Microtubule Assembly Research Articles

The structure of a Tau fragment bound to tubulin prompts new hypotheses on Tau mechanism and oligomerization.

Tau is a protein involved in the regulation of axonal microtubules in neurons. In pathological conditions, it forms filamentous aggregates which are molecular markers of neurodegenerative diseases known as tauopathies. Structures of Tau in fibrils or bound to the microtubule have been reported. We present here a structure of a Tau construct comprising the PHF6 motif, an oligopeptide involved in Tau aggregation, as a complex with tubulin. This Tau fragment binds as a dimer to a new site which, when transposed to the microtubule, would correspond to a pore between protofilaments. These results raise new hypotheses on Tau-induced microtubule assembly and stabilization and on Tau oligomerization.

The Potential Role of PeMAP65-18 in Secondary Cell Wall Formation in Moso Bamboo.

Microtubule-associated proteins (MAPs) play a pivotal role in the assembly and stabilization of microtubules, which are essential for plant cell growth, development, and morphogenesis. A class of plant-specific MAPs, MAP65, plays largely unexplored roles in moso bamboo (Phyllostachys edulis). This study identified 19 PeMAP65 genes in moso bamboo, systematically examining their phylogenetic relationships, conserved motifs, gene structures, collinearity, and cis-acting elements. Analysis of gene expression indicated that PeMAP65s exhibit tissue-specific expression patterns. Functional differentiation was investigated among the members of different PeMAP65 subfamilies according to their expression patterns in different development stages of bamboo shoots. The expression of PeMAP65-18 was positively correlated with the expression of genes involved in secondary cell wall (SCW) biosynthesis. Y1H and Dual-LUC assays demonstrated that the transcription of PeMAP65-18 was upregulated by PeMYB46, a key transcription factor of SCW biosynthesis. The result of subcellular localization showed that PeMAP65-18 was located in cortical microtubules. We speculate that PeMAP65-18 may play a crucial role in the SCW deposition of moso bamboo. This comprehensive analysis of the MAP65 family offers novel insights into the roles of PeMAP65s in moso bamboo, particularly in relation to the formation of SCWs.

DYRK1A interacts with the tuberous sclerosis complex and promotes mTORC1 activity.

DYRK1A, a ubiquitously expressed kinase, is linked to the dominant intellectual developmental disorder, microcephaly, and Down syndrome in humans. It regulates numerous cellular processes such as cell cycle, vesicle trafficking, and microtubule assembly. DYRK1A is a critical regulator of organ growth; however, how it regulates organ growth is not fully understood. Here, we show that the knockdown of DYRK1A in mammalian cells results in reduced cell size, which depends on mTORC1. Using proteomic approaches, we found that DYRK1A interacts with the tuberous sclerosis complex (TSC) proteins, namely TSC1 and TSC2, which negatively regulate mTORC1 activation. Furthermore, we show that DYRK1A phosphorylates TSC2 at T1462, a modification known to inhibit TSC activity and promote mTORC1 activity. We also found that the reduced cell growth upon knockdown of DYRK1A can be rescued by overexpression of RHEB, an activator of mTORC1. Our findings suggest that DYRK1A inhibits TSC complex activity through inhibitory phosphorylation on TSC2, thereby promoting mTORC1 activity. Furthermore, using the Drosophila neuromuscular junction as a model, we show that the mnb, the fly homologs of DYRK1A, is rescued by RHEB overexpression, suggesting a conserved role of DYRK1A in TORC1 regulation.

A potent Bioorganic azapodophyllotoxin derivative Suppresses tumor Progression in Triple negative breast Cancer: An Insight into its Inhibitory effect on tubulin polymerization and Disruptive effect on microtubule assembly

