Regulates Microtubule Stability Research Articles

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.

STMN1 Promotes Tumor Metastasis in Non-small Cell Lung Cancer Through Microtubule-dependent And Nonmicrotubule-dependent Pathways.

The relationship between STMN1 and cancer metastasis is controversial. The purpose of this study was to explore the role and mechanism of STMN1 in NSCLC metastasis. In this study, we reported that STMN1 was highly expressed in NSCLC tissues and associated with poor prognosis. Both in vivo and in vitro functional assays confirmed that STMN1 promoted NSCLC metastasis. Further studies confirmed that STMN1 promoted cell migration by regulating microtubule stability. The results of Co-IP and LC‒MS/MS illustrated that STMN1 interacts with HMGA1. HMGA1 decreases microtubule stability by regulating the phosphorylation level of STMN1 at Ser16 and Ser38 after interacting with STMN1. This result suggested that STMN1 could be activated by HMGA1 to further promote NSCLC metastasis. Meanwhile, it has been found that STMN1 could promote cell migration by activating the p38MAPK/STAT1 signaling pathway, which is not dependent on microtubule stability. However, activating p38MAPK can decrease microtubule stability by promoting the dephosphorylation of STMN1 at ser16. A positive feedback loop was formed between STMN1 and p38MAPK to synergistically promote cell migration. In summary, our study demonstrated that STMN1 could promote NSCLC metastasis through microtubule-dependent and nonmicrotubule-dependent mechanisms. STMN1 has the potential to be a therapeutic target to inhibit metastasis.

17β-estradiol mitigates the inhibition of SH-SY5Y cell differentiation through WNT1 expression

17β-estradiol (E2) and canonical WNT-signaling represent crucial regulatory pathways for microtubule dynamics and synaptic formation. However, it is unclear yet whether E2-induced canonical WNT ligands have significant impact on neurogenic repair under inflammatory condition. In this study, first, we prepared the chronic activated-microglial-conditioned media, known to be comprised of neuro-inflammatory components. Long term exposure of microglial conditioned media to SH-SY5Y cells showed a negative impact on differentiation markers, microtubule associated protein-2 (MAP2) and synaptophysin (SYP), which was successfully rescued by pre and co-treatment of 10 nM 17β-estradiol. The inhibition of estrogen receptors, ERα and ERβ significantly blocked the E2-mediated recovery in the expression of differentiation marker, SYP. Furthermore, the inflammatory inhibition of canonical signaling ligand, WNT1 was also found to be rescued by E2. To our surprise, E2 was unable to replicate this success with β-catenin, which is considered to be the intracellular transducer of canonical WNT signaling. However, WNT antagonist - Dkk1 blocked the E2-mediated recovery in the expression of the differentiation marker, MAP2. Therefore, our data suggests that E2-mediated recovery in SH-SY5Y differentiation follows a divergent pathway from the conventional canonical WNT signaling pathway, which seems to regulate microtubule stability without the involvement of β-catenin. This mechanism provides fresh insight into how estradiol contributes to the restoration of differentiation marker proteins in the context of chronic neuroinflammation.

Slain2 attenuates brain injury following subarachnoid hemorrhage by controlling axonal microtubule structure in mice

Neuronal injury is accountable for the poor outcome of SAH patients. In this study, oxyhemoglobin (oxyHb) was used to treat cultured primary neurons to simulate SAH, while the SAH model was established by vascular puncture in mice. First, proteomics analysis and western blot assays showed Slain2 as an increased factor in neurons exposed to oxyHb treatment, which has been reported to play an important role in axonal development by regulating microtubule stability. Upregulation of neuronal Slain2 was also detected in the murine SAH model compared with sham surgery. In addition, there was no sex difference in the protein level of Slain2 in either the sham-operated or SAH groups. Furthermore, Slain2 overexpression rescued SAH-induced sensorimotor impairments in mice, while Slain2 knockdown had the opposite effect. Finally, Slain2 overexpression rescued SAH-induced axonal injury both in vivo and in vitro, which was exacerbated by Slain2 knockdown. Thus, we demonstrate here that Slain2 acts as an endogenous protective factor of neuronal axonal microtubule structure, which plays a key role in the protection against SAH-induced neuronal axonal injury. Facilitated axonal microtubule structure by Slain2 overexpression may reduce SAH-induced axonal injury and neurobehavioral dysfunction.

UNC-116 and UNC-16 function with the NEKL-3 kinase to promote axon targeting.

