Transmembrane protein TMEM230, regulator of metalloproteins and motor proteins in gliomas and gliosis.

Glial cells provide physical and chemical support and protection for neurons and for the extracellular compartments of neural tissue through secretion of soluble factors, insoluble scaffolds, and vesicles. Additionally, glial cells have regenerative capacity by remodeling their physical microenvironment and changing physiological properties of diverse cell types in their proximity. Various types of aberrant glial and macrophage cells are associated with human diseases, disorders, and malignancy. We previously demonstrated that transmembrane protein, TMEM230 has tissue revascularization and regenerating capacity by its ability to secrete pro-angiogenic factors and metalloproteinases, inducing endothelial cell sprouting and channel formation. In healthy normal neural tissue, TMEM230 is predominantly expressed in glial and marcophate cells, suggesting a prominent role in neural tissue homeostasis. TMEM230 regulation of the endomembrane system was supported by co-expression with RNASET2 (lysosome, mitochondria, and vesicles) and STEAP family members (Golgi complex). Intracellular trafficking and extracellular secretion of glial cellular components are associated with endocytosis, exocytosis and phagocytosis mediated by motor proteins. Trafficked components include metalloproteins, metalloproteinases, glycans, and glycoconjugate processing and digesting enzymes that function in phagosomes and vesicles to regulate normal neural tissue microenvironment, homeostasis, stress response, and repair following neural tissue injury or degeneration. Aberrantly high sustained levels TMEM230 promotes metalloprotein expression, trafficking and secretion which contribute to tumor associated infiltration and hypervascularization of high tumor grade gliomas. Following injury of the central nervous or peripheral systems, transcient regulated upregulation of TMEM230 promotes tissue wound healing, remodeling and revascularization by activating glial and macrophage generated microchannels/microtubules (referred to as vascular mimicry) and blood vessel sprouting and branching. Our results support that TMEM230 may act as a master regulator of motor protein mediated trafficking and compartmentalization of a large class of metalloproteins in gliomas and gliosis.

Plant Kinesin Repertoires Expand with New Domain Architecture and Contract with the Loss of Flagella.

Kinesins are eukaryotic microtubule motor proteins subdivided into conserved families with distinct functional roles. While many kinesin families are widespread in eukaryotes, each organismal lineage maintains a unique kinesin repertoire composed of many families with distinct numbers of genes. Previous genomic surveys indicated that land plant kinesin repertoires differ markedly from other eukaryotes. To determine when repertoires diverged during plant evolution, we performed robust phylogenomic analyses of kinesins in 24 representative plants, two algae, two animals, and one yeast. These analyses show that kinesin repertoires expand and contract coincident with major shifts in the biology of algae and land plants. One kinesin family and five subfamilies, each defined by unique domain architectures, emerged in the green algae. Four of those kinesin groups expanded in ancestors of modern land plants, while six other kinesin groups were lost in the ancestors of pollen-bearing plants. Expansions of different kinesin families and subfamilies occurred in moss and angiosperm lineages. Other kinesin families remained stable and did not expand throughout plant evolution. Collectively these data support a radiation of kinesin domain architectures in algae followed by differential positive and negative selection on kinesins families and subfamilies in different lineages of land plants.

Slippery sequences stall the 26S proteasome at multiple points along the translocation pathway.

In eukaryotes, the ubiquitin-proteasome system is responsible for intracellular protein degradation. Proteins tagged with ubiquitin are recognized by ubiquitin receptors on the 19S regulatory particle (RP) of the 26S proteasome, unfolded, routed through the translocation channel of the RP, and are then degraded in the 20S core particle (CP). Aromatic paddles on the pore-1 loops of the RP's Rpt subunits grip the substrate and pull folded domains into the channel, thereby unfolding them. The sequence that the aromatic paddles grip while unfolding a substrate is therefore expected to influence the extent of unfolding, and low complexity sequences have been shown to interfere with grip. However, the detailed spatial requirements for grip while unfolding proteins, particularly from the N-terminus, remain unknown. We determined how the location of glycine-rich tracts relative to a folded domain impairs unfolding. We find that, in contrast to a previous report, inserting glycine-rich sequences closer to the folded domain reduced unfolding ability more than positioning them further away. Locations that have the biggest effect on unfolding map onto the regions where the aromatic paddles are predicted to interact with the substrate. Effects on unfolding from locations up to 67 amino acids away from the folded domain suggest that there are additional interactions between the substrate and the proteasome beyond the aromatic paddles that facilitate translocation of the substrate. In sum, this study deepens understanding of the mechanical interactions within the substrate channel by mapping the spacing of interactions between the substrate and the proteasome during unfolding.

