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Microtubule-associated protein, MAP1B, is crucial for neuronal morphogenesis and disruptions in MAP1B function are correlated with neurodevelopmental disorders. MAP1B encodes a single polypeptide that is processed into discrete proteins, a heavy chain (HC) and a light chain (LC); however, it is unclear if these two chains operate individually or as a complex within the cell. In vivo studies have characterized the contribution of MAP1B HC and LC to microtubule and actin-based processes, but their molecular mechanisms of action are unknown. Using in vitro reconstitution with purified proteins, we dissect the biophysical properties of the HC and LC and uncover distinct binding behaviors and functional roles for these MAPs. Our biochemical assays indicate that MAP1B HC and LC do not form a constitutive complex, supporting the hypothesis that these proteins operate independently within cells. Both HC and LC inhibit the microtubule motors, kinesin-3, kinesin-4, and dynein, and differentially affect the severing activity of spastin. Notably, MAP1B LC binds to actin filaments in vitro and can simultaneously bind and cross-link actin filaments and microtubules, a function not observed for MAP1B HC. Phosphorylation of MAP1B HC by dual-specificity, tyrosine phosphorylation-regulated kinase 1a negatively regulates its actin-binding activity without significantly affecting its microtubule-binding capacity, suggesting a dynamic contribution of MAP1B HC in cytoskeletal organization. Overall, our study provides new insights into the distinct functional properties of MAP1B HC and LC, underscoring their roles in coordinating cytoskeletal networks during neuronal development.

The microtubule-associated protein, doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for kinase inhibitors. The physiological roles of DCLK1 kinase activity and how it is regulated remain elusive. Here, we analyze the role of mammalian DCLK1 kinase activity in regulating microtubule binding. We found that DCLK1 autophosphorylates a residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation within the doublecortin domains, which abolishes microtubule binding. Therefore, autophosphorylation at specific sites within DCLK1 has diametric effects on the molecule's association with microtubules. Our results suggest a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. These results provide molecular insights for future therapeutic efforts related to DCLK1's role in cancer development and progression.

Many eukaryotic cells distribute their intracellular components asymmetrically through regulated active transport driven by molecular motors along microtubule tracks. While intrinsic and extrinsic regulation of motor activity exists, what governs the overall distribution of activated motor-cargo complexes within cells remains unclear. Here, we utilize in vitro reconstitution of purified motor proteins and non-enzymatic microtubule-associated proteins (MAPs) to demonstrate that MAPs exhibit distinct influences on the motility of the three main classes of transport motors: kinesin-1, kinesin-3, and cytoplasmic dynein. Further, we dissect how combinations of MAPs affect motors and unveil MAP9 as a positive modulator of kinesin-3 motility. From these data, we propose a general "MAP code" that has the capacity to strongly bias directed movement along microtubules and helps elucidate the intricate intracellular sorting observed in highly polarized cells such as neurons.

The microtubule (MT)-associated protein tau regulates the critical growing and shortening behaviors of MTs, and its normal activity is essential for neuronal development and maintenance. Accordingly, aberrant tau action is tightly associated with Alzheimer's disease and is genetically linked to several additional neurodegenerative diseases known as tauopathies. Although tau is known to promote net MT growth and stability, the precise mechanistic details governing its regulation of MT dynamics remain unclear. Here, we have used the slowly-hydrolyzable GTP analog, guanylyl-(α,β)-methylene-diphosphonate (GMPCPP), to examine the structural effects of tau at MT ends that may otherwise be too transient to observe. The addition of both four-repeat (4R) and three-repeat (3R) tau isoforms to pre-formed GMPCPP MTs resulted in the formation of extended, multiprotofilament-wide projections at MT ends. Furthermore, at temperatures too low for assembly of bona fide MTs, both tau isoforms promoted the formation of long spiral ribbons from GMPCPP tubulin heterodimers. In addition, GMPCPP MTs undergoing cold-induced disassembly in the presence of 4R tau (and to a much lesser extent 3R tau) also formed spirals. Finally, three pathological tau mutations known to cause neurodegeneration and dementia were differentially compromised in their abilities to stabilize MT disassembly intermediates. Taken together, we propose that tau promotes the formation/stabilization of intermediate states in MT assembly and disassembly by promoting both longitudinal and lateral tubulin-tubulin contacts. We hypothesize that these activities represent fundamental aspects of tau action that normally occur at the GTP-rich ends of GTP/GDP MTs and that may be compromised in neurodegeneration-causing tau variants.

Tau, a member of the MAP2/tau family of microtubule-associated proteins, stabilizes and organizes axonal microtubules in healthy neurons. In neurodegenerative tauopathies, tau dissociates from microtubules and forms neurotoxic extracellular aggregates. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. Here, we used molecular dynamics, microtubule-binding experiments, and live-cell microscopy, revealing that highly-conserved histidine residues near the C terminus of each microtubule-binding repeat are pH sensors that can modulate tau-microtubule interaction strength within the physiological intracellular pH range. We observed that at low pH (7.5), tau deprotonation decreased binding to microtubules both in vitro and in cells. Electrostatic and hydrophobic characteristics of histidine were both required for tau-microtubule binding, as substitutions with constitutively and positively charged nonaromatic lysine or uncharged alanine greatly reduced or abolished tau-microtubule binding. Consistent with these findings, tau-microtubule binding was reduced in a cancer cell model with increased intracellular pH but was rapidly restored by decreasing the pH to normal levels. These results add detailed insights into the intracellular regulation of tau activity that may be relevant in both normal and pathological conditions.

Compressed sensing (CS) is widely used to accelerate clinical diffusion MRI acquisitions, but it is not widely used in preclinical settings yet. In this study, we optimized and compared several CS reconstruction methods for diffusion imaging. Different undersampling patterns and two reconstruction approaches were evaluated: conventional CS, based on Berkeley Advanced Reconstruction Toolbox (BART-CS) toolbox, and a new kernel low-rank (KLR)-CS, based on kernel principal component analysis and low-resolution-phase (LRP) maps. 3D CS acquisitions were performed at 9.4T using a 4-element cryocoil on mice (wild type and a MAP6 knockout). Comparison metrics were error and structural similarity index measure (SSIM) on fractional anisotropy (FA) and mean diffusivity (MD), as well as reconstructions of the anterior commissure and fornix. Acceleration factors (AF) up to 6 were considered. In the case of retrospective undersampling, the proposed KLR-CS outperformed BART-CS up to AF = 6 for FA and MD maps and tractography. For instance, for AF = 4, the maximum errors were, respectively, 8.0% for BART-CS and 4.9% for KLR-CS, considering both FA and MD in the corpus callosum. Regarding undersampled acquisitions, these maximum errors became, respectively, 10.5% for BART-CS and 7.0% for KLR-CS. This difference between simulations and acquisitions arose mainly from repetition noise, but also from differences in resonance frequency drift, signal-tonoise ratio, and in reconstruction noise. Despite this increased error, fully sampled and AF = 2 yielded comparable results for FA, MD and tractography, and AF = 4 showed minor faults. Altogether, KLR-CS based on LRP maps seems a robust approach to accelerate preclinical diffusion MRI and thereby limit the effect of the frequency drift. [ABSTRACT FROM AUTHOR]

