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Glioma is a highly aggressive and invasive brain tumor with limited treatment options, highlighting the need for novel therapeutic approaches. Kinesin superfamily proteins (KIFs) are a diverse group of motor proteins that play essential roles in cellular processes such as mitosis, intracellular transport, and signal transduction, all of which are crucial for tumorigenesis. This review focuses on the multifaceted role of KIFs in glioma, examining their clinical relevance, contribution to tumor progression, and potential as therapeutic targets. We discuss how KIFs influence key aspects of glioma biology, including cell proliferation, invasion, migration, and metastasis. Furthermore, we explore the regulation of the cell cycle and critical signaling pathways associated with glioma, such as PI3K-Akt, Wnt/β-catenin, and Hedgehog signaling by KIFs. The review also addresses the emerging interplay between KIFs and non-coding RNAs, including circular RNAs (circRNAs) and microRNAs (miRNAs), in glioma progression. Finally, we examine current therapeutic strategies targeting KIFs, including immunotherapy, chemotherapy, and small-molecule inhibitors, and their potential to improve treatment outcomes for glioma patients. By synthesizing these insights, this review underscores the significance of KIFs in glioma pathogenesis and their promise as novel therapeutic targets in the fight against glioma. [ABSTRACT FROM AUTHOR]

Abstract Cleft palate (CP) is a congenital condition characterized by a complex etiology and limited diagnostic and therapeutic options. In this study, we delved into the molecular mechanisms associated with retinoic acid (RA)-induced CP in Kun Ming mice. Proteomic analysis of control and RA-induced CP samples at embryonic day 15.5 revealed 25 upregulated and 19 downregulated proteins. Further analysis identified these differentially expressed proteins (DEPs) as being involved in extracellular matrix organization, actin cytoskeleton, and myosin complex. Moreover, these DEPs were found to be enriched in pathways related to motor protein activity and extracellular matrix-receptor interaction. Protein-protein interaction network analysis identified 10 hub proteins, including motor proteins and ECM-related proteins, which exhibited higher expression levels in CP compared to control tissues. These findings provide insights into the molecular mechanisms underlying CP and highlight potential targets for diagnostic and therapeutic purposes.

Kinesin and dynein-dynactin motors move endosomes and other vesicles bidirectionally along microtubules, a process mainly studied under in vitro conditions. Here, we provide a physiological bidirectional transport model following color-coded, endogenously tagged transport-related proteins as they move through a crowded cellular environment. Late endosomes (LEs) surf bidirectionally on Protrudin-enriched endoplasmic reticulum (ER) membrane contact sites, while hopping and gliding along microtubules and bypassing cellular obstacles, such as mitochondria. During bidirectional transport, late endosomes do not switch between opposing Rab7 GTPase effectors, RILP and FYCO1, or their associated dynein and KIF5B motor proteins, respectively. In the endogenous setting, far fewer motors associate with endosomal membranes relative to effectors, implying coordination of transport with other aspects of endosome physiology through GTPase-regulated mechanisms. We find that directionality of transport is provided in part by various microtubule-associated proteins (MAPs), including MID1, EB1, and CEP169, which recruit Lis1-activated dynein motors to microtubule plus ends for transport of early and late endosomal populations. At these microtubule plus ends, activated dynein motors encounter the dynactin subunit p150glued and become competent for endosomal capture and minus-end movement in collaboration with membrane-associated Rab7-RILP. We show that endosomes surf over the ER through the crowded cell and move bidirectionally under the control of MAPs for motor activation and through motor replacement and capture by endosomal anchors. [Display omitted] • Late endosomes (LEs) surf over the ER network in continuous contact with Protrudin • LEs hop and glide along microtubules to pass mitochondria • Bidirectionally moving LEs do not exchange among Rab7 effectors or motor types • MAPs control the directionality of LE transport through dynein-dynactin assembly Microtubule-linked endosomes move bidirectionally through the crowded cytoplasm, maintaining contacts with the ER. Using endogenously tagged proteins, Jongsma et al. show that bidirectional transport control via the Rab7-effector-motor axis is provided by microtubule-associated proteins (MAPs). [ABSTRACT FROM AUTHOR]

Extreme value analysis (EVA) is a statistical method that studies the properties of extreme values of datasets, crucial for fields like engineering, meteorology, finance, insurance, and environmental science. EVA models extreme events using distributions such as Fréchet, Weibull, or Gumbel, aiding in risk prediction and management. This review explores EVA's application to nanoscale biological systems. Traditionally, biological research focuses on average values from repeated experiments. However, EVA offers insights into molecular mechanisms by examining extreme data points. We introduce EVA's concepts with simulations and review its use in studying motor protein movements within cells, highlighting the importance of in vivo analysis due to the complex intracellular environment. We suggest EVA as a tool for extracting motor proteins' physical properties in vivo and discuss its potential in other biological systems. While there have been only a few applications of EVA to biological systems, it holds promise for uncovering hidden properties in extreme data, promoting its broader application in life sciences. [ABSTRACT FROM AUTHOR]

