Cytoskeletal Assembly Research Articles

Cytoskeletal assembly in axonal outgrowth and regeneration analyzed on the nanoscale

The axonal cytoskeleton is organized in a highly periodic structure, the membrane-associated periodic skeleton (MPS), which is essential to maintain the structure and function of the axon. Here, we use stimulated emission depletion microscopy of primary rat cortical neurons in microfluidic chambers to analyze the temporal and spatial sequence of MPS formation at the distal end of growing axons and during regeneration after axotomy. We demonstrate that the MPS does not extend continuously into the growing axon but develops from patches of periodic βII-spectrin arrangements that grow and coalesce into a continuous scaffold. We estimate that the underlying sequence of assembly, elongation, and subsequent coalescence of periodic βII-spectrin patches takes around 15 h. Strikingly, we find that development of the MPS occurs faster in regenerating axons after axotomy and note marked differences in the morphology of the growth cone and adjacent axonal regions between regenerating and unlesioned axons. Moreover, we find that inhibition of the spectrin-cleaving enzyme calpain accelerates MPS formation in regenerating axons and increases the number of regenerating axons after axotomy. Taken together, we provide here a detailed nanoscale analysis of MPS development in growing axons.

Toward the cellular-scale simulation of motor-driven cytoskeletal assemblies.

The cytoskeleton - a collection of polymeric filaments, molecular motors, and crosslinkers - is a foundational example of active matter, and in the cell assembles into organelles that guide basic biological functions. Simulation of cytoskeletal assemblies is an important tool for modeling cellular processes and understanding their surprising material properties. Here, we present aLENS (a Living Ensemble Simulator), a novel computational framework designed to surmount the limits of conventional simulation methods. We model molecular motors with crosslinking kinetics that adhere to a thermodynamic energy landscape, and integrate the system dynamics while efficiently and stably enforcing hard-body repulsion between filaments. Molecular potentials are entirely avoided in imposing steric constraints. Utilizing parallel computing, we simulate tens to hundreds of thousands of cytoskeletal filaments and crosslinking motors, recapitulating emergent phenomena such as bundle formation and buckling. This simulation framework can help elucidate how motor type, thermal fluctuations, internal stresses, and confinement determine the evolution of cytoskeletal active matter.

DAPLE orchestrates apical actomyosin assembly from junctional polarity complexes.

Establishment of apicobasal polarity and the organization of the cytoskeleton must operate coordinately to ensure proper epithelial cell shape and function. However, the precise molecular mechanisms by which polarity complexes directly instruct the cytoskeletal machinery to determine cell shape are poorly understood. Here, we define a mechanism by which the PAR polarity complex (PAR3-PAR6-aPKC) at apical cell junctions leads to efficient assembly of the apical actomyosin network to maintain epithelial cell morphology. We found that the PAR polarity complex recruits the protein DAPLE to apical cell junctions, which in turn triggers a two-pronged mechanism that converges upon assembly of apical actomyosin. More specifically, DAPLE directly recruits the actin-stabilizing protein CD2AP to apical junctions and, concomitantly, activates heterotrimeric G protein signaling in a GPCR-independent manner to favor RhoA-myosin activation. These observations establish DAPLE as a direct molecular link between junctional polarity complexes and the formation of apical cytoskeletal assemblies that support epithelial cell shape.

Modeling contractility kit mediated mechanosensation and cytoskeletal network assembly

The mechanobiome consists of a meshwork of macromolecules that determines cellular mechanical properties and enables mechanosensation, facilitating shape-change processes such as proliferation, polarization, migration, and cancer metastasis. Myosin II and cortexillin I, critical elements of the mechanobiome, pre-assemble in the cytoplasm into complexes known as mechano-contractility kits (MCKs). IQGAP scaffolding proteins regulate the mechano-responsiveness of the cortexillin I-myosin II elements within MCKs.

Cooperative dynamics of curvature-sensing proteins control cytoskeletal assembly near the plasma membrane

The plasma membrane, demarcating the shape of a cell, works together with the cellular cytoskeleton to carry out many important functions such as mechanosensing. To study these phenomena, we have recently introduced a microscopic computational model, membrane-MEDYAN, which enables simulations of complex cytoskeleton-membrane dynamics. In the current work, we introduced a capability of treating stochastic reaction-diffusion processes on the membrane manifold, as well as adsorption and desorption exchange of proteins between the membrane and the enclosed cytosolic volume.