Functional characterization of calmodulin-like proteins, CML13 and CML14, as novel light chains of Arabidopsis class VIII myosins.

Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck region to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a myosin VIII. Here, we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among CMLs tested for binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with red fluorescent protein-myosin fusion proteins containing IQ and tail domains of myosin VIIIs. In vitro actin motility assays using recombinant myosin VIIIs demonstrated that CaM, CML13, and CML14 function as light chains. Suppression of CML13 or CML14 expression using RNA silencing resulted in a shortened-hypocotyl phenotype, similar to that observed in a quadruple myosin mutant, myosin viii4KO. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains.

Autoinhibited kinesin-1 adopts a hierarchical folding pattern.

Conventional kinesin-1 is the primary anterograde motor in cells for transporting cellular cargo. While there is a consensus that the C-terminal tail of kinesin-1 inhibits motility, the molecular architecture of a full-length autoinhibited kinesin-1 remains unknown. Here, we combine crosslinking mass spectrometry (XL-MS), electron microscopy (EM), and AlphaFold structure prediction to determine the architecture of the full-length autoinhibited kinesin-1 homodimer (kinesin-1 heavy chain [KHC]) and kinesin-1 heterotetramer (KHC bound to kinesin light chain 1 [KLC1]). Our integrative analysis shows that kinesin-1 forms a compact, bent conformation through a break in coiled-coil 3. Moreover, our XL-MS analysis demonstrates that kinesin light chains stabilize the folded inhibited state rather than inducing a new structural state. Using our structural model, we show that disruption of multiple interactions between the motor, stalk, and tail domains is required to activate the full-length kinesin-1. Our work offers a conceptual framework for understanding how cargo adaptors and microtubule-associated proteins relieve autoinhibition to promote activation.

A mathematical model for axonal transport of large cargo vesicles.

In this study, we consider axonal transport of large cargo vesicles characterised by transient expansion of the axon shaft. Our goal is to formulate a mathematical model which captures the dynamic mechanical interaction of such cargo vesicles with the membrane associated periodic cytoskeletal structure (MPS). It consists of regularly spaced actin rings that are transversal to the longitudinal direction of the axon and involved in the radial contraction of the axon. A system of force balance equations is formulated by which we describe the transversal rings as visco-elastic Kelvin-Voigt elements. In a homogenisation limit, we reformulate the model as a free boundary problem for the interaction of the submembranous MPS with the large vesicle. We derive a non-linear force-velocity relation as a quasi-steady state solution. Computationally we analyse the vesicle size dependence of the transport speed and use an asymptotic approximation to formulate it as a power law that can be tested experimentally.

In Silico Prediction of MYO1C-Rhodopsin Interactions and Its Significance in Protein Localization and Visual Function.

Rods and cones are photoreceptor neurons in the retina that are required for visual sensation in vertebrates, where proper protein localization and compartmentalization are critical for phototransduction and visual function. In human retinal diseases, improper protein transport to the outer segment (OS) or mislocalization of proteins to the inner segment (IS) could lead to impaired visual responses and photoreceptor cell degeneration, causing a loss of visual function. We showed involvement of an unconventional motor protein, MYO1C, in the proper localization of rhodopsin to the OS, where loss of MYO1C in a mammalian model caused mislocalization of rhodopsin to IS and cell bodies, leading to progressively severe retinal phenotypes. In this study, using modeling and docking analysis, we aimed to identify the protein-protein interaction sites between MYO1C and Rhodopsin to establish a hypothesis that a physical interaction between these proteins is necessary for the proper trafficking of rhodopsin and visual function.

