Microtubule Disassembly Research Articles

Decoupling actin assembly from microtubule disassembly by TBC1D3C-mediated direct GEF-H1 activation.

Actin and microtubules are essential cytoskeletal components and coordinate their dynamics through multiple coupling and decoupling mechanisms. However, how actin and microtubule dynamics are decoupled remains incompletely understood. Here, we identified TBC1D3C as a new regulator that can decouple actin filament assembly from microtubule disassembly. We showed that TBC1D3C induces the release of GEF-H1 from microtubules into the cytosol without perturbing microtubule arrays, leading to RhoA activation and actin filament assembly. Mechanistically, we found that TBC1D3C directly binds to GEF-H1, disrupting its interaction with the Tctex-DIC-14-3-3 complex and thereby displacing GEF-H1 from microtubules independently of microtubule disassembly. Super-resolution microscopy and live-cell imaging further confirmed that TBC1D3C triggers GEF-H1 release and actin filament assembly while maintaining microtubule integrity. Therefore, our findings demonstrated that TBC1D3C functions as a direct GEF activator and a novel regulator in decoupling actin assembly from microtubule disassembly, providing new insights into cytoskeletal regulation.

Characterization of Intrinsically Disordered Proteins in Healthy and Diseased States by Nuclear Magnetic Resonance.

Intrinsically Disordered Proteins (IDPs) are active in different cellular procedures like ordered assembly of chromatin and ribosomes, interaction with membrane, protein, and ligand binding, molecular recognition, binding, and transportation via nuclear pores, microfilaments and microtubules process and disassembly, protein functions, RNA chaperone, and nucleic acid binding, modulation of the central dogma, cell cycle, and other cellular activities, post-translational qualification and substitute splicing, and flexible entropic linker and management of signaling pathways. The intrinsic disorder is a precise structural characteristic that permits IDPs/IDPRs to be involved in both one-to-many and many-to-one signaling. IDPs/IDPRs also exert some dynamical and structural ordering, being much less constrained in their activities than folded proteins. Nuclear magnetic resonance (NMR) spectroscopy is a major technique for the characterization of IDPs, and it can be used for dynamic and structural studies of IDPs. This review was carried out to discuss intrinsically disordered proteins and their different goals, as well as the importance and effectiveness of NMR in characterizing intrinsically disordered proteins in healthy and diseased states.

Memantine alleviates cognitive impairment and hippocampal morphology injury in a mouse model of chronic alcohol exposure

Alcohol-related cognitive impairment (ARCI) is highly prevalent among patients with alcohol abuse and dependence. The pathophysiology of ARCI, pivotal for refined therapeutic approaches, is not fully elucidated, posing a risk of progression to severe neurological sequelae such as Korsakoff's syndrome (KS) and Alcohol-Related Dementia (ARD). This study ventures into the underlying mechanisms of chronic alcohol-induced neurotoxicity, notably glutamate excitotoxicity and cytoskeletal disruption, and explores the therapeutic potential of Memantine, a non-competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor known for its neuroprotective effect against excitotoxicity. Our investigation centers on the efficacy of Memantine in mitigating chronic alcohol-induced cognitive and hippocampal damages in vivo. Male C57BL/6J mice were subjected to 30 % (v/v, 6.0 g/kg) ethanol via intragastric administration alongside Memantine co-treatment (10 mg/kg/day, intraperitoneally) for six weeks. The assessment involved Y maze, Morris water maze, and novel object recognition tests to evaluate spatial and recognition memory deficits. Histopathological evaluations of the hippocampus were conducted to examine the extent of alcohol-induced morphological changes and the potential protective effect of Memantine. The findings reveal that Memantine significantly improves chronic alcohol-compromised cognitive functions and mitigates hippocampal pathological changes, implicating a moderating effect on the disassembly of actin cytoskeleton and microtubules in the hippocampus, induced by chronic alcohol exposure. Our results underscore Memantine's capability to attenuate chronic alcohol-induced cognitive and hippocampal morphological harm may partly through regulating cytoskeleton dynamics, offering valuable insights into innovative therapeutic strategies for ARCI.

CEP41, a ciliopathy-linked centrosomal protein, regulates microtubule assembly and cell proliferation.

