High-confidence 3D template matching for cryo-electron tomography.

Visual proteomics attempts to build atlases of the molecular content of cells but the automated annotation of cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures of macromolecules. However, their applicability remains limited in terms of both the abundance and size of the molecular targets. Here we show that the performance of TM is greatly improved by using template-specific search parameter optimization and by including higher-resolution information. We establish a TM pipeline with systematically tuned parameters for the automated, objective and comprehensive identification of structures with confidence 10 to 100-fold above the noise level. We demonstrate high-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes and microtubules, and individual subunits inside crowded eukaryotic cells. We provide software tools for the generic implementation of our method that is broadly applicable towards realizing visual proteomics.

FGF13 enhances the function of TRPV1 by stabilizing microtubules and regulates acute and chronic itch.

Itching is an aversive somatosensation that triggers the desire to scratch. Transient receptor potential (TRP) channel proteins are key players in acute and chronic itch. However, whether the modulatory effect of fibroblast growth factor 13 (FGF13) on acute and chronic itch is associated with TRP channel proteins is unclear. Here, we demonstrated that conditional knockout of Fgf13 in dorsal root ganglion neurons induced significant impairment in scratching behaviors in response to acute histamine-dependent and chronic dry skin itch models. Furthermore, FGF13 selectively regulated the function of the TRPV1, but not the TRPA1 channel on Ca2+ imaging and electrophysiological recordings, as demonstrated by a significant reduction in neuronal excitability and current density induced by TRPV1 channel activation, whereas TRPA1 channel activation had no effect. Changes in channel currents were also verified in HEK cell lines. Subsequently, we observed that selective modulation of TRPV1 by FGF13 required its microtubule-stabilizing effect. Furthermore, in FGF13 knockout mice, only the overexpression of FGF13 with a tubulin-binding domain could rescue TRP channel function and the impaired itch behavior. Our findings reveal a novel mechanism by which FGF13 is involved in TRPV1-dependent itch transduction and provide valuable clues for alleviating pathological itch syndrome.

Prolonged depletion of profilin 1 or F-actin causes an adaptive response in microtubules.

In addition to its well-established role in actin assembly, profilin 1 (PFN1) has been shown to bind to tubulin and alter microtubule growth. However, whether PFN1's predominant control over microtubules in cells occurs through direct regulation of tubulin or indirectly through the polymerization of actin has yet to be determined. Here, we manipulated PFN1 expression, actin filament assembly, and actomyosin contractility and showed that reducing any of these parameters for extended periods of time caused an adaptive response in the microtubule cytoskeleton, with the effect being significantly more pronounced in neuronal processes. All the observed changes to microtubules were reversible if actomyosin was restored, arguing that PFN1's regulation of microtubules occurs principally through actin. Moreover, the cytoskeletal modifications resulting from PFN1 depletion in neuronal processes affected microtubule-based transport and mimicked phenotypes that are linked to neurodegenerative disease. This demonstrates how defects in actin can cause compensatory responses in other cytoskeleton components, which in turn significantly alter cellular function.

NEKL-4 regulates microtubule stability and mitochondrial health in ciliated neurons.

Ciliopathies are often caused by defects in the ciliary microtubule core. Glutamylation is abundant in cilia, and its dysregulation may contribute to ciliopathies and neurodegeneration. Mutation of the deglutamylase CCP1 causes infantile-onset neurodegeneration. In C. elegans, ccpp-1 loss causes age-related ciliary degradation that is suppressed by a mutation in the conserved NEK10 homolog nekl-4. NEKL-4 is absent from cilia, yet it negatively regulates ciliary stability via an unknown, glutamylation-independent mechanism. We show that NEKL-4 was mitochondria-associated. Additionally, nekl-4 mutants had longer mitochondria, a higher baseline mitochondrial oxidation state, and suppressed ccpp-1∆ mutant lifespan extension in response to oxidative stress. A kinase-dead nekl-4(KD) mutant ectopically localized to ccpp-1∆ cilia and rescued degenerating microtubule doublet B-tubules. A nondegradable nekl-4(PEST∆) mutant resembled the ccpp-1∆ mutant with dye-filling defects and B-tubule breaks. The nekl-4(PEST∆) Dyf phenotype was suppressed by mutation in the depolymerizing kinesin-8 KLP-13/KIF19A. We conclude that NEKL-4 influences ciliary stability by activating ciliary kinesins and promoting mitochondrial homeostasis.