Crystal structure of the stalk region of axonemal inner-arm dynein-d reveals unique features in the coiled-coil and microtubule-binding domain.

Axonemal dynein is an ATP-dependent microtubular motor protein responsible for cilia and flagella beating, and its dysfunction can cause diseases such as primary ciliary dyskinesia and sperm dysmotility. Despite its biological importance, structure-based mechanisms underlying axonemal dynein motors remain unclear. Here, we determined the X-ray crystal structure of the human inner-arm dynein-d (DNAH1) stalk region, which contains a long antiparallel coiled-coil and a microtubule-binding domain (MTBD), at 2.7 Å resolution. Notably, differences in the relative orientation of the coiled-coil and MTBD in comparison with other dyneins, as well as the diverse orientations of the MTBD flap region among various isoforms, lead us to propose a 'spike shoe model' with an altered stepping angle for the interaction between IAD-d and microtubules. Based on these findings, we discuss isoform-specific functions of the axonemal dynein stalk MTBDs.

KIF1A-Associated Neurological Disorder: An Overview of a Rare Mutational Disease.

KIF1A-associated neurological diseases (KANDs) are a group of inherited conditions caused by changes in the microtubule (MT) motor protein KIF1A as a result of KIF1A gene mutations. Anterograde transport of membrane organelles is facilitated by the kinesin family protein encoded by the MT-based motor gene KIF1A. Variations in the KIF1A gene, which primarily affect the motor domain, disrupt its ability to transport synaptic vesicles containing synaptophysin and synaptotagmin leading to various neurological pathologies such as hereditary sensory neuropathy, autosomal dominant and recessive forms of spastic paraplegia, and different neurological conditions. These mutations are frequently misdiagnosed because they result from spontaneous, non-inherited genomic alterations. Whole-exome sequencing (WES), a cutting-edge method, assists neurologists in diagnosing the illness and in planning and choosing the best course of action. These conditions are simple to be identified in pediatric and have a life expectancy of 5-7 years. There is presently no permanent treatment for these illnesses, and researchers have not yet discovered a medicine to treat them. Scientists have more hope in gene therapy since it can be used to cure diseases brought on by mutations. In this review article, we discussed some of the experimental gene therapy methods, including gene replacement, gene knockdown, symptomatic gene therapy, and cell suicide gene therapy. It also covered its clinical symptoms, pathogenesis, current diagnostics, therapy, and research advances currently occurring in the field of KAND-related disorders. This review also explained the impact that gene therapy can be designed in this direction and afford the remarkable benefits to the patients and society.

Rotary mechanism of V/A-ATPases-how is ATP hydrolysis converted into a mechanical step rotation in rotary ATPases?

V/A-ATPase is a rotary molecular motor protein that produces ATP through the rotation of its central rotor. The soluble part of this protein, the V1 domain, rotates upon ATP hydrolysis. However, the mechanism by which ATP hydrolysis in the V1 domain couples with the mechanical rotation of the rotor is still unclear. Cryo-EM snapshot analysis of V/A-ATPase indicated that three independent and simultaneous catalytic events occurred at the three catalytic dimers (ABopen, ABsemi, and ABclosed), leading to a 120° rotation of the central rotor. Besides the closing motion caused by ATP bound to ABopen, the hydrolysis of ATP bound to ABsemi drives the 120° step. Our recent time-resolved cryo-EM snapshot analysis provides further evidence for this model. This review aimed to provide a comprehensive overview of the structure and function of V/A-ATPase from a thermophilic bacterium, one of the most well-studied rotary ATPases to date.

High-Resolution Tracking of Dynein-Dynactin-BicD2 Complexes.