Triple negative breast cancer (TNBC) has long been a challenging disease owing to its high aggressive behaviour, poor prognosis and its limited treatment options. The growing demand of new therapeutics against TNBC enables us to examine the therapeutic efficiency of an emerging class of anticancer compounds, azapodophyllotoxin derivative (HTDQ), a nitrogen analogue of podophyllotoxin, using different biochemical, spectroscopic and computational approaches. The anticancer activities of HTDQ are studied by performing MTT assay in a dose depended manner on Triple negative breast cancer cells using MDA–MB-468 and MDA-MB-231 cell lines with IC50 value 937 nM and 1.13 µM respectively while demonstrating minimal effect on normal epithelial cells. The efficacy of HTDQ was further tested in 3D tumour spheroids formed by the human TNBC cell line MDA-MB468 and also the murine MMTV positive TNBC cell line 4 T1. The shrinkage that observed in the tumor spheroid clearly indicates that HTDQ remarkably decreases the growth of tumor spheroid thereby affirming its cytotoxicity. The 2D cell viability assay shows significant morphological alteration that possibly caused by the cytoskeleton disturbances. Hence the binding interaction of HTDQ with cytoskeleton protein tubulin, its effect on tubulin polymerisation as well as depolymerisation of preformed microtubules along with the conformational alternation in the protein itself have been investigated in detail. Moreover, the apoptotic effects of HTDQ have been examined using a range of apoptotic markers. HTDQ-treated cancer cells showed increased expression of cleaved PARP-1 and pro-caspase-3, suggesting activation of the apoptosis process. HTDQ also upregulated pro-apoptotic Bax expression while inhibiting anti-apoptotic Bcl2 expression, supporting its ability to induce apoptosis in cancer cells. Hence the consolidated biochemical and spectroscopic research described herein may provide enormous information to use azapodophyllotoxin as promising anticancer therapeutics for TNBC cells.

Citrinin disrupts microtubule assembly in cardiac cells: Impact on mitochondrial organization and function

Citrinin (CTN) is a mycotoxin commonly present in various foods and feeds worldwide, as well as dietary supplements in Asian countries, but the risks and cellular mechanisms associated with its cardiotoxicity remains unclear. In this study, RNA-seq analysis of CTN-treated H9c2 cardiac cells demonstrated significant perturbations in pathways related to microtubule cytoskeleton and mitochondrial network organization. CTN disrupted microtubule polymerization and downregulated mRNA levels of microtubule-assembling genes, Map2 and Tpx2, in H9c2 cardiac cells. Additionally, CTN interfered with the distribution of mitochondrial network along the microtubules, leading to the accumulation of dysfunctional mitochondria characterized by elevated superoxide levels and reduced membrane potential. This disruption also caused the buildup of lysosomes and ubiquitinated proteins, which hindered waste clearance in microtubule-disassembled H9c2 cells. Molecular docking analysis indicated that CTN could bind to the colchicine binding site on β-tubulin, thereby mimicking the microtubule-disrupting effect of colchicine. This study provides morphological, transcriptomic, and mechanistic evidence to elucidate the cardiotoxic mechanisms of CTN, which involve the dysregulated microtubule network, subsequent mitochondrial mislocalization, and impaired proteolysis of damaged proteins/organelles in cardiac cells. Our findings may enhance the fundamental understanding and facilitate future risk assessment of CTN.

Paclitaxel and Vinblastine Increase Plasminogen Activator Inhibitor-1 Production in Human Breast Cancer MCF-7 Cells but Not MDA-MB-231 Cells.

Cancer chemotherapy increases the risk of thrombosis; however, the mechanisms underlying this thrombosis are not completely understood. Plasminogen activator inhibitor (PAI)-1 is a key molecule in the fibrinolytic system that inhibits tissue plasminogen activator and urokinase, which converts plasminogen into plasmin; therefore, excess PAI-1 increases the risk of thrombosis. In this study, we investigated whether temporary treatment of the human luminal A-type breast cancer cell line MCF-7 with antitumor drugs clinically used for breast cancer therapy promotes PAI-1 production. Treatment of MCF-7 cells with paclitaxel (PTX), a microtubule-stabilizing antitumor drug, at 1 µM for 2 h elevated the PAI-1 concentration of the conditioned medium at 48 h after treatment but not in those treated with tamoxifen and cyclophosphamide. Microtubule assembly inhibitors vinblastine (VBT) and vincristine (VCT) also increased the PAI-1 concentration in the conditioned medium. PAI-1 (SERPINE1) expression was upregulated in MCF-7 cells after PTX, VBT, and VCT treatment; this increase in expression persisted for eight days. In contrast, PAI-1 production in MDA-MB-231 cells treated with PTX, VBT, or VCT did not increase with increasing PAI-1 concentration. This study demonstrated that temporary low-dose treatment with microtubule-associated anticancer drugs increased PAI-1 release from MCF-7 cells but not from MDA-MB-231 cells. These results indicate that chemotherapy against luminal A-type breast cancer using microtubule-associated drugs may cause thrombosis through the inhibition of the fibrinolytic system by PAI-1.