FgPfn participates in vegetative growth, sexual reproduction, pathogenicity, and fungicides sensitivity via affecting both microtubules and actin in the filamentous fungus Fusarium graminearum.

Fusarium head blight (FHB), caused by Fusarium graminearum species complexes (FGSG), is an epidemic disease in wheat and poses a serious threat to wheat production and security worldwide. Profilins are a class of actin-binding proteins that participate in actin depolymerization. However, the roles of profilins in plant fungal pathogens remain largely unexplored. Here, we identified FgPfn, a homolog to profilins in F. graminearum, and the deletion of FgPfn resulted in severe defects in mycelial growth, conidia production, and pathogenicity, accompanied by marked disruptions in toxisomes formation and deoxynivalenol (DON) transport, while sexual development was aborted. Additionally, FgPfn interacted with Fgα1 and Fgß2, the significant components of microtubules. The organization of microtubules in the ΔFgPfn was strongly inhibited under the treatment of 0.4 µg/mL carbendazim, a well-known group of tubulin interferers, resulting in increased sensitivity to carbendazim. Moreover, FgPfn interacted with both myosin-5 (FgMyo5) and actin (FgAct), the targets of the fungicide phenamacril, and these interactions were reduced after phenamacril treatment. The deletion of FgPfn disrupted the normal organization of FgMyo5 and FgAct cytoskeleton, weakened the interaction between FgMyo5 and FgAct, and resulting in increased sensitivity to phenamacril. The core region of the interaction between FgPfn and FgAct was investigated, revealing that the integrity of both proteins was necessary for their interaction. Furthermore, mutations in R72, R77, R86, G91, I101, A112, G113, and D124 caused the non-interaction between FgPfn and FgAct. The R86K, I101E, and D124E mutants in FgPfn resulted in severe defects in actin organization, development, and pathogenicity. Taken together, this study revealed the role of FgPfn-dependent cytoskeleton in development, DON production and transport, fungicides sensitivity in F. graminearum.

Poc1 bridges basal body inner junctions to promote triplet microtubule integrity and connections.

Basal bodies (BBs) are conserved eukaryotic structures that organize cilia. They are comprised of nine, cylindrically arranged, triplet microtubules (TMTs) connected to each other by inter-TMT linkages which stabilize the structure. Poc1 is a conserved protein important for BB structural integrity in the face of ciliary forces transmitted to BBs. To understand how Poc1 confers BB stability, we identified the precise position of Poc1 in the Tetrahymena BB and the effect of Poc1 loss on BB structure. Poc1 binds at the TMT inner junctions, stabilizing TMTs directly. From this location, Poc1 also stabilizes inter-TMT linkages throughout the BB, including the cartwheel pinhead and the inner scaffold. The full localization of the inner scaffold protein Fam161A requires Poc1. As ciliary forces are increased, Fam161A is reduced, indicative of a force-dependent molecular remodeling of the inner scaffold. Thus, while not essential for BB assembly, Poc1 promotes BB interconnections that establish an architecture competent to resist ciliary forces.

MARK2 phosphorylates KIF13A at a 14-3-3 binding site to polarize vesicular transport of transferrin receptor within dendrites.

Neurons regulate the microtubule-based transport of certain vesicles selectively into axons or dendrites to ensure proper polarization of function. The mechanism of this polarized vesicle transport is still not fully elucidated, though it is known to involve kinesins, which drive anterograde transport on microtubules. Here, we explore how the kinesin-3 family member KIF13A is regulated such that vesicles containing transferrin receptor (TfR) travel only to dendrites. In experiments involving live-cell imaging, knockout of KIF13A, BioID assay, we found that the kinase MARK2 phosphorylates KIF13A at a 14-3-3 binding motif, strengthening interaction of KIF13A with 14-3-3 such that it dissociates from TfR-containing vesicles, which therefore cannot enter axons. Overexpression of KIF13A or knockout of MARK2 leads to axonal transport of TfR-containing vesicles. These results suggest a unique kinesin-based mechanism for polarized transport of vesicles to dendrites.