The adapter dynactin and the activator BicD2 associate with dynein to form the highly motile dynein-dynactin-BicD2 (DDB) complex. In single-molecule assays, DDB displays processive runs, diffusive episodes, and transient pauses. The switching rates and durations of the different phases can be determined by tracking gold nanoparticle-labeled DDB complexes with interferometric scattering (iSCAT) microscopy and using an algorithm to separate out different motility phases. Here we describe methods for purifying DDB complexes from brain lysate, labeling with gold nanoparticles, imaging by iSCAT, and analyzing the resulting trajectories.

Tetraploidy-linked sensitization to CENP-E inhibition in human cells.

Tetraploidy is a hallmark of cancer cells, and tetraploidy-selective cell growth suppression is a potential strategy for targeted cancer therapy. However, how tetraploid cells differ from normal diploids in their sensitivity to anti-proliferative treatments remains largely unknown. In this study, we found that tetraploid cells are significantly more susceptible to inhibitors of a mitotic kinesin (CENP-E) than are diploids. Treatment with a CENP-E inhibitor preferentially diminished the tetraploid cell population in a diploid-tetraploid co-culture at optimum conditions. Live imaging revealed that a tetraploidy-linked increase in unsolvable chromosome misalignment caused substantially longer mitotic delay in tetraploids than in diploids upon moderate CENP-E inhibition. This time gap of mitotic arrest resulted in cohesion fatigue and subsequent cell death, specifically in tetraploids, leading to tetraploidy-selective cell growth suppression. In contrast, the microtubule-stabilizing compound paclitaxel caused tetraploidy-selective suppression through the aggravation of spindle multipolarization. We also found that treatment with a CENP-E inhibitor had superior generality to paclitaxel in its tetraploidy selectivity across a broader spectrum of cell lines. Our results highlight the unique properties of CENP-E inhibitors in tetraploidy-selective suppression and their potential use in the development of tetraploidy-targeting interventions in cancer.

Road-blocker HSP disease mutation disrupts pre-organization for ATP hydrolysis in kinesin through a second sphere control.

Kinesin motor proteins perform several essential cellular functions powered by the adenosine triphosphate (ATP) hydrolysis reaction. Several single-point mutations in the kinesin motor protein KIF5A have been implicated to hereditary spastic paraplegia disease (HSP), a lethal neurodegenerative disease in humans. In earlier studies, we have shown that a series of HSP-related mutations can impair the kinesin's long-distance displacement or processivity by modulating the order-disorder transition of the linker connecting the heads to the coiled coil. On the other hand, the reduction of kinesin's ATP hydrolysis reaction rate by a distal asparagine-to-serine mutation is also known to cause HSP disease. However, the molecular mechanism of the ATP hydrolysis reaction in kinesin by this distal mutation is still not fully understood. Using classical molecular dynamics simulations combined with quantum mechanics/molecular mechanics calculations, the pre-organization geometry required for optimal hydrolysis in kinesin motor bound to α/ß-tubulin is determined. This optimal geometry has only a single salt-bridge (of the possible two) between Arg203-Glu236, putting a reactive water molecule at a perfect position for hydrolysis. Such geometry is also needed to create the appropriate configuration for proton translocation during ATP hydrolysis. The distal asparagine-to-serine mutation is found to disrupt this optimal geometry. Therefore, the current study along with our previous one demonstrates how two different effects on kinesin dynamics (processivity and ATP hydrolysis), caused by a different set of genotypes, can give rise to the same phenotype leading to HSP disease.

Understanding protein translocation across chloroplast membranes: Translocons and motor proteins.

