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Microtubules are dynamic cytoskeletal structures involved in several cellular functions, such as intracellular trafficking, cell division and motility. More than other cell types, neurons rely on the proper functioning of microtubules to conduct their activities and achieve complex morphologies. Pathogenic variants in genes encoding for a and ß-tubulins, the structural subunits of microtubules, give rise to a wide class of neurological disorders collectively known as "tubulinopathies" and mainly involving a wide and overlapping range of brain malformations resulting from defective neuronal proliferation, migration, differentiation and axon guidance. Although tubulin mutations have been classically linked to neurodevelopmental defects, growing evidence demonstrates that perturbations of tubulin functions and activities may also drive neurodegeneration. In this study, we causally link the previously unreported missense mutation p.I384N in TUBA1A, one of the neuron-specific a-tubulin isotype I, to a neurodegenerative disorder characterized by progressive spastic paraplegia and ataxia. We demonstrate that, in contrast to the p.R402H substitution, which is one of the most recurrent TUBA1A pathogenic variants associated to lissencephaly, the present mutation impairs TUBA1A stability, reducing the abundance of TUBA1A available in the cell and preventing its incorporation into microtubules. We also show that the isoleucine at position 384 is an amino acid residue, which is critical for a-tubulin stability, since the introduction of the p.I384N substitution in three different tubulin paralogs reduces their protein level and assembly into microtubules, increasing their propensity to aggregation. Moreover, we demonstrate that the inhibition of the proteasome degradative systems increases the protein levels of TUBA1A mutant, promoting the formation of tubulin aggregates that, as their size increases, coalesce into inclusions that precipitate within the insoluble cellular fraction. Overall, our data describe a novel pathogenic effect of p.I384N mutation that differs from the previously described substitutions in TUBA1A, and expand both phenotypic and mutational spectrum related to this gene. [ABSTRACT FROM AUTHOR]

Microtubules (MTs) are large cytoskeletal polymers, composed of αβ- tubulin heterodimers, capable of stochastically converting from polymerizing to depolymerizing states and vice versa. Depolymerization is coupled with hydrolysis of guanosine triphosphate (GTP) within β- tubulin. Hydrolysis is favored in the MT lattice compared to a free heterodimer with an experimentally observed rate increase of 500- to 700- fold, corresponding to an energetic barrier lowering of 3.8 to 4.0 kcal/mol. Mutagenesis studies have implicated α- tubulin residues, α:E254 and α:D251, as catalytic residues completing the β- tubulin active site of the lower heterodimer in the MT lattice. The mechanism for GTP hydrolysis in the free heterodimer, however, is not understood. Additionally, there has been debate concerning whether the GTP- state lattice is expanded or compacted relative to the GDP state and whether a "compacted" GDP- state lattice is required for hydrolysis. In this work, extensive quantum mechanics/molecular mechanics simulations with transition- tempered metadynamics free- energy sampling of compacted and expanded interdimer complexes, as well as a free heterodimer, have been carried out to provide clear insight into the GTP hydrolysis mechanism. α:E254 was found to be the catalytic residue in a compacted lattice, while in the expanded lattice, disruption of a key salt bridge interaction renders α:E254 less effective. The simulations reveal a barrier decrease of 3.8 ± 0.5 kcal/mol for the compacted lattice compared to a free heterodimer, in good agreement with experimental kinetic measurements. Additionally, the expanded lattice barrier was found to be 6.3 ± 0.5 kcal/mol higher than compacted, demonstrating that GTP hydrolysis is variable with lattice state and slower at the MT tip. [ABSTRACT FROM AUTHOR]

During cell division, kinetochores link chromosomes to spindle microtubules. The Ndc80 complex, a crucial microtubule binder, populates each kinetochore with dozens of copies. Whether adjacent Ndc80 complexes cooperate to promote microtubule binding remains unclear. Here we demonstrate that the Ndc80 loop, a short sequence that interrupts the Ndc80 coiled‐coil at a conserved position, folds into a more rigid structure than previously assumed and promotes direct interactions between full‐length Ndc80 complexes on microtubules. Mutations in the loop impair these Ndc80‐Ndc80 interactions, prevent the formation of force‐resistant kinetochore‐microtubule attachments, and cause cells to arrest in mitosis for hours. This arrest is not due to an inability to recruit the kinetochore‐microtubule stabilizing SKA complex and cannot be overridden by mutations in the Ndc80 tail that strengthen microtubule attachment. Thus, loop‐mediated organization of adjacent Ndc80 complexes is crucial for stable end‐on kinetochore‐microtubule attachment and spindle assembly checkpoint satisfaction. Synopsis: Multiple copies of the extended Ndc80 complex mediate microtubule binding at each kinetochore. This study finds that homotypic interactions between Ndc80 complexes mediated by a loop region enable stable attachment of kinetochores to dynamic microtubule ends, explaining the crucial role of the Ndc80 loop during mitosis. Structural analysis of conserved interruptions in the long Ndc80 coiled‐coil shows rigid folding of the Ndc80 loop.The loop segment promotes Ndc80‐Ndc80 interactions on microtubules.End‐on kinetochore‐microtubule attachment is impaired by point mutations in the loop.The Ndc80 loop and Ndc80 tail contribute synergystically to chromosome congression. [ABSTRACT FROM AUTHOR]

The γ-tubulin complex (γTuC) is a widely conserved microtubule nucleator, but some of its components, namely GCP4, GCP5 and GCP6 (also known as TUBGCP4, TUBGCP5 and TUBGCP6, respectively), have not been detected in Caenorhabditis elegans. Here, we identified two γTuC-associated proteins in C. elegans, GTAP-1 and GTAP-2, for which apparent orthologs were detected only in the genus Caenorhabditis. GTAP-1 and GTAP-2 were found to localize at centrosomes and the plasma membrane of the germline, and their centrosomal localization was interdependent. In early C. elegans embryos, whereas the conserved γTuC component MZT-1 (also known as MOZART1 and MZT1) was essential for the localization of centrosomal γ-tubulin, depletion of GTAP-1 and/or GTAP-2 caused up to 50% reduction of centrosomal γ-tubulin and precocious disassembly of spindle poles during mitotic telophase. In the adult germline, GTAP-1 and GTAP-2 contributed to efficient recruitment of the γTuC to the plasma membrane. Depletion of GTAP-1, but not of GTAP-2, severely disrupted both the microtubule array and the honeycomb-like structure of the adult germline. We propose that GTAP-1 and GTAP-2 are unconventional components of the γTuC that contribute to the organization of both centrosomal and non-centrosomal microtubules by targeting the γTuC to specific subcellular sites in a tissue-specific manner. [ABSTRACT FROM AUTHOR]