In cells, microtubule location, length, and dynamics are regulated by a host of microtubule-associated proteins and enzymes that read where to bind and act based on the microtubule "tubulin code," which is predominantly encoded in the tubulin carboxy-terminal tail (CTT). Katanin is a highly conserved AAA ATPase enzyme that binds to the tubulin CTTs to remove dimers and sever microtubules. We have previously demonstrated that short CTT peptides are able to inhibit katanin severing. Here, we examine the effects of CTT sequences on this inhibition activity. Specifically, we examine CTT sequences found in nature, alpha1A (TUBA1A), detyrosinated alpha1A, Δ2 alpha1A, beta5 (TUBB/TUBB5), beta2a (TUBB2A), beta3 (TUBB3), and beta4b (TUBB4b). We find that these natural CTTs have distinct abilities to inhibit, most noticeably beta3 CTT cannot inhibit katanin. Two non-native CTT tail constructs are also unable to inhibit, despite having 94% sequence identity with alpha1 or beta5 sequences. Surprisingly, we demonstrate that poly-E and poly-D peptides are capable of inhibiting katanin significantly. An analysis of the hydrophobicity of the CTT constructs indicates that more hydrophobic polypeptides are less inhibitory than more polar polypeptides. These experiments not only demonstrate inhibition, but also likely interaction and targeting of katanin to these various CTTs when they are part of a polymerized microtubule filament. [ABSTRACT FROM AUTHOR]

The reorganization of microtubules in mitosis, meiosis, and development requires the microtubule-severing activity of katanin. Katanin is a heterodimer composed of an ATPase associated with diverse cellular activities (AAA) subunit and a regulatory subunit. Microtubule severing requires ATP hydrolysis by katanin's conserved AAA ATPase domains. Whereas other AAA ATPases form stable hexamers, we show that katanin forms only a monomer or dimers of heterodimers in solution. Katanin oligomers consistent with hexamers of heterodimers or heterododecamers were only observed for an ATP hydrolysis-deficient mutant in the presence of ATP. X-ray structures of katanin's AAA ATPase in monomeric nucleotide-free and pseudo-oligomeric ADP-bound states revealed conformational changes in the AAA subdomains that explained the structural basis for the instability of the katanin heterododecamer. We propose that the rapid dissociation of katanin AAA oligomers may lead to an autoinhibited state that prevents inappropriate microtubule severing or that cyclical disassembly into heterodimers may critically contribute to the microtubule-severing mechanism.

Hsp70 chaperones bind to various protein substrates for folding, trafficking, and degradation. Considerable structural information is available about how prokaryotic Hsp70 (DnaK) binds substrates, but less is known about mammalian Hsp70s, of which there are 13 isoforms encoded in the human genome. Here, we report the interaction between the human Hsp70 isoform heat shock cognate 71-kDa protein (Hsc70 or HSPA8) and peptides derived from the microtubule-associated protein Tau, which is linked to Alzheimer's disease. For structural studies, we used an Hsc70 construct (called BETA) comprising the substrate-binding domain but lacking the lid. Importantly, we found that truncating the lid does not significantly impair Hsc70's chaperone activity or allostery in vitro Using NMR, we show that BETA is partially dynamically disordered in the absence of substrate and that binding of the Tau sequence GKVQIINKKG (with a KD = 500 nm) causes dramatic rigidification of BETA. NOE distance measurements revealed that Tau binds to the canonical substrate-binding cleft, similar to the binding observed with DnaK. To further develop BETA as a tool for studying Hsc70 interactions, we also measured BETA binding in NMR and fluorescent competition assays to peptides derived from huntingtin, insulin, a second Tau-recognition sequence, and a KFERQ-like sequence linked to chaperone-mediated autophagy. We found that the insulin C-peptide binds BETA with high affinity (KD < 100 nm), whereas the others do not (KD > 100 μm). Together, our findings reveal several similarities and differences in how prokaryotic and mammalian Hsp70 isoforms interact with different substrate peptides.

The superior cervical ganglion (SCG) is part of the autonomic nervous system providing sympathetic innervation to the head and neck, and has been regularly used to prepare postnatal neuronal cultures for cell biological studies. We found that during development these neurons change tau expression from the low molecular weight (LMW) isoforms to Big tau, with the potential to affect functions associated with tau such as microtubule dynamic and axonal transport. Big tau contains the large 4a exon that transforms tau from LMW isoforms of 45–60 kDa to 110 kDa. We describe tau expression during postnatal development reporting that the transition from LMW tau to Big tau which started at late embryonic stages is completed by about 4–5 weeks postnatally. We confirmed the presence of Big tau in dissociated postnatal SCG neurons making them an ideal system to study the function of Big tau in neurons. We used SCG explants to examine the response of SCG neurons to lesion and found that Big tau expression returned gradually along the regrowing neurites suggesting that it does not drives regeneration, but facilitates the structure/function of mature SCG neurons. The structural/functional roles of Big tau remain unknown, but it is intriguing that neurons that express Big tau appear less vulnerable to tauopathies. [ABSTRACT FROM AUTHOR]

Aneuploidy eggs are a common cause of human infertility, spontaneous abortion, or trisomy syndromes. The spindle assembly checkpoint (SAC) plays a crucial role in preventing aneuploidy in oocytes, yet it is unclear if additional mechanisms exist to ensure oocyte adherence to this checkpoint. It is now revealed that the microtubule-associated protein NUSAP can prevent oocytes from evading the SAC and regulate the speed of the cell cycle. Mechanistically, the study identifies NUSAP as a novel stabilizer of the E3 ubiquitin ligase APC/C CDH1 , protecting CDH1 from SCF BTRC -mediated degradation. Depletion of NUSAP reduces CDH1 protein level, leading to abnormal spindle assembly and chromosome alignment, and disrupting the balance of cell cycle proteins. This misregulated balance causes oocytes to evade the SAC. Consequently, these abnormal oocytes not only fail to arrest at metaphase but also accelerate the cell process, ultimately resulting in the production of aneuploid eggs. Together, the findings not only clarify the existence of mechanisms that ensure oocytes compliance with the spindle assembly checkpoint but also expand the new functions of NUSAP beyond its role as a microtubule- associated protein., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Microtubule associated protein 2 (MAP2) interacts with the regulatory protein 14-3-3ζ in a cAMP-dependent protein kinase (PKA) phosphorylation dependent manner. Using selective phosphorylation, calorimetry, nuclear magnetic resonance, chemical crosslinking, and X-ray crystallography, we characterized interactions of 14-3-3ζ with various binding regions of MAP2c. Although PKA phosphorylation increases the affinity of MAP2c for 14-3-3ζ in the proline rich region and C-terminal domain, unphosphorylated MAP2c also binds the dimeric 14-3-3ζ via its microtubule binding domain and variable central domain. Monomerization of 14-3-3ζ leads to the loss of affinity for the unphosphorylated residues. In neuroblastoma cell extract, MAP2c is heavily phosphorylated by PKA and the proline kinase ERK2. Although 14-3-3ζ dimer or monomer do not interact with the residues phosphorylated by ERK2, ERK2 phosphorylation of MAP2c in the C-terminal domain reduces the binding of MAP2c to both oligomeric variants of 14-3-3ζ., (© 2025 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Microtubules are filamentous structures that play a critical role in a diverse array of cellular functions including, mitosis, nuclear translocation, trafficking of organelles and cell shape. They are composed of α/β-tubulin heterodimers which are encoded by a large multigene family that has been implicated in an umbrella of disease states collectively known as the tubulinopathies. De novo mutations in different tubulin genes are known to cause lissencephaly, microcephaly, polymicrogyria, motor neuron disease, and female infertility. The diverse clinical features associated with these maladies have been attributed to the expression pattern of individual tubulin genes, as well as their distinct Functional repertoire. Recent studies, however, have highlighted the impact of tubulin mutations on microtubule-associated proteins (MAPs). MAPs can be classified according to their effect on microtubules and include polymer stabilizers (e.g., tau, MAP2, doublecortin), destabilizers (e.g., spastin, katanin), plus-end binding proteins (e.g., EB1-3, XMAP215, CLASPs) and motor proteins (e.g., dyneins, kinesins). In this review we analyse mutation-specific disease mechanisms that influence MAP binding and their phenotypic consequences, and discuss methods by which we can exploit genetic variation to identify novel MAPs.