Centrosomal proteins play pivotal roles in orchestrating microtubule dynamics, and their dysregulation leads to disorders, including cancer and ciliopathies. Understanding the multifaceted roles of centrosomal proteins is vital to comprehend their involvement in disease development. Here, we report novel cellular functions of CEP41, a centrosomal and ciliary protein implicated in Joubert syndrome. We show that CEP41 is an essential microtubule-associated protein with microtubule-stabilizing activity. Purified CEP41 binds to preformed microtubules, promotes microtubule nucleation and suppresses microtubule disassembly. When overexpressed in cultured cells, CEP41 localizes to microtubules and promotes microtubule bundling. Conversely, shRNA-mediated knockdown of CEP41 disrupts the interphase microtubule network and delays microtubule reassembly, emphasizing its role in microtubule organization. Further, we demonstrate that the association of CEP41 with microtubules relies on its conserved rhodanese homology domain (RHOD) and the N-terminal region. Interestingly, a disease-causing mutation in the RHOD domain impairs CEP41-microtubule interaction. Moreover, depletion of CEP41 inhibits cell proliferation and disrupts cell cycle progression, suggesting its potential involvement in cell cycle regulation. These insights into the cellular functions of CEP41 hold promise for unraveling the impact of its mutations in ciliopathies.

Autonomous assembly and disassembly of gliding molecular robots regulated by a DNA-based molecular controller.

In recent years, there has been a growing interest in engineering dynamic and autonomous systems with robotic functionalities using biomolecules. Specifically, the ability of molecular motors to convert chemical energy to mechanical forces and the programmability of DNA are regarded as promising components for these systems. However, current systems rely on the manual addition of external stimuli, limiting the potential for autonomous molecular systems. Here, we show that DNA-based cascade reactions can act as a molecular controller that drives the autonomous assembly and disassembly of DNA-functionalized microtubules propelled by kinesins. The DNA controller is designed to produce two different DNA strands that program the interaction between the microtubules. The gliding microtubules integrated with the controller autonomously assemble to bundle-like structures and disassemble into discrete filaments without external stimuli, which is observable by fluorescence microscopy. We believe this approach to be a starting point toward more autonomous behavior of motor protein-based multicomponent systems with robotic functionalities.

Inhibition of microtubule function prevents hypoxia-induced glycolysis

In normoxic conditions where oxygen supply exceeds demand, mammalian cells produce 38 molecules of adenosine triphosphate (ATP) per molecule of glucose through cellular respiration which is primarily oxygen-dependent. In hypoxia where oxygen demand exceeds supply, the cell enters a bioenergetic crisis due to the decrease in oxygen-dependent ATP synthesis. The cell relies on overcoming this deficiency in ATP through upregulation of glycolytic gene expression via the hypoxia inducible factor 1-a (HIF-1a). However, it is highly unlikely, if not impossible, that the bioenergetic requirements of the hypoxic cell depend solely on the increased expression of glycolytic enzymes distributed randomly in the cytoplasm. We have recently shown that in response to hypoxia, glycolytic enzymes coalesce to form a glycolytic metabolon to effciently increase ATP production in response to this metabolic challenge (Kierans SJ, et al., 2023). While the function of this metabolon has been characterised, the mechanisms underpinning formation, scaffolding, and intracellular distribution remains poorly understood. Here we investigate the possible role of the cytoskeleton as a scaffold or transport system. Microtubules are a primary method of intracellular transport and may alter the energy distribution strategies of cells in bioenergetic crisis. It is hypothesized that the microtubular network transports glycolytic enzymes and/or facilitates increased glycolytic ATP production by promoting metabolon formation to induce glycolysis in hypoxia. Through western blots, it was successfully shown that upon exposure to 1% oxygen, caco2 intestinal epithelial cells induce a robust hypoxic response by stabilising HIF-1a. To investigate the role of the microtubules, two microtubule inhibitors were used: nocodazole, a microtubule assembly and disassembly inhibitor and dynarrestin, a dynein-1 motor transport protein inhibitor. Non-toxic concentrations of nocodazole and dynarrestin were determined using cell viability assays (trypan blue and resazurin assays). Upon exposure to 24 hours of hypoxia, caco2 cells elicit a statistically significant increase in intracellular lactate (185.9% ± 7.5% SEM, n = 3), which was then supressed when treated with nocodazole or dynarrestin (20.6% ± 10.2% SEM and 74.9% ± 8.87 SEM respectively, n = 3). These results thus implicate a role for the transport and structural capabilities of the microtubules in hypoxia-induced glycolysis. Kierans, S. J., Fagundes, R. R., Malkov, M. I., Sparkes, R., Dillon, E. T., Smolenski, A.,... & Taylor, C. T. (2023). Hypoxia induces a glycolytic complex in intestinal epithelial cells independent of HIF-1-driven glycolytic gene expression. Proceedings of the National Academy of Sciences, 120(35), e2208117120. University College Dublin, School of Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Regulation of Microtubule Stability in Pulmonary Microvascular Endothelial Cells in Rats with Severe Acute Pancreatitis: Qingyi Decoction is a Potential CDK5 Inhibitor.