Ninein domains required for its localization, association with partners dynein and ensconsin, and microtubule organization.

Ninein (Nin) is a microtubule (MT) anchor at the subdistal appendages of mother centrioles and the pericentriolar material (PCM) of centrosomes that also functions to organize MTs at noncentrosomal MT-organizing centers (ncMTOCs). In humans, the NIN gene is mutated in Seckel syndrome, an inherited developmental disorder. Here, we dissect the protein domains involved in Nin's localization and interactions with dynein and ensconsin (ens/MAP7) and show that the association with ens cooperatively regulates MT assembly in Drosophila fat body cells. We define domains of Nin responsible for its localization to the ncMTOC on the fat body cell nuclear surface, localization within the nucleus, and association with Dynein light intermediate chain (Dlic) and ens, respectively. We show that Nin's association with ens synergistically regulates MT assembly. Together, these findings reveal novel features of Nin function and its regulation of a ncMTOC.

NP-Collabs: Investigating Counterion-Mediated Bridges in the Multiply Phosphorylated Tau-R2 Repeat.

Tau is an intrinsically disordered (IDP) microtubule-associated protein (MAP) that plays a key part in microtubule assembly and organization. The function of tau can be regulated by multiple phosphorylation sites. These post-translational modifications are known to decrease the binding affinity of tau for microtubules, and abnormal tau phosphorylation patterns are involved in Alzheimer's disease. Using all-atom molecular dynamics simulations, we compared the conformational landscapes explored by the tau R2 repeat domain (which comprises a strong tubulin binding site) in its native state and with multiple phosphorylations on the S285, S289, and S293 residues, with four different standard force field (FF)/water model combinations. We find that the different parameters used for the phosphate groups (which can be more or less flexible) in these FFs and the specific interactions between bulk cations and water lead to the formation of a specific type of counterion bridge, termed nP-collab (for nphosphate collaboration, with n being an integer), where counterions form stable structures binding with two or three phosphate groups simultaneously. The resulting effect of nP-collabs on the tau-R2 conformational space differs when using sodium or potassium cations and is likely to impact the peptide overall dynamics and how this MAP interacts with tubulins. We also investigated the effect of phosphoresidue spacing and ionic concentration by modeling polyalanine peptides containing two phosphoserines located one-six residues apart. Three new metrics specifically tailored for IDPs (proteic Menger curvature, local curvature, and local flexibility) were introduced, which allow us to fully characterize the impact of nP-collabs on the dynamics of disordered peptides at the residue level.

Arl2 GTPase associates with the centrosomal protein Cdk5rap2 to regulate cortical development via microtubule organization.

ADP ribosylation factor-like GTPase 2 (Arl2) is crucial for controlling mitochondrial fusion and microtubule assembly in various organisms. Arl2 regulates the asymmetric division of neural stem cells in Drosophila via microtubule growth. However, the function of mammalian Arl2 during cortical development was unknown. Here, we demonstrate that mouse Arl2 plays a new role in corticogenesis via regulating microtubule growth, but not mitochondria functions. Arl2 knockdown (KD) leads to impaired proliferation of neural progenitor cells (NPCs) and neuronal migration. Arl2 KD in mouse NPCs significantly diminishes centrosomal microtubule growth and delocalization of centrosomal proteins Cdk5rap2 and γ-tubulin. Moreover, Arl2 physically associates with Cdk5rap2 by in silico prediction using AlphaFold multimer, which was validated by co-immunoprecipitation and proximity ligation assay. Remarkably, Cdk5rap2 overexpression significantly rescues the neurogenesis defects caused by Arl2 KD. Therefore, Arl2 plays an important role in mouse cortical development through microtubule growth via the centrosomal protein Cdk5rap2.