Subcellular organelles in eukaryotes are surrounded by lipid membranes. In an endomembrane system, vesicle trafficking is the primary mechanism for the delivery of organellar proteins to specific organelles. However, organellar proteins for chloroplasts, mitochondria, the nucleus, and peroxisomes that are translated in the cytosol are directly imported into their target organelles. Chloroplasts are a plant-specific organelle with outer and inner envelope membranes, a dual-membrane structure that is similar to mitochondria. Interior chloroplast proteins translated by cytosolic ribosomes are thus translocated through TOC and TIC complexes (translocons in the outer and inner envelope of chloroplasts, respectively), with stromal ATPase motor proteins playing a critical role in pulling pre-proteins through these import channels. Over the last three decades, the identity and function of TOC/TIC components and stromal motor proteins have been actively investigated, which has shed light on the action mechanisms at a molecular level. However, there remains some disagreement over the exact composition of TIC complexes and genuine stromal motor proteins. In this review, we discuss recent findings on the mechanisms by which proteins are translocated through TOC/TIC complexes and discuss future prospects for this field of research.

1-Indanone retards cyst development in ADPKD mouse model by stabilizing tubulin and down-regulating anterograde transport of cilia.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Cyst development in ADPKD involves abnormal epithelial cell proliferation, which is affected by the primary cilia-mediated signal transduction in the epithelial cells. Thus, primary cilium has been considered as a therapeutic target for ADPKD. Since ADPKD exhibits many pathological features similar to solid tumors, we investigated whether targeting primary cilia using anti-tumor agents could alleviate the development of ADPKD. Twenty-four natural compounds with anti-tumor activity were screened in MDCK cyst model, and 1-Indanone displayed notable inhibition on renal cyst growth without cytotoxicity. This compound also inhibited cyst development in embryonic kidney cyst model. In neonatal kidney-specific Pkd1 knockout mice, 1-Indanone remarkably slowed down kidney enlargement and cyst expansion. Furthermore, we demonstrated that 1-Indanone inhibited the abnormal elongation of cystic epithelial cilia by promoting tubulin polymerization and significantly down-regulating expression of anterograde transport motor protein KIF3A and IFT88. Moreover, we found that 1-Indanone significantly down-regulated ciliary coordinated Wnt/ß-catenin, Hedgehog signaling pathways. These results demonstrate that 1-Indanone inhibits cystic cell proliferation by reducing abnormally prolonged cilia length in cystic epithelial cells, suggesting that 1-Indanone may hold therapeutic potential to retard cyst development in ADPKD.

IFT-A structure reveals carriages for membrane protein transport into cilia.

Intraflagellar transport (IFT) trains are massive molecular machines that traffic proteins between cilia and the cell body. Each IFT train is a dynamic polymer of two large complexes (IFT-A and -B) and motor proteins, posing a formidable challenge to mechanistic understanding. Here, we reconstituted the complete human IFT-A complex and obtained its structure using cryo-EM. Combined with AlphaFold prediction and genome-editing studies, our results illuminate how IFT-A polymerizes, interacts with IFT-B, and uses an array of ß-propeller and TPR domains to create "carriages" of the IFT train that engage TULP adaptor proteins. We show that IFT-A⋅TULP carriages are essential for cilia localization of diverse membrane proteins, as well as ICK-the key kinase regulating IFT train turnaround. These data establish a structural link between IFT-A's distinct functions, provide a blueprint for IFT-A in the train, and shed light on how IFT evolved from a proto-coatomer ancestor.

Unconventional Myosins from Caenorhabditis elegans as a Probe to Study Human Orthologues.

Unconventional myosins are a superfamily of actin-based motor proteins that perform a number of roles in fundamental cellular processes, including (but not limited to) intracellular trafficking, cell motility, endocytosis, exocytosis and cytokinesis. 40 myosins genes have been identified in humans, which belong to different 12 classes based on their domain structure and organisation. These genes are widely expressed in different tissues, and mutations leading to loss of function are associated with a wide variety of pathologies while over-expression often results in cancer. Caenorhabditis elegans (C. elegans) is a small, free-living, non-parasitic nematode. ~38% of the genome of C. elegans has predicted orthologues in the human genome, making it a valuable tool to study the function of human counterparts and human diseases. To date, 8 unconventional myosin genes have been identified in the nematode, from 6 different classes with high homology to human paralogues. The hum-1 and hum-5 (heavy chain of an unconventional myosin) genes encode myosin of class I, hum-2 of class V, hum-3 and hum-8 of class VI, hum-6 of class VII and hum-7 of class IX. The hum-4 gene encodes a high molecular mass myosin (307 kDa) that is one of the most highly divergent myosins and is a member of class XII. Mutations in many of the human orthologues are lethal, indicating their essential properties. However, a functional characterisation for many of these genes in C. elegans has not yet been performed. This article reviews the current knowledge of unconventional myosin genes in C. elegans and explores the potential use of the nematode to study the function and regulation of myosin motors to provide valuable insights into their role in diseases.