Explore the therapeutic effects and regulatory mechanism of Qingyi Decoction (QYD) on severe acute pancreatitis (SAP) associated acute lung injury (ALI). We identified the constituents absorbed into the blood of QYD based on a network pharmacological strategy. The differentially expressed genes from the GEO database were screened to identify the critical targets of QYD treatment of SAP-ALI. The SAP-ALI rat model was constructed.Some methods were used to evaluate the efficacy and mechanism of QYD in treating SAP-ALI. LPS-stimulated pulmonary microvascular endothelial cell injury simulated the SAP-induced pulmonary endothelial injury model. We further observed the therapeutic effect of QYD and CDK5 plasmid transfection on endothelial cell injury. 18 constituents were absorbed into the blood, and 764 targets were identified from QYD, 25 of which were considered core targets for treating SAP-ALI. CDK5 was identified as the most critical gene. The results of differential expression analysis showed that the mRNA expression level of CDK5 in the blood of SAP patients was significantly up-regulated compared with that of healthy people. Animal experiments have demonstrated that QYD can alleviate pancreatic and lung injury inflammatory response and reduce the upregulation of CDK5 in lung tissue. QYD or CDK5 inhibitors could decrease the expression of NFAT5 and GEF-H1, and increase the expression of ACE-tub in SAP rat lung tissue. Cell experiments proved that QYD could inhibit the expression of TNF-α and IL-6 induced by LPS. Immunofluorescence results suggested that QYD could alleviate the cytoskeleton damage of endothelial cells, and the mechanism might be related to the inhibition of CDK5-mediated activation of NFAT5, GEF-H1, and ACE-tub. CDK5 has been identified as a critical target for pulmonary endothelial injury of SAP-ALI. QYD may partially alleviate microtubule disassembly by targeting the CDK5/NFAT5/GEF-H1 signaling pathway, thus relieving SAP-induced pulmonary microvascular endothelial cell injury.

Cytokinetic abscission requires actin-dependent microtubule severing

Cell division is completed by the abscission of the intercellular bridge connecting the daughter cells. Abscission requires the polymerization of an ESCRT-III cone close to the midbody to both recruit the microtubule severing enzyme spastin and scission the plasma membrane. Here, we found that the microtubule and the membrane cuts are two separate events that are regulated differently. Using HeLa cells, we uncovered that the F-actin disassembling protein Cofilin-1 controls the disappearance of a transient pool of branched F-actin which is precisely assembled at the tip of the ESCRT-III cone shortly before the microtubule cut. Functionally, Cofilin-1 and Arp2/3-mediated branched F-actin favor abscission by promoting local severing of the microtubules but do not participate later in the membrane scission event. Mechanistically, we propose that branched F-actin functions as a physical barrier that limits ESCRT-III cone elongation and thereby favors stable spastin recruitment. Our work thus reveals that F-actin controls the timely and local disassembly of microtubules required for cytokinetic abscission.

Male meiotic spindle poles are stabilized by TACC3 and cKAP5/chTOG differently from female meiotic or somatic mitotic spindles in mice.