KIF5C is a kinesin-1 heavy chain that has been associated with neurodevelopmental disorders. Although the roles of kinesin-1 in axon transport are well known, little is known about how it regulates axon targeting. We report that UNC-116/KIF5C functions with the NEKL-3/NEK6/7 kinase to promote axon targeting in Caenorhabditis elegans. Loss of UNC-116 causes the axon to overshoot its target and UNC-116 gain-of-function causes premature axon termination. We find that loss of the UNC-16/JIP3 kinesin-1 cargo adaptor disrupts axon termination, but loss of kinesin-1 light chain function does not affect axon termination. Genetic analysis indicates that UNC-16 functions with the NEKL-3 kinase to promote axon termination. Consistent with this observation, imaging experiments indicate that loss of UNC-16 and UNC-116 disrupt localization of NEKL-3 in the axon. Moreover, genetic interactions suggest that NEKL-3 promotes axon termination by functioning with RPM-1, a ubiquitin ligase that regulates microtubule stability in the growth cone. These observations support a model where UNC-116 functions with UNC-16 to promote localization of NEKL-3 in the axon. NEKL-3, in turn, functions with the RPM-1 ubiquitin ligase to promote axon termination.

In Silico Exploration of Microtubule Agent Griseofulvin and Its Derivatives Interactions with Different Human β-Tubulin Isotypes

Tubulin isotypes are known to regulate microtubule stability and dynamics, as well as to play a role in the development of resistance to microtubule-targeted cancer drugs. Griseofulvin is known to disrupt cell microtubule dynamics and cause cell death in cancer cells through binding to tubulin protein at the taxol site. However, the detailed binding mode involved molecular interactions, and binding affinities with different human β-tubulin isotypes are not well understood. Here, the binding affinities of human β-tubulin isotypes with griseofulvin and its derivatives were investigated using molecular docking, molecular dynamics simulation, and binding energy calculations. Multiple sequence analysis shows that the amino acid sequences are different in the griseofulvin binding pocket of βI isotypes. However, no differences were observed at the griseofulvin binding pocket of other β-tubulin isotypes. Our molecular docking results show the favorable interaction and significant affinity of griseofulvin and its derivatives toward human β-tubulin isotypes. Further, molecular dynamics simulation results show the structural stability of most β-tubulin isotypes upon binding to the G1 derivative. Taxol is an effective drug in breast cancer, but resistance to it is known. Modern anticancer treatments use a combination of multiple drugs to alleviate the problem of cancer cells resistance to chemotherapy. Our study provides a significant understanding of the involved molecular interactions of griseofulvin and its derivatives with β-tubulin isotypes, which may help to design potent griseofulvin analogues for specific tubulin isotypes in multidrug-resistance cancer cells in future.

A live-cell marker to visualize the dynamics of stable microtubules throughout the cell cycle.

The microtubule (MT) cytoskeleton underlies processes such as intracellular transport and cell division. Immunolabeling for posttranslational modifications of tubulin has revealed the presence of different MT subsets, which are believed to differ in stability and function. Whereas dynamic MTs can readily be studied using live-cell plus-end markers, the dynamics of stable MTs have remained obscure due to a lack of tools to directly visualize these MTs in living cells. Here, we present StableMARK (Stable Microtubule-Associated Rigor-Kinesin), a live-cell marker to visualize stable MTs with high spatiotemporal resolution. We demonstrate that a rigor mutant of Kinesin-1 selectively binds to stable MTs without affecting MT organization and organelle transport. These MTs are long-lived, undergo continuous remodeling, and often do not depolymerize upon laser-based severing. Using this marker, we could visualize the spatiotemporal regulation of MT stability before, during, and after cell division. Thus, this live-cell marker enables the exploration of different MT subsets and how they contribute to cellular organization and transport.

Characterizing tubulin interactions with the proline rich region of tau.