The microtubule targeting agent ST-401 triggers cell death in interphase and prevents the formation of polyploid giant cancer cells.

Microtubule targeting agents (MTAs) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells escape death in mitosis, exit mitosis and become malignant polyploid giant cancer cells (PGCC). Considering the low number of cancer cells undergoing mitosis in tumor tissues, killing them in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule (MT) assembly, preferentially kills cancer cells in interphase as opposed to mitosis, a cell death mechanism that avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces a transient integrated stress response, reduces energy metabolism, and promotes mitochondria fission. This cell response may underly death in interphase and avoid the development of PGCC. Considering that ST-401 is a brain-penetrant MTA, we validated these results in glioblastoma cell lines and found that ST-401 also reduces energy metabolism and promotes mitochondria fission in GBM sensitive lines. Thus, brain-penetrant mild inhibitors of MT assembly, such as ST-401, that induce death in interphase through a previously unanticipated antitumor mechanism represent a potentially transformative new class of therapeutics for the treatment of GBM.

Stable GDP-tubulin islands rescue dynamic microtubules.

Microtubules are dynamic polymers that interconvert between phases of growth and shrinkage, yet they provide structural stability to cells. Growth involves hydrolysis of GTP-tubulin to GDP-tubulin, which releases energy that is stored within the microtubule lattice and destabilizes it; a GTP cap at microtubule ends is thought to prevent GDP subunits from rapidly dissociating and causing catastrophe. Here, using in vitro reconstitution assays, we show that GDP-tubulin, usually considered inactive, can itself assemble into microtubules, preferentially at the minus end, and promote persistent growth. GDP-tubulin-assembled microtubules are highly stable, displaying no detectable spontaneous shrinkage. Strikingly, islands of GDP-tubulin within dynamic microtubules stop shrinkage events and promote rescues. Microtubules thus possess an intrinsic capacity for stability, independent of accessory proteins. This finding provides novel mechanisms to explain microtubule dynamics.

Transcription factor 4 promotes increased corneal endothelial cellular migration by altering microtubules in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a complex corneal disease characterized by the progressive decline and morphological changes of corneal endothelial cells (CECs) that leads to corneal edema and vision loss. The most common mutation in FECD is an intronic CTG repeat expansion in transcription factor 4 (TCF4) that leads to its altered expression. Corneal endothelial wound healing occurs primarily through cell enlargement and migration, and FECD CECs have been shown to display increased migration speeds. In this study, we aim to determine whether TCF4 can promote cellular migration in FECD CECs. We generated stable CEC lines derived from FECD patients that overexpressed different TCF4 isoforms and investigated epithelial-to-mesenchymal (EMT) expression, morphological analysis and cellular migration speeds. We found that full length TCF4-B isoform overexpression promotes cellular migration in FECD CECs in an EMT-independent manner. RNA-sequencing identified several pathways including the negative regulation of microtubules, with TUBB4A (tubulin beta 4A class IVa) as the top upregulated gene. TUBB4A expression was increased in FECD ex vivo specimens, and there was altered expression of cytoskeleton proteins, tubulin and actin, compared to normal healthy donor ex vivo specimens. Additionally, there was increased acetylation and detyrosination of microtubules in FECD supporting that microtubule stability is altered in FECD and could promote cellular migration. Future studies could be aimed at investigating if targeting the cytoskeleton and microtubules would have therapeutic potential for FECD by promoting cellular migration and regeneration.

Arabidopsis α-Aurora kinase plays a role in cytokinesis through regulating MAP65-3 association with microtubules at phragmoplast midzone.