The MAPT gene encoding the microtubule-associated protein tau can generate multiple isoforms by alternative splicing giving rise to proteins which are differentially expressed in specific areas of the nervous system and at different developmental stages. Tau plays important roles in modulating microtubule dynamics, axonal transport, synaptic plasticity, and DNA repair, and has also been associated with neurodegenerative diseases (tauopathies) including Alzheimer’s disease and frontotemporal dementia. A unique high-molecular-weight isoform of tau, originally found to be expressed in the peripheral nervous system and projecting neurons, has been termed Big tau and has been shown to uniquely contain the large exon 4a that significantly increases the size and 3D structure of tau. With little progress since the original discovery of Big tau, more than 25 years ago, we have now completed a comprehensive comparative study to analyze the structure of the MAPT gene against available databases with respect to the composition of the tau exons as they evolved from early vertebrates to primates and human. We focused the analysis on the evolution of the 4a exon variants and their homology relative to humans. We discovered that the 4a exon defining Big tau appears to be present early in vertebrate evolution as a large insert that dramatically changed the size of the tau protein with low sequence conservation despite a stable size range of about 250aa, and in some species a larger 4a-L exon of 355aa. We suggest that 4a exon variants evolved independently in different species by an exonization process using new alternative splicing to address the growing complexities of the evolving nervous systems. Thus, the appearance of a significantly larger isoform of tau independently repeated itself multiple times during evolution, accentuating the need across vertebrate species for an elongated domain that likely endows Big tau with novel physiological functions as well as properties related to neurodegeneration. [ABSTRACT FROM AUTHOR]

The evolutionarily conserved MAP65 family proteins bundle anti-parallel microtubules (MTs). In Arabidopsis thaliana, mutations in the MAP65-3 gene lead to serious defects in MT organization in the phragmoplast and cause failures in cytokinesis. However, the functions of other ArabidopsisMAP65 isoforms are largely unknown. MAP65 functions were analyzed based on genetic interactions among different map65 mutations. Live-cell imaging and immunolocalization experiments revealed dynamic activities of two closely related MAP65 proteins in dividing cells. The map65-4 mutation caused synthetic lethality with map65-3 although map65-4 alone did not cause a noticeable phenotype. Furthermore, the introduction of an extra copy of the MAP65-4 gene significantly suppressed defects in cytokinesis and seedling growth caused by map65-3 because of restoring MT engagement in the spindle midzone. During mitosis, MAP65-4 first appeared at the preprophase band and persisted at the cortical division site afterwards. It was also concentrated on MTs in the spindle midzone and the phragmoplast. In the absence of MAP65-3, MAP65-4 exhibited greatly enhanced localization in the midzone of developing phragmoplast. Therefore, we have uncovered redundant but differential contributions of MAP65-3 and MAP65-4 to engaging and bundling anti-parallel MTs in the phragmoplast and disclosed a novel action of MAP65-4 at the cortical cell division site.

The MAPT gene encoding the microtubule-associated protein tau can generate multiple isoforms by alternative splicing giving rise to proteins which are differentially expressed in specific areas of the nervous system and at different developmental stages. Tau plays important roles in modulating microtubule dynamics, axonal transport, synaptic plasticity, and DNA repair, and has also been associated with neurodegenerative diseases (tauopathies) including Alzheimer’s disease and frontotemporal dementia. A unique high-molecular-weight isoform of tau, originally found to be expressed in the peripheral nervous system and projecting neurons, has been termed Big tau and has been shown to uniquely contain the large exon 4a that significantly increases the size and 3D structure of tau. With little progress since the original discovery of Big tau, more than 25 years ago, we have now completed a comprehensive comparative study to analyze the structure of the MAPT gene against available databases with respect to the composition of the tau exons as they evolved from early vertebrates to primates and human. We focused the analysis on the evolution of the 4a exon variants and their homology relative to humans. We discovered that the 4a exon defining Big tau appears to be present early in vertebrate evolution as a large insert that dramatically changed the size of the tau protein with low sequence conservation despite a stable size range of about 250aa, and in some species a larger 4a-L exon of 355aa. We suggest that 4a exon variants evolved independently in different species by an exonization process using new alternative splicing to address the growing complexities of the evolving nervous systems. Thus, the appearance of a significantly larger isoform of tau independently repeated itself multiple times during evolution, accentuating the need across vertebrate species for an elongated domain that likely endows Big tau with novel physiological functions as well as properties related to neurodegeneration.

The major cause of death in cancer patients is strongly associated with metastasis. While much remains to be understood, microtubule-associated proteins (MAPs) have shed light on metastatic progression’s molecular mechanisms. In this review article, we focus on the role of MAPs in cancer aggressiveness, particularly cancer metastasis activity. Increasing evidence has shown that a growing number of MAP member proteins might be fundamental regulators involved in altering microtubule dynamics, contributing to cancer migration, invasion, and epithelial-to-mesenchymal transition. MAP types have been established according to their microtubule-binding site and function in microtubule-dependent activities. We highlight that altered MAP expression was commonly found in many cancer types and related to cancer progression based on available evidence. Furthermore, we discuss and integrate the relevance of MAPs and related molecular signaling pathways in cancer metastasis. Our review provides a comprehensive understanding of MAP function on microtubules. It elucidates how MAPs regulate cancer progression, preferentially in metastasis, providing substantial scientific information on MAPs as potential therapeutic targets and prognostic markers for cancer management.