Tau is a microtubule-associated protein thought to function in the regulation of microtubule stability and dynamics. Normally, tau plays an important role in modulating axonal microtubules in neurons, where it is highly expressed. Intracellular tau aggregates are found in a broad class of disorders, including Alzheimer's Disease, termed tauopathies. As an intrinsically disordered protein, tau lacks stable secondary and tertiary structure, and this structural disorder is maintained even when binding to soluble tubulin and microtubules. Multiple tau-tubulin binding sites have also been identified, spanning the proline-rich region (PRR), microtubule binding repeats (MTBR: R1-R4), and pseudo-repeat, R′. Although dozens of post-translational modifications have been identified on tau, phosphorylation, and specifically hyperphosphorylation, of tau is correlated with disease and alterations to microtubule binding. Intriguingly, potential phosphorylation sites also cluster with high frequency within the PRR. Our lab has recently demonstrated that the isolated PRR both binds tubulin and has tubulin polymerization capacity. Here, we use fluorescence correlation spectroscopy (FCS) in conjunction with bulk polymerization measurements to characterize the impact of phosphomimic mutations in the PRR on tubulin binding and polymerization. We find that single phosphomimics affect tubulin binding nominally, with T231E having the most significant impact. However, as phosphorylation events rarely occur in isolation on tau, we also tested combinatorial effects. We found that multiple phosphomimics alter tubulin binding and microtubule polymerization. Furthermore, we used mass spectrometry to map the regions of the PRR involved in tubulin binding. Together these measurements provide insight into previously overlooked relevance of tau's PRR in functional interactions with tubulin.

The OPEN STOMATA1-SPIRAL1 module regulates microtubule stability during abscisic acid-induced stomatal closure in Arabidopsis.

Drought stress triggers abscisic acid (ABA) signaling in guard cells and induces stomatal closure to prevent water loss in land plants. Stomatal movement is accompanied by reorganization of the cytoskeleton. Cortical microtubules disassemble in response to ABA, which is required for stomatal closure. However, how ABA signaling regulates microtubule disassembly is unclear, and the microtubule-associated proteins (MAPs) involved in this process remain to be identified. In this study, we show that OPEN STOMATA 1 (OST1), a central component in ABA signaling, mediates microtubule disassembly during ABA-induced stomatal closure in Arabidopsis thaliana. We identified the MAP SPIRAL1 (SPR1) as the substrate of OST1. OST1 interacts with and phosphorylates SPR1 at Ser6, which promotes the disassociation of SPR1 from microtubules and facilitates microtubule disassembly. Compared with the wild type, the spr1 mutant exhibited significantly greater water loss and reduced ABA responses, including stomatal closure and microtubule disassembly in guard cells. These phenotypes were restored by introducing the phosphorylated active form of SPR1. Our findings demonstrate that SPR1 positively regulates microtubule disassembly during ABA-induced stomatal closure, which depends on OST1-mediated phosphorylation. These findings reveal a specific connection between a core component of ABA signaling and MAPs.

Dynamin-2 regulates microtubule stability via an endocytosis-independent mechanism.

Microtubule stability and dynamics regulations are essential for vital cellular processes, such as microtubule-dependent axonal transport. Dynamin is involved in many membrane fission events, such as clathrin-mediated endocytosis. The ubiquitously expressed dynamin-2 has been reported to regulate microtubule stability. However, the underlying molecular mechanisms remain unclear. This study aimed to investigate the roles of intrinsic properties of dynamin-2 on microtubule regulation by rescue experiments. A heterozygous DNM2 mutation in HeLa cells was generated, and an increase in the level of stabilized microtubules in these heterozygous cells was observed. The expression of wild-type dynamin-2 in heterozygous cells reduced stabilized microtubules. Conversely, the expression of self-assembly-defective mutants of dynamin-2 in the heterozygous cells failed to decrease stabilized microtubules. This indicated that the self-assembling ability of dynamin-2 is necessary for regulating microtubule stability. Moreover, the heterozygous cells expressing the GTPase-defective dynamin-2 mutant, K44A, reduced microtubule stabilization, similar to the cells expressing wild-type dynamin-2, suggesting that GTPase activity of dynamin-2 is not essential for regulating microtubule stability. These results showed that the mechanism of microtubule regulation by dynamin-2 is diverse from that of endocytosis.

Kinase Activity of PAR1b, Which Mediates Nuclear Translocation of the BRCA1 Tumor Suppressor, Is Potentiated by Nucleic Acid-Mediated PAR1b Multimerization.

PAR1b is a cytoplasmic serine/threonine kinase that controls cell polarity and cell–cell interaction by regulating microtubule stability while mediating cytoplasmic-to-nuclear translocation of BRCA1. PAR1b is also a cellular target of the CagA protein of Helicobacter pylori, which leads to chronic infection causatively associated with the development of gastric cancer. The CagA-PAR1b interaction inactivates the kinase activity of PAR1b and thereby dampens PAR1b-mediated BRCA1 phosphorylation, which reduces the level of nuclear BRCA1 and thereby leads to BRCAness and BRCAness-associated genome instability underlying gastric carcinogenesis. While PAR1b can multimerize within the cells, little is known about the mechanism and functional role of PAR1b multimerization. We found in the present study that PAR1b was multimerized in vitro by binding with nucleic acids (both single- and double-stranded DNA/RNA) via the spacer region in a manner independent of nucleic-acid sequences, which markedly potentiated the kinase activity of PAR1b. Consistent with these in vitro observations, cytoplasmic introduction of double-stranded DNA or expression of single-stranded RNA increased the PAR1b kinase activity in the cells. These findings indicate that the cytoplasmic DNA/RNA contribute to nuclear accumulation of BRCA1 by constitutively activating/potentiating cytoplasmic PAR1b kinase activity, which is subverted in gastric epithelial cells upon delivery of H. pylori CagA oncoprotein.

