in glu-tubulin as a marker of MT stability and changes in MT dynamics. Restoration of glu-tubulin fraction with ttl KD led to a rescue of NaV function in zebrafish larval hearts. Taken together, MAPRE2 loss-of-function may contribute to BrS pathogenesis via a novel paradigm of disrupting MT stability and dynamics.

Endoplasmic reticulum membranes are continuously required to maintain mitotic spindle size and forces.

Membrane organelle function, localization, and proper partitioning upon cell division depend on interactions with the cytoskeleton. Whether membrane organelles also impact the function of cytoskeletal elements remains less clear. Here, we show that acute disruption of the ER around spindle poles affects mitotic spindle size and function in Drosophila syncytial embryos. Acute ER disruption was achieved through the inhibition of ER membrane fusion by the dominant-negative cytoplasmic domain of atlastin. We reveal that when centrosome-proximal ER membranes are disrupted, specifically at metaphase, mitotic spindles become smaller, despite no significant changes in microtubule dynamics. These smaller spindles are still able to mediate sister chromatid separation, yet with decreased velocity. Furthermore, by inducing mitotic exit, we found that nuclear separation and distribution are affected by ER disruption. Our results suggest that ER integrity around spindle poles is crucial for the maintenance of mitotic spindle shape and pulling forces. In addition, ER integrity also ensures nuclear spacing during syncytial divisions.

Preprint Highlight: Interphase microtubule disassembly is a signaling cue that drives cell rounding at mitotic entry.

Metaphase cell rounding is an important step during cell division; however, the cell cycle regulation of the ezrin, radixin, and moesin (ERM) proteins which drive cell rounding is not well understood. Using a small molecule screen, the authors unexpectedly found that microtubule disassembly activates ERM proteins and cell rounding. The authors determined that microtubule disassembly acts as a cell cycle signal to activate ERM proteins via GEF-H1 and the kinase SLK. This new model suggests that as cells transition from interphase to metaphase, the observed change in microtubule dynamics is not only important for spindle assembly but also helps drive cortical actomyosin remodeling to enable cell rounding.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

The mitotic spindle protein CKAP2 potently increases formation and stability of microtubules.

Cells increase microtubule dynamics to make large rearrangements to their microtubule cytoskeleton during cell division. Changes in microtubule dynamics are essential for the formation and function of the mitotic spindle, and misregulation can lead to aneuploidy and cancer. Using in vitro reconstitution assays we show that the mitotic spindle protein Cytoskeleton-Associated Protein 2 (CKAP2) has a strong effect on nucleation of microtubules by lowering the critical tubulin concentration 100-fold. CKAP2 increases the apparent rate constant ka of microtubule growth by 50-fold and increases microtubule growth rates. In addition, CKAP2 strongly suppresses catastrophes. Our results identify CKAP2 as the most potent microtubule growth factor to date. These finding help explain CKAP2's role as an important spindle protein, proliferation marker, and oncogene.

Spatiotemporal changes in microtubule dynamics during dendritic morphogenesis

ABSTRACT Dendritic morphogenesis requires dynamic microtubules (MTs) to form a coordinated cytoskeletal network during development. Dynamic MTs are characterized by their number, polarity and speed of polymerization. Previous studies described a correlation between anterograde MT growth and terminal branch extension in Drosophila dendritic arborization (da) neurons, suggesting a model that anterograde MT polymerization provides a driving force for dendritic branching. We recently found that the Ste20-like kinase Tao specifically regulates dendritic branching by controlling the number of dynamic MTs in a kinase activity-dependent fashion, without affecting MT polarity or speed. This finding raises the interesting question of how MT dynamics affects dendritic morphogenesis, and if Tao kinase activity is developmentally regulated to coordinate MT dynamics and dendritic morphogenesis. We explored the possible correlation between MT dynamics and dendritic morphogenesis together with the activity changes of Tao kinase in C1da and C4da neurons during larval development. Our data show that spatiotemporal changes in the number of dynamic MTs, but not polarity or polymerization speed, correlate with dendritic branching and Tao kinase activity. Our findings suggest that Tao kinase limits dendritic branching by controlling the abundance of dynamic MTs and we propose a novel model on how regulation of MT dynamics might influence dendritic morphogenesis.