Ta2O5-doped TiO2 nanotubes on Ti6Al4V alloy with improved cytocompatibility to endothelial cells

Microscopic topography and chemical composition of implant surface play critical roles in cellular response. In this work, Ta2O5-doped TiO2 nanotubes (Ta2O5/TiO2 NTs) were prepared on Ti6Al4V (TC4) to improve its cytocompatibility to endothelial cells. Firstly, a Ta diffusion layer on TC4 (Ta/TC4) was designed by plasma surface Ta-alloying. Then, a Ta2O5/TiO2 NTs film was grown on Ta/TC4 by anodization. The surface morphology, phase composition, and microstructure of the obtained samples were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and transmission electron microscopy (TEM). Ta incorporation leads to the lamellar microstructure (α'+β) of Ta/TC4 and the lamellar distribution of Ta2O5/TiO2 NTs. In vitro assays demonstrate that the synergistic effect of Ta2O5 addition and biomimetic nanostructure is responsible for the enhanced proliferation, adhesion, cytoskeletal assembly, and spreading of endothelial cells on Ta2O5/TiO2 NTs. The excellent cytocompatibility of Ta2O5/TiO2 NTs highlights its potential in biomedical applications.

Protein Assembly by Design.

Proteins are nature's primary building blocks for the construction of sophisticated molecular machines and dynamic materials, ranging from protein complexes such as photosystem II and nitrogenase that drive biogeochemical cycles to cytoskeletal assemblies and muscle fibers for motion. Such natural systems have inspired extensive efforts in the rational design of artificial protein assemblies in the last two decades. As molecular building blocks, proteins are highly complex, in terms of both their three-dimensional structures and chemical compositions. To enable control over the self-assembly of such complex molecules, scientists have devised many creative strategies by combining tools and principles of experimental and computational biophysics, supramolecular chemistry, inorganic chemistry, materials science, and polymer chemistry, among others. Owing to these innovative strategies, what started as a purely structure-building exercise two decades ago has, in short order, led to artificial protein assemblies with unprecedented structures and functions and protein-based materials with unusual properties. Our goal in this review is to give an overview of this exciting and highly interdisciplinary area of research, first outlining the design strategies and tools that have been devised for controlling protein self-assembly, then describing the diverse structures of artificial protein assemblies, and finally highlighting the emergent properties and functions of these assemblies.

Can we talk about myoblast fusion?

Can we talk about myoblast fusion?

In silico mechanistic model of microtubule assembly inhibition by selective chromone derivatives

Microtubules and actin filaments are cytoskeletal protein assemblies that are engaged in motility, mitosis, and regulate cell signalling with an essential role in the control of protein synthesis and accordingly, cell growth. Inhibition of cytoskeletal organelles, in particular tubulin polymerization, is important in cancer therapy due to their antimitotic role.Considering protein structure variations, we conducted a molecular docking study followed by a series of molecular dynamics (MD) simulations at different binding sites to investigate how a known α-/β-tubulin dimerization inhibitor, Glaziovianin A, and its analogue Gatastatin target human γ-tubulin subunit. The results of our MD simulations reproduced these protein-ligand interactions, demonstrating that Glaziovianin A is selective against β-tubulin's colchicine site and Gatastatin hits γ-tubulin's GTP binding site.Further analyses shed light on the reasons for subunit-focused selectivity for these structurally similar compounds, provided mechanistic insights into some of the tubulin functions such as the conformational transitions and subsequent microtubule disruption. The information combined from energy and structural analysis of MD, with all represented validation steps, and detailed study of specific protein-protein interaction features potentially could facilitate the early identification of γ-tubulin-specific inhibitors.

Mutation-specific non-canonical pathway of PTEN as a distinct therapeutic target for glioblastoma

PTEN is one of the most frequently altered tumor suppressor genes in malignant tumors. The dominant-negative effect of PTEN alteration suggests that the aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hotspot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that overexpress these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes (‘edge mutations’) localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties regarding their subcellular localization. Further, some missense mutations (‘edge mutations’) in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.