The structure of the γ-TuRC at the microtubule minus end - not just one solution.

In cells, microtubules (MTs) assemble from α/β-tubulin subunits at nucleation sites containing the γ-tubulin ring complex (γ-TuRC). Within the γ-TuRC, exposed γ-tubulin molecules act as templates for MT assembly by interacting with α/β-tubulin. The vertebrate γ-TuRC is scaffolded by γ-tubulin-interacting proteins GCP2-6 arranged in a specific order. Interestingly, the γ-tubulin molecules in the γ-TuRC deviate from the cylindrical geometry of MTs, raising the question of how the γ-TuRC structure changes during MT nucleation. Recent studies on the structure of the vertebrate γ-TuRC attached to the end of MTs came to varying conclusions. In vitro assembly of MTs, facilitated by an α-tubulin mutant, resulted in a closed, cylindrical γ-TuRC showing canonical interactions between all γ-tubulin molecules and α/β-tubulin subunits. Conversely, native MTs formed in a frog extract were capped by a partially closed γ-TuRC, with some γ-tubulin molecules failing to align with α/β-tubulin. This review discusses these outcomes, along with the broader implications.

Genomic inbreeding analysis reveals resilience and genetic diversity in Indian yak populations

The yak (Bos grunniens), renowned for its adaptability to extreme cold and hypoxic conditions, stands as a remarkable domestic animal crucial for sustaining livelihoods in harsh climates. We conducted a comprehensive analysis of the whole genome sequence data from three distinct Indian yak populations: Arunachali yak (n = 10), Himachali yak (n = 10), and Ladakhi yak (n = 10). The genomic data for Indian yaks were meticulously generated by our laboratory and compared with their Chinese counterpart, the Jinchuan yak (n = 8), for a more nuanced understanding. Our investigation revealed a total of 37,437 runs of homozygosity (ROH) segments in 34 animals representing four distinct yak populations. The Jinchuan yak population exhibited the highest proportion, constituting 80.8 % of total ROHs, predominantly as small segments (<0.1 Mb), accounting for 63 % of the overall ROHs. Further analysis uncovered a significantly higher degree of inbreeding in Chinese yaks compared to their Indian counterparts. The Indian yak populations, in contrast, demonstrated relatively lower and consistent levels of inbreeding. Moreover, we identified ROH hotspots that covered at least 60 % of individuals in our study, indicating their pivotal role in environmental adaptation. A total of five hotspot regions were detected, housing genes such as ENSBGRG00000015023 (WNT2), YIPF4, SPAST, TLN2, and DSG4. These genes are associated with traits including hair follicle initiation, nutrient stress response, microtubule assembly, development of cardiac muscle, hair follicle, and coat color. This observation strongly suggests that there is substantial selection acting on these genes, emphasizing their important role in environmental adaptation among yak populations.

A tubulin-binding protein that preferentially binds to GDP-tubulin and promotes GTP exchange.

α- and β-tubulin form heterodimers, with GTPase activity, that assemble into microtubules. Like other GTPases, the nucleotide-bound state of tubulin heterodimers controls whether the molecules are in a biologically active or inactive state. While α-tubulin in the heterodimer is constitutively bound to GTP, β-tubulin can be bound to either GDP (GDP-tubulin) or GTP (GTP-tubulin). GTP-tubulin hydrolyzes its GTP to GDP following assembly into a microtubule and, upon disassembly, must exchange its bound GDP for GTP to participate in subsequent microtubule polymerization. Tubulin dimers have been shown to exhibit rapid intrinsic nucleotide exchange in vitro, leading to a commonly accepted belief that a tubulin guanine nucleotide exchange factor (GEF) may be unnecessary in cells. Here, we use quantitative binding assays to show that BuGZ, a spindle assembly factor, binds tightly to GDP-tubulin, less tightly to GTP-tubulin, and weakly to microtubules. We further show that BuGZ promotes the incorporation of GTP into tubulin using a nucleotide exchange assay. The discovery of a tubulin GEF suggests a mechanism that may aid rapid microtubule assembly dynamics in cells.