The α-Aurora kinase is a crucial regulator of spindle microtubule organization during mitosis in plants. Here, we report a post-mitotic role for α-Aurora in reorganizing the phragmoplast microtubule array. In Arabidopsis thaliana, α-Aurora relocated from spindle poles to the phragmoplast midzone, where it interacted with the microtubule cross-linker MAP65-3. In a hypomorphic α-Aurora mutant, MAP65-3 was detected on spindle microtubules, followed by a diffuse association pattern across the phragmoplast midzone. Simultaneously, phragmoplast microtubules remained belatedly in a solid disk array before transitioning to a ring shape. Microtubules at the leading edge of the matured phragmoplast were often disengaged, accompanied by conspicuous retentions of MAP65-3 at the phragmoplast interior edge. Specifically, α-Aurora phosphorylated two residues towards the C-terminus of MAP65-3. Mutation of these residues to alanines resulted in an increased association of MAP65-3 with microtubules within the phragmoplast. Consequently, the expansion of the phragmoplast was notably slower compared to wild-type cells or cells expressing a phospho-mimetic variant of MAP65-3. Moreover, mimicking phosphorylation reinstated disrupted MAP65-3 behaviors in plants with compromised α-Aurora function. Overall, our findings reveal a mechanism in which α-Aurora facilitates cytokinesis progression through phosphorylation-dependent restriction of MAP65-3 associating with microtubules at the phragmoplast midzone.

Microtubule dependent sorting of actin-binding proteins in mitosis.

The patterns of Formin B and of the Arp2/3 complex formed during mitosis were studied in a mutant of Dictyostelium discoideum that produces multinucleate cells, which divide by the ingression of unilateral cleavage furrows. During cytokinesis the cells of this mutant remain spread on a glass surface where they generate a planar pattern based on the sorting-out of actin-binding proteins. During anaphase, Formin B and Arp2/3 became localized to the regions of microtubule asters around the centrosomes; Formin B in particular in the form of round, quite uniformly covered areas. These areas have been shown to be depleted of myosin II and the actin-filament crosslinker cortexillin, and to be avoided by cleavage furrows on their path into the cell.

A novel conductive microtubule hydrogel for electrical stimulation of chronic wounds based on biological electrical wires.

Electrical stimulation (ES) is considered a promising therapy for chronic wounds via conductive dressing. However, the lack of a clinically suitable conductive dressing is a serious challenge. In this study, a suitable conductive biomaterial with favorable biocompatibility and conductivity was screened by means of an inherent structure derived from the body based on electrical conduction in vivo. Ions condensed around the surface of the microtubules (MTs) derived from the cell's cytoskeleton are allowed to flow in the presence of potential differences, effectively forming a network of biological electrical wires, which is essential to the bioelectrical communication of cells. We hypothesized that MT dressing could improve chronic wound healing via the conductivity of MTs applied by ES. We first developed an MT-MAA hydrogel by a double cross-linking method using UV and calcium chloride to improve chronic wound healing by ES. In vitro studies showed good conductivity, mechanical properties, biocompatibility, and biodegradability of the MT-MAA hydrogel, as well as an elevated secretion of growth factors with enhanced cell proliferation and migration ability in response to ES. The in vivo experimental results from a full-thickness diabetic wound model revealed rapid wound closure within 7d in C57BL/6J mice, and the wound bed dressed by the MT-MAA hydrogel was shown to have promoted re-epithelization, enhanced angiogenesis, accelerated nerve growth, limited inflammation phases, and improved antibacterial effect under the ES treatment. These preclinical findings suggest that the MT-MAA hydrogel may be an ideal conductive dressing for chronic wound healing. Furthermore, biomaterials based on MTs may be also promising for treating other diseases.

An interphase actin wave promotes mitochondrial content mixing and organelle homeostasis.

Across the cell cycle, mitochondrial dynamics are regulated by a cycling wave of actin polymerization/depolymerization. In metaphase, this wave induces actin comet tails on mitochondria that propel these organelles to drive spatial mixing, resulting in their equitable inheritance by daughter cells. In contrast, during interphase the cycling actin wave promotes localized mitochondrial fission. Here, we identify the F-actin nucleator/elongator FMNL1 as a positive regulator of the wave. FMNL1-depleted cells exhibit decreased mitochondrial polarization, decreased mitochondrial oxygen consumption, and increased production of reactive oxygen species. Accompanying these changes is a loss of hetero-fusion of wave-fragmented mitochondria. Thus, we propose that the interphase actin wave maintains mitochondrial homeostasis by promoting mitochondrial content mixing. Finally, we investigate the mechanistic basis for the observation that the wave drives mitochondrial motility in metaphase but mitochondrial fission in interphase. Our data indicate that when the force of actin polymerization is resisted by mitochondrial tethering to microtubules, as in interphase, fission results.