Diverse DNA and RNA viruses utilize cytoskeletal networks to efficiently enter, replicate, and exit the host cell, while evading host immune responses. It is well established that the microtubule (MT) network is commonly hijacked by viruses to traffic to sites of replication after entry and to promote egress from the cell. However, mounting evidence suggests that the MT network is also a key regulator of host immune responses to infection. At the same time, viruses have acquired mechanisms to manipulate and/or usurp MT networks to evade these immune responses. Central to most interactions of viruses with the MT network are virally encoded microtubule-associated proteins (MAPs) that bind to MTs directly or indirectly. These MAPs associate with MTs and other viral or cellular MAPs to regulate various aspects of the MT network, including MT dynamics, MT-dependent transport via motor proteins such as kinesins and dyneins, and MT-dependent regulation of innate immune responses. In this review, we examine how viral MAP interactions with the MT network facilitate viral replication and immune evasion. [ABSTRACT FROM AUTHOR]

The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2-9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2-8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.

Tau is a microtubule-associated protein, which is highly expressed in the central nervous system as well as ocular neurons and stabilizes microtubule structure. It is a phospho-protein being moderately phosphorylated under physiological conditions but its abnormal hyperphosphorylation or some post-phosphorylation modifications would result in a pathogenic condition, microtubule dissociation, and aggregation. The aggregates can induce neuroinflammation and trigger some pathogenic cascades, leading to neurodegeneration. Taking these together, targeting pathogenic tau employing tau immunotherapy may be a promising therapeutic strategy in fighting with cerebral and ocular neurodegenerative disorders.

The microtubule-associated protein, doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for kinase inhibitors. The physiological roles of DCLK1 kinase activity and how it is regulated remain elusive. Here, we analyze the role of mammalian DCLK1 kinase activity in regulating microtubule binding. We found that DCLK1 autophosphorylates a residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation within the doublecortin domains, which abolishes microtubule binding. Therefore, autophosphorylation at specific sites within DCLK1 has diametric effects on the molecule’s association with microtubules. Our results suggest a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. These results provide molecular insights for future therapeutic efforts related to DCLK1’s role in cancer development and progression.

Tau is a microtubule-associated protein for which the physiological functions remain a topic of vigorous investigation. Additionally, tau is a central player in the pathogenesis of several diseases such as Alzheimer’s disease and several frontotemporal dementias. A critical variable to understanding tau in physiological and disease contexts is its normal localization within cells of the adult CNS. Tau is often described as an axon-specific (or enriched) and neuron-specific protein with little to no expression in glial cells, all of which are untrue. Understanding normal tau distribution also impacts interpretation of experimental results and hypotheses regarding its role in disease. Thus, we set out to help clarify the normal localization of tau in the adult CNS of middle-aged rats and rhesus macaque using the hippocampus as a representative brain structure. The physiological concentration of tau in the rat hippocampus was 6.6 μM and in white matter was 3.6 μM as determined by quantitative sandwich ELISAs. We evaluated the cellular localization of tau using multiple tau-specific antibodies with epitopes to different regions, including Tau1, Tau5, Tau7, R1, and two novel primate-specific antibodies NT9 and NT15. In the rat and monkey, tau was localized within the somatodendritic and axonal compartments, as well as a subset of neuronal nuclei. Semi-quantitative fluorescence intensity measurements revealed that depending on the specific reagent used the somatodendritic tau is relatively equal to, higher than, or lower than axonal tau, highlighting differential labeling of tau with various antibodies despite its distribution throughout the neuron. Tau was strongly expressed in mature oligodendrocytes and displayed little to no expression in oligodendrocyte precursor cells, astrocytes or microglia. Collectively, the data indicate tau is ∼3 – 7 μM under physiological conditions, is not specifically enriched in axons, and is normally found in both neurons and mature oligodendrocytes in the adult CNS. The full landscape of tau distribution is not revealed by all antibodies suggesting availability of the epitopes is different within specific neuronal compartments. These findings set the stage for better understanding normal tau distributions and interpreting data regarding the presence of tau in different compartments or cell types within disease conditions.

CENP-E (centromere protein E) and CENP-F (centromere protein F), also known as mitosin, are large, multi-functional proteins associated with the outer kinetochore. CENP-E features a well-characterized kinesin motor domain at its N-terminus and a second microtubule-binding domain at its C-terminus of unknown function. CENP-F is important for the formation of proper kinetochore-microtubule attachment and, similar to CENP-E, contains two microtubule-binding domains at its termini. While the importance of these proteins is known, the details of their interactions with microtubules have not yet been investigated. We have biochemically and structurally characterized the microtubule-binding properties of the amino- and carboxyl-terminal domains of CENP-F as well as the carboxyl-terminal (non-kinesin) domain of CENP-E. CENP-E's C-terminus and CENP-F's N-terminus bind microtubules with similar affinity to the well-characterized Ndc80 complex, while CENP-F's C-terminus shows much lower affinity. Electron microscopy analysis reveals that all of these domains engage the microtubule surface in a disordered manner, suggesting that these factors have no favored binding geometry and may allow for initial side-on attachments early in mitosis.

Tau is a microtubule-associated protein for which the physiological functions remain a topic of vigorous investigation. Additionally, tau is a central player in the pathogenesis of several diseases such as Alzheimer's disease and several frontotemporal dementias. A critical variable to understanding tau in physiological and disease contexts is its normal localization within cells of the adult CNS. Tau is often described as an axon-specific (or enriched) and neuron-specific protein with little to no expression in glial cells, all of which are untrue. Understanding normal tau distribution also impacts interpretation of experimental results and hypotheses regarding its role in disease. Thus, we set out to help clarify the normal localization of tau in the adult CNS of middle-aged rats and rhesus macaque using the hippocampus as a representative brain structure. The physiological concentration of tau in the rat hippocampus was 6.6 μM and in white matter was 3.6 μM as determined by quantitative sandwich ELISAs. We evaluated the cellular localization of tau using multiple tau-specific antibodies with epitopes to different regions, including Tau1, Tau5, Tau7, R1, and two novel primate-specific antibodies NT9 and NT15. In the rat and monkey, tau was localized within the somatodendritic and axonal compartments, as well as a subset of neuronal nuclei. Semi-quantitative fluorescence intensity measurements revealed that depending on the specific reagent used the somatodendritic tau is relatively equal to, higher than, or lower than axonal tau, highlighting differential labeling of tau with various antibodies despite its distribution throughout the neuron. Tau was strongly expressed in mature oligodendrocytes and displayed little to no expression in oligodendrocyte precursor cells, astrocytes or microglia. Collectively, the data indicate tau is ∼3 – 7 μM under physiological conditions, is not specifically enriched in axons, and is normally found in both neurons and mature oligodendrocytes in the adult CNS. The full landscape of tau distribution is not revealed by all antibodies suggesting availability of the epitopes is different within specific neuronal compartments. These findings set the stage for better understanding normal tau distributions and interpreting data regarding the presence of tau in different compartments or cell types within disease conditions. [ABSTRACT FROM AUTHOR]