Tubular epithelial cells are routinely exposed to severe changes in osmolarity. Although the autophagic activity of cells is an indispensable process to maintain cellular homeostasis and respond to stressors, the effect of hyperosmotic stress on autophagic activity in tubular epithelial cells remains unknown. The aim of this study was to determine the effect of hyperosmotic stress on autophagy in rat kidney tubular epithelial cells focusing on the role of actin and microtubule cytoskeletons. Normal rat kidney (NRK)-52E cells exposed to mannitol-induced hyperosmotic stress. As a result, NRK-52E cells showed elevated protein levels of the autophagosome marker LC3-II, indicating enhancement of the autophagic flux. Hyperosmotic stress also transiently decreased cell volume and caused the reorganization of actin and microtubule cytoskeletal structures in NRK-52E cells. The inhibition of the actin cytoskeleton reorganization by cytochalasin D impaired the increase in the levels of LC3-II; however, disassembly of the microtubules following treatment with nocodazole did not affect the increase. These results indicate that hyperosmotic stress can induce autophagy mediated by the reorganization of the actin cytoskeleton in tubular epithelial cells. • Hyperosmotic stress enhances autophagic flux in proximal tubular epithelial cells. • Transient cell volume reduction by hyperosmolarity causes actin reorganization. • Inhibition of actin reorganization impairs the hyperosmolarity-induced autophagy. [ABSTRACT FROM AUTHOR]

Environmental and occupational exposure to hexavalent chromium, Cr(VI), causes female reproductive failures and infertility. Cr(VI) is used in more than 50 industries and is a group A carcinogen, mutagenic and teratogenic, and a male and female reproductive toxicant. Our previous findings indicate that Cr(VI) causes follicular atresia, trophoblast cell apoptosis, and mitochondrial dysfunction in metaphase II (MII) oocytes. However, the integrated molecular mechanism of Cr(VI)-induced oocyte defects is not understood. The current study investigates the mechanism of Cr(VI) in causing meiotic disruption of MII oocytes, leading to oocyte incompetence in superovulated rats. Postnatal day (PND) 22 rats were treated with potassium dichromate (1 and 5 ppm) in drinking water from PND 22–29 and superovulated. MII oocytes were analyzed by immunofluorescence, and images were captured by confocal microscopy and quantified by Image-Pro Plus software, Version 10.0.5. Our data showed that Cr(VI) increased microtubule misalignment (~9 fold), led to missegregation of chromosomes and bulged and folded actin caps, increased oxidative DNA (~3 fold) and protein (~9–12 fold) damage, and increased DNA double-strand breaks (~5–10 fold) and DNA repair protein RAD51 (~3–6 fold). Cr(VI) also induced incomplete cytokinesis and delayed polar body extrusion. Our study indicates that exposure to environmentally relevant doses of Cr(VI) caused severe DNA damage, distorted oocyte cytoskeletal proteins, and caused oxidative DNA and protein damage, resulting in developmental arrest in MII oocytes. [ABSTRACT FROM AUTHOR]

The role of G2 and S phase‐expressed‐1 (GTSE1), a microtubule‐localized protein, in non‐small‐cell lung cancer (NSCLC) remains unknown. We explored its role in NSCLC growth. GTSE1 was detected in NSCLC tissues and cell lines using quantitative real‐time polymerase chain reaction. The clinical significance of GTSE1 levels was evaluated. Biological and apoptotic effects of GTSE1 were evaluated using transwell, cell‐scratch, and MTT assays, and flow cytometry and western blotting, respectively. Its association with cellular microtubules was shown by western blotting and immunofluorescence. GTSE1 expression was upregulated in NSCLC tissues and cell lines. GTSE1 levels correlated with lymph node metastasis. Higher GTSE1 mRNA expression correlated with shorter progression‐free survival. GTSE1‐knockdown decreased proliferation, colony formation, invasion, and migration of NSCLC cells, and inhibited tau and stathmin‐1 microtubule‐associated protein expression, via the extracellular‐regulated protein kinase/mitogen‐activated protein kinase (ERK/MAPK) signaling pathway, and microtubule disruption. GTSE1 may promote NSCLC growth by regulating tau and stathmin‐1 through the ERK/MAPK signaling pathway. [ABSTRACT FROM AUTHOR]

During eukaryotic cell division, organelles are distributed between daughter cells through a dynamic process to ensure that cells can differentiate and perform their functions correctly. Uncovering the mode of lipid droplet (LD) distribution may help reveal the mechanism of membrane remodeling during cell division and lipid droplet function. Our results showed that LDs were equally distributed in both daughter cells during cytokinesis. Further experiments demonstrated that the key factor regulating the movement of LDs is the microtubule (MT)-resident protein KIF5B. Because the KIF5B structure lacks a hydrophilic region, we believe that there are proteins that mediate the interaction between LDs and KIF5B. Mass spectrometric detection of KIF5B-interacting proteins on the surface of LDs demonstrated that LDs were first wrapped by intermediate filaments forming a meshwork and then contacted with MTs to mediate lipid droplet movement during cytokinesis. Disruption of the homogeneous distribution of LDs may hinder cell proliferation and even lead to apoptosis. [ABSTRACT FROM AUTHOR]

Background: Morphological properties of tissues and organs rely on cell growth. The growth of plant cells is determined by properties of a tough outer cell wall that deforms anisotropically in response to high turgor pressure. Cortical microtubules bias the mechanical anisotropy of a cell wall by affecting the trajectories of cellulose synthases in the wall that polymerize cellulose microfibrils. The microtubule cytoskeleton is often oriented in one direction at cellular length-scales to regulate growth direction, but the means by which cellular-scale microtubule patterns emerge has not been well understood. Correlations between the microtubule orientation and tensile forces in the cell wall have often been observed. However, the plausibility of stress as a determining factor for microtubule patterning has not been directly evaluated to date. Results: Here, we simulated how different attributes of tensile forces in the cell wall can orient and pattern the microtubule array in the cortex. We implemented a discrete model with transient microtubule behaviors influenced by local mechanical stress in order to probe the mechanisms of stress-dependent patterning. Specifically, we varied the sensitivity of four types of dynamic behaviors observed on the plus end of microtubules – growth, shrinkage, catastrophe, and rescue – to local stress. Then, we evaluated the extent and rate of microtubule alignments in a two-dimensional computational domain that reflects the structural organization of the cortical array in plant cells. Conclusion: Our modeling approaches reproduced microtubule patterns observed in simple cell types and demonstrated that a spatial variation in the magnitude and anisotropy of stress can mediate mechanical feedback between the wall and of the cortical microtubule array. [ABSTRACT FROM AUTHOR]

Tauopathies are a group of neurodegenerative diseases, which include frontotemporal dementia (FTD) and Alzheimer's disease (AD), broadly defined by the development of tau brain aggregates. Both missense and splicing tau mutations can directly cause early onset FTD. Tau protein is a microtubuleassociated protein that stabilizes and regulates microtubules, but this function can be disrupted in disease states. One contributing factor is the balance of different tau isoforms, which can be categorized into either three repeat (3R) or four repeat (4R) isoforms based on the number of microtubule-binding repeats that are expressed. Imbalance of 3R and 4R isoforms in either direction can cause FTD and neurodegeneration. There is also increasing evidence that 3R tauopathies such as Pick's disease form tau aggregates predominantly comprised of 3R isoforms and these can present differently from 4R and mixed 3R/4R tauopathies. In this study, multiple mutations in 3R tau were assessed for MT binding properties and prionlike aggregation propensity. Different missense tau mutations showed varying effects on MT binding depending on molecular location and properties. Of the mutations that were surveyed, S356T tau is uniquely capable of prion-like seeded aggregation and forms extensive Thioflavin positive aggregates. This unique prion-like tau strain will be useful to model 3R tau aggregation and will contribute to the understanding of diverse presentations of different tauopathies. [ABSTRACT FROM AUTHOR]