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.

Eosinophil cytolysis on Immunoglobulin G is associated with microtubule formation and suppression of rho-associated protein kinase signalling.

The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Cell-free eosinophil granules are found in tissues in eosinophilic diseases, including asthma. This suggests that eosinophils have lysed and released cellular content, likely harming tissues. The present study explores the mechanism of CD32- and αMß2 integrin-dependent eosinophil cytolysis of IL3-primed blood eosinophils seeded on heat-aggregated immunoglobulin G (HA-IgG). Cytoskeletal events and signalling pathways potentially involved in cytolysis were assessed using inhibitors. The level of activation of the identified events and pathways involved in cytolysis was measured. In addition, the links between these identified pathways and changes in degranulation (exocytosis) and adhesion were analysed. Cytolysis of IL3-primed eosinophils was dependent on the production of reactive oxygen species (ROS) and downstream phosphorylation of p-38 MAPK. In addition, formation of microtubule (MT) arrays was necessary for cytolysis and was accompanied by changes in MT dynamics as measured by phosphorylation status of stathmin and microtubule-associated protein 4 (MAP4), the latter of which was regulated by ROS production. Reduced ROCK signalling preceded cytolysis, which was associated with eosinophil adhesion and reduced migration. In this CD32- and αMß2 integrin-dependent adhesion model, lysing eosinophils exhibit reduced migration and ROCK signalling, as well as both MT dynamic changes and p-38 phosphorylation downstream of ROS production. We propose that interfering with these pathways would modulate eosinophil cytolysis and subsequent eosinophil-driven tissue damage.

The focal adhesion scaffold protein Hic-5 regulates vimentin organization in fibroblasts.

Focal adhesion (FA)-stimulated reorganization of the F-actin cytoskeleton regulates cellular size, shape, and mechanical properties. However, FA cross-talk with the intermediate filament cytoskeleton is poorly understood. Genetic ablation of the FA-associated scaffold protein Hic-5 in mouse cancer-associated fibroblasts (CAFs) promoted a dramatic collapse of the vimentin network, which was rescued following EGFP-Hic-5 expression. Vimentin collapse correlated with a loss of detergent-soluble vimentin filament precursors and decreased vimentin S72/S82 phosphorylation. Additionally, fluorescence recovery after photobleaching analysis indicated impaired vimentin dynamics. Microtubule (MT)-associated EB1 tracking and Western blotting of MT posttranslational modifications indicated no change in MT dynamics that could explain the vimentin collapse. However, pharmacological inhibition of the RhoGTPase Cdc42 in Hic-5 knockout CAFs rescued the vimentin collapse, while pan-formin inhibition with SMIFH2 promoted vimentin collapse in Hic-5 heterozygous CAFs. Our results reveal novel regulation of vimentin organization/dynamics by the FA scaffold protein Hic-5 via modulation of RhoGTPases and downstream formin activity.

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.

Spastin is a dual-function enzyme that severs microtubules and promotes their regrowth to increase the number and mass of microtubules

The remodeling of the microtubule cytoskeleton underlies dynamic cellular processes, such as mitosis, ciliogenesis, and neuronal morphogenesis. An important class of microtubule remodelers comprises the severases-spastin, katanin, and fidgetin-which cut microtubules into shorter fragments. While severing activity might be expected to break down the microtubule cytoskeleton, inhibiting these enzymes in vivo actually decreases, rather increases, the number of microtubules, suggesting that severases have a nucleation-like activity. To resolve this paradox, we reconstituted Drosophila spastin in a dynamic microtubule assay and discovered that it is a dual-function enzyme. In addition to its ATP-dependent severing activity, spastin is an ATP-independent regulator of microtubule dynamics that slows shrinkage and increases rescue. We observed that spastin accumulates at shrinking ends; this increase in spastin concentration may underlie the increase in rescue frequency and the slowdown in shortening. The changes in microtubule dynamics promote microtubule regrowth so that severed microtubule fragments grow, leading to an increase in the number and mass of microtubules. A mathematical model shows that spastin's effect on microtubule dynamics is essential for this nucleation-like activity: spastin switches microtubules into a state where the net flux of tubulin onto each polymer is positive, leading to the observed exponential increase in microtubule mass. This increase in the microtubule mass accounts for spastin's in vivo phenotypes.