Transient Interphase Microtubules Appear in Differentiating Sponge Cells.

Microtubules are an indispensable component of all eukaryotic cells due to their role in mitotic spindle formation, yet their organization and number can vary greatly in the interphase. The last common ancestor of all eukaryotes already had microtubules and microtubule motor proteins moving along them. Sponges are traditionally regarded as the oldest animal phylum. Their body does not have a clear differentiation into tissues, but it contains several distinguishable cell types. The choanocytes stand out among them and are responsible for creating a flow of water with their flagella and increasing the filtering and feeding efficiency of the sponge. Choanocyte flagella contain microtubules, but thus far, observing a developed system of cytoplasmic microtubules in non-flagellated interphase sponge cells has been mostly unsuccessful. In this work, we combine transcriptomic analysis, immunofluorescence, and electron microscopy with time-lapse recording to demonstrate that microtubules appear in the cytoplasm of sponge cells only when transdifferentiation processes are activated. We conclude that dynamic cytoplasmic microtubules in the cells of sponges are not a persistent but rather a transient structure, associated with cellular plasticity.

Map-1a regulates Sertoli cell BTB dynamics through the cytoskeletal organization of microtubule and F-actin.

Microtubule-associated protein 1a (Map1a) is a microtubule (MT) regulatory protein that binds to the MT protofilaments in mammalian cells to promote MT stabilization. Maps work with MT cleavage proteins and other MT catastrophe-inducing proteins to confer MT dynamics to support changes in the Sertoli cell shape to sustain spermatogenesis. However, no functional studies are found in the literature to probe its role in spermatogenesis. Using an RNAi approach, coupled with the use of toxicant-induced testis (in vivo)- and Sertoli cell (in vitro)-injury models, RNA-Seq analysis, transcriptome profiling, and relevant bioinformatics analysis, immunofluorescence analysis, and pertinent biochemical assays for cytoskeletal organization, we have delineated the functional role of Map1a in Sertoli cells and testes. Map1a was shown to support MT structural organization, and its knockdown (KD) also perturbed the structural organization of actin, vimentin, and septin cytoskeletons as these cytoskeletons are intimately related, working in concert to support spermatogenesis. More importantly, cadmium-induced Sertoli cell injury that perturbed the MT structural organization across the cell cytoplasm was associated with disruptive changes in the distribution of Map1a and a surge in p-p38-MAPK (phosphorylated p38-mitogen-activated protein kinase) expression but not total p38-MAPK. These findings thus support the notion that p-p38-MAPK activation is involved in cadmium-induced Sertoli cell injury. This conclusion was supported by studies using doramapimod, a specific p38-MAPK phosphorylation (activation) inhibitor, which was capable of restoring the cadmium-induced disruptive structural organization of MTs across the Sertoli cell cytoplasm. In summary: this study provides mechanistic insights regarding restoration of toxicant-induced Sertoli cell and testis injury and male infertility.

Systematic profiling of Taxol resistance and sensitivity to tubulin missence mutations at molecular and cellular levels.