Cytoskeletal networks usually refer to cellular structures that are comprised of microtubules, actin filaments, intermediate filaments, and their associated accessory proteins and motor proteins, facilitating a range of cellular functions such as cell motion, division and growth. In addition to the narrow definition of “cytoskeleton” that provides frame support to an otherwise fluidic cell, cytoskeletal polymers play an important role in many other cellular functions. For example, bacteria flagella and spindle apparatus are essentially microtubules in different forms. In living cells, there are also hundreds of proteins and biochemical factors regulating the structure and dynamics of such networks, which makes it extremely difficult to elucidate the physical mechanisms behind these processes. In recent years, study of in vitro cytoskeletal polymer-motor protein networks built from purified protein components not only helps to understand the fundamental principles of nonequilibrium self-organization and dynamic behavior of cytoskeletal polymers and motor proteins on the subcellular level, but also sheds light on the design of active matter system and active machines that may operate far from equilibrium with life-like behaviors and functions. One notable success is the artificial active nematics built upon microtubules and kinesin motors, in which active stresses are used to generate macroscopic active flow and guide materials assembly. The active stress and order emergence of materials organization can also be tuned by a set of external parameters, such as external magnetic fields and light-activated proteins, in addition to the concentration of protein building blocks, ATP, and crowding agents. In this review, we focus on in vitro cytoskeleton-motor protein networks based on purified components including tubulin, actin, kinesin, and myosin, emphasizing on the non-equilibrium nature of microtubule and F-actin polymerization, generation of active stress and formation of dynamic networks, as well as the self-organization and dynamic behavior of subcellular structures on a larger scale. We conclude with the application of such networks in the study of active matter and artificial cells. [ABSTRACT FROM AUTHOR]

Background and purpose: Microtubule defects are a common feature in several neurodegenerative disorders, including hereditary spastic paraplegia. The most frequent form of hereditary spastic paraplegia is caused by mutations in the SPG4/SPAST gene, encoding the microtubule severing enzyme spastin. To date, there is no effective therapy available but spastin‐enhancing therapeutic approaches are emerging; thus prognostic and predictive biomarkers are urgently required. Methods: An automated, simple, fast and non‐invasive cell imaging‐based method was developed to quantify microtubule cytoskeleton organization changes in lymphoblastoid cells and peripheral blood mononuclear cells. Results: It was observed that lymphoblastoid cells and peripheral blood mononuclear cells from individuals affected by SPG4‐hereditary spastic paraplegia show a polarized microtubule cytoskeleton organization. In a pilot study on freshly isolated peripheral blood mononuclear cells, our method discriminates SPG4‐hereditary spastic paraplegia from healthy donors and other hereditary spastic paraplegia subtypes. In addition, it is shown that our method can detect the effects of spastin protein level changes. Conclusions: These findings open the possibility of a rapid, non‐invasive, inexpensive test useful to recognize SPG4‐hereditary spastic paraplegia subtype and evaluate the effects of spastin‐enhancing drug in non‐neuronal cells. [ABSTRACT FROM AUTHOR]

Whispering gallery mode (WGM) microtubule resonators are widely used in highly sensitive sensing applications due to their ultra-high quality factor Q and extremely small mode size. Capillary microtubules, as sensing devices, have the advantages of low reagent sample consumption, a large specific surface area to enhance analytical sensitivity, and a fast mass transfer rate to shorten analysis time. In recent years, research scholars have conducted extensive and in-depth studies on the optimization of microtubule resonators. However, there are still several problems that limit the development of the technology applications and solutions that need to be explored. Therefore, this paper analyzes the current research status of resonant sensing technology in microtubule whispering gallery, analyzes key problems, and points out research priorities and methods in coupled resonance technology and microtubule structure optimization. Finally, suggestions for future development are made. Capillary microcavity Whispering gallery mode (WGM) resonators with high Q and small mode volume are well suited for applications in high sensitivity sensing. Such as biosensing, temperature sensing, pressure sensing, flow rate sensing, strain sensing, and magnetic sensing, etc. The article analyzes the key technologies of capillary microcavity WGM resonant sensing, focusing on the coupled resonant technology to excite capillary WGM, capillary structure parameter optimization and sensor packaging, and provides an outlook on the future development. [ABSTRACT FROM AUTHOR]

Cell biology is moving from observing molecules to controlling them in real time, a critical step towards a mechanistic understanding of how cells work. Initially developed from light-gated ion channels to control neuron activity, optogenetics now describes any genetically encoded protein system designed to accomplish specific light-mediated tasks. Recent photosensitive switches use many ingenious designs that bring spatial and temporal control within reach for almost any protein or pathway of interest. This next generation optogenetics includes light-controlled protein-protein interactions and shape-shifting photosensors, which in combination with live microscopy enable acute modulation and analysis of dynamic protein functions in living cells. We provide a brief overview of various types of optogenetic switches. We then discuss how diverse approaches have been used to control cytoskeleton dynamics with light through Rho GTPase signaling, microtubule and actin assembly, mitotic spindle positioning and intracellular transport and highlight advantages and limitations of different experimental strategies.

Oligodendrocytes in the central nervous system (CNS) produce myelin sheaths that insulate axons to facilitate efficient electrical conduction. These myelin sheaths contain lamellar microtubules that enable vesicular transport into the inner sheath. Mechanistically, oligodendrocytes rely on Golgi outpost organelles and the associated protein tubulin polymerization promoting protein (TPPP) to nucleate or form new microtubules outside of the cell body. Consequently, elongation of lamellar microtubules is defective in Tppp knock-out (KO) mice, which have thinner and shorter myelin sheaths. We now explore the behavioral phenotypes of Tppp KO mice using a number of different assays. In open-field assays, Tppp KO mice display similar activity levels and movement patterns as wild-type mice, indicating that they do not display anxiety behavior. However, Tppp KO mice lack fear responses by two types of assays, traditional fear-conditioning assays and looming fear assays, which test for innate fear responses. Deficits in fear conditioning, which is a memory-dependent task, as well as in spatial memory tests, support possible short-term memory defects in Tppp KO mice. Together, our experiments indicate a connection between CNS myelination and behavioral deficits.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light and electron microscopy, in situ cryo-electron tomography, and subtomogram analysis, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase and kinase are in close proximity, with the GTPase closer to the microtubule surface, whereas the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to generate models of previously unsolved structures in their cellular environment.