Dynein efficiently navigates the dendritic cytoskeleton to drive the retrograde trafficking of BDNF/TrkB signaling endosomes.

The efficient transport of cargoes within axons and dendrites is critical for neuronal function. Although we have a basic understanding of axonal transport, much less is known about transport in dendrites. We used an optogenetic approach to recruit motor proteins to cargo in real time within axons or dendrites in hippocampal neurons. Kinesin-1, a robust axonal motor, moves cargo less efficiently in dendrites. In contrast, cytoplasmic dynein efficiently navigates both axons and dendrites; in both compartments, dynamic microtubule plus ends enhance dynein-dependent transport. To test the predictions of the optogenetic assay, we examined the contribution of dynein to the motility of an endogenous dendritic cargo and found that dynein inhibition eliminates the retrograde bias of BDNF/TrkB trafficking. However, inhibition of microtubule dynamics has no effect on BDNF/TrkB motility, suggesting that dendritic kinesin motors may cooperate with dynein to drive the transport of signaling endosomes into the soma. Collectively our data highlight compartment-specific differences in kinesin activity that likely reflect specialized tuning for localized cytoskeletal determinants, whereas dynein activity is less compartment specific but is more responsive to changes in microtubule dynamics.

Role of Signal-Regulated Changes in Microtubule Stability Dynamics through &amp;lt;i&amp;gt;α&amp;lt;/i&amp;gt;-Tubulin Phosphorylation on Ser/Tyr in Modulation of Salivary Gland Matrix Metalloproteinase-9 (MMP-9) Secretion in Response to &amp;lt;i&amp;gt;Porphyromonas gingivalis&amp;lt;/i&amp;gt; and Ghrelin

Up-regulation in salivary gland acinar cell MMP-9 secretion in response to proinflammatory challenge by periodontopathic bacterium, P. gingivalis relays heavily on the factors that influence the protein processing at the level of endoplasmic reticulum-to-Golgi trafficking, and occurs in concert with the changes in the stability dynamics of the major cytoskeleton polymeric structures, microtubules (MTs). In this study, we report that P. gingivalis LPS-elicited induction in the acinar cell MMP-9 secretion is accompanied by the enhancement in MT stabilization, while the modulatory influence of peptide hormone, ghrelin, is associated with MT destabilization. Further, we reveal that the changes in MT dynamics induced by the LPS and ghrelin occur through signal-regulated α-tubulin phosphorylation on Ser/Tyr. The LPS effect is reflected in a marked increase in PKCδ-mediated α-tubulin phosphorylation on Ser, whereas the modulatory influence of ghrelin on MT dynamics is manifested in by SFK-dependent phosphorylation of α-tubulin on Tyr. Moreover, we show that that the changes in MT dynamics, conferred by the LPS and ghrelin, affect the Golgi localization of GTP-Arf1 and the recruitment and activation of PKD2. The findings underscore the role of signal-regulated changes in MT stability dynamics through PKCδ/SFK-mediated α-tubulin phosphorylation on Ser/Tyr in controlling the salivary gland acinar cell MMP-9 secretion in response to P. gingivalis LPS as well as its modulation by ghrelin.

Bisphenol A and its analogues disrupt centrosome cycle and microtubule dynamics in prostate cancer.