Skeletal dysplasias are group of rare genetic diseases resulting from mutations in genes encoding structural proteins of the cartilage extracellular matrix (ECM), signaling molecules, transcription factors, epigenetic modifiers, and several intracellular proteins. Cell division, organelle maintenance, and intracellular transport are all orchestrated by the cytoskeleton-associated proteins, and intracellular processes affected through microtubule-associated movement are important for the function of skeletal cells. Among microtubule-associated motor proteins, kinesins in particular have been shown to play a key role in cell cycle dynamics, including chromosome segregation, mitotic spindle formation, and ciliogenesis, in addition to cargo trafficking, receptor recycling, and endocytosis. Recent studies highlight the fundamental role of kinesins in embryonic development and morphogenesis and have shown that mutations in kinesin genes lead to several skeletal dysplasias. However, many questions concerning the specific functions of kinesins and their adaptor molecules as well as specific molecular mechanisms in which the kinesin proteins are involved during skeletal development remain unanswered. Here we present a review of the skeletal dysplasias resulting from defects in kinesins and discuss the involvement of kinesin proteins in the molecular mechanisms that are active during skeletal development. [ABSTRACT FROM AUTHOR]

An array of motor proteins consumes chemical energy in setting up the architectures of chromosomes. Here, we explore how the structure of ideal polymer chains is influenced by two classes of motors. The first class which we call "swimming motors" acts to propel the chromatin fiber through three-dimensional space. They represent a caricature of motors such as RNA polymerases. Previously, they have often been described by adding a persistent flow onto Brownian diffusion of the chain. The second class of motors, which we call "grappling motors" caricatures the loop extrusion processes in which segments of chromatin fibers some distance apart are brought together. We analyze these models using a self-consistent variational phonon approximation to a many-body Master equation incorporating motor activities. We show that whether the swimming motors lead to contraction or expansion depends on the susceptibility of the motors, that is, how their activity depends on the forces they must exert. Grappling motors in contrast to swimming motors lead to long-ranged correlations that resemble those first suggested for fractal globules and that are consistent with the effective interactions inferred by energy landscape analyses of Hi-C data on the interphase chromosome. [ABSTRACT FROM AUTHOR]

Background: Oligospermia is one of the most common reasons for male infertility which is troubling numerous couples of child-bearing age. This investigation scrutinizes the implications and mechanistic underpinnings of ursolic acid's effect on busulfan-induced oligospermia in mouse models. Methods: A singular intraperitoneal injection of busulfan at a dosage of 30 mg/kg induced oligospermia. Two weeks subsequent to this induction, mice were subjected to various dosages of ursolic acid (10, 30, and 50 mg/kg body weight, respectively) on a daily basis for four consecutive weeks. Following this treatment period, a meticulous analysis of epididymal sperm parameters, encompassing concentration and motility, was conducted using a computer-assisted sperm analysis system. The histopathology of the mice testes was performed utilizing hematoxylin and eosin staining, and the cytoskeleton regeneration of the testicular tissues was analyzed via immunofluorescent staining. Serum hormone levels, including testosterone, luteinizing hormone, and follicle-stimulating hormone, as well as reactive oxygen species levels (inclusive of reactive oxygen species and malondialdehyde), were gauged employing specific enzyme-linked immunosorbent assay kits. Differentially expressed genes of testicular mRNA between the oligospermia-induced group and the various ursolic acid treatment groups were identified through RNA sequencing analysis. Results: The results revealed that a dosage of 50 mg/kg ursolic acid treatment could increase the concentration of epididymal sperm in oligospermia mice, promote the recovery of testicular morphology, regulate hormone levels and ameliorate oxidative damage. The mechanism research results indicated that ursolic acid increased the expression level of genes related to motor proteins in oligospermia mice. [ABSTRACT FROM AUTHOR]

Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20° tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change. [ABSTRACT FROM AUTHOR]

Membrane nanotubes (NTs) are dynamic communication channels connecting spatially separated cells even over long distances and promoting the transport of different cellular cargos. NTs are also involved in the intercellular spread of different pathogens and the deterioration of some neurological disorders. Transport processes via NTs may be controlled by cytoskeletal elements. NTs are frequently observed membrane projections in numerous mammalian cell lines, including various immune cells, but their functional significance in the 'antibody factory' B cells is poorly elucidated. Here, we report that as active channels, NTs of B-lymphoma cells can mediate bidirectional mitochondrial transport, promoted by the cooperation of two different cytoskeletal motor proteins, kinesin along microtubules and myosin VI along actin, and bidirectional transport processes are also supported by the heterogeneous arrangement of the main cytoskeletal filament systems of the NTs. We revealed that despite NTs and axons being different cell extensions, the mitochondrial transport they mediate may exhibit significant similarities. Furthermore, we found that microtubules may improve the stability and lifespan of B-lymphoma-cell NTs, while F-actin strengthens NTs by providing a structural framework for them. Our results may contribute to a better understanding of the regulation of the major cells of humoral immune response to infections. [ABSTRACT FROM AUTHOR]