Transforming acidic acid coiled-coil protein 3 (TACC3) and cytoskeleton associated protein 5 (cKAP5; or colonic hepatic tumor overexpressed gene, chTOG) are vital for spindle assembly and stabilization initiated through TACC3 Aurora-A kinase interaction. Here, TACC3 and cKAP5/chTOG localization with monospecific antibodies is investigated in eGFP-centrin-2- expressing mouse meiotic spermatocytes. Both proteins bind spermatocyte spindle poles but neither kinetochore nor interpolar microtubules, unlike in mitotic mouse fibroblasts or female meiotic oocyte spindles. Spermatocytes do not display a liquid-like spindle domain (LISD), although fusing them into maturing oocytes generates LISD-like TACC3 condensates around sperm chromatin but sparse microtubule assembly. Microtubule inhibitors do not reduce TACC3 and cKAP5/chTOG spindle pole binding. MLN 8237 Aurora-A kinase inhibitor removes TACC3, not cKAP5/chTOG, disrupting spindle organization, chromosome alignment, and impacting spindle pole γ-tubulin intensity. The LISD disruptor 1,6-hexanediol abolished TACC3 in spermatocytes, impacting spindle bipolarity and chromosome organization. Cold microtubule disassembly and rescue experiments in the presence of 1,6-hexanediol reinforce the concept that spermatocyte TACC3 spindle pole presence is not required for spindle pole microtubule assembly. Collectively, meiotic spermatocytes without a LISD localize TACC3 and cKAP5/chTOG exclusively at spindle poles to support meiotic spindle pole stabilization during male meiosis, different from either female meiosis or mitosis.

Centriole elimination during Caenorhabditis elegans oogenesis initiates with loss of the central tube protein SAS-1.

In most metazoans, centrioles are lost during oogenesis, ensuring that the zygote is endowed with the correct number of two centrioles, which are paternally contributed. How centriole architecture is dismantled during oogenesis is not understood. Here, we analyze with unprecedent detail the ultrastructural and molecular changes during oogenesis centriole elimination in Caenorhabditis elegans. Centriole elimination begins with loss of the so-called central tube and organelle widening, followed by microtubule disassembly. The resulting cluster of centriolar proteins then disappears gradually, usually moving in a microtubule- and dynein-dependent manner to the plasma membrane. Our analysis indicates that neither Polo-like kinases nor the PCM, which modulate oogenesis centriole elimination in Drosophila, do so in C. elegans. Furthermore, we demonstrate that the central tube protein SAS-1 normally departs initially from the organelle, which loses integrity earlier in sas-1 mutants. Overall, our work provides novel mechanistic insights regarding the fundamental process of oogenesis centriole elimination.

Quantitative analysis of peroxisome tracks using a Hidden Markov Model

Diffusion and mobility are essential for cellular functions, as molecules are usually distributed throughout the cell and have to meet to interact and perform their function. This also involves the cytosolic migration of cellular organelles. However, observing such diffusion and interaction dynamics is challenging due to the high spatial and temporal resolution required and the accurate analysis of the diffusional tracks. The latter is especially important when identifying anomalous diffusion events, such as directed motions, which are often rare. Here, we investigate the migration modes of peroxisome organelles in the cytosol of living cells. Peroxisomes predominantly migrate randomly, but occasionally they bind to the cell's microtubular network and perform directed migration, which is difficult to quantify, and so far, accurate analysis of switching between these migration modes is missing. We set out to solve this limitation by experiments and analysis with high statistical accuracy. Specifically, we collect temporal diffusion tracks of thousands of individual peroxisomes in the HEK 293 cell line using two-dimensional spinning disc fluorescence microscopy at a high acquisition rate of 10 frames/s. We use a Hidden Markov Model with two hidden states to (1) automatically identify directed migration segments of the tracks and (2) quantify the migration properties for comparison between states and between different experimental conditions. Comparing different cellular conditions, we show that the knockout of the peroxisomal membrane protein PEX14 leads to a decrease in the directed movement due to a lowered binding probability to the microtubule. However, it does not eradicate binding, highlighting further microtubule-binding mechanisms of peroxisomes than via PEX14. In contrast, structural changes of the microtubular network explain perceived eradication of directed movement by disassembly of microtubules by Nocodazole-treatment.

Why kiss-and-hop explains that tau does not stabilize microtubules and does not interfere with axonal transport (at physiological conditions).

Tau is a microtubule-associated protein that is enriched in the axonal process of neurons. Post-translational modifications of tau have been implicated in the development of tauopathies characterized by defects in axonal transport, neuronal atrophy, and microtubule disassembly. Although tau is almost quantitatively bound to microtubules under physiological conditions, it does not significantly affect axonal transport. Furthermore, acute or chronic tau deficiency does not result in significant destabilization of neuronal microtubules, challenging the classical view that disease-related tau modifications directly cause axonal microtubule collapse. Here, we discuss how the rapid interaction kinetics of the tau-microtubule interaction, which we previously termed the kiss-and-hop interaction, explains why tau does not affect microtubule-dependent axonal transport but still allows tau to modulate microtubule polymerization. In contrast, tau modifications that slow down the kinetics of the tau-microtubule interaction and increase the residence time of tau at a microtubule interaction site can disrupt axonal transport and cause dendritic atrophy. We discuss the consequences of such a gain-of-toxicity mechanism in terms of the development of disease-modulating drugs that target the tau protein.