During embryonic development and cancer metastasis, migratory cells must establish stable connections with new partners at their destinations. Here, we establish the Drosophila border cells as a model for this multistep process. During oogenesis, border cells delaminate from the follicular epithelium and migrate. When they reach their target, the oocyte, they undergo a stereotypical series of steps to adhere to it, then connect with another migrating epithelium. We identify gap-junction-forming innexin proteins as critical. Surprisingly, the channel function is dispensable. Instead, Innexins 2 and 3 function within the border cells, and Innexin 4 functions within the germline, to regulate microtubules. The microtubule-dependent border cell-oocyte interaction is essential to brace the cells against external morphogenetic forces. Thus, we establish an experimental model and use genetic, thermogenetic, and live-imaging approaches to uncover the contributions of Innexins and microtubules to a cell-biological process important in development and cancer.

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.

Promoting axon regeneration in the central and peripheral nervous system is of clinical importance in neural injury and neurodegenerative diseases. Both pro- and antiregeneration factors are being identified. We previously reported that the Rtca mediated RNA repair/splicing pathway restricts axon regeneration by inhibiting the nonconventional splicing of Xbp1 mRNA under cellular stress. However, the downstream effectors remain unknown. Here, through transcriptome profiling, we show that the tubulin polymerization-promoting protein (TPPP) ringmaker/ringer is dramatically increased in Rtca-deficient Drosophila sensory neurons, which is dependent on Xbp1. Ringer is expressed in sensory neurons before and after injury, and is cell-autonomously required for axon regeneration. While loss of ringer abolishes the regeneration enhancement in Rtca mutants, its overexpression is sufficient to promote regeneration both in the peripheral and central nervous system. Ringer maintains microtubule stability/dynamics with the microtubule-associated protein futsch/MAP1B, which is also required for axon regeneration. Furthermore, ringer lies downstream from and is negatively regulated by the microtubule-associated deacetylase HDAC6, which functions as a regeneration inhibitor. Taken together, our findings suggest that ringer acts as a hub for microtubule regulators that relays cellular status information, such as cellular stress, to the integrity of microtubules in order to instruct neuroregeneration.

Steroid hormones control various aspects of brain development and behavior in metazoans, but how they enter the central nervous system (CNS) through the blood-brain barrier (BBB) remains poorly understood. It is generally believed that steroid hormones freely diffuse through the plasma membrane of the BBB cells to reach the brain [1], because of the predominant "simple diffusion" model of steroid hormone transport across cell membranes. Recently, however, we challenged the simple diffusion model by showing that a Drosophila organic anion-transporting polypeptide (OATP), which we named Ecdysone Importer (EcI), is required for cellular uptake of the primary insect steroid hormone ecdysone [2]. As ecdysone is first secreted into the hemolymph before reaching the CNS [3], our finding raised the question of how ecdysone enters the CNS through the BBB to exert its diverse role in Drosophila brain development. Here, we demonstrate in the Drosophila BBB that EcI is indispensable for ecdysone entry into the CNS to facilitate brain development. EcI is highly expressed in surface glial cells that form the BBB, and EcI knockdown in the BBB suppresses ecdysone signaling within the CNS and blocks ecdysone-mediated neuronal events during development. In an ex vivo culture system, the CNS requires EcI in the BBB to incorporate ecdysone from the culture medium. Our results suggest a transporter-mediated mechanism of steroid hormone entry into the CNS, which may provide important implications in controlling brain development and behavior by regulating steroid hormone permeability across the BBB.

Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.

Traditional microtubule inhibitors fail to significantly enhance the effect of colorectal cancer; hence, new and efficient strategies are necessary. In this study, a supramolecular nanoreactor (DOC@TA-Fe3+) based on tannic acid (TA), iron ion (Fe3+), and docetaxel (DOC) with microtubule inhibition, reactive oxygen species (ROS) generation, and glutathione peroxidase 4 (GPX4) inhibition, is prepared for ferroptosis/apoptosis treatment. After internalization by CT26 cells, the DOC@TA-Fe3+ nanoreactor escapes from the lysosomes to release payloads. The subsequent Fe3+/Fe2+ conversion mediated by TA reducibility can trigger the Fenton reaction to enhance the ROS concentration. Additionally, Fe3+ can consume glutathione to repress the activity of GPX4 to induce ferroptosis. Meanwhile, the released DOC controls microtubule dynamics to activate the apoptosis pathway. The superior in vivo antitumor efficacy of DOC@TA-Fe3+ nanoreactor in terms of tumor growth inhibition and improved survival is verified in CT26 tumor-bearing mouse model. Therefore, the nanoreactor can act as an effective apoptosis and ferroptosis inducer for application in colorectal cancer therapy.

Dendritic morphogenesis requires dynamic microtubules (MTs) to form a coordinated cytoskeletal network during development. Dynamic MTs are characterized by their number, polarity and speed of polymerization. Previous studies described a correlation between anterograde MT growth and terminal branch extension in Drosophila dendritic arborization (da) neurons, suggesting a model that anterograde MT polymerization provides a driving force for dendritic branching. We recently found that the Ste20-like kinase Tao specifically regulates dendritic branching by controlling the number of dynamic MTs in a kinase activity-dependent fashion, without affecting MT polarity or speed. This finding raises the interesting question of how MT dynamics affects dendritic morphogenesis, and if Tao kinase activity is developmentally regulated to coordinate MT dynamics and dendritic morphogenesis. We explored the possible correlation between MT dynamics and dendritic morphogenesis together with the activity changes of Tao kinase in C1da and C4da neurons during larval development. Our data show that spatiotemporal changes in the number of dynamic MTs, but not polarity or polymerization speed, correlate with dendritic branching and Tao kinase activity. Our findings suggest that Tao kinase limits dendritic branching by controlling the abundance of dynamic MTs and we propose a novel model on how regulation of MT dynamics might influence dendritic morphogenesis.

Sulforaphane and its metabolites (SFNs) cause apoptosis in cancers and could be potential anti-cancer drugs. We focused on investigating the underlying mechanisms through which SFNs inhibit cancers. First, SFNs cause microtubule disruption by phosphorylated MAPK1/ERK2-MAPK3/ERK1-mediated activation of 26S proteasome leading to a microtubule-associated protein degradation and microtubule depolymerization. Second, SFNs cause the accumulation of autophagosomes and mitophagosomes via blocking their fusion with lysosomes. These changes might be involved in multiple signaling pathways. High-performance liquid chromatography-tandem mass spectrometry showed that SFN regulates the expression of lipoproteins; highly expressed FASN (fatty acid synthase) correlates with cancer malignancy and poor prognosis. More, SFN lowers the expressions of FASN, ACACA (acetyl-CoA carboxylase alpha), and ACLY (ATP citrate lyase) by activating the 26S proteasome; SFN inhibits the interactions of TUBA/α-tubulin with FASN, ACACA or ACLY; SFN also reduces the production of intracellular fatty acids; knockdown of FASN increases mitochondrial abnormality and apoptosis. Moreover, SFN decreases the expressions of mitophagy-associated proteins BNIP3L/NIX and BNIP3 and the interaction between BNIP3L/NIX and LC3-II/-I and upregulates mitochondria-associated LC3-II/-I. Therefore, SFN might cause apoptosis via inhibiting the microtubule-mediated lipoprotein activity and the fusion of lysosomes with autophagosomes and mitophagosomes.