A transient radial cortical microtubule array primes cell division in Arabidopsis

Although the formation of new walls during plant cell division tends to follow maximal tensile stress direction, analyses of individual cells over time reveal a much more variable behavior. The origin of such variability as well as the exact role of interphasic microtubule behavior before cell division have remained mysterious so far. To approach this question, we took advantage of the Arabidopsis stem, where the tensile stress pattern is both highly anisotropic and stable. Although cortical microtubules (CMTs) generally align with maximal tensile stress, we detected a specific time window, ca. 3 h before cell division, where cells form a radial pattern of CMTs. This microtubule array organization preceded preprophase band (PPB) formation, a transient CMT array predicting the position of the future division plane. It was observed under different growth conditions and was not related to cell geometry or polar auxin transport. Interestingly, this cortical radial pattern correlated with the well-documented increase of cytoplasmic microtubule accumulation before cell division. This radial organization was prolonged in cells of the trm678 mutant, where CMTs are unable to form a PPB. Whereas division plane orientation in trm678 is noisier, we found that cell division symmetry was in contrast less variable between daughter cells. We propose that this "radial step" reflects a trade-off in robustness for two essential cell division attributes: symmetry and orientation. This involves a "reset" stage in G2, where an increased cytoplasmic microtubule accumulation transiently disrupts CMT alignment with tissue stress.

Regulatable assembly of synthetic microtubule architectures using engineered microtubule-associated protein-IDR condensates

Microtubule filaments are assembled into higher-order structures using microtubule-associated proteins. However, synthetic MAPs that direct the formation of new structures are challenging to design, as nanoscale biochemical activities must be organized across micron length-scales. Here, we develop modular MAP-IDR condensates (synMAPs) that enable inducible assembly of higher-order microtubule structures for synthetic exploration invitro and in mammalian cells. synMAPs harness a small microtubule-binding domain from oligodendrocytes (TPPP) whose activity we show can be rewired by interaction with unrelated condensate-forming IDR sequences. This combination is sufficient to allow synMAPs to self-organize multivalent structures that bind and bridge microtubules into higher-order architectures. By regulating the connection between the microtubule-binding domain and condensate-forming components of a synMAP, the formation of these structures can be triggered by small molecules or cell-signaling inputs. We systematically test a panel of synMAP circuit designs to define how the assembly of these synthetic microtubule structures can be controlled at the nanoscale (via microtubule-binding affinity) and microscale (via condensate formation). synMAPs thus provide a modular starting point for the design of higher-order microtubule systems and an experimental testbed for exploring condensate-directed mechanisms of higher-order microtubule assembly from the bottom-up.

IQGAP1 domesticates macrophages to favor mycobacteria survival via modulating NF-κB signal and augmenting VEGF secretion

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), still ranks among the leading causes of annual human death by infectious disease. Mtb has developed several strategies to survive for years at a time within the host despite the presence of a robust immune response, including manipulating the progression of the inflammatory response and forming granulomatous lesions. Here we demonstrate that IQGAP1, a highly conserved scaffolding protein, compartmentalizes and coordinates multiple signaling pathways in macrophages infected with Mycobacterium marinum (Mm or M.marinum), the closest relative of Mtb. Upregulated IQGAP1 ultimately suppresses TNF-α production by repressing the MKK3 signal and reducing NF-κBp65 translocation, deactivating the p38MAPK pathway. Accordingly, IQGAP1 silencing and overexpression significantly alter p38MAPK activity by modulating the production of phosphorylated MKK3 during mycobacterial infection. Pharmacological inhibition of IQGAP1-associated microtubule assembly not only alleviates tissue damage caused by M.marinum infection but also significantly decreases the production of VEGF—a critical player for granuloma-associated angiogenesis during pathogenic mycobacterial infection. Similarly, IQGAP1 silencing in Mm-infected macrophages diminishes VEGF production, while IQGAP1 overexpression upregulates VEGF. Our data indicate that mycobacteria induce IQGAP1 to hijack NF-κBp65 activation, preventing the expression of proinflammatory cytokines as well as promoting VEGF production during infection and granuloma formation. Thus, therapies targeting host IQGAP1 may be a promising strategy for treating tuberculosis, particularly in drug-resistant diseases.