Nitrosylation of β2-Tubulin Promotes Microtubule Disassembly and Differentiated Cardiomyocyte Beating in Ischemic Mice.

Beating cardiomyocyte regeneration therapies have revealed as alternative therapeutics for heart transplantation. Nonetheless, the importance of nitric oxide (NO) in cardiomyocyte regeneration has been widely suggested, little has been reported concerning endogenous NO during cardiomyocyte differentiation. Here, we used P19CL6 cells and a Myocardiac infarction (MI) model to confirm NO-induced protein modification and its role in cardiac beating. Two tyrosine (Tyr) residues of β2-tubulin (Y106 and Y340) underwent nitrosylation (Tyr-NO) by endogenously generated NO during cardiomyocyte differentiation from pre-cardiomyocyte-like P19CL6 cells. Tyr-NO-β2-tubulin mediated the interaction with Stathmin, which promotes microtubule disassembly, and was prominently observed in spontaneously beating cell clusters and mouse embryonic heart (E11.5d). In myocardial infarction mice, Tyr-NO-β2-tubulin in transplanted cells was closely related with cardiac troponin-T expression with their functional recovery, reduced infarct size and thickened left ventricular wall. This is the first discovery of a new target molecule of NO, β2-tubulin, that can promote normal cardiac beating and cardiomyocyte regeneration. Taken together, we suggest therapeutic potential of Tyr-NO-β2-tubulin, for ischemic cardiomyocyte, which can reduce unexpected side effect of stem cell transplantation, arrhythmogenesis.

Chemical Underpinnings of Alzheimer’s Disease Symptoms and Causative Factors: A Systematic Review

The brain depends on a complex network of chemical interactions to maintain its homeostasis and functionality. Accordingly, the pathology of neurodegenerative diseases, such as Alzheimer’s disease (AD), has been shown to disrupt the chemical mechanisms that support the brain to carry out its degenerative effect. However, the nature of these disruptions remain elusive. AD is biologically defined by the deposition of Amyloid-Beta (Aβ) plaques and tau neurofibrillary tangles, and has been shown to be strongly correlated with the expression of the Apolipoprotein E4 (ApoE4) allele. Recent findings in the field of neurochemistry indicate the chemical underpinnings of hallmark signs of AD. This review will discuss the process of pathogenesis of AD, from the formation to the causal effects of AD markers, through a chemical lens. The fibrillization of Aβ isoforms as well as the fluent molecular mixing of tau isoforms and hyperphosphorylation of tau observed in mouse lines are discussed as precursor processes to Aβ plaques and tau neurofibrillary tangles. Furthermore, the pathology of Aβ plaques through redox chemistry in the brain, tau neurofibrillary tangles through microtubule disassembly, and ApoE4 through reactive oxygen species (ROS) formation are also presented. Current therapeutic approaches targeted towards specific facets of AD, such as Aducanumab, have found moderate success in treatment, and this progress indicates neurochemical pathways as a potential target for future therapeutic procedures to counteract the degenerative effects of AD.

Prediction of anti-microtubular target proteins of tubulins and their interacting proteins using Gene Ontology tools