Alzheimer’s disease is known as one of the “top ten killers in the world”. Due to lack of effective therapy at present， early pathological changes have captivated widespread attention. Axonal transport dysfunction has been reported as an early pathological feature of many neurodegenerative diseases. However， multiple factors can cause axonal transport dysfunction. In this article， the relationship between axonal transport dysfunction caused by kinesins， microtubules and mitochondria and Alzheimer’s disease was discussed， aiming to provide new ideas for the prevention and treatment of Alzheimer’s disease by in-depth study on axonal transport mechanism of neure.

Uterine leiomyosarcoma is an aggressive soft tissue tumor. Stathmin, a phosphoprotein that modulates microtubule dynamics, is highly expressed in many malignancies including leiomyosarcoma. The microtubule-depolymerizing agent eribulin has been recently approved for treating malignant soft tissue tumors. Although eribulin inhibits microtubule polymerization, little is known about the relationship between eribulin treatment and stathmin dynamics. In this study, we explored the role of stathmin expression in the action of eribulin in leiomyosarcoma cells. Eribulin induced phosphorylation of stathmin and reduced expression of subunits A and C of protein phosphatase 2A (PP2A) in a leiomyosarcoma cell line. The PP2A activator FTY720 reduced levels of phosphorylated stathmin. Eribulin decreased stathmin protein levels without affecting stathmin mRNA expression. Furthermore, stathmin knockdown attenuated the inhibitory effects of eribulin on cell viability, whereas stathmin overexpression enhanced the anti-proliferative effect of eribulin. Eribulin-resistant leiomyosarcoma cell lines had enhanced expression of the class Ⅰ β-tubulin TUBB1, multi-drug resistance 1 protein MDR1 and breast cancer-resistance protein BCRP, and decreased expression of stathmin. Taken together, these results suggest that stathmin expression modulates the pharmacological efficacy of eribulin in uterine leiomyosarcoma cells.

Griseofulvin was considered an effective agent for cancer therapy in past decades. Although the negative effects of griseofulvin on microtubule stability are known, the exact target and mechanism of action in plants remain unclear. Here, we used trifluralin, a well-known herbicide targeting microtubules, as a reference and revealed the differences in root tip morphology, reactive oxygen species production (ROS), microtubule dynamics, and transcriptome analysis between Arabidopsis treated with griseofulvin and trifluralin to elucidate the mechanism of root growth inhibition by griseofulvin. Like trifluralin, griseofulvin inhibited root growth and caused significant swelling of the root tip due to cell death induced by ROS. However, the presence of griseofulvin and trifluralin caused cell swelling in the transition zone (TZ) and meristematic zone (MZ) of root tips, respectively. Further observations revealed that griseofulvin first destroyed cortical microtubules in the cells of the TZ and early elongation zone (EZ) and then gradually affected the cells of other zones. The first target of trifluralin is the microtubules in the root MZ cells. Transcriptome analysis showed that griseofulvin mainly affected the expression of microtubule-associated protein (MAP) genes rather than tubulin genes, whereas trifluralin significantly suppressed the expression of αβ-tubulin genes. Finally, it was proposed that griseofulvin could first reduce the expression of MAP genes, meanwhile increasing the expression of auxin and ethylene-related genes to disrupt microtubule alignment in root tip TZ and early EZ cells, induce dramatic ROS production, and cause severe cell death, eventually leading to cell swelling in the corresponding zones and inhibition of root growth. [ABSTRACT FROM AUTHOR]

Active nematics represent a relatively new class of materials with liquid crystalline properties that rely on an energy source to maintain their structure and dynamics. In this paper, we focus on the well-known microtubule-kinesin-based active nematic, powered by chemical energy as ATP (Adenosine triphosphate), and report on how ATP depletion impacts active nematic defect dynamics and texture. Using fluorescence microscope video imaging, we measure the time averaged mean defect separations and root mean square velocities of the active flows as a function of time. We also characterise textural changes and the growth of void space in the network after ATP depletion using an image binarization technique. [ABSTRACT FROM AUTHOR]

• A TEV-based system was developed to control tau cleavage in cells. • The simultaneous effects of the N- and C-terminal fragments can be examined. • Tau fragments show aberrant localizations with respect to the cytoskeleton. • N-terminal fragments partially relocate to the nucleus. In several forms of dementia, such as Alzheimer's disease, the cytoskeleton-associated protein tau undergoes proteolysis, giving rise to fragments that have a toxic impact on neuronal homeostasis. How these fragments interact with cellular structures, in particular with the cytoskeleton, is currently incompletely understood. Here, we developed a method, derived from a Tobacco Etch Virus (TEV) protease system, to induce controlled cleavage of tau at specific sites. Five tau proteins containing specific TEV recognition sites corresponding to pathological proteolytic sites were engineered, and tagged with GFP at one end and mCherry at the other. After a controlled cleavage to produce GFP-N-terminal and C-terminal-mCherry fragments, we followed the fate of tau fragments in cells. Our results showed that whole engineered tau proteins associate with the cytoskeleton similarly to the non-modified tau, whereas tau fragments adopted different localizations with respect to the actin and microtubule cytoskeletons. These distinct localizations were confirmed by expressing each separate fragment in cells. Some cleavages – in particular cleavages at amino-acid positions 124 or 256 – displayed a certain level of cellular toxicity, with an unusual relocalization of the N-terminal fragments to the nucleus. Based on the data presented here, inducible cleavage of tau by the TEV protease appears to be a valuable tool to reproduce tau fragmentation in cells and study the resulting consequences on cell physiology. [ABSTRACT FROM AUTHOR]

HSP-SPAST is the most common form of hereditary spastic paraplegia (HSP), a neurodegenerative disease causing lower limb spasticity. Previous studies using HSP-SPAST patient-derived induced pluripotent stem cell cortical neurons have shown that patient neurons have reduced levels of acetylated a-tubulin, a form of stabilized microtubules, leading to a chain of downstream effects eventuating in increased vulnerability to axonal degeneration. Noscapine treatment rescued these downstream effects by restoring the levels of acetylated a-tubulin in patient neurons. Here we show that HSP-SPAST patient non-neuronal cells, peripheral blood mononuclear cells (PBMCs), also have the disease-associated effect of reduced levels of acetylated a-tubulin. Evaluation of multiple PBMC subtypes showed that patient T cell lymphocytes had reduced levels of acetylated a-tubulin. T cells make up to 80% of all PBMCs and likely contributed to the effect of reduced acetylated a-tubulin levels seen in overall PBMCs. We further showed that mouse administered orally with increasing concentrations of noscapine exhibited a dose-dependent increase of noscapine levels and acetylated a-tubulin in the brain. A similar effect of noscapine treatment is anticipated in HSP-SPAST patients. To measure acetylated a-tubulin levels, we used a homogeneous time resolved fluorescence technologybased assay. This assay was sensitive to noscapine-induced changes in acetylated a-tubulin levels in multiple sample types. The assay is high throughput and uses nano-molar protein concentrations, making it an ideal assay for evaluation of noscapine-induced changes in acetylated a-tubulin levels. This study shows that HSP-SPAST patient PBMCs exhibit disease-associated effects. This finding can help expedite the drug discovery and testing process. [ABSTRACT FROM AUTHOR]