Interaction of Plasmodium falciparum apicortin with \u03b1- and \u03b2-tubulin is critical for parasite growth and survival

Cytoskeletal structures of Apicomplexan parasites are important for parasite replication, motility, invasion to the host cell and survival. Apicortin, an Apicomplexan specific protein appears to be a crucial factor in maintaining stability of the parasite cytoskeletal assemblies. However, the function of apicortin, in terms of interaction with microtubules still remains elusive. Herein, we have attempted to elucidate the function of Plasmodium falciparum apicortin by monitoring its interaction with two main components of parasite microtubular structure, α-tubulin-I and β-tubulin through in silico and in vitro studies. Further, a p25 domain binding generic drug Tamoxifen (TMX), was used to disrupt PfApicortin-tubulin interactions which led to the inhibition in growth and progression of blood stage life cycle of P. falciparum.

Wnt5A-Mediated Actin Organization Regulates Host Response to Bacterial Pathogens and Non-Pathogens.

Wnt5A signaling facilitates the killing of several bacterial pathogens, but not the non-pathogen E. coli DH5α. The basis of such pathogen vs. non-pathogen distinction is unclear. Accordingly, we analyzed the influence of Wnt5A signaling on pathogenic E. coli K1 in relation to non-pathogenic E. coli K12-MG1655 and E. coli DH5α eliminating interspecies variability from our study. Whereas cell internalized E. coli K1 disrupted cytoskeletal actin organization and multiplied during Wnt5A depletion, rWnt5A mediated activation revived cytoskeletal actin assembly facilitating K1 eradication. Cell internalized E. coli K12-MG1655 and E. coli DH5α, which did not perturb actin assembly appreciably, remained unaffected by rWnt5A treatment. Phagosomes prepared separately from Wnt5A conditioned medium treated K1 and K12-MG1655 infected macrophages revealed differences in the relative levels of actin and actin network promoting proteins, upholding that the Wnt5A-Actin axis operates differently for internalized pathogen and non-pathogen. Interestingly, exposure of rWnt5A treated K1 and K12-MG1655/DH5α infected macrophages to actin assembly inhibitors reversed the scenario, blocking killing of K1, yet promoting killing of both K12-MG1655 and DH5α. Taken together, our study illustrates that the state of activation of the Wnt5A/Actin axis in the context of the incumbent bacteria is crucial for directing host response to infection.

Advances in research on the regulatory mechanism of NHE1 in tumors (Review)

Tumors pose a major threat to human health and present with difficulties that modern medicine has yet to overcome. It has been demonstrated that the acid-base balance of the tumor microenvironment is closely associated with the dynamic balance in the human body and that it regulates several processes, such as cell proliferation and differentiation, intracellular enzyme activity, and cytoskeletal assembly and depolymerization. It has been well established that the regulation of intra- and extracellular pH depends on a series of functional ion transporters and hydrogen ion channels, such as the Na+/H+ exchanger (NHE) protein and thee Cl/HCO3- exchange protein, among which the NHE1 member of the NHE family has been attracting increasing attention in recent years, particularly in studies on the correlation between pH regulation and tumors. NHE1 is a housekeeping gene encoding a protein that is widely expressed on the surface of all plasma membranes. Due to its functional domain, which determines the pHi at its N-terminus and C-terminus, NHE1 is involved in the regulation of the cellular pH microenvironment. It has been reported in the literature that NHE1 can regulate cell volume, participate in the transmembrane transport of intracellular and extracellular ions, affect cell proliferation and apoptosis, and regulate cell behavior and cell cycle progression; however, research on the role of NHE1 in tumorigenesis and tumor development in various systems is at its early stages. The aim of the present study was to review the current research on the correlation between the NHE family proteins and various systemic tumors, in order to indicate a new direction for antitumor drug development with the pH microenvironment as the target.