Background Microtubule-associated protein light chains (LC) 3A and 3B are the structural proteins of the autophagosomal membrane widely used as endogenous autophagy markers. LC3A and LC3B autophagosomes reportedly have a distinct subcellular localization yet their role in the transition from premalignant to malignant phase remains unclear. This exploratory study aimed to investigate the expression of autophagy-related proteins LC3A and LC3B in oral premalignant lesions (OPL) and oral squamous cell carcinoma (OSCC) cases. Methodology A cross-sectional study was conducted on 100 OPL and 39 OSCC samples. OPL samples comprised both dysplastic and non-dysplastic lesions. The expression of LC3A and LC3B markers was evaluated in the study samples using immunohistochemistry and associated with dysplasia in OPL and with invasive OSCC versus OPL. Fisher's exact test was used for statistical analysis. Results There was a higher ratio of LC3A positivity in non-dysplastic OPL (31/38) compared to dysplastic premalignant lesions (36/62, p=0.017). There was a higher ratio of LC3B positivity in dysplastic OPL (16/62) compared to non-dysplastic lesions (4/38) with a trend towards statistical significance (p=0.075). There was no statistical difference in the ratio of LC3A positivity between OSCC (23/39) and premalignant (67/100) lesions, while the ratio of LC3B marker positivity was higher in OSCC cases (18/39) relative to premalignant lesions (20/100, p=0.003). Conclusion Autophagy-related proteins LC3A and LC3B may have different roles to play in a disease context manner. LC3A is likely to be negatively associated with dysplasia in OPL while LC3B expression is positively associated with carcinogenesis of OSCC, possibly including dysplasia., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Institutional Ethics Committee of All India Institute of Medical Sciences, Rishikesh issued approval AIIMS/IEC/19/724. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Wakhloo et al.)

Tau protein (MAPT) is classified as a microtubule-associated protein (MAP) and is believed to regulate the axonal microtubule arrangement. It belongs to the tau/MAP2/MAP4 family of MAPs that have a similar microtubule binding region at their carboxy-terminal half. In tauopathies, such as Alzheimer's disease, tau is distributed more in the somatodendritic compartment, where it aggregates into filamentous structures, the formation of which correlates with cognitive impairments in patients. While microtubules are the dominant interaction partners of tau under physiological conditions, tau has many additional interaction partners that can contribute to its physiological and pathological role. In particular, the amino-terminal non-microtubule binding domain (N-terminal projection region, NTR) of tau interacts with many partners that are involved in membrane organization. The NTR contains intrinsically disordered regions (IDRs) that show a strong evolutionary increase in the disorder and may have been the basis for the development of new, tau-specific interactions. In this review we discuss the functional organization of the tau protein and the special features of the tau non-microtubule binding region also in the connection with the results of Tau KO models. We consider possible physiological and pathological functions of tau's non-microtubule interactions, which could indicate that interactions mediated by tau's NTR and regulated by far-reaching functional interactions of the PRR and the extreme C-terminus of tau contribute to the pathological processes.

Abstract Background The skeletal muscle fiber has a specific and precise intracellular organization which is at the basis of an efficient muscle contraction. Microtubules are long known to play a major role in the function and organization of many cells, but in skeletal muscle, the contribution of the microtubule cytoskeleton to the efficiency of contraction has only recently been studied. The microtubule network is dynamic and is regulated by many microtubule-associated proteins (MAPs). In the present study, the role of the MAP6 protein in skeletal muscle organization and function has been studied using the MAP6 knockout mouse line. Methods The presence of MAP6 transcripts and proteins was shown in mouse muscle homogenates and primary culture using RT-PCR and western blot. The in vivo evaluation of muscle force of MAP6 knockout (KO) mice was performed on anesthetized animals using electrostimulation coupled to mechanical measurement and multimodal magnetic resonance. The impact of MAP6 deletion on microtubule organization and intracellular structures was studied using immunofluorescent labeling and electron microscopy, and on calcium release for muscle contraction using Fluo-4 calcium imaging on cultured myotubes. Statistical analysis was performed using Student’s t test or the Mann-Whitney test. Results We demonstrate the presence of MAP6 transcripts and proteins in skeletal muscle. Deletion of MAP6 results in a large number of muscle modifications: muscle weakness associated with slight muscle atrophy, alterations of microtubule network and sarcoplasmic reticulum organization, and reduction in calcium release. Conclusion Altogether, our results demonstrate that MAP6 is involved in skeletal muscle function. Its deletion results in alterations in skeletal muscle contraction which contribute to the global deleterious phenotype of the MAP6 KO mice. As MAP6 KO mouse line is a model for schizophrenia, our work points to a possible muscle weakness associated to some forms of schizophrenia.

Doublecortin (DCX) is a neuronal microtubule‐associated protein (MAP) indispensable for brain development. Its flexibly linked doublecortin (DC) domains—NDC and CDC—mediate microtubule (MT) nucleation and stabilization, but it is unclear how. Using high‐resolution time‐resolved cryo‐EM, we mapped NDC and CDC interactions with tubulin at different MT polymerization stages and studied their functional effects on MT dynamics using TIRF microscopy. Although coupled, each DC repeat within DCX appears to have a distinct role in MT nucleation and stabilization: CDC is a conformationally plastic module that appears to facilitate MT nucleation and stabilize tubulin–tubulin contacts in the nascent MT lattice, while NDC appears to be favored along the mature lattice, providing MT stabilization. Our structures of MT‐bound DC domains also explain in unprecedented detail the DCX mutation‐related brain defects observed in the clinic. This modular composition of DCX reflects a common design principle among MAPs where pseudo‐repeats of tubulin/MT binding elements chaperone or stabilize distinct conformational transitions to regulate distinct stages of MT dynamic instability. Synopsis: Doublecortin nucleates and stabilizes microtubules via two distinct DC domains that differentially interact with distinct stages of microtubule assembly. This mode of action likely represents the general principle of cytoskeletal dynamics regulation by modular proteins. Each doublecortin DC domain is involved at a distinct stage of microtubule assemblyThe conformationally plastic CDC stabilizes early microtubule nucleation eventsThe more stable NDC binds to the microtubule lattice to protect against catastrophePseudo‐repeats within MAPs act as chaperones for tubulin arrays in dynamic polymers [ABSTRACT FROM AUTHOR]

Tau protein (MAPT) is classified as a microtubule-associated protein (MAP) and is believed to regulate the axonal microtubule arrangement. It belongs to the tau/MAP2/MAP4 family of MAPs that have a similar microtubule binding region at their carboxy-terminal half. In tauopathies, such as Alzheimer's disease, tau is distributed more in the somatodendritic compartment, where it aggregates into filamentous structures, the formation of which correlates with cognitive impairments in patients. While microtubules are the dominant interaction partners of tau under physiological conditions, tau has many additional interaction partners that can contribute to its physiological and pathological role. In particular, the amino-terminal non-microtubule binding domain (N-terminal projection region, NTR) of tau interacts with many partners that are involved in membrane organization. The NTR contains intrinsically disordered regions (IDRs) that show a strong evolutionary increase in the disorder and may have been the basis for the development of new, tau-specific interactions. In this review we discuss the functional organization of the tau protein and the special features of the tau non-microtubule binding region also in the connection with the results of Tau KO models. We consider possible physiological and pathological functions of tau's non-microtubule interactions, which could indicate that interactions mediated by tau's NTR and regulated by far-reaching functional interactions of the PRR and the extreme C-terminus of tau contribute to the pathological processes. [ABSTRACT FROM AUTHOR]