BackgroundTubulins are highly conserved globular proteins involved in stabilization of cellular cytoskeletal microtubules during cell cycle. Different isoforms of tubulins are differentially expressed in various cell types, and their protein–protein interactions (PPIs) analysis will help in identifying the anti-microtubular drug targets for cancer and neurological disorders. Numerous web-based PPIs analysis methods are recently being used, and in this paper, I used Gene Ontology (GO) tools, e.g., Stringbase, ProteomeHD, GeneMANIA, and ShinyGO, to identify anti-microtubular target proteins by selecting strongly interacting proteins of tubulins. ResultsI used 6 different human tubulin isoforms (two from each of α-, β-, and γ-tubulin) and found several thousands of node-to-node protein interactions (highest 4956 in GeneMANIA) and selected top 10 strongly interacting node-to-node interactions with highest score, which included 7 tubulin family protein and 6 non-tubulin family proteins (total 13). Functional enrichment analysis indicated a significant role of these 13 proteins in nucleation, polymerization or depolymerization of microtubules, membrane tethering and docking, dorsal root ganglion development, mitotic cycle, and cytoskeletal organization. I found γ-tubulins (TUBG1, TUBGCP4, and TUBBGCP6) were known to contribute majorly for tubulin-associated functions followed by α-tubulin (TUBA1A) and β-tubulins (TUBB AND TUBB3). In PPI results, I found several non-tubular proteins interacting with tubulins, and six of them (HTT, DPYSL2, SKI, UNC5C, NINL, and DDX41) were found closely associated with their functions. ConclusionsIncreasing number of regulatory proteins and subpopulation of tubulin proteins are being reported with poor understanding in their association with microtubule assembly and disassembly. The functional enrichment analysis of tubulin isoforms using recent GO tools resulted in identification of γ-tubulins playing a key role in microtubule functions and observed non-tubulin family of proteins HTT, DPYSL2, SKI, UNC5C, NINL, and DDX41 strongly interacting functional proteins of tubulins. The present study yields a promising model system using GO tools to narrow down tubulin-associated proteins as a drug target in cancer, Alzheimer’s, neurological disorders, etc.

PARP deficiency causes hypersensitivity to Taxol through oxidative stress induced DNA damage

Taxol is an antitumor drug derived from the bark of the Pacific Yew tree that inhibits microtubule disassembly, resulting in cell cycle arrest in late G2 and M phases. Additionally, Taxol increases cellular oxidative stress by generating reactive oxygen species. We hypothesized that the inhibition of specific DNA repair machinery/mechanisms would increase cellular sensitivity to the oxidative stress capacity of Taxol. Initial screening using Chinese hamster ovary (CHO) cell lines demonstrated that base excision repair deficiency, especially PARP deficiency, caused cellular Taxol hypersensitivity. Taxane diterpenes-containing Taxus yunnanensis extract also showed hypertoxicity in PARP deficient cells, which was consistent with other microtubule inhibitors like colcemid, vinblastine, and vincristine. Acute exposure of 50 nM Taxol treatment induced both significant cytotoxicity and M-phase arrest in PARP deficient cells, but caused neither significant cytotoxicity nor late G2-M cell cycle arrest in wild type cells. Acute exposure of 50 nM Taxol treatment induced oxidative stress and DNA damage. The antioxidant Ascorbic acid 2 glucoside partially reduced the cytotoxicity of Taxol in PARP deficient cell lines. Finally, the PARP inhibitor Olaparib increased cytotoxicity of Taxol in wild type CHO cells and two human cancer cell lines. Our study clearly demonstrates that cytotoxicity of Taxol would be enhanced by inhibiting PARP function as an enzyme implicated in DNA repair for oxidative stress.

Tubulysins are Essential for the Preying of Ciliates by Myxobacteria.

Tubulysins are bioactive secondary metabolites produced by myxobacteria that promote microtubule disassembly. Microtubules are required for protozoa such as Tetrahymena to form cilia and flagella. To study the role of tubulysins in myxobacteria, we co-cultured myxobacteria and Tetrahymena. When 4000 Tetrahymena thermophila and 5.0×108 myxobacteria were added to 1 ml of CYSE medium and co-cultured for 48 h, the population of T. thermophila increased to more than 75,000. However, co-culturing tubulysin-producing myxobacteria, including Archangium gephyra KYC5002, with T. thermophila caused the population of T. thermophila to decrease from 4000 to less than 83 within 48 h. Almost no dead bodies of T. thermophila were observed in the culture medium. Co-culturing of T. thermophila and the A. gephyra KYC5002 strain with inactivation of the tubulysin biosynthesis gene led to the population of T. thermophila increasing to 46,667. These results show that in nature, most myxobacteria are preyed upon by T. thermophila, but some myxobacteria prey on and kill T. thermophila using tubulysins. Adding purified tubulysin A to T. thermophila changed the cell shape from ovoid to spherical and caused cell surface cilia to disappear.