Background Oligospermia is one of the most common reasons for male infertility which is troubling numerous couples of child-bearing age. This investigation scrutinizes the implications and mechanistic underpinnings of ursolic acid’s effect on busulfan-induced oligospermia in mouse models. Methods A singular intraperitoneal injection of busulfan at a dosage of 30 mg/kg induced oligospermia. Two weeks subsequent to this induction, mice were subjected to various dosages of ursolic acid (10, 30, and 50 mg/kg body weight, respectively) on a daily basis for four consecutive weeks. Following this treatment period, a meticulous analysis of epididymal sperm parameters, encompassing concentration and motility, was conducted using a computer-assisted sperm analysis system. The histopathology of the mice testes was performed utilizing hematoxylin and eosin staining, and the cytoskeleton regeneration of the testicular tissues was analyzed via immunofluorescent staining. Serum hormone levels, including testosterone, luteinizing hormone, and follicle-stimulating hormone, as well as reactive oxygen species levels (inclusive of reactive oxygen species and malondialdehyde), were gauged employing specific enzyme-linked immunosorbent assay kits. Differentially expressed genes of testicular mRNA between the oligospermia-induced group and the various ursolic acid treatment groups were identified through RNA sequencing analysis. Results The results revealed that a dosage of 50 mg/kg ursolic acid treatment could increase the concentration of epididymal sperm in oligospermia mice, promote the recovery of testicular morphology, regulate hormone levels and ameliorate oxidative damage. The mechanism research results indicated that ursolic acid increased the expression level of genes related to motor proteins in oligospermia mice.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that belong to the group of endopeptidases or matrixins. They are able to cleave a plethora of substrates, including components of the extracellular matrix and cell-surface-associated proteins, as well as intracellular targets. Accordingly, MMPs play key roles in a variety of physiological and pathological processes, such as tissue homeostasis and cancer cell invasion. MMP activity is exquisitely regulated at several levels, including pro-domain removal, association with inhibitors, intracellular trafficking and transport via extracellular vesicles. Moreover, the regulation of MMP activity is currently being rediscovered for the development of respective therapies for the treatment of cancer, as well as infectious, inflammatory and neurological diseases. In this Cell Science at a Glance article and the accompanying poster, we present an overview of the current knowledge regarding the regulation of MMPactivity, the intra- and extra-cellular trafficking pathways of these enzymes and their diverse groups of target proteins, as well as their impact on health and disease. [ABSTRACT FROM AUTHOR]

Biological muscles mainly consist of motor proteins that play essential roles and have a hierarchical structure covered with collagen that can transmit the tension to bones via tendons. The artificial reconstruction of motor proteins is highly expected from the viewpoint of engineering and biology. Although artificial muscles have been developed via synthetic and biological molecular motors, there is a considerable gap between the components of artificial and biological muscles, resulting in a significant obstacle to further scale‐up and force transfer through macroscale structures. To overcome these obstacles via bioderived biomolecules, this study proposes a collagen‐actomyosin hybrid soft actuator inspired by the structure of biological muscles. Collagen gel entrapping actomyosin can play roles in maintaining the actuator's shape and transmitting the tensile force. The generated forces reach the micro‐newton range, enabling the actuation of millimeter‐scale mechanical components. These properties may be helpful for the fabrication of soft robotic systems with advanced functionalities. [ABSTRACT FROM AUTHOR]

Kinesin motor proteins play crucial roles in anterograde transport of cargo vesicles in neurons, moving them along axons from the cell body towards the synaptic region. Not only the transport force and velocity of single motor protein, but also the number of kinesin molecules involved in transporting a specific cargo, is pivotal for synapse formation. This collective transport by multiple kinesins ensures stable and efficient cargo transport in neurons. Abnormal increases or decreases in the number of engaged kinesin molecules per cargo could potentially act as biomarkers for neurodegenerative diseases such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis (ALS), spastic paraplegia, polydactyly syndrome, and virus transport disorders. We review here a model constructed using physical measurements to quantify the number of kinesin molecules associated with their cargo, which could shed light on the molecular mechanisms of neurodegenerative diseases related to axonal transport. • Multiple motor proteins, such as kinesin and dynein, collaborate in axonal transport. • The decrease and increase in the number of motor proteins can lead to neurological disorders. • A method for measuring the number of motor proteins is proposed, based on a physical theorem. [ABSTRACT FROM AUTHOR]

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

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.

Cytoplasmic dynein is an AAA+ motor that drives the transport of many intracellular cargoes towards the minus end of microtubules (MTs). Previous in vitro studies characterized isolated dynein as an exceptionally weak motor that moves slowly and diffuses on an MT. Recent studies altered this view by demonstrating that dynein remains in an autoinhibited conformation on its own, and processive motility is activated when it forms a ternary complex with dynactin and a cargo adaptor. This complex assembles more efficiently in the presence of Lis1, providing an explanation for why Lis1 is a required cofactor for most cytoplasmic dynein-driven processes in cells. This review describes how dynein motility is activated and regulated by cargo adaptors and accessory proteins.

The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer-binding, and mechanical elements. Such communication is particularly complex and not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule-binding domain (MTBD) that is separated from the AAA ring by a ~ 135 Å coiled-coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule-independent hyperactive ATPase activity, and nucleotide-independent low affinity for microtubules. Cryo-electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide-dependent conformational changes occur normally in one-half of the AAA ring, but are disrupted in the other half. The large-scale linker conformational change observed in the wild-type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring.

Like other cells in the body, nerve cells need many proteins and substances to maintain homeostasis. As we know, the transcription and translation of proteins and necessary cellular substances occurs in the cell nucleus. The nucleus of nerve cell is located in the cell body. Another part of the nerve cell is “Axon”, which has a long structure. Even in some nerve cells axon’s length reaches up to 1000 mm. On the other hand, all parts of the neuron need substances and proteins synthesized in nucleus locating in the cell body. Therefore, a mechanism is necessary to express the movement of materials from nucleus along the axon. The movement of materials along the axon is called ‘Axoplasmic Transport’. It seems that disturbances in axoplasmic transport can cause various neuronal problems. The purpose of this study is to investigate the mechanism of axoplasmic transport and its types; moreover, the possible effect of exercise on this transition will be discussed.