Giardia lamblia, a widespread parasitic protozoan, attaches to the host gastrointestinal epithelium by using the ventral disc, a complex microtubule (MT) organelle. The ‘cup-like’ disc is formed by a spiral MT array that scaffolds numerous disc-associated proteins (DAPs) and higher-order protein complexes. In interphase, the disc is hyperstable and has limited MT dynamics; however, it remains unclear how DAPs confer these properties. To investigate mechanisms of hyperstability, we confirmed the disc-specific localization of over 50 new DAPs identified by using both a disc proteome and an ongoing GFP localization screen. DAPs localize to specific disc regions and many lack similarity to known proteins. By screening 14 CRISPRi-mediated DAP knockdown (KD) strains for defects in hyperstability and MT dynamics, we identified two strains – DAP5188KD and DAP6751KD – with discs that dissociate following high-salt fractionation. Discs in the DAP5188KD strain were also sensitive to treatment with the MT-polymerization inhibitor nocodazole. Thus, we confirm here that at least two of the 87 known DAPs confer hyperstable properties to the disc MTs, and we anticipate that other DAPs contribute to disc MT stability, nucleation and assembly. [ABSTRACT FROM AUTHOR]

There are three subfamilies of patatin-related phospholipase A (pPLA) group of genes: pPLAI, pPLAII, and pPLAIII. Among the four members of pPLAIIIs (α, β, γ, δ), the overexpression of three isoforms (α, β, and δ) displayed distinct morphological growth patterns, in which the anisotropic cell expansion was disrupted. Here, the least studied pPLAIIIγ was characterized, and it was found that the overexpression of pPLAIIIγ in Arabidopsis resulted in longitudinally reduced cell expansion patterns, which are consistent with the general phenotype induced by pPLAIIIs overexpression. The microtubule-associated protein MAP18 was found to be enriched in a pPLAIIIδ overexpressing line in a previous study. This indicates that factors, such as microtubules and ethylene biosynthesis, are involved in determining the radial cell expansion patterns. Microtubules have long been recognized to possess functional key roles in the processes of plant cells, including cell division, growth, and development, whereas ethylene treatment was reported to induce the reorientation of microtubules. Thus, the possible links between the altered anisotropic cell expansion and microtubules were studied. Our analysis revealed changes in the transcriptional levels of microtubule-associated genes, as well as phospholipase D (PLD) genes, upon the overexpression of pPLAIIIγ. Overall, our results suggest that the longitudinally reduced cell expansion observed in pPLAIIIγ overexpression is driven by microtubules via transcriptional modulation of the PLD and MAP genes. The altered transcripts of the genes involved in ethylene-biosynthesis in pPLAIIIγOE further support the conclusion that the typical phenotype is derived from the link with microtubules.

Microtubules in the mitotic spindle are organised by microtubule-associated proteins. In the late stage of mitosis, spindle microtubules are robustly organised through bundling by the antiparallel microtubule bundler Ase1/PRC1. In early mitosis, however, it is not well characterised as to whether spindle microtubules are actively bundled, as Ase1 does not particularly localise to the spindle at that stage. Here we show that the conserved microtubule-associated protein CLASP (fission yeast Peg1/Cls1) facilitates bundling of spindle microtubules in early mitosis. The peg1 mutant displayed a fragile spindle with unbundled microtubules, which eventually resulted in collapse of the metaphase spindle and abnormal segregation of chromosomes. Peg1 is known to be recruited to the spindle by Ase1 to stabilise antiparallel microtubules in late mitosis. However, we demonstrate that the function of Peg1 in early mitosis does not rely on Ase1. The unbundled spindle phenotype of the peg1 mutant was not seen in the ase1 mutant, and Peg1 preferentially localised to the spindle even in early mitosis unlike Ase1. Moreover, artificial overexpression of Ase1 in the peg1 mutant partially suppressed unbundled microtubules. We thus conclude that Peg1 bundles microtubules in early mitosis, in a distinct manner from its conventional Ase1-dependent functions in other cell cycle stages.

Tau is a neuronal microtubule-associated protein (MAP) that is involved in the regulation of axonal microtubule assembly. However, as a protein with intrinsically disordered regions (IDRs), tau also interacts with many other partners in addition to microtubules. Phosphorylation at selected sites modulates tau’s various intracellular interactions and regulates the properties of IDRs. In Alzheimer’s disease (AD) and other tauopathies, tau exhibits pathologically increased phosphorylation (hyperphosphorylation) at selected sites and aggregates into neurofibrillary tangles (NFTs). By bioinformatics means, we tested the hypothesis that the sequence of tau has changed during the vertebrate evolution in a way that novel interactions developed and also the phosphorylation pattern was affected, which made tau prone to the development of tauopathies. We report that distinct regions of tau show functional specialization in their molecular interactions. We found that tau’s amino-terminal region, which is involved in biological processes related to “membrane organization” and “regulation of apoptosis,” exhibited a strong evolutionary increase in protein disorder providing the basis for the development of novel interactions. We observed that the predicted phosphorylation sites have changed during evolution in a region-specific manner, and in some cases the overall number of phosphorylation sites increased owing to the formation of clusters of phosphorylatable residues. In contrast, disease-specific hyperphosphorylated sites remained highly conserved. The data indicate that novel, non-microtubule related tau interactions developed during evolution and suggest that the biological processes, which are mediated by these interactions, are of pathological relevance. Furthermore, the data indicate that predicted phosphorylation sites in some regions of tau, including a cluster of phosphorylatable residues in the alternatively spliced exon 2, have changed during evolution. In view of the “antagonistic pleiotropy hypothesis” it may be worth to take disease-associated phosphosites with low evolutionary conservation as relevant biomarkers into consideration.