The Structure of Tau Proteins and Its Role in Neurodegenerative Disease

Abundant in the nerve cells of the central nervous system, tau proteins primarily serve to help maintain the stabilization of microtubules in axons (Michalicova et al., 2020). These microtubules are essential for the rigid structure of the cell and allow for intracellular transport of nutrients and other molecules (Parker et al., 2014). Tau proteins, using polymerization, regulate microtubule stability by binding between the tubulin heterodimers, which form the microtubules. Irregular interaction between tau proteins and microtubules can lead to associated neurodegenerative diseases called tauopathies. While there are many tauopathies, such as frontotemporal dementia with parkinsonism‐17 and Pick disease, Alzheimer’s is one of the most prevalent and studied diseases associated with tau (Gao et al., 2018). Tau protein aggression causes neurofibrillary tangles that deteriorate the microtubules’ structure, thus eventually blocking the synaptic communication between neurons (NIH National Institute on Aging, 2017). Over the past few years, numerous research has been conducted for potential tau immunotherapies, raising hopes for a possible solution (Congdon & Sigurdsson, 2018). Microtubule‐stabilizing drugs may also be instrumental in easing neurodegenerative diseases (Kadavath et al., 2015). We will, therefore, investigate the relationship between tau proteins and the development of these diseases.

The evolution of microtubule associated proteins \u2013 a reference proteomic perspective

Microtubule associated proteins (MAPs), defined as proteins that bind microtubules but are not molecular motors or severing enzymes, play a key role in regulating microtubule stability in neurons. Existing studies of the evolutionary relationships between these proteins are limited to genomic data from a small number of species. We therefore used a large collection of publicly available reference-quality eukaryotic proteomes to carry out a phylogenetic analysis of microtubule associated proteins in both vertebrates and invertebrates. Complete or near-complete reference quality proteomes were obtained from Uniprot. Microtubule associated proteins were identified using InterProtScan, aligned using MUSCLE and then phylogenetic trees constructed using the WAG algorithm. We identified 889 proteins with tubulin binding domains, of which 663 were in eukaryotes, including 168 vertebrates and 64 invertebrates. The vertebrate proteins separated into three families, resembling human MAP 2, MAP4 and MAPT, respectively, while invertebrate MAPs clustered separately. We found significant variation in number of microtubule associated proteins and number of microtubule binding domains between taxa, with fish and mollusks having an unexpectedly high number of MAPs and binding domains, respectively. Our findings represent a novel analysis of the evolution of microtubule associated proteins based on publicly available proteomics data sets. We were able to confirm the phylogeny of MAPs identified based on more limited genomic analyses, and in addition, derived several novel insights on the structure and function of MAPs.

Acetylated α-tubulin K394 regulates microtubule stability to shape the growth of axon terminals

Microtubules are essential to neuron shape and function. Acetylation of tubulin has the potential to directly tune the behavior and function of microtubules in cells. Although proteomic studies have identified several acetylation sites in α-tubulin, the effects of acetylation at these sites remains largely unknown. This includes the highly conserved residue lysine 394 (K394), which is located at the αβ-tubulin dimer interface. Using a fly model, we show that α-tubulin K394 is acetylated in the nervous system and is an essential residue. We found that an acetylation-blocking mutation in endogenous α-tubulin, K394R, perturbs the synaptic morphogenesis of motoneurons and reduces microtubule stability. Intriguingly, the K394R mutation has opposite effects on the growth of two functionally and morphologically distinct motoneurons, revealing neuron-type-specific responses when microtubule stability is altered. Eliminating the deacetylase HDAC6 increases K394 acetylation, and the over-expression of HDAC6 reduces microtubule stability similar to the K394R mutant. Thus, our findings implicate α-tubulin K394 and its acetylation in the regulation of microtubule stability and suggest that HDAC6 regulates K394 acetylation during synaptic morphogenesis.