Humans are increasingly exposed to structural analogues of bisphenol A (BPA), as BPA is being replaced by these compounds in BPA-free consumer products. We have previously shown that chronic and developmental exposure to BPA is associated with increased prostate cancer (PCa) risk in human and animal models. Here, we examine whether exposure of PCa cells (LNCaP, C4-2) to low-dose BPA and its structural analogues (BPS, BPF, BPAF, TBBPA, DMBPA and TMBPA) affects centrosome amplification (CA), a hallmark of cancer initiation and progression. We found that exposure to BPA, BPS, DMBPA and TBBPA, in descending order, increased the number of cells with CA, in a non-monotonic dose-response manner. Furthermore, cells treated with BPA and their analogues initiated centrosome duplication at 8 h after release from serum starvation, significantly earlier in G-1 phase than control cells. This response was attended by earlier release of nucleophosmin from unduplicated centrosomes. BPA-exposed cells exhibited increased expression of cyclin-dependent kinase CDK6 and decreased expression of CDK inhibitors (p21Waf1/CIP1 and p27KIP1). Using specific antagonists for estrogen/androgen receptors, CA in the presence of BPA or its analogues was likely to be mediated via ESR1 signaling. Change in microtubule dynamics was observed on exposure to these analogues, which, for BPA, was accompanied by increased expression of centrosome-associated protein CEP350 Similar to BPA, chronic treatment of cells with DMBPA, but not other analogues, resulted in the enhancement of anchorage-independent growth. We thus conclude that selected BPA analogues, similar to BPA, disrupt centrosome function and microtubule organization, with DMBPA displaying the broadest spectrum of cancer-promoting effects.

Cytokinesis defect in BY-2 cells caused by ATP-competitive kinase inhibitors

ABSTRACTCytokinesis is last but not least in cell division as it completes the formation of the two cells. The main role in cell plate orientation and expansion have been assigned to microtubules and kinesin proteins. However, recently we reported severe cytokinesis defect in BY-2 cells not accompanied by changes in microtubules dynamics. Here we also confirmed that distribution of kinesin NACK1 is not the cause of cytokinesis defect. We further explored inhibition of the cell plate expansion by ATP-competitive inhibitors. Two different inhibitors, 5-Iodotubercidin and ML-7 resulted in a very similar phenotype, which indicates that they target same protein cascade. Interestingly, in our previous study we showed that 5-Iodotubercidin treatment affects concentration of actin filaments on the cell plate, while ML-7 is inhibitor of myosin light chain kinase. Although not directly, it indicates importance of actomyosin complex in plant cytokinesis.

Helicobacter pylori-induced changes in microtubule dynamics conferred by α-tubulin phosphorylation on Ser/Tyr mediate gastric mucosal secretion of matrix metalloproteinase-9 (MMP-9) and its modulation by ghrelin.

Regulation of matrix metalloproteinase-9 (MMP-9) secretion in response to proinflammatory challenge remains under a strict control of factors that affect the stability dynamics of the major cytoskeleton polymeric structures, microtubules (MTs). In this study, we report that H. pylori LPS-elicited induction gastric mucosal MMP-9 secretion is accompanied by the enhancement in MT stabilization as evidenced by the increase in α-tubulin acetylation and detyrosination while the modulatory influence of hormone, ghrelin, is associated with MT destabilization and reflected in a decrease in α-tubulin acetylation and detyrosination. Further, we reveal that the LPS-induced enhancement in MT stabilization and up-regulation in MMP-9 secretion as well as the modulatory influence of ghrelin occur with the involvement of PKCδ and SFK. The LPS effect is reflected in a marked increase in PKCδ-mediated α-tubulin phosphorylation on Ser, while the modulatory effect of ghrelin on MT dynamics and MMP-9 secretion is manifested by the SFK-dependent phosphorylation of α-tubulin on Tyr. Moreover, the changes in α-tubulin phosphorylation and MT stabilization dynamics occur in concert with the Golgi recruitment and activation of PKD2 and Arf-GEF. The findings demonstrate that the enhancement in gastric mucosal MMP-9 secretion in response to H. pylori and its modulation by ghrelin are the result of changes in MT dynamics conferred by PKCδ/SFK- mediated α-tubulin Ser/Tyr phosphorylation.