Motor proteins are key players in exerting spatiotemporal control over the intracellular location of membrane-bound compartments, including endosomes containing cargo. In this Review, we focus on how motors and their cargo adaptors regulate positioning of cargoes from the earliest stages of endocytosis and through the two main intracellular itineraries: (1) degradation at the lysosome or (2) recycling back to the plasma membrane. In vitro and cellular (in vivo) studies on cargo transport thus far have typically focussed independently on either the motor proteins and adaptors, or membrane trafficking. Here, we will discuss recent studies to highlight what is known about the regulation of endosomal vesicle positioning and transport by motors and cargo adaptors. We also emphasise that in vitro and cellular studies are often performed at different scales, from single molecules to whole organelles, with the aim to provide a perspective on the unified principles of motor-driven cargo trafficking in living cells that can be learned from these differing scales. [ABSTRACT FROM AUTHOR]

F1-ATPase is a motor protein that couples the rotation of its rotary β subunit with ATP synthesis or hydrolysis. Single-molecule experiments indicate that nucleotide binding and release events occur almost simultaneously during the synthesis cycle, allowing the energy gain due to spontaneous binding of ADP to one catalytic β subunit to be directly harnessed for driving the release of ATP from another rather than being dissipated as heat. Here, we examine the unknown mechanism of this coupling that is critical for an exceptionally high mechanochemical efficiency of F1-ATPase by means of all-atom free-energy simulations. We find that nondissipative and kinetically fast progression of the motor in the synthesis direction requires a concerted conformational change involving the closure of the ADP-binding β subunit followed by the gradual opening of the ATP-releasing β subunit over the course of the 30 to 40 rotary substep of the β subunit. This rotary substep, preceding the ATP-dependent metastable state, allows for the recovery of a large portion of the ADP binding energy in the conformation of ATP-bound β that gradually adopts the low-affinity conformation, captured also by the recent cryo-EM structure of this elusive state. The release of ATP from this nearly open conformation leads to its further opening, which enables the progression of the motor to the next catalytic metastable state. Our simulations explain this energy conversion mechanism in terms of intersubunit and ligand-protein interactions. [ABSTRACT FROM AUTHOR]

Motor proteins are powered by nucleotide hydrolysis and exert mechanical work to carry out many fundamental biological tasks. To ensure their correct and efficient performance, the motors' activities are allosterically regulated by additional factors that enhance or suppress their NTPase activity. Here, we review two highly conserved mechanisms of ATP hydrolysis activation and repression operating in motor proteins-the glutamate switch and the arginine finger-and their associated regulatory factors. We examine the implications of these regulatory mechanisms in proteins that are formed by multiple ATPase subunits. We argue that the regulatory mechanisms employed by motor proteins display features similar to those described in small GTPases, which require external regulatory elements, such as dissociation inhibitors, exchange factors and activating proteins, to switch the protein's function 'on' and 'off'. Likewise, similar regulatory roles are taken on by the motor's substrate, additional binding factors, and even adjacent subunits in multimeric complexes. However, in motor proteins, more than one regulatory factor and the two mechanisms described here often underlie the machine's operation. Furthermore, ATPase regulation takes place throughout the motor's cycle, which enables a more complex function than the binary 'active' and 'inactive' states.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

The role of mechanical signals in the proper functioning of organisms is increasingly recognised, and every cell senses physical forces and responds to them. These forces are generated both from outside the cell or via the sophisticated force-generation machinery of the cell, the cytoskeleton. All regions of the cell are connected via mechanical linkages, enabling the whole cell to function as a mechanical system. In this Review, we define some of the key concepts of how this machinery functions, highlighting the critical requirement for mechanosensory proteins, and conceptualise the coupling of mechanical linkages to mechanochemical switches that enables forces to be converted into biological signals. These mechanical couplings provide a mechanism for how mechanical crosstalk might coordinate the entire cell, its neighbours, extending into whole collections of cells, in tissues and in organs, and ultimately in the coordination and operation of entire organisms. Consequently, many diseases manifest through defects in this machinery, which we map onto schematics of the mechanical linkages within a cell. This mapping approach paves the way for the identification of additional linkages between mechanosignalling pathways and so might identify treatments for diseases, where mechanical connections are affected by mutations or where individual force-regulated components are defective. [ABSTRACT FROM AUTHOR]

Optical tweezers (OT) provide a noninvasive approach for delivering minute physical forces to targeted objects. Controlling such forces in living cells or in vitro preparations allows for the measurement and manipulation of numerous processes relevant to the form and function of cells. As such, OT have made important contributions to our understanding of the structures of proteins and nucleic acids, the interactions that occur between microscopic structures within cells, the choreography of complex processes such as mitosis, and the ways in which cells interact with each other. In this review, we highlight recent contributions made to the field of cell biology using OT and provide basic descriptions of the physics, the methods, and the equipment that made these studies possible. Optical tweezers (OT) can measure or apply minute forces inside cells noninvasively. Recent developments in OT technology have allowed insights into the structure, function, and behavior of cells. Particular progress has been made in understanding mitotic machinery, cellular viscoelasticity, and metastasis. Emerging methods in optical physics promise to extend the breadth and utility of OT in the years to come. [ABSTRACT FROM AUTHOR]