Microtubule-targeting agents (MTAs) bind to one of several distinct sites in the tubulin dimer, the subunit of microtubules. The binding affinities of MTAs may vary by several orders of magnitude, even for MTAs that specifically bind to a particular site. The first drug binding site discovered in tubulin was the colchicine binding site (CBS), which has been known since the discovery of the tubulin protein. Although highly conserved throughout eukaryotic evolution, tubulins show diversity in their sequences between tubulin orthologs (inter-species sequence differences) and paralogs (intraspecies differences, such as tubulin isotypes). The CBS is promiscuous and binds to a broad range of structurally distinct molecules that can vary in size, shape, and affinity. This site remains a popular target for the development of new drugs to treat human diseases (including cancer) and parasitic infections in plants and animals. Despite the rich knowledge about the diversity of tubulin sequences and the structurally distinct molecules that bind to the CBS, a pattern has yet to be found to predict the affinity of new molecules that bind to the CBS. In this commentary, we briefly discuss the literature evidencing the coexistence of the varying binding affinities for drugs that bind to the CBS of tubulins from different species and within species. We also comment on the structural data that aim to explain the experimental differences observed in colchicine binding to the CBS of β-tubulin class VI (TUBB1) compared to other isotypes. [ABSTRACT FROM AUTHOR]

Traumatic axonal injury (TAI) accounts for a large proportion of the mortality of traumatic brain injury (TBI). The diagnosis of TAI is currently of limited use for medicolegal purposes. It is known that axons in TAI are diffusely damaged by secondary processes other than direct head injury. However, the physiopathological mechanism of TAI is still elusive. The present study used RGD peptide, an antagonist of the mechanotransduction protein integrin, to explore the role of integrin-transmitted mechanical signalling in the pathogenesis of rat TAI. The rats were subjected to a linearly accelerating load, and changes in beta-amyloid precursor protein (β-APP) expression, skeleton ultrastructure, skeleton protein neurofilament light (NF-L), and α-tubulin in the brainstem were observed, indicating that RGD could relieve the severity of axonal injury in TAI rats. In addition, the expression of β-integrin was stronger and centralized in the brainstem of the deceased died from TAI compared to other nonviolent causes. This study examined the pathophysiology and biomechanics of TAI and assessed the role of integrin in the injury of microtubules and neurofilaments in TAI. Thus, we propose that integrin-mediated cytoskeletal injury plays an important role in TAI and that integrin has the potential as a biomarker for TAI. [ABSTRACT FROM AUTHOR]

Diffusion is a pervasive process present in a broad spectrum of cellular reactions. Its mathematical description has existed for nearly two centuries and permits the construction of simple rules for evaluating the characteristic timescales of diffusive processes and some of their determinants. Although the term diffusion originally referred to random motions in three-dimensional (3D) media, several biological diffusion processes in lower dimensions have been reported. One-dimensional (1D) diffusions have been reported, for example, for translocations of various proteins along DNA or protein (e.g., microtubule) lattices and translation of helical peptides along the coiled-coil interface. Two-dimensional (2D) diffusion has been shown for dynamics of proteins along membranes. The microscopic mechanisms of these 1–3D diffusions may vary significantly depending on the nature of the diffusing molecules, the substrate, and the interactions between them. In this review, we highlight some key examples of 1–3D biomolecular diffusion processes and illustrate the roles that electrostatic interactions and intrinsic disorder may play in modulating these processes. [ABSTRACT FROM AUTHOR]

Microtubules are cytoskeletal cell elements that also build flagella and cilia. Moreover, these structures participate in spermatogenesis and form a microtubular manchette during spermiogenesis. The present study aims to assess the influence of propyzamide, a microtubule-disrupting agent, on alga Chara vulgaris spermatids during their differentiation by means of immunofluorescent and electron microscopy methods. Propyzamide blocks the functioning of the β-tubulin microtubule subunit, which results in the creation of a distorted shape of a sperm nucleus at some stages. Present ultrastructural studies confirm these changes. In nuclei, an altered chromatin arrangement and nuclear envelope fragmentation were observed in the research as a result of incorrect nucleus–cytoplasm transport behavior that disturbed the action of proteolytic enzymes and the chromatin remodeling process. In the cytoplasm, large autolytic vacuoles and the dilated endoplasmic reticulum (ER) system, as well as mitochondria, were revealed in the studies. In some spermatids, the arrangement of microtubules present in the manchette was disturbed and the structure was also fragmented. The observations made in the research at present show that, despite some differences in the manchette between Chara and mammals, and probably also in the alga under study, microtubules participate in the intramanchette transport (IMT) process, which is essential during spermatid differentiation. In the present study, the effect of propyzamide on Chara spermiogenesis is also presented for the first time; however, the role of microtubule-associated proteins in this process still needs to be elucidated in the literature. [ABSTRACT FROM AUTHOR]

The measurement of cytoskeletal features can provide valuable insights into cell biology. In recent years, digital image analysis of cytoskeletal features has become an important research tool for quantitative evaluation of cytoskeleton organization. In this study, we examined the utility of a supervised machine learning approach with digital image analysis to distinguish different cellular organizational patterns. We focused on the jigsaw puzzle-shaped pavement cells of Arabidopsis thaliana. Measurements of three features of cortical microtubules in these cells (parallelness, density, and the coefficient of variation of the intensity distribution of fluorescently labeled cytoskeletons [as an indicator of microtubule bundling]) were obtained from microscopic images. A random forest machine learning model was then used with these images to differentiate mutant and wild type, and Taxol-treated and control cells. Using these three metrics, we were able to distinguish wild type from bpp125 triple mutant cells, with approximately 80% accuracy; classification accuracy was 88% for control and Taxol-treated cells. Different features contributed most to the classification, namely, coefficient of variation for the wild-type/mutant cells and parallelness for the Taxol-treated/control cells. The random forest method used enabled quantitative evaluation of the contribution of features to the classification, and partial dependence plots showed the relationships between metric values and classification accuracy. While further improvements to the method are needed, our small-scale analysis shows the potential for this approach in large-scale screening analyses. [ABSTRACT FROM AUTHOR]