Interdependence of Filament Nucleation, Elongation and Crosslinking in Actin Bundle Assembly

A network of actin-binding proteins tightly regulates each stage of actin assembly, including filament nucleation, elongation and crosslinking. These processes are integrated together to assemble higher-order actin structures specialized for specific cellular roles. Actin bundles are composed of unbranched filaments that are crosslinked in parallel or anti-parallel orientations by bundling proteins. These structures form the basis of a number of cytoskeletal assemblies, including the contractile ring, stress fibers, and filopodia. Aberrant regulation of actin bundles and their associated proteins is linked to diseases such as focal segmental glomerulosclerosis and metastatic cancers. Yet despite their importance, the dynamic properties of actin bundles are not well understood. To gain insight into the mechanism of bundle assembly, we investigated how the processes of nucleation, elongation, and crosslinking influence each other. We utilized formins, which function as both nucleators and actin polymerases, to elucidate the interdependence between nucleation and elongation. We found that, whereas filament nucleation necessarily precedes and promotes filament elongation, the dependence of both processes on actin monomer availability gives rise to competition that ultimately favors nucleation over elongation. To investigate the relationship between elongation and crosslinking, we generated pools of actin filaments of uniform lengths and analyzed their kinetics of crosslinking by the bundling protein fascin. We found that fascin crosslinks short filaments more slowly than long filaments, suggesting that filament elongation plays a role in regulating filament bundling. We propose that elongating filaments undergo changes in curvature and rotational diffusion that modulate the probability of filament alignment and ultimately dictate the efficiency of fascin-mediated crosslinking. This work suggests that upregulation of fascin might alter the mechanical properties of bundled actin structures by promoting the incorporation of short filaments.

Disease-Associated Tau Phosphorylation Hinders Tubulin Assembly within Tau Condensates.

Cellular condensation of intrinsically disordered proteins (IDPs) through liquid–liquid phase separation (LLPS) allows dynamic compartmentalization and regulation of biological processes. The IDP tau, which promotes the assembly of microtubules and is hyperphosphorylated in Alzheimer's disease, undergoes LLPS in solution and on the surface of microtubules. Little is known, however, about the influence of tau phosphorylation on its ability to nucleate microtubule bundles in conditions of tau LLPS. Herein, we show that unmodified tau as well as tau phosphorylated at disease‐associated epitopes condense into liquid‐like droplets. Although tubulin partitioned into and reached high concentrations inside all tau droplets, it was unable to grow into microtubules form the inside of droplets formed by tau phosphorylated at the AT180 epitope (T231/S235). In contrast, neither phosphorylation of tau in the repeat domain nor at its tyrosine residues inhibited the assembly of tubulin from tau droplets. Because LLPS of IDPs has been shown to promote different types of cytoskeletal assembly, our study suggests that IDP phosphorylation might be a broadly used mechanism for the modulation of condensate‐mediated cytoskeletal assembly.

CBIO-04. MUTATION-SPECIFIC NON-CANONICAL PATHWAY OF PTEN AS A DISTINCT THERAPEUTIC TARGET FOR GLIOBLASTOMA

Abstract The dominant-negative effect of PTEN mutation has been described previously, suggesting that aberrant gain of function attributed to mutation might be more disastrous than deletion in respect to malignant potential. In present study, we explored the functional implications of hot spot mutations of PTEN in GBM tumors. Subcellular location of PTEN is important for its distinct function and spatial distribution within the cytoplasm is known to be associated with cellular locomotion. We evaluated the subcellular compartmentalization of different PTEN mutants and found that some PTEN mutants located at cellular edges of chemotaxing cells. Moreover, these PTEN mutations exhibited invasive phenotype, which was not disrupted by PI3K inhibitor, but microtubule inhibitors. This finding suggests that cytoskeletal assembly as a novel non-canonical pathway of PTEN, thus unraveling a novel therapeutic vulnerability of PTEN. Mutation-specific therapeutic options should be considered in treating GBM patients with PTEN mutations.

Lysine acetylation of cytoskeletal proteins: Emergence of an actin code.

Reversible lysine acetylation of nuclear proteins such as histones is a long-established important regulatory mechanism for chromatin remodeling and transcription. In the cytoplasm, acetylation of a number of cytoskeletal proteins, including tubulin, cortactin, and the formin mDia2, regulates both cytoskeletal assembly and stability. More recently, acetylation of actin itself was revealed to regulate cytoplasmic actin polymerization through the formin INF2, with downstream effects on ER-to-mitochondrial calcium transfer, mitochondrial fission, and vesicle transport. This finding raises the possibility that actin acetylation, along with other post-translational modifications to actin, might constitute an "actin code," similar to the "histone code" or "tubulin code," controlling functional shifts to these central cellular proteins. Given the multiple roles of actin in nuclear functions, its modifications might also have important roles in gene expression.