Molecular motors move processively along cytoskeletal filaments by stepping of their motor domains (MDs). Observation of how the MDs step relative to each other reveals the mechanism of motor processivity and various gating mechanisms used by motors to coordinate the catalytic cycles of their MDs. This chapter will discuss developments in simultaneous observation of the stepping motions of the two MDs of processive motors using two-color single-particle tracking microscopy.Techniques presented: FIONA, multicolor tracking/image registration., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Tunnelling nanotubes (TNTs) are membranous connections that represent a unique type of intercellular communication in different cell types. They are associated with cell physiology and cancer pathology. The possible existence of tunnelling nanotubes communication between urothelial cancer and normal cells has not yet been elucidated. Therefore, we analyzed TNTs formed by T24 cells (human invasive cancer urothelial cells) and normal porcine urothelial (NPU) cells, which serve as surrogate models for healthy human urothelial cells. Monocultures and cocultures of NPU and T24 cells were established and analyzed using live-cell imaging, optical tweezers, fluorescence microscopy, and scanning electron microscopy. TNTs of NPU cells differed significantly from tunnelling nanotubes of T24 cells in number, length, diameter, lipid composition, and elastic properties. Membrane domains enriched in cholesterol/sphingomyelin were present in tunnelling nanotubes of T24 cells but not in NPU cells. The tunnelling nanotubes in T24 cells were also easier to bend than the tunnelling nanotubes in NPU cells. The tunnelling nanotubes of both cell types were predominantly tricytoskeletal, and contained actin filaments, intermediate filaments, and microtubules, as well as the motor proteins myosin Va, dynein, and kinesin 5B. Mitochondria were transported within tunnelling nanotubes in living cells, and were colocalized with microtubules and the microtubule-associated protein dynamin 2. In cocultures, heterocellular tunnelling nanotubes were formed between NPU cells and T24 cells and vice versa. The presence of connexin 43 at the end of urothelial tunnelling nanotubes suggests a junctional connection and the involvement of tunnelling nanotube in signal transduction. In this study, we established a novel urothelial cancer-normal coculture model and showed cells in the minority tend to form tunnelling nanotubes with cells in the majority. The condition with cancer cells in the minority is an attractive model to mimic the situation after surgical resection with remaining cancer cells and may help to understand cancer progression and recurrence. Our results shed light on the biological activity of tunnelling nanotubes and have the potential to advance the search for anticancer drugs that target tunnelling nanotubes.

Cilia are eukaryotic organelles essential for movement, signaling or sensing. Primary cilia act as antennae to sense a cell's environment and are involved in a wide range of signaling pathways essential for development. Motile cilia drive cell locomotion or liquid flow around the cell. Proper functioning of both types of cilia requires a highly orchestrated bi-directional transport system, intraflagellar transport (IFT), which is driven by motor proteins, kinesin-2 and IFT dynein. In this review, we explore how IFT is regulated in cilia, focusing from three different perspectives on the issue. First, we reflect on how the motor track, the microtubule-based axoneme, affects IFT. Second, we focus on the motor proteins, considering the role motor action, cooperation and motor-train interaction plays in the regulation of IFT. Third, we discuss the role of kinases in the regulation of the motor proteins. Our goal is to provide mechanistic insights in IFT regulation in cilia and to suggest directions of future research. [ABSTRACT FROM AUTHOR]

During cell division, cross-linking motors determine the architecture of the spindle, a dynamic microtubule network that segregates the chromosomes in eukaryotes. It is unclear how motors with opposite directionality coordinate to drive both contractile and extensile behaviors in the spindle. Particularly, the impact of different cross-linker designs on network self-organization is not understood, limiting our understanding of self-organizing structures in cells but also our ability to engineer new active materials. Here, we use experiment and theory to examine active microtubule networks driven by mixtures of motors with opposite directionality and different cross-linker design. We find that although the kinesin-14 HSET causes network contraction when dominant, it can also assist the opposing kinesin-5 KIF11 to generate extensile networks. This bifunctionality results from HSET's asymmetric design, distinct from symmetric KIF11. These findings expand the set of rules underlying patterning of active microtubule assemblies and allow a better understanding of motor cooperation in the spindle. [ABSTRACT FROM AUTHOR]

Dyrk1a triplication in Down's syndrome and its overexpression in Alzheimer's disease suggest a role for increased DYRK1A activity in the abnormal metabolism of APP. Transport defects are early phenotypes in the progression of Alzheimer's disease, which lead to APP processing impairments. However, whether DYRK1A regulates the intracellular transport and delivery of APP in human neurons remains unknown. From a proteomic dataset of human cerebral organoids treated with harmine, a DYRK1A inhibitor, we found expression changes in protein clusters associated with the control of microtubule-based transport and in close interaction with the APP vesicle. Live imaging of APP axonal transport in human-derived neurons treated with harmine or overexpressing a dominant negative DYRK1A revealed a reduction in APP vesicle density and enhanced the stochastic behavior of retrograde vesicle transport. Moreover, harmine increased the fraction of slow segmental velocities and changed speed transitions supporting a DYRK1A-mediated effect in the exchange of active motor configuration. Contrarily, the overexpression of DYRK1A in human polarized neurons increased the axonal density of APP vesicles and enhanced the processivity of retrograde APP. In addition, increased DYRK1A activity induced faster retrograde segmental velocities together with significant changes in slow to fast anterograde and retrograde speed transitions, suggesting the facilitation of the active motor configuration. Our results highlight DYRK1A as a modulator of the axonal transport machinery driving APP intracellular distribution in neurons, and stress DYRK1A inhibition as a putative therapeutic intervention to restore APP axonal transport in Down's syndrome and Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Bacteriophage T7 gp4 helicase has served as a model system for understanding mechanisms of hexameric replicative helicase translocation. The mechanistic basis of how nucleoside 50-triphosphate hydrolysis and translocation of gp4 helicase are coupled is not fully resolved. Here, we used a thermodynamically benchmarked coarse-grained protein force field, Associative memory, Water mediated, Structure and Energy Model (AWSEM), with the single-stranded DNA (ssDNA) force field 3SPN.2C to investigate gp4 translocation. We found that the adenosine 50-triphosphate (ATP) at the subunit interface stabilizes the subunit-subunit interaction and inhibits subunit translocation. Hydrolysis of ATP to adenosine 50-diphosphate enables the translocation of one subunit, and new ATP binding at the new subunit interface finalizes the subunit translocation. The LoopD2 and the N-terminal primase domain provide transient protein-protein and protein-DNA interactions that facilitate the large-scale subunit movement. The simulations of gp4 helicase both validate our coarse-grained protein-ssDNA force field and elucidate the molecular basis of replicative helicase translocation. [ABSTRACT FROM AUTHOR]

Molecular motors move processively along cytoskeletal filaments through stepping of their catalytic head domains. Observation of the stepping movement of the heads reveals the mechanism of motor processivity and how they coordinate the cycles of the catalytic heads during processive motility. This chapter will discuss recent developments in simultaneous observation of the stepping motions of the two heads using multicolor single particle tracking microscopy.