Cancer is one of the most common causes of human death worldwide; thus, numerous therapies, including chemotherapy, have been and are being continuously developed. In cancer cells, an aberrant mitotic spindle—a microtubule-based structure necessary for the equal splitting of genetic material between daughter cells—leads to genetic instability, one of the hallmarks of cancer. Thus, the building block of microtubules, tubulin, which is a heterodimer formed from α- and β-tubulin proteins, is a useful target in anti-cancer research. The surface of tubulin forms several pockets, i.e., sites that can bind factors that affect microtubules' stability. Colchicine pockets accommodate agents that induce microtubule depolymerization and, in contrast to factors that bind to other tubulin pockets, overcome multi-drug resistance. Therefore, colchicine-pocket-binding agents are of interest as anti-cancer drugs. Among the various colchicine-site-binding compounds, stilbenoids and their derivatives have been extensively studied. Herein, we report systematic studies on the antiproliferative activity of selected stilbenes and oxepine derivatives against two cancer cell lines—HCT116 and MCF-7—and two normal cell lines—HEK293 and HDF-A. The results of molecular modeling, antiproliferative activity, and immunofluorescence analyses revealed that compounds 1a, 1c, 1d, 1i, 2i, 2j, and 3h were the most cytotoxic and acted by interacting with tubulin heterodimers, leading to the disruption of the microtubular cytoskeleton. [ABSTRACT FROM AUTHOR]

Microtubules are highly dynamic polymers of α/β‐tubulin that represent major components of the cytoskeleton and have been established as an attractive druggable target of tumors. Taxol, also known as Paclitaxel, is a microtubule‐stabilizing agent that binds stoichiometrically to a specific site in β‐tubulin, where is out of but nearby the interacting interface of α/β‐tubulin complex, to improve the complex stability through an allosterically regulatory mechanism. In this study, the systematic binding profile of taxol and its 22 structurally diverse, medicinally relevant analogues (termed as taxalogues) to the specific site of β‐tubulin as well as their effects on the α/β‐tubulin complex stability were created and investigated by using structural modeling, dynamics simulation, and energetics analysis. Two helices H1 and H2 of β‐tubulin were identified as key hotspots at the α/β‐tubulin interface to mediate the binding event of β‐tubulin to α‐tubulin. Taxalogue binding can trigger a conformational displacement in the H1 and H2 to elicit the stabilization (or destabilization) of α/β‐tubulin. However, strong taxalogue binding potency to β‐tubulin does not mean effective α/β‐tubulin stabilization; there is only an indirect, moderate correlation between them. Cell viability assay revealed that the taxalogue‐induced α/β‐tubulin stabilization, but not the taxalogue binding, directly influences the antitumor activity of taxalogues. The activity increases in the order: SB‐T‐1214 < docetaxel < taxol < larotaxel < cisplatin < cabazitaxel. [ABSTRACT FROM AUTHOR]

Purpose: Mutations in the β-tubulin isotype, TUBB8, can cause female infertility. Although several mutations of TUBB8 have been reported, the full spectrum for guiding genetics counseling still needs to be further explored. Here, we sought to identify novel variants in TUBB8 and their phenotypic effects on microtubule network structure in vitro. Methods: Whole-exome sequence analysis was performed in two families with infertility to detect pathogenic variants, with validation by Sanger sequencing. All gene variants and protein structures were predicted in silico. Cells were transfected with wild-type and mutants, and immunofluorescence analysis was performed to visualize microtubule network changes. Results: We detected a novel compound heterozygous mutation, c.915_916delCC (p.Arg306Serfs*21) and c.82C > T (p.His28Tyr), and a benign heterozygous variant c.1286C > T (p.Thr429Met) in TUBB8 in the two families. Female patients with p.Arg306Serfs*21 and p.His28Tyr were infertile with early embryonic developmental arrest. The female patient with p.Thr429Met gave birth to a healthy baby in the second in vitro fertilization frozen embryo transfer cycle. The p.Arg306Serfs*21 mutation was predicted to cause large structural alteration in the TUBB8 protein and was confirmed to produce a truncated and trace protein by western blot analysis. Immunofluorescence analysis of transfected HeLa cells showed that p.Arg306Serfs*21 significantly disrupted microtubule structure. Conclusions: Our findings expand the known mutational spectrum of TUBB8 associated with early embryonic developmental arrest and female infertility. [ABSTRACT FROM AUTHOR]

Dendritic morphology underlies the source and processing of neuronal signal inputs. Morphology can be broadly described by two types of geometric characteristics. The first is dendrogram topology, defined by the length and frequency of the arbor branches; the second is spatial embedding, mainly determined by branch angles and straightness. We have previously demonstrated that microtubules and actin filaments are associated with arbor elongation and branching, fully constraining dendrogram topology. Here, we relate the local distribution of these two primary cytoskeletal components with dendritic spatial embedding. We first reconstruct and analyze 167 sensory neurons from the Drosophila larva encompassing multiple cell classes and genotypes. We observe that branches with a higher microtubule concentration tend to deviate less from the direction of their parent branch across all neuron types. Higher microtubule branches are also overall straighter. F-actin displays a similar effect on angular deviation and branch straightness, but not as consistently across all neuron types as microtubule. These observations raise the question as to whether the associations between cytoskeletal distributions and arbor geometry are sufficient constraints to reproduce type-specific dendritic architecture. Therefore, we create a computational model of dendritic morphology purely constrained by the cytoskeletal composition measured from real neurons. The model quantitatively captures both spatial embedding and dendrogram topology across all tested neuron groups. These results suggest a common developmental mechanism regulating diverse morphologies, where the local cytoskeletal distribution can fully specify the overall emergent geometry of dendritic arbors. [ABSTRACT FROM AUTHOR]

Blood platelets are small non-nucleated cellular fragments that prevent and stop hemorrhages. They are produced in the bone marrow by megakaryocytes through megakaryopoiesis. This intricate process involves profound microtubule rearrangements culminating in the formation of a unique circular sub-membranous microtubule array, the marginal band, which supports the typical disc-shaped morphology of platelets. Mechanistically, these processes are thought to be controlled by a specific tubulin code. In this review, we summarize the current knowledge on the key isotypes, notably β1-, α4A- and α8-tubulin, and putative post-translational modifications, involved in platelet and marginal band formation. Additionally, we provide a provisional list of microtubule-associated proteins (MAPs) involved in these processes and a survey of tubulin variants identified in patients presenting defective platelet production. A comprehensive characterization of the platelet tubulin code and the identification of essential MAPs may be expected in the near future to shed new light on a very specialized microtubule assembly process with applications in platelet diseases and transfusion. • Blood platelets harbor a unique circular microtubule scaffold called the marginal band. • Platelet biogenesis and marginal band formation are controlled by a specialized tubulin code. • Tubulin isotypes including TUBB1, TUBA4A and TUBA8 are crucial in these two processes. • Post-translational modifications of tubulin and microtubule-associated proteins (MAPs) may regulate platelet biology. [ABSTRACT FROM AUTHOR]

Protists are an exceptionally diverse group of mostly single-celled eukaryotes. The organization of the microtubular cytoskeleton in protists from various evolutionary lineages has different levels of sophistication, from a network of microtubules (MTs) supporting intracellular trafficking as in Dictyostelium , to complex structures such as basal bodies and cilia/flagella enabling cell motility, and lineage-specific adaptations such as the ventral disc in Giardia. MTs building these diverse structures have specific properties partly due to the presence of tubulin post-translational modifications (PTMs). Among them there are highly evolutionarily conserved PTMs: acetylation, detyrosination, (poly)glutamylation and (poly)glycylation. In some protists also less common tubulin PTMs were identified, including phosphorylation, methylation, Δ2-, Δ5- of α-tubulin, polyubiquitination, sumoylation, or S-palmitoylation. Not surprisingly, several single-celled organisms become models to study tubulin PTMs, including their effect on MT properties and discovery of the modifying enzymes. Here, we briefly summarize the current knowledge on tubulin PTMs in unicellular eukaryotes and highlight key findings in protists as model organisms. [ABSTRACT FROM AUTHOR]