Enhancing mask activity in dopaminergic neurons extends lifespan in flies.

Dopaminergic neurons (DANs) are essential modulators for brain functions involving memory formation, reward processing, and decision‐making. Here I demonstrate a novel and important function of the DANs in regulating aging and longevity. Overexpressing the putative scaffolding protein Mask in two small groups of DANs in flies can significantly extend the lifespan in flies and sustain adult locomotor and fecundity at old ages. This Mask‐induced beneficial effect requires dopaminergic transmission but cannot be recapitulated by elevating dopamine production alone in the DANs. Independent activation of Gαs in the same two groups of DANs via the drug‐inducible DREADD system also extends fly lifespan, further indicating the connection of specific DANs to aging control. The Mask‐induced lifespan extension appears to depend on the function of Mask to regulate microtubule (MT) stability. A structure–function analysis demonstrated that the ankyrin repeats domain in the Mask protein is both necessary for regulating MT stability (when expressed in muscles and motor neurons) and sufficient to prolong longevity (when expressed in the two groups of DANs). Furthermore, DAN‐specific overexpression of Unc‐104 or knockdown of p150Glued, two independent interventions previously shown to impact MT dynamics, also extends lifespan in flies. Together, these data demonstrated a novel DANs‐dependent mechanism that, upon the tuning of their MT dynamics, modulates systemic aging and longevity in flies.

α-tubulin tail modifications regulate microtubule stability through selective effector recruitment, not changes in intrinsic polymer dynamics.

Microtubules are non-covalent polymers of αβ-tubulin dimers. Posttranslational processing of the intrinsically disordered C-terminal α-tubulin tail produces detyrosinated and Δ2-tubulin. Although these are widely employed as proxies for stable cellular microtubules, their effect (and of the α-tail) on microtubule dynamics remains uncharacterized. Using recombinant, engineered human tubulins, we now find that neither detyrosinated nor Δ2-tubulin affect microtubule dynamics, while the α-tubulin tail is an inhibitor of microtubule growth. Consistent with the latter, molecular dynamics simulations show the α-tubulin tail transiently occluding the longitudinal microtubule polymerization interface. The marked differential invivo stabilities of the modified microtubule subpopulations, therefore, must result exclusively from selective effector recruitment. We find that tyrosination quantitatively tunes CLIP-170 density at the growing plus end and that CLIP170 and EB1 synergize to selectively upregulate the dynamicity of tyrosinated microtubules. Modification-dependent recruitment of regulators thereby results in microtubule subpopulations with distinct dynamics, a tenet of the tubulin code hypothesis.

Novel function of N-acetyltransferase for microtubule stability and JNK signaling in Drosophila organ development

Regulation of microtubule stability is crucial for the maintenance of cell structure and function. While the acetylation of α-tubulin lysine 40 by acetylase has been implicated in the regulation of microtubule stability, the in vivo functions of N-terminal acetyltransferases (NATs) involved in the acetylation of N-terminal amino acids are not well known. Here, we identify an N-terminal acetyltransferase, Mnat9, that regulates cell signaling and microtubule stability in Drosophila Loss of Mnat9 causes severe developmental defects in multiple tissues. In the wing imaginal disc, Mnat9 RNAi leads to the ectopic activation of c-Jun N-terminal kinase (JNK) signaling and apoptotic cell death. These defects are suppressed by reducing the level of JNK signaling. Overexpression of Mnat9 can also inhibit JNK signaling. Mnat9 colocalizes with mitotic spindles, and its loss results in various spindle defects during mitosis in the syncytial embryo. Furthermore, overexpression of Mnat9 enhances microtubule stability. Mnat9 is physically associated with microtubules and shows a catalytic activity in acetylating N-terminal peptides of α- and β-tubulin in vitro. Cell death and tissue loss in Mnat9-depleted wing discs are restored by reducing the severing protein Spastin, suggesting that Mnat9 protects microtubules from its severing activity. Remarkably, Mnat9 mutated in the acetyl-CoA binding site is as functional as its wild-type form. We also find that human NAT9 can rescue Mnat9 RNAi phenotypes in flies, indicating their functional conservation. Taken together, we propose that Mnat9 is required for microtubule stability and regulation of JNK signaling to promote cell survival in developing Drosophila organs.