α-Synuclein and tau are abundant multifunctional brain proteins that are mainly expressed in the presynaptic and axonal compartments of neurons, respectively. Previous works have revealed that intracellular deposition of α-synuclein and/or tau causes many neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. Despite intense investigation, the normal physiological functions and roles of α-synuclein and tau are still unclear, owing to the fact that mice with knockout of either of these proteins do not present apparent phenotypes. Interestingly, the co-occurrence of α-synuclein and tau aggregates was found in post-mortem brains with synucleinopathies and tauopathies, some of which share similarities in clinical manifestations. Furthermore, the direct interaction of α-synuclein with tau is considered to promote the fibrillization of each of the proteins in vitro and in vivo. On the other hand, our recent findings have revealed that α-synuclein and tau are cooperatively involved in brain development in a stage-dependent manner. These findings indicate strong cross-talk between the two proteins in physiology and pathology. In this review, we provide a summary of the recent findings on the functional roles of α-synuclein and tau in the physiological conditions and pathogenesis of neurodegenerative diseases. A deep understanding of the interplay between α-synuclein and tau in physiological and pathological conditions might provide novel targets for clinical diagnosis and therapeutic strategies to treat neurodegenerative diseases., Competing Interests: None

Microtubules grow not only from the centrosome but also from various noncentrosomal microtubule-organizing centers (MTOCs), including the nuclear envelope (NE) and pre-existing microtubules. The evolutionarily conserved proteins Mto1/CDK5RAP2 and Alp14/TOG/XMAP215 have been shown to be involved in promoting microtubule nucleation. However, it has remained elusive as to how the microtubule nucleation promoting factors are specified to various noncentrosomal MTOCs, particularly the NE, and how these proteins coordinate to organize microtubule assembly. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe , efficient interphase microtubule growth from the NE requires Alp7/TACC, Alp14/TOG/XMAP215, and Mto1/CDK5RAP2. The absence of Alp7, Alp14, or Mto1 compromises microtubule regrowth on the NE in cells undergoing microtubule repolymerization. We further demonstrate that Alp7 and Mto1 interdependently localize to the NE in cells without microtubules and that Alp14 localizes to the NE in an Alp7 and Mto1-dependent manner. Tethering Mto1 to the NE in cells lacking Alp7 partially restores microtubule number and the efficiency of microtubule generation from the NE. Hence, our study delineates that Alp7, Alp14, and Mto1 work in concert to regulate interphase microtubule regrowth on the NE. [ABSTRACT FROM AUTHOR]

Unlike animal cells, plant cells do not possess centrosomes that serve as microtubule organizing centers; how microtubule arrays are organized throughout plant morphogenesis remains poorly understood. We report here that Arabidopsis INCREASED PETAL GROWTH ANISOTROPY 1 (IPGA1), a previously uncharacterized microtubule-associated protein, regulates petal growth and shape by affecting cortical microtubule organization. Through a genetic screen, we showed that IPGA1 loss-of-function mutants displayed a phenotype of longer and narrower petals, as well as increased anisotropic cell expansion of the petal epidermis in the late phases of flower development. Map-based cloning studies revealed that IPGA1 encodes a previously uncharacterized protein that colocalizes with and directly binds to microtubules. IPGA1 plays a negative role in the organization of cortical microtubules into parallel arrays oriented perpendicular to the axis of cell elongation, with the ipga1-1 mutant displaying increased microtubule ordering in petal abaxial epidermal cells. The IPGA1 family is conserved among land plants and its homologs may have evolved to regulate microtubule organization. Taken together, our findings identify IPGA1 as a novel microtubule-associated protein and provide significant insights into IPGA1-mediated microtubule organization and petal growth anisotropy. [ABSTRACT FROM AUTHOR]

Tau is a microtubule-associated protein, which is highly expressed in the central nervous system as well as ocular neurons and stabilizes microtubule structure. It is a phospho-protein being moderately phosphorylated under physiological conditions but its abnormal hyperphosphorylation or some post-phosphorylation modifications would result in a pathogenic condition, microtubule dissociation, and aggregation. The aggregates can induce neuroinflammation and trigger some pathogenic cascades, leading to neurodegeneration. Taking these together, targeting pathogenic tau employing tau immunotherapy may be a promising therapeutic strategy in fighting with cerebral and ocular neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Tau is a neuronal microtubule-associated protein (MAP) that is involved in the regulation of axonal microtubule assembly. However, as a protein with intrinsically disordered regions (IDRs), tau also interacts with many other partners in addition to microtubules. Phosphorylation at selected sites modulates tau's various intracellular interactions and regulates the properties of IDRs. In Alzheimer's disease (AD) and other tauopathies, tau exhibits pathologically increased phosphorylation (hyperphosphorylation) at selected sites and aggregates into neurofibrillary tangles (NFTs). By bioinformatics means, we tested the hypothesis that the sequence of tau has changed during the vertebrate evolution in a way that novel interactions developed and also the phosphorylation pattern was affected, which made tau prone to the development of tauopathies. We report that distinct regions of tau show functional specialization in their molecular interactions. We found that tau's amino-terminal region, which is involved in biological processes related to "membrane organization" and "regulation of apoptosis," exhibited a strong evolutionary increase in protein disorder providing the basis for the development of novel interactions. We observed that the predicted phosphorylation sites have changed during evolution in a region-specific manner, and in some cases the overall number of phosphorylation sites increased owing to the formation of clusters of phosphorylatable residues. In contrast, disease-specific hyperphosphorylated sites remained highly conserved. The data indicate that novel, non-microtubule related tau interactions developed during evolution and suggest that the biological processes, which are mediated by these interactions, are of pathological relevance. Furthermore, the data indicate that predicted phosphorylation sites in some regions of tau, including a cluster of phosphorylatable residues in the alternatively spliced exon 2, have changed during evolution. In view of the "antagonistic pleiotropy hypothesis" it may be worth to take disease-associated phosphosites with low evolutionary conservation as relevant biomarkers into consideration. [ABSTRACT FROM AUTHOR]

Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs. [Display omitted] • C. elegans MAPH-9 localizes exclusively to MTDs • MAPH-9 localization is in part mediated by tubulin polyglutamylation • maph-9 deletion perturbs MTD ultrastructure, motor movement, and cilia function • MAP9 is conserved in metazoans and localizes to the proximal cilium in mammals Tran et al. identify a role for the microtubule-associated protein MAP9/MAPH-9 in cilia, which are specialized cellular appendages composed of microtubule doublets. They discovered that C. elegans MAPH-9 localizes exclusively to microtubule doublets, shaping doublet architecture and enabling cilia function. Mammalian MAP9 also localized to cilia, and this localization suggests a conserved role. [ABSTRACT FROM AUTHOR]

Viruses are entirely dependent on their ability to infect a host cell in order to replicate. To reach their site of replication as rapidly and efficiently as possible following cell entry, many have evolved elaborate mechanisms to hijack the cellular transport machinery to propel themselves across the cytoplasm. Long-range movements have been shown to involve motor proteins along microtubules (MTs) and direct interactions between viral proteins and dynein and/or kinesin motors have been well described. Although less well-characterized, it is also becoming increasingly clear that non-motile microtubule-associated proteins (MAPs), including structural MAPs of the MAP1 and MAP2 families, and microtubule plus-end tracking proteins (+TIPs), can also promote viral trafficking in infected cells, by mediating interaction of viruses with filaments and/or motor proteins, and modulating filament stability. Here we review our current knowledge on non-motile MAPs, their role in the regulation of cytoskeletal dynamics and in viral trafficking during the early steps of infection.