Fission yeast undergoes premeiotic nuclear oscillation, which is dependent on microtubules and is driven by cytoplasmic dynein. Although the molecular mechanisms have been analyzed, how a robust oscillation is generated despite the dynamic behaviors of microtubules has yet to be elucidated. Here, we show that the oscillation exhibits cell length‐dependent frequency and requires a balance between microtubule and viscous drag forces, as well as proper microtubule dynamics. Comparison of the oscillations observed in living cells with a simulation model based on microtubule dynamic instability reveals that the period of oscillation correlates with cell length. Genetic alterations that reduce cargo size suggest that the nuclear movement depends on viscous drag forces. Deletion of a gene encoding Kinesin‐8 inhibits microtubule catastrophe at the cell cortex and results in perturbation of oscillation, indicating that nuclear movement also depends on microtubule dynamic instability. Our findings link numerical parameters from the simulation model with cellular functions required for generating the oscillation and provide a basis for understanding the physical properties of microtubule‐dependent nuclear movements. Synopsis: In fission yeast, the meiotic nucleus exhibits oscillatory movement, which is driven by cytoplasmic dynein. A model that considers various microtubule properties, including pulling and pushing forces, recaptures robust nuclear oscillations. A model considering microtubule pulling and pushing forces recapitulates the premeiotic nuclear oscillation in fission yeast.Period of the spindle pole body (SPB) oscillation correlates with cell length.Speed of the SPB movement is limited by viscous drag force associated with nuclear volume.Kinesin‐8 family protein Klp6 contributes to efficient oscillation by controlling microtubule catastrophe. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) outcomes vary greatly among individuals, but most of the variation remains unexplained. Using a Drosophila melanogaster TBI model and 178 genetically diverse lines from the Drosophila Genetic Reference Panel (DGRP), we investigated the role that genetic variation plays in determining TBI outcomes. Following injury at 20–27 days old, DGRP lines varied considerably in mortality within 24 h (“early mortality”). Additionally, the disparity in early mortality resulting from injury at 20–27 vs 0–7 days old differed among DGRP lines. These data support a polygenic basis for differences in TBI outcomes, where some gene variants elicit their effects by acting on aging-related processes. Our genome-wide association study of DGRP lines identified associations between single nucleotide polymorphisms in Lissencephaly-1 (Lis-1) and Patronin and early mortality following injury at 20–27 days old. Lis-1 regulates dynein, a microtubule motor required for retrograde transport of many cargoes, and Patronin protects microtubule minus ends against depolymerization. While Patronin mutants did not affect early mortality, Lis-1 compound heterozygotes (Lis-1x /Lis-1y ) had increased early mortality following injury at 20–27 or 0–7 days old compared with Lis-1 heterozygotes (Lis-1x /+), and flies that survived 24 h after injury had increased neurodegeneration but an unaltered lifespan, indicating that Lis-1 affects TBI outcomes independently of effects on aging. These data suggest that Lis-1 activity is required in the brain to ameliorate TBI outcomes through effects on axonal transport, microtubule stability, and other microtubule proteins, such as tau, implicated in chronic traumatic encephalopathy, a TBI-associated neurodegenerative disease in humans. [ABSTRACT FROM AUTHOR]

Mitotic cell division requires that kinetochores form microtubule attachments that can segregate chromosomes and control mitotic progression via the spindle assembly checkpoint. During prometaphase, kinetochores shed a domain called the fibrous corona as microtubule attachments form. This shedding is mediated, in part, by the minus-end directed motor dynein, which 'strips' cargoes along K-fibre microtubules. Despite its essentiality, little is known about how dynein stripping is regulated and how it responds to attachment maturation. Lis1 (also known as PAFAH1B1) is a conserved dynein regulator that is mutated in the neurodevelopmental disease lissencephaly. Here, we have combined loss-of-function studies, high-resolution imaging and separation-of-function mutants to define how Lis1 contributes to dynein-mediated corona stripping in HeLa cells. Cells depleted of Lis1 fail to disassemble the corona and showa delay in metaphase as a result of persistent checkpoint activation. Furthermore, we find that although kinetochore-tethered Lis1-dynein is required for error-free microtubule attachment, the contribution of Lis1 to corona disassembly can be mediated by a cytoplasmic pool. These findings support the idea that Lis1 drives dynein function at kinetochores to ensure corona disassembly and prevent chromosome mis-segregation. [ABSTRACT FROM AUTHOR]

Microtubules are critical for a variety of important functions in eukaryotic cells. During intracellular trafficking, molecular motor proteins of the kinesin superfamily drive the transport of cellular cargoes by stepping processively along the microtubule surface. Traditionally, the microtubule has been viewed as simply a track for kinesin motility. New work is challenging this classic view by showing that kinesin-1 and kinesin-4 proteins can induce conformational changes in tubulin subunits while they are stepping. These conformational changes appear to propagate along the microtubule such that the kinesins can work allosterically through the lattice to influence other proteins on the same track. Thus, the microtubule is a plastic medium through which motors and other microtubule-associated proteins (MAPs) can communicate. Furthermore, stepping kinesin-1 can damage the microtubule lattice. Damage can be repaired by the incorporation of new tubulin subunits, but too much damage leads to microtubule breakage and disassembly. Thus, the addition and loss of tubulin subunits are not restricted to the ends of the microtubule filament but rather, the lattice itself undergoes continuous repair and remodeling. This work leads to a new understanding of how kinesin motors and their microtubule tracks engage in allosteric interactions that are critical for normal cell physiology. [ABSTRACT FROM AUTHOR]

The precise regulation of microtubule length during mitosis is essential to assemble and position the mitotic spindle and segregate chromosomes. The kinesin-13 Kif2C or MCAK acts as a potent microtubule depolymerase that diffuses short distances on microtubules, whereas the kinesin-8 Kif18b is a processive motor with weak depolymerase activity. However, the individual activities of these factors cannot explain the dramatic increase in microtubule dynamics in mitosis. Using in vitro reconstitution and single-molecule imaging, we demonstrate that Kif18b, MCAK and the plus-end tracking protein EB3 (also known as MAPRE3) act in an integrated manner to potently promote microtubule depolymerization at very low concentrations. We find that Kif18b can transport EB3 and MCAK and promotes their accumulation to microtubule plus ends through multivalent weak interactions. Together, our work defines the mechanistic basis for a cooperative Kif18b-MCAK-EB network at microtubule plus ends, that acts to efficiently shorten and regulate microtubules in mitosis, essential for correct chromosome segregation. [ABSTRACT FROM AUTHOR]