Contributions of microtubule dynamics and transport to presynaptic and postsynaptic functions.

Microtubules (MT) are elongated, tubular, cytoskeletal structures formed from polymerization of tubulin dimers. They undergo continuous cycles of polymerization and depolymerization, primarily at their plus ends, termed dynamic instability. Although this is an intrinsic property of MTs, there are a myriad of MT-associated proteins that function in regulating MT dynamic instability and other dynamic processes that shape the MT array. Additionally, MTs assemble into long, semi-rigid structures which act as substrates for long-range, motor-driven transport of many different types of cargoes throughout the cell. Both MT dynamics and motor-based transport play important roles in the function of every known type of cell. Within the last fifteen years many groups have shown that MT dynamics and transport play ever-increasing roles in the neuronal function of mature neurons. Not only are neurons highly polarized cells, but they also connect with one another through synapses to form complex networks. Here we will focus on exciting studies that have illuminated how MTs function both pre-synaptically in axonal boutons and post-synaptically in dendritic spines. It is becoming clear that MT dynamics and transport both serve important functions in synaptic plasticity. Thus, it is not surprising that disruption of MTs, either through hyperstabilization or destabilization, has profound consequences for learning and memory. Together, the studies described here suggest that MT dynamics and transport play key roles in synaptic function and when disrupted result in compromised learning and memory.

KLC4 shapes axon arbors during development and mediates adult behavior.

Development of elaborate and polarized neuronal morphology requires precisely regulated transport of cellular cargos by motor proteins such as kinesin-1. Kinesin-1 has numerous cellular cargos which must be delivered to unique neuronal compartments. The process by which this motor selectively transports and delivers cargo to regulate neuronal morphogenesis is poorly understood, although the cargo-binding kinesin light chain (KLC) subunits contribute to specificity. Our work implicates one such subunit, KLC4, as an essential regulator of axon branching and arborization pattern of sensory neurons during development. Using live imaging approaches in klc4 mutant zebrafish, we show that KLC4 is required for stabilization of nascent axon branches, proper microtubule (MT) dynamics, and endosomal transport. Furthermore, KLC4 is required for proper tiling of peripheral axon arbors: in klc4 mutants, peripheral axons showed abnormal fasciculation, a behavior characteristic of central axons. This result suggests that KLC4 patterns axonal compartments and helps establish molecular differences between central and peripheral axons. Finally, we find that klc4 mutant larva are hypersensitive to touch and adults show anxiety-like behavior in a novel tank test, implicating klc4 as a new gene involved in stress response circuits.

Effect of detachment of motor protein from track on its transport.

The transportation of the cargoes in biological cells is primarily driven by the motor proteins on filamentous protein tracks. The stochastic nature of the motion of motor protein often leads to its spontaneous detachment from the track. We formulate a mathematical model to study the effect of the detachment of motor protein on its track bound transport. We calculate two quantities: the distance traveled by the motor protein in given time, and the average time taken by a single motor protein to reach a target distance. Expectedly, both of these quantities decrease with the increasing detachment rate if the motor velocity is kept fixed. However, the existing experimental data suggest that a change in the detachment rate also affects the velocity of the motor protein. This relation between motor protein speed and its detachment rate results in a non-monotonic dependence on the distance traveled in fixed time and transport rate to a fixed distance. Therefore, we demonstrate that optimal motor speeds can be identified for the time and distance controlled conditions.