We are studying the changes in the organization of the cytoskeleton which accompany expression of differentiated neuronal morphology. Of particular interest is the elaboration of growth cones, the motile domains of the neuronal plasma membrane, and the cytoskeletal structures that underlie them. A candidate for a component of the growth cone cytoskeleton of cultured hippocampal neurons is the antigen recognized by the monoclonal antibody, 13H9 (Birgbauer and Solomon, J Cell Biol 109:1609-1620, 1989; Goslin et al., J Cell Biol 109:1621-1631, 1989). That antibody binds strongly to growth cones, but barely stains neurites. The characterization of the antigen, both biochemical and microscopic, suggests that it may interact with microfilaments and microtubules. We have established that 13H9 recognizes a subset of the isoforms of ezrin (unpublished results). Here, we describe the properties and localization of ezrin isoforms in differentiating neuronal cells, using two in vitro systems and developing spinal cord. In embryonal carcinoma cells, both the abundance of ezrin and the proportion of ezrin associated with the cytoskeletal fraction increase upon induction of neuronal differentiation with retinoic acid. In the neuronal cells within such cultures, the 13H9-positive forms of ezrin are enriched in the growth cone, while the bulk of ezrin identified by a polyclonal antibody shows no specific localization. In mouse DRG neurons, 13H9 staining is asymmetrically distributed along the edges of the complex growth cones of these cells. Staining of developing spinal cord in rat embryos also demonstrates that the 13H9-positive forms of ezrin do not colocalize with the majority of ezrin.(ABSTRACT TRUNCATED AT 250 WORDS)

We are studying the changes in the organization of the cytoskeleton which accompany expression of differentiated neuronal morphology. Of particular interest is the elaboration of growth cones, the motile domains of the neuronal plasma membrane, and the cytoskeletal structures that underlie them. A candidate for a component of the growth cone cytoskeleton of cultured hippocampal neurons is the antigen recognized by the monoclonal antibody, 13H9 (Birgbauer and Solomon, J Cell Biol 109:1609-1620, 1989; Goslin et al., J Cell Biol 109:1621-1631, 1989). That antibody binds strongly to growth cones, but barely stains neurites. The characterization of the antigen, both biochemical and microscopic, suggests that it may interact with microfilaments and microtubules. We have established that 13H9 recognizes a subset of the isoforms of ezrin (unpublished results). Here, we describe the properties and localization of ezrin isoforms in differentiating neuronal cells, using two in vitro systems and developing spinal cord. In embryonal carcinoma cells, both the abundance of ezrin and the proportion of ezrin associated with the cytoskeletal fraction increase upon induction of neuronal differentiation with retinoic acid. In the neuronal cells within such cultures, the 13H9-positive forms of ezrin are enriched in the growth cone, while the bulk of ezrin identified by a polyclonal antibody shows no specific localization. In mouse DRG neurons, 13H9 staining is asymmetrically distributed along the edges of the complex growth cones of these cells. Staining of developing spinal cord in rat embryos also demonstrates that the 13H9-positive forms of ezrin do not colocalize with the majority of ezrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Tau, a primary component of microtubule-associated protein, promotes microtubule assembly and/or disassembly and maintains the stability of the microtubule structure. Although the importance of tau in neurodegenerative diseases has been well demonstrated, whether tau is involved in peripheral nerve regeneration remains unknown. In the current study, we obtained sciatic nerve tissue from adult rats 0, 1, 4, 7, and 14 days after sciatic nerve crush and examined tau mRNA and protein expression levels and the location of tau in the sciatic nerve following peripheral nerve injury. The results from our quantitative reverse transcription polymerase chain reaction analysis showed that compared with the uninjured control sciatic nerve, mRNA expression levels for both tau and tau tubulin kinase 1, a serine/threonine kinase that regulates tau phosphorylation, were decreased following peripheral nerve injury. Our western blot assay results suggested that the protein expression levels of tau and phosphorylated tau initially decreased 1 day post nerve injury but then gradually increased. The results of our immunohistochemical labeling showed that the location of tau protein was not altered by nerve injury. Thus, these results showed that the expression of tau was changed following sciatic nerve crush, suggesting that tau may be involved in peripheral nerve repair and regeneration.

Microtubule associated protein 11 (MAP11, previously termed C7orf43) encodes a highly conserved protein whose function is unknown. Through genome-wide linkage analysis combined with whole exome sequencing, we demonstrate that human autosomal recessive primary microcephaly is caused by a truncating mutation in MAP11. Moreover, homozygous MAP11-orthologue CRISPR/Cas9 knock-out zebrafish presented with microcephaly and decreased neuronal proliferation, recapitulating the human phenotype. We demonstrate that MAP11 is ubiquitously transcribed with high levels in brain and cerebellum. Immunofluorescence and co-immunoprecipitation studies in SH-SY5Y cells showed that MAP11 associates with mitotic spindles, co-localizing and physically associating with α-tubulin during mitosis. MAP11 expression precedes α-tubulin in gap formation of cell abscission at the midbody and is co-localized with PLK1, a key regulator of cytokinesis, at the edges of microtubule extensions of daughter cells post cytokinesis abscission, implicating a role in mitotic spindle dynamics and in regulation of cell abscission during cytokinesis. Finally, lentiviral-mediated silencing of MAP11 diminished SH-SY5Y cell viability, reducing proliferation rather than affecting apoptosis. Thus, MAP11 encodes a microtubule-associated protein that plays a role in spindle dynamics and cell division, in which mutations cause microcephaly in humans and zebrafish. [ABSTRACT FROM AUTHOR]

Background and Aims Trees constantly experience wind, perceive resulting mechanical cues, and modify their growth and development accordingly. Previous studies have demonstrated that multiple bending treatments trigger ovalization of the stem and the formation of flexure wood in gymnosperms, but ovalization and flexure wood have rarely been studied in angiosperms, and none of the experiments conducted so far has used multidirectional bending treatments at controlled intensities. Assuming that bending involves tensile and compressive strain, we hypothesized that different local strains may generate specific growth and wood differentiation responses. Methods Basal parts of young poplar stems were subjected to multiple transient controlled unidirectional bending treatments during 8 weeks, which enabled a distinction to be made between the wood formed under tensile or compressive flexural strains. This set-up enabled a local analysis of poplar stem responses to multiple stem bending treatments at growth, anatomical, biochemical and molecular levels. Key Results In response to multiple unidirectional bending treatments, poplar stems developed significant cross-sectional ovalization. At the tissue level, some aspects of wood differentiation were similarly modulated in the compressed and stretched zones (vessel frequency and diameter of fibres without a G-layer), whereas other anatomical traits (vessel diameter, G-layer formation, diameter of fibres with a G-layer and microfibril angle) and the expression of fasciclin-encoding genes were differentially modulated in the two zones. Conclusions This work leads us to propose new terminologies to distinguish the 'flexure wood' produced in response to multiple bidirectional bending treatments from wood produced under transient tensile strain (tensile flexure wood; TFW) or under transient compressive strain (compressive flexure wood; CFW). By highlighting similarities and differences between tension wood and TFW and by demonstrating that plants could have the ability to discriminate positive strains from negative strains, this work provides new insight into the mechanisms of mechanosensitivity in plants. [ABSTRACT FROM AUTHOR]