DRD1 downregulation contributes to mechanical stretch-induced lung endothelial barrier dysfunction

Rationale: The lung-protective effects of dopamine and its role in the pathology of ventilator-induced lung injury (VILI) are emerging. However, the underlying mechanisms are still largely unknown.Objective: To investigate the contribution of dopamine receptor dysregulation in the pathogenesis of VILI and therapeutic potential of dopamine D1 receptor (DRD1) agonist in VILI.Methods: The role of dopamine receptors in mechanical stretch-induced endothelial barrier dysfunction and lung injury was studied in DRD1 knockout mice, in isolated mouse lung vascular endothelial cells (MLVECs), and in lung samples from patients who underwent pulmonary lobectomy with mechanical ventilation for different time periods.Measurements and Main Results: DRD1 was downregulated in both surgical patients and mice exposed to mechanical ventilation. Prophylactic administration of dopamine or DRD1 agonist attenuated mechanical stretch-induced lung endothelial barrier dysfunction and lung injury. By contrast, pulmonary knockdown or global knockout of DRD1 exacerbated these effects. Prophylactic administration of dopamine attenuated mechanical stretch-induced α-tubulin deacetylation and subsequent endothelial hyperpermeability through DRD1 signaling. We identified that cyclic stretch-induced glycogen-synthase-kinase-3β activation led to phosphorylation and activation of histone deacetylase 6 (HDAC6), which resulted in deacetylation of α-tubulin. Upon activation, DRD1 signaling attenuated mechanical stretch-induced α-tubulin deacetylation and subsequent lung endothelial barrier dysfunction through cAMP/exchange protein activated by cAMP (EPAC)-mediated inactivation of HDAC6.Conclusions: This work identifies a novel protective role for DRD1 against mechanical stretch-induced lung endothelial barrier dysfunction and lung injury. Further study of the mechanisms involving DRD1 in the regulation of microtubule stability and interference with DRD1/cAMP/EPAC/HDAC6 signaling may provide insight into therapeutic approaches for VILI.

Ex-vivo Microtubule Stability Assay Using Drosophila Wing Disc.

Regulation of microtubule stability is crucial for diverse biological processes, including cell division, morphogenesis, and signaling. Various in vitro assays for microtubule stability have been developed to identify and characterize proteins involved in controlling microtubule stability. Here, we introduce a simple ex-vivo assay for identifying potential microtubule regulators in the wing imaginal disc of Drosophila melanogaster. This assay utilizes silicon rhodamine-tubulin (SiR-Tub) as a cell-permeable fluorogenic dye for labeling microtubules. In an attempt to increase the sensitivity of the screen, we designed an assay using a sensitized microtubule condition. Wing discs are treated with SiR-Tub followed by demecolcine, a microtubule inhibitor, to partially label impaired microtubules. Under this sensitized condition, we can test whether overexpression or downregulation of a gene can enhance or suppress the weakened SiR-Tub labeling. This assay allows highly sensitive detection of microtubules in developing larval tissues. Hence, it provides a useful tool for identifying new microtubule regulators in both unfixed and fixed imaginal discs in Drosophila. This strategy may also be applied to characterize microtubule regulators in tissues from other model organisms. Graphic abstract: Graphical summary of Ex-vivo microtubule stability assay using Drosophila wing disc.

NEAT1 regulates microtubule stabilization via FZD3/GSK3β/P-tau pathway in SH-SY5Y cells and APP/PS1 mice.

Nuclear paraspeckles assembly transcript 1 (NEAT1) is a well-known long noncoding RNA (lncRNA) with various functions in different physiological and pathological processes. Notably, aberrant NEAT1 expression is implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer’s disease (AD). However, the molecular mechanism of NEAT1 in AD remains poorly understood. In this study, we investigated that NEAT1 regulated microtubules (MTs) polymerization via FZD3/GSK3β/p-tau pathway. Downregulation of NEAT1 inhibited Frizzled Class Receptor 3 (FZD3) transcription activity by suppressing H3K27 acetylation (H3K27Ac) at the FZD3 promoter. Our data also demonstrated that P300, an important histone acetyltransferases (HAT), recruited by NEAT1 to bind to FZD3 promoter and mediated its transcription via regulating histone acetylation. In addition, according to immunofluorescence staining of MTs, metformin, a medicine for the treatment of diabetes mellitus, rescued the reduced length of neurites detected in NEAT1 silencing cells. We suspected that metformin may play a neuroprotective role in early AD by increasing NEAT1 expression and through FZD3/GSK3β/p-tau pathway. Collectively, NEAT1 regulates microtubule stabilization via FZD3/GSK3β/P-tau pathway and influences FZD3 transcription activity in the epigenetic way.