Depolymerization of tubulin as the main molecular mechanism of the cytotoxic and antitumor activity of pyrrole-containing heterocyclic compounds

Introduction. Microtubules are highly dynamic polymers of α, β-tubulin dimers involves in a broad spectrum of the processes, such as intracellular transport and cell proliferation. This makes them an attractive molecular target for anti-cancer therapies. Substances that affect the dynamic state of tubulin microtubules are known as the mitotic poisons that are effectiveand widely used in the chemotherapy of various tumors. Mitotic poisons are able to interfere with polymerization (stabilization) or depolymerization of tubulin, which in turn leads to the arrest of cells in the M-phase (named as a mitotic catastrophe) and their subsequent death via activation of apoptotic mechanisms. However, the effectiveness of MP-based therapies is gradually decreasing over the time due to development of multiple drug resistance mechanisms in cancer cells. Thus, development of novel compounds selectively targeting tubulin and effectively overcoming multiple drugresistance phenotype in cancer is an urgent need in current oncology. Aim. To examine the cytotoxic and antitumor activities of several pyrrole-containing heterocyclic compounds (EPC-91, EPC-92 and PCA-93) against cancer cell lines with epithelial and mesenchymal origin, including those with multiple drug resistance phenotype. Materials and methods. Studies were performed on parental human cancer cell lines – triple-negative breast cancer HCC1806, gastrointestinal stromal tumor GIST T-1, osteosarcoma SaOS-2, – sensitive to chemotherapy (paclitaxel, doxorubicin) and their resistant sublines (HCC1806 Tx-R, GIST T-1 Tx-R, SaOS-2 Dox-R), as well as on murine colorectal adenocarcinoma cell line Colon-26, exhibiting primary resistance to the aforementioned chemotherapeutic agents. Results. The cytotoxic activities of EPC-91 and PCA-93 were due to their abilities to depolymerize tubulin. The results of immunofluorescence microscopy and Western blotting indicated that the compounds disrupt assembly of tubulin microtubules and prevent polymerization of α-tubulin in cancer cells. Inhibition of tubulin polymerizations led to significant increasein number of round-shaped and phospho-histone 3 (e. g. mitotic) cells, followed by their death through apoptosis. PCA-93 also exhibited potent anti-tumor effect against Colon-26 cells due to its anti-proliferative and proapoptotic activities. Conclusion. The data shown here illustrates potent cytotoxic activities of EPC-91 and PCA-93 against multiple cancer cell lines in vitro including those with multiple drug resistance phenotype. Similarly, PCA-93 was found to be highly effective against Colon-26 cell in vivo, thereby illustrating the attractive platform for the development of novel pyrrole-based agents exhibiting potent anti-tumor activities.

Oligodendrocyte Dysfunction in Tauopathy: A Less Explored Area in Tau-Mediated Neurodegeneration.

Aggregation of the microtubule-associated protein tau (MAPT) is the hallmark pathology in a spectrum of neurodegenerative disorders collectively called tauopathies. Physiologically, tau is an inherent neuronal protein that plays an important role in the assembly of microtubules and axonal transport. However, disease-associated mutations of this protein reduce its binding to the microtubule components and promote self-aggregation, leading to formation of tangles in neurons. Tau is also expressed in oligodendrocytes, where it has significant developmental roles in oligodendrocyte maturation and myelin synthesis. Oligodendrocyte-specific tau pathology, in the form of fibrils and coiled coils, is evident in major tauopathies including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease (PiD). Multiple animal models of tauopathy expressing mutant forms of MAPT recapitulate oligodendroglial tau inclusions with potential to cause degeneration/malfunction of oligodendrocytes and affecting the neuronal myelin sheath. Till now, mechanistic studies heavily concentrated on elucidating neuronal tau pathology. Therefore, more investigations are warranted to comprehensively address tau-induced pathologies in oligodendrocytes. The present review provides the current knowledge available in the literature about the intricate relations between tau and oligodendrocytes in health and diseases.

