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

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

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.

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.

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.

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.

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.

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.

Microtubule-Targeting Agents: Disruption of the Cellular Cytoskeleton as a Backbone of Ovarian Cancer Therapy.

Microtubules are dynamic polymers composed of α- and ß-tubulin heterodimers. Microtubules are universally conserved among eukaryotes and participate in nearly every cellular process, including intracellular trafficking, replication, polarity, cytoskeletal shape, and motility. Due to their fundamental role in mitosis, they represent a classic target of anti-cancer therapy. Microtubule-stabilizing agents currently constitute a component of the most effective regimens for ovarian cancer therapy in both primary and recurrent settings. Unfortunately, the development of resistance continues to present a therapeutic challenge. An understanding of the underlying mechanisms of resistance to microtubule-active agents may facilitate the development of novel and improved approaches to this disease.

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.

9-fold symmetry is not essential for centriole elongation and formation of new centriole-like structures.

As daughter centrioles assemble during G2, they recruit conserved Ana3/RTTN followed by its partner Rcd4/PPP1R35. Together, this contributes to the subsequent recruitment of Ana1/CEP295, required for the centriole's conversion to a centrosome. Here, we show that Rcd4/PPP1R35 is also required to maintain 9-fold centriole symmetry in the Drosophila male germline; its absence causes microtubule triplets to disperse into a reduced number of doublet or singlet microtubules. rcd4-null mutant spermatocytes display skinny centrioles that elongate normally and localize centriolar components correctly. Mutant spermatocytes also have centrioles of normal girth that splay at their proximal ends when induced to elongate by Ana1 overexpression. Skinny and splayed spermatid centrioles can still recruit a proximal centriole-like (PCL) structure marking a capability to initiate features of centriole duplication in developing sperm. Thus, stable 9-fold symmetry of microtubule triplets is not essential for centriole growth, correct longitudinal association of centriole components, and aspects of centriole duplication.

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.

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.

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.

Tubulin Complexity in Cancer and Metastasis.

Tubulin plays a fundamental role in cellular function and as the subject for microtubule-active agents in the treatment of ovarian cancer. Microtubule-binding proteins (e.g., tau, MAP1/2/4, EB1, CLIP, TOG, survivin, stathmin) and posttranslational modifications (e.g., tyrosination, deglutamylation, acetylation, glycation, phosphorylation, polyamination) further diversify tubulin functionality and may permit additional opportunities to understand microtubule behavior in disease and to develop microtubule-modifying approaches to combat ovarian cancer. Tubulin-based structures that project from suspended ovarian cancer cells known as microtentacles may contribute to metastatic potential of ovarian cancer cells and could represent an exciting novel therapeutic target.

Vimentin is a key regulator of cell mechanosensing through opposite actions on actomyosin and microtubule networks.

The cytoskeleton is a complex network of interconnected biopolymers consisting of actin filaments, microtubules, and intermediate filaments. These biopolymers work in concert to transmit cell-generated forces to the extracellular matrix required for cell motility, wound healing, and tissue maintenance. While we know cell-generated forces are driven by actomyosin contractility and balanced by microtubule network resistance, the effect of intermediate filaments on cellular forces is unclear. Using a combination of theoretical modeling and experiments, we show that vimentin intermediate filaments tune cell stress by assisting in both actomyosin-based force transmission and reinforcement of microtubule networks under compression. We show that the competition between these two opposing effects of vimentin is regulated by the microenvironment stiffness. These results reconcile seemingly contradictory results in the literature and provide a unified description of vimentin's effects on the transmission of cell contractile forces to the extracellular matrix.

Uncovering kinesin dynamics in neurites with MINFLUX.

Neurons grow neurites of several tens of micrometers in length, necessitating active transport from the cell body by motor proteins. By tracking fluorophores as minimally invasive labels, MINFLUX is able to quantify the motion of those proteins with nanometer/millisecond resolution. Here we study the substeps of a truncated kinesin-1 mutant in primary rat hippocampal neurons, which have so far been mainly observed on polymerized microtubules deposited onto glass coverslips. A gentle fixation protocol largely maintains the structure and surface modifications of the microtubules in the cell. By analyzing the time between the substeps, we identify the ATP-binding state of kinesin-1 and observe the associated rotation of the kinesin-1 head in neurites. We also observed kinesin-1 switching microtubules mid-walk, highlighting the potential of MINFLUX to study the details of active cellular transport.

Competition between physical search and a weak-to-strong transition rate-limits kinesin binding times.

The self-organization of cells relies on the profound complexity of protein-protein interactions. Challenges in directly observing these events have hindered progress toward understanding their diverse behaviors. One notable example is the interaction between molecular motors and cytoskeletal systems that combine to perform a variety of cellular functions. In this work, we leverage theory and experiments to identify and quantify the rate-limiting mechanism of the initial association between a cargo-bound kinesin motor and a microtubule track. Recent advances in optical tweezers provide binding times for several lengths of kinesin motors trapped at varying distances from a microtubule, empowering the investigation of competing models. We first explore a diffusion-limited model of binding. Through Brownian dynamics simulations and simulation-based inference, we find this simple diffusion model fails to explain the experimental binding times, but an extended model that accounts for the ADP state of the molecular motor agrees closely with the data, even under the scrutiny of penalizing for additional model complexity. We provide quantification of both kinetic rates and biophysical parameters underlying the proposed binding process. Our model suggests that a typical binding event is limited by ADP state rather than physical search. Lastly, we predict how these association rates can be modulated in distinct ways through variation of environmental concentrations and physical properties.