The G protein-coupled receptors (GPCRs) are plasma membrane proteins that function as sensors of changes in the internal and external milieux and play essential roles in health and disease. They are targets of hormones, neurotransmitters, local hormones (autacoids), and a large proportion of the drugs currently used as therapeutics and for "recreational" purposes. Understanding how these receptors signal and are regulated is fundamental for progress in areas such as physiology and pharmacology. This review will focus on what is currently known about their structure, the molecular events that trigger their signaling, and their trafficking to endosomal compartments. GPCR phosphorylation and its role in desensitization (signaling switching) are also discussed. It should be mentioned that the volume of information available is enormous given the large number and variety of GPCRs. However, knowledge is fragmentary even for the most studied receptors, such as the adrenergic receptors. Therefore, we attempt to present a panoramic view of the field, conscious of the risks and limitations (such as oversimplifications and incorrect generalizations). We hope this will provoke further research in the area. It is currently accepted that GPCR internalization plays a role signaling events. Therefore, the processes that allow them to internalize and recycle back to the plasma membrane are briefly reviewed. The functions of cytoskeletal elements (mainly actin filaments and microtubules), the molecular motors implicated in receptor trafficking (myosin, kinesin, and dynein), and the GTPases involved in GPCR internalization (dynamin) and endosomal sorting (Rab proteins), are discussed. The critical role phosphoinositide metabolism plays in regulating these events is also depicted. [ABSTRACT FROM AUTHOR]

The process of chromosome congression and alignment is at the core of mitotic fidelity. In this review, we discuss distinct spatial routes that the chromosomes take to align during prometaphase, which are characterized by distinct biomolecular requirements. Peripheral polar chromosomes are an intriguing case as their alignment depends on the activity of kinetochore motors, polar ejection forces, and a transition from lateral to end-on attachments to microtubules, all of which can result in the delayed alignment of these chromosomes. Due to their undesirable position close to and often behind the spindle pole, these chromosomes may be particularly prone to the formation of erroneous kinetochore-microtubule interactions, such as merotelic attachments. To prevent such errors, the cell employs intricate mechanisms to preposition the spindle poles with respect to chromosomes, ensure the formation of end-on attachments in restricted spindle regions, repair faulty attachments by error correction mechanisms, and delay segregation by the spindle assembly checkpoint. Despite this protective machinery, there are several ways in which polar chromosomes can fail in alignment, mis-segregate, and lead to aneuploidy. In agreement with this, polar chromosomes are present in certain tumors and may even be involved in the process of tumorigenesis. [ABSTRACT FROM AUTHOR]

Bending of cilia and flagella occurs when axonemal dynein molecules on one side of the axoneme produce force and move toward the microtubule (MT) minus end. These dyneins are then pulled back when the axoneme bends in the other direction, meaning oscillatory back and forth movement of dynein during repetitive bending of cilia/flagella. There are various factors that may regulate the dynein activity, e.g. the nexin-dynein regulatory complex, radial spokes, and central apparatus. In order to understand the basic mechanism of dynein’s oscillatory movement, we constructed a simple model system composed of MTs, outer-arm dyneins, and crosslinks between the MTs made of DNA origami. Electron microscopy (EM) showed pairs of parallel MTs crossbridged by patches of regularly arranged dynein molecules bound in two different orientations, depending on which of the MTs their tails bind to. The oppositely oriented dyneins are expected to produce opposing forces when the pair of MTs have the same polarity. Optical trapping experiments showed that the dynein-MT-DNA-origami complex actually oscillates back and forth after photolysis of caged ATP. Intriguingly, the complex, when held at one end, showed repetitive bending motions. The results show that a simple system composed of ensembles of oppositely oriented dyneins, MTs, and inter-MT crosslinkers, without any additional regulatory structures, has an intrinsic ability to cause oscillation and repetitive bending motions.

Nervous system function requires intracellular transport of channels, receptors, mRNAs, and other cargo throughout complex neuronal morphologies. Local signals such as synaptic input can regulate cargo trafficking, motivating the leading conceptual model of neuron-wide transport, sometimes called the 'sushi-belt model' (Doyle and Kiebler, 2011). Current theories and experiments are based on this model, yet its predictions are not rigorously understood. We formalized the sushi belt model mathematically, and show that it can achieve arbitrarily complex spatial distributions of cargo in reconstructed morphologies. However, the model also predicts an unavoidable, morphology dependent tradeoff between speed, precision and metabolic efficiency of cargo transport. With experimental estimates of trafficking kinetics, the model predicts delays of many hours or days for modestly accurate and efficient cargo delivery throughout a dendritic tree. These findings challenge current understanding of the efficacy of nucleus-to-synapse trafficking and may explain the prevalence of local biosynthesis in neurons.