Biomimetic mineralized microenvironment stiffness regulated BMSCs osteogenic differentiation through cytoskeleton mediated mechanical signaling transduction

Construction of biomimetic microenvironment is vital to understand the relationship between matrix mechanical cues and cell fate, as well as to explore potential tissue engineering scaffolds for clinical application. In this study, through the enzymatic mineralizable collagen hydrogel system, we established the biomimetic bone matrix which was capable of realizing mechanical regulation independent of mineralization by incorporation of phosphorylated molecules (vinylphosphonic acid, VAP). Then, based on the biomimetic mineralized matrix with same composition but significantly different mechanical stiffness, we further investigated the effect of matrix stiffness on osteogenic differentiation of bone marrow stromal cells (BMSCs). The results clearly demonstrated that biomimetic mineralized microenvironment with higher mechanical strength promoted osteogenic differentiation of BMSCs. Further mechanism analysis demonstrated that the mineralized hydrogel with higher stiffness promoted cytoskeletal assembly, which enhanced the expression and nuclear colocalization of YAP and RUNX2, thereby promoted the osteogenic differentiation of stem cells. This study supplies a promising material platform not only for bone tissue engineering but also for exploring the mechanism of biomimetic bone matrix mechanics on osteogenesis.

Tumor Microenvironment‐Activatable Nanoenzymes for Mechanical Remodeling of Extracellular Matrix and Enhanced Tumor Chemotherapy

AbstractIncreased tissue stiffness is a hallmark of cancer and promotes tumor progression. It is hypothesized that decreased tumorous stress may aid or sensitize chemotherapies. To overcome extracellular matrix (ECM) stiffening and fulfill sensitized chemotherapy in one nanosystem, a reactive oxygen species‐activatable nanoenzyme (SP‐NE) based on a dendritic polyglycerol scaffold, integrating collagenase and paclitaxel (PTX) prodrug, is constructed. The dense and tough ECM is highly remitted by SP‐NE in the tumor microenvironment (TME) mimicking gelatin hydrogel models, which causes cell shrinkage, disorders cytoskeletal constructions, and subsequently enhances chemotherapeutic efficacy. ECM softening via SP‐NE downregulates mechanotransduction signaling pathways of integrin‐focal adhesion kinase (FAK)‐Ras homolog family member A (RhoA) implicated in cytoskeletal assembly, and integrin‐FAK‐phosphorylated extracellular signal regulated kinase (pERK 1/2) mediating mitosis. Notably, this programmed nanosystem in human breast MCF‐7 tumor‐bearing mice models displays a significant relief of ECM stress from 4300 to 1200 Pa and results in 87.1% suppression of tumor growth at a low PTX dosage of 3 mg kg−1. The attenuated expression of the key players RhoA and pERK 1/2 involved in cellular mechano‐sensing is further verified in vivo. This study thus provides a new and potential nanoplatform to selectively decrease TME stiffness for enhanced chemotherapy.

MTOR plays a pivotal role in multiple processes of enamel organ development principally through the mTORC1 pathway and in part via regulating cytoskeleton dynamics

We herein report that deletion of mTOR in dental epithelia caused defective development of multiple cell layers of the enamel organ, which culminated in tooth malformation and cystogenesis. Specifically, cells of the stellate reticulum and stratum intermedium were poorly formed, resulting in cystic changes. The pre-ameloblasts failed to elongate along the apical-basal axis and persisted vigorous expression of Sox2 and P63, which are normally downregulated during cytodifferentiation. Expression of amelogenic markers was also attenuated in mutants. Cell proliferation and cell sizes in mutants were significantly reduced over time. Importantly, we found reduced amounts and aberrant aggregations of cytoskeletal components in mutants, along with attenuated expression of cytoskeleton regulator Cdc42, whose epithelial deletion causes a similar phenotype. Moreover, disruption of actin assembly in an organ culture system affected cell proliferation and cytodifferentiation of tooth germs, supporting a causative role of mTOR-regulated cytoskeleton dynamics for the observed phenotype of mTOR mutant mice. In further support of this view, we showed that mTOR overactivation caused increased cytoskeletal component synthesis and assembly, along with accelerated cytodifferentiation in the enamel organ. Finally, we demonstrated that mTOR regulated enamel organ development principally through the mTORC1 pathway.