Effects of P301L-TAU on post-translational modifications of microtubules in human iPSC-derived cortical neurons and TAU transgenic mice.

JOURNAL/nrgr/04.03/01300535-202508000-00025/figure1/v/2024-09-30T120553Z/r/image-tiff TAU is a microtubule-associated protein that promotes microtubule assembly and stability in the axon. TAU is missorted and aggregated in an array of diseases known as tauopathies. Microtubules are essential for neuronal function and regulated via a complex set of post-translational modifications, changes of which affect microtubule stability and dynamics, microtubule interaction with other proteins and cellular structures, and mediate recruitment of microtubule-severing enzymes. As impairment of microtubule dynamics causes neuronal dysfunction, we hypothesize cognitive impairment in human disease to be impacted by impairment of microtubule dynamics. We therefore aimed to study the effects of a disease-causing mutation of TAU (P301L) on the levels and localization of microtubule post-translational modifications indicative of microtubule stability and dynamics, to assess whether P301L-TAU causes stability-changing modifications to microtubules. To investigate TAU localization, phosphorylation, and effects on tubulin post-translational modifications, we expressed wild-type or P301L-TAU in human MAPT -KO induced pluripotent stem cell-derived neurons (iNeurons) and studied TAU in neurons in the hippocampus of mice transgenic for human P301L-TAU (pR5 mice). Human neurons expressing the longest TAU isoform (2N4R) with the P301L mutation showed increased TAU phosphorylation at the AT8, but not the p-Ser-262 epitope, and increased polyglutamylation and acetylation of microtubules compared with endogenous TAU-expressing neurons. P301L-TAU showed pronounced somatodendritic presence, but also successful axonal enrichment and a similar axodendritic distribution comparable to exogenously expressed 2N4R-wildtype-TAU. P301L-TAU-expressing hippocampal neurons in transgenic mice showed prominent missorting and tauopathy-typical AT8-phosphorylation of TAU and increased polyglutamylation, but reduced acetylation, of microtubules compared with non-transgenic littermates. In sum, P301L-TAU results in changes in microtubule PTMs, suggestive of impairment of microtubule stability. This is accompanied by missorting and aggregation of TAU in mice but not in iNeurons. Microtubule PTMs/impairment may be of key importance in tauopathies.

Dual spatio-temporal regulation of axon growth and microtubule dynamics by RhoA signaling pathways.

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA-mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK signaling, which restrained growth cone microtubule assembly and protrusion.

Doublecortin-like kinase is required for cnidocyte development in Nematostella vectensis

The complex morphology of neurons requires precise control of their microtubule cytoskeleton. This is achieved by microtubule-associated proteins (MAPs) that regulate the assembly and stability of microtubules, and transport of molecules and vesicles along them. While many of these MAPs function in all cells, some are specifically or predominantly involved in regulating microtubules in neurons. Here we use the sea anemone Nematostella vectensis as a model organism to provide new insights into the early evolution of neural microtubule regulation. As a cnidarian, Nematostella belongs to an outgroup to all bilaterians and thus occupies an informative phylogenetic position for reconstructing the evolution of nervous system development. We identified an ortholog of the microtubule-binding protein doublecortin-like kinase (NvDclk1) as a gene that is predominantly expressed in neurons and cnidocytes (stinging cells), two classes of cells belonging to the neural lineage in cnidarians. A transgenic NvDclk1 reporter line revealed an elaborate network of neurite-like processes emerging from cnidocytes in the tentacles and the body column. A transgene expressing NvDclk1 under the control of the NvDclk1 promoter suggests that NvDclk1 localizes to microtubules and therefore likely functions as a microtubule-binding protein. Further, we generated a mutant for NvDclk1 using CRISPR/Cas9 and show that the mutants fail to generate mature cnidocytes. Our results support the hypothesis that the elaboration of programs for microtubule regulation occurred early in the evolution of nervous systems.