Eg5 is a kinesin motor protein that is responsible for bipolar spindle formation and plays a crucial role during mitosis. Loss of Eg5 function leads to the formation of monopolar spindles, followed by mitotic arrest, and subsequent cell death. Several cell‐permeable small molecules have been reported to inhibit Eg5 and some have been evaluated as anticancer agents. We now describe the design, synthesis, and biological evaluation of photoswitchable variants with five different pharmacophores. Our lead compound Azo‐EMD is a cell permeable azobenzene that inhibits Eg5 more potently in its light‐induced cis form. This activity decreased the velocity of Eg5 in single‐molecule assays, promoted formation of monopolar spindles, and led to mitotic arrest in a light dependent way. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system with complex determinants, including genetic and non-genetic factors. A key pathological signature of ALS is the cytoplasmic mislocalization and aggregation of TDP-43 in affected motor neurons, which is found in 97% of cases. Recent reports have shown that mitochondrial dysfunction plays a significant role in motor neuron degeneration in ALS, and TDP-43 modulates several mitochondrial transcripts. In this study, we used induced pluripotent stem cell-derived motor neurons from ALS patients with TDP-43 mutations and a transgenic TDP-43 M337V mouse model to determine how TDP-43 mutations alter mitochondrial function and axonal transport. We detected significantly reduced mitochondrial respiration and ATP production in patient induced pluripotent stem cell-derived motor neurons, linked to an interaction between TDP-43 M337V with ATPB and COX5A. A downstream reduction in speed of retrograde axonal transport in patient induced pluripotent stem cell-derived motor neurons was detected, which correlated with downregulation of the motor protein complex, DCTN1/dynein. Overexpression of DCTN1 in patient induced pluripotent stem cell-derived motor neurons significantly increased the percentage of retrograde travelling mitochondria and reduced the percentage of stationary mitochondria. This study shows that ALS induced pluripotent stem cell-derived motor neurons with mutations in TDP-43 have deficiencies in essential mitochondrial functions with downstream effects on retrograde axonal transport, which can be partially rescued by DCTN1 overexpression., Competing Interests: The authors report no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Nature has generated sophisticated and very efficient molecular motors, employed for nanoscale transport at the intracellular level. As a complementary tool to nanofluidics, these motors have been envisioned for nanotechnological devices. In order to pave the way for such applications, a thorough understanding of the mechanisms governing these motors is needed. Because of the complexity of their in vivo functions, this understanding is best acquired in vitro, where functional parameters can independently be controlled. I will report on work in my group that studies and harnesses the transport properties of molecular motors on functionalized structures of reduced dimensionality such as carbon nanotubes,1 lithographically designed electrodes,2 microwires,3 loops4 and swarms.5 In addition, I will show results that demonstrate the potential of this work for biomedical advances.6

Mitotic cell divisions ensure stable transmission of genetic information from a mother to daughter cells in a series of generations. To ensure this crucial task is accomplished, the cell forms a bipolar structure called the mitotic spindle that divides sister chromatids to the opposite sides of the dividing mother cell. After successful establishment of stable attachments of microtubules to chromosomes and inspection of connections between them, at the heart of mitosis, the cell starts the process of segregation. This spectacular moment in the life of a cell is termed anaphase, and it involves two distinct processes: depolymerization of microtubules bound to chromosomes, which is also known as anaphase A, and elongation of the spindle or anaphase B. Both processes ensure physical separation of disjointed sister chromatids. In this chapter, we review the mechanisms of anaphase B spindle elongation primarily in mammalian systems, combining different pioneering ideas and concepts with more recent findings that shed new light on the force generation and regulation of biochemical modules operating during spindle elongation. Finally, we present a comprehensive model of spindle elongation that includes structural, biophysical, and molecular aspects of anaphase B. • Overview of spindle elongation dynamics in the context of different mitotic phases. • Synopsis of pioneering ideas and recent structural literature on metaphase and anaphase spindles in human cells. • Dissection of force-generation mechanisms during anaphase B and experimental support for their existence in mammals. • Description of changes in protein localization after metaphase-to-anaphase transition for a large set of spindle proteins. • Detailed molecular models of key aspects of anaphase B and their temporal regulation. [ABSTRACT FROM AUTHOR]

The dynamics and interactions of cellular organelles underlie many aspects of cellular functioning. Until recently, assessment of organelle dynamics has been primarily observational or required whole-cell perturbations to assess the implications of altered organelle motility and positioning. However, thanks to recently developed and optimized intervention strategies, we now have the ability to control organelles in their unperturbed state, altering organelle positioning, membrane trafficking pathways, as well as organelle interactions. This can be performed both globally and locally, giving fine control over the range, reversibility, and extent of organelle dynamics. Here, we describe how these tools are currently used for controlling organelles and give insight into the exciting future of this emerging field. [ABSTRACT FROM AUTHOR]

Reports from neural cell cultures and experimental animal studies provide evidence of age- and disease-related changes in retrograde transport of spent or misfolded proteins destined for degradation or recycling. However, few studies address these issues in human brain from those who either age without dementia and overt neuropathology, or succumb to Alzheimer's; especially as such propensity may be influenced by APOE genotype. We studied the expression and distribution of the dynein subunit dynactin-P50, the β amyloid precursor protein (βAPP), and hyperphosphorylated tau (P-tau) in tissues and tissue sections of brains from non-demented, neuropathology-free patients and from Alzheimer patients, with either APOE ε3,3 or APOE ε4,4. We found that advanced age in patients without dementia or neuropathological change was associated with coordinated increases in dynactin-P50 and βAPP in neurons in pyramidal layers of the hippocampus. In contrast, in Alzheimer's, βAPP and dynactin were significantly reduced. Furthermore, the dynactin-P50 and βAPP that was present was located primarily in dystrophic neurites in Aβ plaques. Tissues from Alzheimer patients with APOE ε3,3 had less P-tau, more βAPP, dynactin-P50, and synaptophysin than did tissues from Alzheimer patients carrying APOE ε4,4. It is logical to conclude, then, that as neurons age successfully, there is coordination between retrograde delivery and maintenance and repair, as well as between retrograde delivery and degradation and/or recycling of spent proteins. The buildup of proteins slated for repair, synaptic viability, transport, and re-cycling in neuron soma and dystrophic neurites suggest a loss of this coordination in Alzheimer neurons. Inheritance of APOE ε3,3 rather than APOE ε4,4, is associated with neuronal resilience, suggestive of better repair capabilities, more synapses, more efficient transport, and less hyperphosphorylation of tau. We conclude that even in disease the ε3 allele is neuroprotective.