Cytosolic JNK-dependent microtubule reassembly protects Jurkat leukemia cells from selenite-induced apoptosis

BackgroundSelenite at high dosage exhibits great potential in curing tumors. It has been shown that selenite inhibits tumor growth through regulation of microtubule dynamics, however, the exact underlying mechanisms remained to be fully elucidated. Methods & resultsWestern blots were carried out to evaluate expression level of different molecules. Our current study discovered that selenite induced microtubule disassembly, cell cycle arrest and finally resulted in apoptosis in Jurkat leukemia cells, while during this process disassembled tubulins were re-organized after long-term exposure to selenite. Furthermore, JNK was activated in the cytoplasm of selenite-treated Jurkat cells, and inhibition of JNK activity successfully prevented the process of microtubule re-assembly. Moreover, inactivation of JNK further enhanced selenite-induced cell cycle arrest and apoptosis. According to the results from cell counting-8 assay, blockage of microtubule re-assembly by colchicine further inhibited Jurkat cell viability after exposure to selenite. Experiments in a xenograft model also proved that selenite could alter JNK activity, destroy microtubule structure and inhibit cell division in vivo. Moreover, TP53, MAPT and YWHAZ were identified to be three most confident interactors that link JNK to microtubule assembly using PPIs analysis. ConclusionOur study indicated that cytosolic JNK-dependent microtubule re-organization took a protective function during selenite-induced apoptosis, while inhibition of this process would finally enhance the anti-tumor effect of selenite.

Transient changes in endothelial and epithelial cell cytoskeleton and morphology upon olfactory receptor signaling

Olfactory receptors (ORs) are expressed in various non-nasal tissues; however, their physiological roles are largely unknown. ORs are G-protein coupled receptors (GPCRs), and there is a well-established link between different classes of G-proteins and cytoskeletal structure changes affecting cellular morphology that has been unexplored after odorant binding. Thus, the present study was conducted to determine if odorant binding in non-olfactory cells causes cytoskeletal changes that lead to cell morphological changes. To this end, human umbilical vein endothelial cells (HUVECs), which express OR10J5, and human keratinocyte (HaCaT) cells, which express OR2AT4, were used to conduct this study. Using these two different cell lines, it was shown that odorant addition, lyral and Sandalore, respectively, caused a dose-dependent increase in cAMP. Furthermore, HUVECs exposed to lyral exhibited a disassembly of the tubulin-containing microtubules and an increase in cortical actin microfilaments, whereas HaCaT cell exposure to Sandalore exhibited both a disassembly of the tubulin-containing microtubules and cortical actin microfilaments. Even though the endothelial and epithelial cell lines had different cytoskeletal responses to odorant exposure, both exhibited a change in cellular morphology as detected by a decrease in electrical resistance using Electric Cell-substrate Impedance Sensing (ECIS; Applied Biophysics, Inc., Troy, NY). Interestingly, these odorant-induced cytoskeletal and morphological changes occurred rapidly (under 1 hour) but returned to normal after 20 hours, highlighting the complexity of olfactory signaling, which may involve desensitization of the signaling pathway and/or internalization of the odorant receptor. Future experiments will further elucidate the olfactory receptor signaling mechanism in endothelial and epithelial cells so that their physiological role can be fully understood. Funded by Army Research Office STTR #W911NF-19-P-0014 and W911NF-20-C-0014, the SUNY Cortland Undergraduate Research Council, and Research and Sponsored Programs. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Causes, costs and consequences of kinesin motors communicating through the microtubule lattice.

Microtubules are critical for a variety of important functions in eukaryotic cells. During intracellular trafficking, molecular motor proteins of the kinesin superfamily drive the transport of cellular cargoes by stepping processively along the microtubule surface. Traditionally, the microtubule has been viewed as simply a track for kinesin motility. New work is challenging this classic view by showing that kinesin-1 and kinesin-4 proteins can induce conformational changes in tubulin subunits while they are stepping. These conformational changes appear to propagate along the microtubule such that the kinesins can work allosterically through the lattice to influence other proteins on the same track. Thus, the microtubule is a plastic medium through which motors and other microtubule-associated proteins (MAPs) can communicate. Furthermore, stepping kinesin-1 can damage the microtubule lattice. Damage can be repaired by the incorporation of new tubulin subunits, but too much damage leads to microtubule breakage and disassembly. Thus, the addition and loss of tubulin subunits are not restricted to the ends of the microtubule filament but rather, the lattice itself undergoes continuous repair and remodeling. This work leads to a new understanding of how kinesin motors and their microtubule tracks engage in allosteric interactions that are critical for normal cell physiology.