Taxol (paclitaxel) is the first approved microtubule-stabilizing agent (MSA) by binding stoichiometrically to tubulin, which is considered to be one of the most significant advances in first-line chemotherapy against diverse tumors. However, a large number of residue missence mutations harboring in the tubulin have been observed to cause acquired drug resistance, largely limiting the clinical application of Taxol and its analogs in chemotherapy. A systematic investigation of the intermolecular interactions between the Taxol and various tubulin mutants would help to establish a comprehensive picture of drug response to tubulin mutations in clinical treatment of cancer, and to design new MSA agents with high potency and selectivity to overcome drug resistance. In this study, we described an integration of in silico analysis and in vitro assay (iSiV) to profile Taxol against a panel of 149 clinically observed, cancer-associated missence mutations in ß-tubulin at molecular and cellular levels, aiming to a systematic understanding of molecular mechanism and biological implication underlying drug resistance and sensitivity conferring from tubulin mutations. It is revealed that the Taxol-resistant mutations can be classified into three types: (I) nonbonded interaction broken due to mutation, (II) steric hindrance caused by mutation, and (III) conformational change upon mutation. In addition, we identified three new Taxol-resistant mutations (C239Y, T274I, and R320P) that can largely reduce the binding affinity of Taxol to tubulin at molecular level, in which the T274I and R320P were observed to considerably impair the antitumor activity of Taxol at cellular level. Moreover, a novel drug-susceptible mutation (M363T) was also identified, which improves Taxol affinity by 2.6-fold and decreases Taxol antitumor EC50 values from 29.4 to 18.7 µM.

ATP Concentration-Dependent Fractions of One-Head-Bound and Two-Head-Bound States of the Kinesin Motor during Its Chemomechanical Coupling Cycle.

Kinesin is a typical motor protein that can use the chemical energy of ATP hydrolysis to step processively on microtubules, alternating between one-head-bound and two-head-bound states. Some published experimental results showed that the duration of the one-head-bound state increases greatly with a decrease in ATP concentration, whereas the duration of the two-head-bound state is independent of ATP concentration, indicating that ATP binding occurs in the one-head-bound state. On the contrary, other experimental results showed that the duration of the two-head-bound state increases greatly with a decrease in ATP concentration, whereas the duration of the one-head-bound state increases slightly with a decrease in ATP concentration, indicating that ATP binding occurs mainly in the two-head-bound state. Here, we explain consistently and quantitatively these contradictory experimental results, resolving the controversy that is critical to the chemomechanical coupling mechanism of the kinesin motor.

Miro GTPases at the Crossroads of Cytoskeletal Dynamics and Mitochondrial Trafficking.

Miro GTPases are key components in the machinery responsible for transporting mitochondria and peroxisomes along microtubules, and also play important roles in regulating calcium homeostasis and organizing contact sites between mitochondria and the endoplasmic reticulum. Moreover, Miro GTPases have been shown to interact with proteins that actively regulate cytoskeletal organization and dynamics, suggesting that these GTPases participate in organizing cytoskeletal functions and organelle transport. Derailed mitochondrial transport is associated with neuropathological conditions such as Parkinson's and Alzheimer's diseases. This review explores our recent understanding of the diverse roles of Miro GTPases under cytoskeletal control, both under normal conditions and during the course of human diseases such as neuropathological disorders.

Modeling Studies of the Mechanism of Context-Dependent Bidirectional Movements of Kinesin-14 Motors.

Kinesin-14s, a subfamily of the large superfamily of kinesin motor proteins, function mainly in spindle assembly and maintenance during mitosis and meiosis. KlpA from Aspergillus nidulans and GiKIN14a from Giardia intestinalis are two types of kinesin-14s. Available experimental results puzzlingly showed that while KlpA moves preferentially toward the minus end in microtubule-gliding setups and inside parallel microtubule overlaps, it moves preferentially toward the plus end on single microtubules. More puzzlingly, the insertion of an extra polypeptide linker in the central region of the neck stalk switches the motility direction of KlpA on single microtubules to the minus end. Prior experimental results showed that GiKIN14a moves preferentially toward the minus end on single microtubules in either tailless or full-length forms. The tail not only greatly enhances the processivity but also accelerates the ATPase rate and velocity of GiKIN14a. The insertion of an extra polypeptide linker in the central region of the neck stalk reduces the ATPase rate of GiKIN14a. However, the underlying mechanism of these puzzling dynamical features for KlpA and GiKIN14a is unclear. Here, to understand this mechanism, the dynamics of KlpA and GiKIN14a were studied theoretically on the basis of the proposed model, incorporating potential changes between the kinesin head and microtubule, as well as the potential between the tail and microtubule. The theoretical results quantitatively explain the available experimental results and provide predicted results. It was found that the elasticity of the neck stalk determines the directionality of KlpA on single microtubules and affects the ATPase rate and velocity of GiKIN14a on single microtubules.