The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3' single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase.

The organization of micron-sized, multi-microtubule arrays from individual microtubules is essential for diverse cellular functions. The microtubule polymer is largely viewed as a passive building block during the organization process. An exception is the ‘tubulin code’ where alterations to tubulin at the amino acid level can influence the activity of microtubule-associated proteins. Recent studies reveal that micron-scale geometrical features of individual microtubules and polymer networks, such as microtubule length, overlap length, contact angle, and lattice defects, can also regulate the activity of microtubule-associated proteins and modulate polymer dynamics. We discuss how the interplay between such geometrical properties of the microtubule lattice and the activity of associated proteins direct multiple aspects of array organization, from microtubule nucleation and coalignment to specification of array dimensions and remodeling of dynamic networks. The mechanisms reviewed here highlight micron-sized features of microtubules as critical parameters to be routinely investigated in the study of microtubule self-organization.

Cin8, the Saccharomyces cerevisiae kinesin-5, has an essential role in mitosis. In in vitro motility assays, tetrameric and dimeric Cin8 constructs showed bidirectional motility in response to ionic strength or Cin8 motor density. However, whether property-switching directionality is present in a monomeric form of Cin8 is unknown. Here we engineered monomeric Cin8 constructs with and without the Cin8-specific ∼99 residues in the loop 8 domain and examined the directionality of these constructs using an in vitro polarity-marked microtubule gliding assay within the range of the motor density or ionic strength. We found that both monomeric constructs showed only plus end-directed activity over the ranges measured, which suggested that minus end-directed motility driven by Cin8 is necessary for at least dimeric forms. Using an in vitro microtubule corkscrewing assay, we also found that monomeric Cin8 corkscrewed microtubules around their longitudinal axes with a constant left-handed pitch. Overall, our results imply that plus-end-directed and left-handed motor activity comprise the intrinsic properties of the Cin8 motor domain as with other monomeric N-kinesins. • Monomeric form of Cin8 kinesin-5 generates only plus end-directed force. • Deletion of the unique insertion in monomeric Cin8 does not cause bi-directionality. • Cin8 directional reversal requires at least linked pairs of motor domains. • Monomeric Cin8 molecules cause microtubules to corkscrew with a left-handed pitch. • Kinesin motor domain might have intrinsic plus-ended and left-handed directionality. [ABSTRACT FROM AUTHOR]

Single-molecule technologies have expanded our ability to detect biological events individually, in contrast to ensemble biophysical technologies, where the result provides averaged information. Recent developments in atomic force microscopy have not only enabled us to distinguish the heterogeneous phenomena of individual molecules, but also allowed us to view up to the resolution of a single covalent bond. Similarly, optical tweezers, due to their versatility and precision, have emerged as a potent technique to dissect a diverse range of complex biological processes, from the nanomechanics of ClpXP protease–dependent degradation to force-dependent processivity of motor proteins. Despite the advantages of optical tweezers, the time scales used in this technology were inconsistent with physiological scenarios, which led to the development of magnetic tweezers, where proteins are covalently linked with the glass surface, which in turn increases the observation window of a single biomolecule from minutes to weeks. Unlike optical tweezers, magnetic tweezers use magnetic fields to impose torque, which makes them convenient for studying DNA topology and topoisomerase functioning. Using modified magnetic tweezers, researchers were able to discover the mechanical role of chaperones, which support their substrate proteinsby pulling them during translocation and assist their native folding as a mechanical foldase. In this article, we provide a focused review of many of these new roles of single-molecule technologies, ranging from single bond breaking to complex chaperone machinery, along with the potential to design mechanomedicine, which would be a breakthrough in pharmacological interventions against many diseases. [ABSTRACT FROM AUTHOR]

Many cellular processes rely on the cell’s ability to transport material to and from the nucleus. Networks consisting of many microtubules and actin filaments are key to this transport. Recently, the inhibition of intracellular transport has been implicated in neurodegenerative diseases such as Alzheimer’s disease and Amyotrophic Lateral Sclerosis. Furthermore, microtubules may contain so-called defective regions where motor protein velocity is reduced due to accumulation of other motors and microtubule-associated proteins. In this work, we propose a new mathematical model describing the motion of motor proteins on microtubules which incorporate a defective region. We take a mean-field approach derived from a first principle lattice model to study motor protein dynamics and density profiles. In particular, given a set of model parameters we obtain a closed-form expression for the equilibrium density profile along a given microtubule. We then verify the analytic results using mathematical analysis on the discrete model and Monte Carlo simulations. This work will contribute to the fundamental understanding of inhomogeneous microtubules providing insight into microscopic interactions that may result in the onset of neurodegenerative diseases. Our results for inhomogeneous microtubules are consistent with prior work studying the homogeneous case. [ABSTRACT FROM AUTHOR]