Cytoskeleton Network Research Articles

Proteomic Analyses for the Global S-Nitrosylated Proteins in the Brain Tissues of Different Human Prion Diseases.

Human prion diseases are fatal neurodegenerative disorders characterized by neuronal damage in brain. Protein S-nitrosylation, the covalent adduction of a NO to cysteine, plays a role in human brain biology, and brain dysfunction is a prominent feature of prion disease, yet the direct brain targets of S-nitrosylation are largely unknown. We described the first proteomic analysis of global S-nitrosylation in brain tissues of sporadic Creutzfeldt-Jakob disease (sCJD), fatal familial insomnia (FFI), and genetic CJD with a substitution of valine for glycine at codon 114 of the prion protein gene (G114V gCJD) accompanying with normal control with isobaric tags for relative and absolute quantitation (iTRAQ) combined with a nano-HPLC/Q-Exactive mass spectrometry platform. In parallel, we used several approaches to provide quality control for the experimentally defined S-nitrosylated proteins. A total of 1509 S-nitrosylated proteins (SNO-proteins) were identified, and data are available via ProteomeXchange with identifier PXD002813. The cerebellum tissues appeared to contain more commonly differentially expressed SNO-proteins (DESPs) than cortex of sCJD, FFI, and gCJD. Three selected SNO-proteins were verified by Western blots, consistent with proteomics assays. Gene ontology analysis showed that more up-regulated DESPs were involved in metabolism, cell cytoskeleton/structure, and immune system both in the cortex and cerebellum, while more down-regulated ones in both regions were involved in cell cytoskeleton/structure, cell-cell communication, and miscellaneous function protein. Pathway analysis suggested that systemic lupus erythematosus, pathogenic Escherichia coli infection, and extracellular matrix-receptor interaction were the most commonly affected pathways, which were identified from at least two different diseases. Using STRING database, the network of immune system and cell cytoskeleton and structure were commonly identified in the context of the up-regulated and down-regulated DESPs, respectively, both in the cortex and cerebellum. Our study thus have implications for understanding the molecular mechanisms of human prion diseases related to abnormal protein S-nitrosylation and pave the way for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases.

Single-cell mechanics – An experimental–computational method for quantifying the membrane–cytoskeleton elasticity of cells

The membrane–cytoskeleton system plays a major role in cell adhesion, growth, migration, and differentiation. F-actin filaments, cross-linkers, binding proteins that bundle F-actin filaments to form the actin cytoskeleton, and integrins that connect the actin cytoskeleton network to the cell plasma membrane and extracellular matrix are major cytoskeleton constituents. Thus, the cell cytoskeleton is a complex composite that can assume different shapes. Atomic force microscopy (AFM)-based techniques have been used to measure cytoskeleton material properties without much attention to cell shape. A recently developed surface chemical patterning method for long-term single-cell culture was used to seed individual cells on circular patterns. A continuum-based cell model, which uses as input the force–displacement response obtained with a modified AFM setup and relates the membrane–cytoskeleton elastic behavior to the cell geometry, while treating all other subcellular components suspended in the cytoplasmic liquid (gel) as an incompressible fluid, is presented and validated by experimental results. The developed analytical–experimental methodology establishes a framework for quantifying the membrane–cytoskeleton elasticity of live cells. This capability may have immense implications in cell biology, particularly in studies seeking to establish correlations between membrane–cytoskeleton elasticity and cell disease, mortality, differentiation, and migration, and provide insight into cell infiltration through nonwoven fibrous scaffolds. The present method can be further extended to analyze membrane–cytoskeleton viscoelasticity, examine the role of other subcellular components (e.g., nucleus envelope) in cell elasticity, and elucidate the effects of mechanical stimuli on cell differentiation and motility. Statement of SignificanceThis is the first study to decouple the membrane–cytoskeleton elasticity from cell stiffness and introduce an effective approach for measuring the elastic modulus. The novelty of this study is the development of new technology for quantifying the elastic stiffness of the membrane–cytoskeleton system of cells. This capability could have immense implications in cell biology, particularly in establishing correlations between various cell diseases, mortality, and differentiation with membrane–cytoskeleton elasticity, examining through-tissue cell migration, and understanding cell infiltration in porous scaffolds. The present method can be further extended to analyze membrane–cytoskeleton viscous behavior, identify the contribution of other subcellular components (e.g., nucleus envelope) to load sharing, and elucidate mechanotransduction effects due to repetitive compressive loading and unloading on cell differentiation and motility.

Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium

Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane.

Role of cytoskeleton network in anisosmotic volume changes of intact and permeabilized A549 cells

Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant 86Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2+. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2+-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel.

To be EndMT or not to be, that is the question in pulmonary hypertension.

To be EndMT or not to be, that is the question in pulmonary hypertension.

Functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus

Porcine deltacoronavirus (PDCoV) is a newly discovered enterotropic swine coronavirus that causes enteritis and diarrhea in piglets. Like other coronaviruses, PDCoV commonly contains 4 major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. Among these, the N protein is known to be the most abundant and multifunctional viral component. Therefore, as the first step toward understanding the biology of PDCoV, the present study investigated functional characteristics and expression dynamics of host proteins in a stable porcine cell line constitutively expressing the PDCoV N protein. Similar to N proteins of other coronaviruses, the PDCoV N protein was found to interact with itself to form non-covalently linked oligomers and was mainly localized to the nucleolus. We then assessed alterations in production levels of proteins in the N-expressing PK (PK-PDCoV-N) cells at different time points by means of proteomic analysis. According to the results of high-resolution two-dimensional gel electrophoresis, a total of 43 protein spots were initially found to be differentially expressed in PK-PDCoV-N cells in comparison with control PK cells. Of these spots, 10 protein spots showed a statistically significant alteration, including 8 up-regulated and 2 down-regulated protein spots and were picked for subsequent protein identification by peptide mass fingerprinting following matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The affected cellular proteins that we identified in this study were classified into the functional groups involved in various cellular processes such as cell division, metabolism, the stress response, protein biosynthesis and transport, cytoskeleton networks and cell communication. Notably, two members of the heat shock protein 70 family were found to be up-regulated in PK-PDCoV-N cells. These proteomic data will provide insights into the specific cellular response to the N protein during PDCoV infection.

Cellular and molecular markers in monitoring the fate of lymphoid cell culture from Penaeus monodon Fabricius (1798).

Lymphoid cell culture from penaeid shrimps has gained much acceptance as an in vitro platform to facilitate research on the development of prophylaxis, and therapeutic strategies against viruses and for cell line development. However, lymphoid cells can be used as platform for in vitro research, only if they are in metabolically and mitotically active state in vitro with unaltered cell surface receptors. Through this study, we addressed the response of lymphoid cells to a new microenvironment at cellular and molecular levels; including the study of mitotic events, DNA synthesis, expression profile of cell cycle genes, cytoskeleton organization, metabolic activity and viral susceptibility. The S-phase entry and synthesis of new DNA was recorded by immunoflourescent technique. Cdc2, CycA, CycB, EF-1α and BUB3 genes involved in cell cycle were studied in both the cells and tissue, of which EF-1α showed an elevated expression in cells in vitro (∼ 19.7%). Cytoskeleton network of the cell was examined by studying the organization of actin filaments. As the markers for metabolic status, mitochondrial dehydrogenase, protein synthesis and glucose assimilation by the cells were also assessed. Viral susceptibility of the cell was determined using WSSV to confirm the preservation of cellular receptors. This study envisages to strengthen the shrimp cell line research and to bring forth lymphoid cell culture system as a 'model' in vitro system for shrimp and crustaceans altogether.

A Spatial Model of Insulin-Granule Dynamics in Pancreatic β-Cells.

Insulin secretion from pancreatic β-cells in response to sudden glucose stimulation is biphasic. Prolonged secretion in vivo requires synthesis, delivery to the plasma membrane (PM) and exocytosis of insulin secretory granules (SGs). Here, we provide the first agent-based space-resolved model for SG dynamics in pancreatic β-cells. Using recent experimental data, we consider a single β-cell with identical SGs moving on a phenomenologically represented cytoskeleton network. A single exocytotic machinery mediates SG exocytosis on the PM. This novel model reproduces the measured spatial organization of SGs and insulin secretion patterns under different stimulation protocols. It proposes that the insulin potentiation effect and the rising second-phase secretion are mainly due to the increasing number of docking sites on the PM. Furthermore, it shows that 6 min after glucose stimulation, the 'newcomer' SGs are recruited from a region within less than 600 nm from the PM.

Actin cytoskeleton contributes to the elastic modulus of embryonic tendon during early development.

Tendon injuries are common and heal poorly. Strategies to regenerate or replace injured tendons are challenged by an incomplete understanding of normal tendon development. Our previous study showed that embryonic tendon elastic modulus increases as a function of developmental stage. Inhibition of enzymatic collagen crosslink formation abrogated increases in tendon elastic modulus at late developmental stages, but did not affect increases in elastic modulus of early stage embryonic tendons. Here, we aimed to identify potential contributors to the mechanical properties of these early stage embryonic tendons. We characterized tendon progenitor cells in early stage embryonic tendons, and the influence of actin cytoskeleton disruption on tissue elastic modulus. Cells were closely packed in embryonic tendons, and did not change in density during early development. We observed an organized network of actin filaments that seemed contiguous between adjacent cells. The actin filaments exhibited a crimp pattern with a period and amplitude that matched the crimp of collagen fibers at each developmental stage. Chemical disruption of the actin cytoskeleton decreased tendon tissue elastic modulus, measured by atomic force microscopy. Our results demonstrate that early developmental stage embryonic tendons possess a well organized actin cytoskeleton network that contributes significantly to tendon tissue mechanical properties.

Schip1 is a novel podocyte foot process protein that mediates actin cytoskeleton rearrangements and forms a complex with Nherf2 and ezrin.

BackgroundPodocyte foot process effacement accompanied by actin cytoskeleton rearrangements is a cardinal feature of many progressive human proteinuric diseases.ResultsBy microarray profiling of mouse glomerulus, SCHIP1 emerged as one of the most highly enriched transcripts. We detected Schip1 protein in the kidney glomerulus, specifically in podocytes foot processes. Functionally, Schip1 inactivation in zebrafish by morpholino knock-down results in foot process disorganization and podocyte loss leading to proteinuria. In cultured podocytes Schip1 localizes to cortical actin-rich regions of lamellipodia, where it forms a complex with Nherf2 and ezrin, proteins known to participate in actin remodeling stimulated by PDGFβ signaling. Mechanistically, overexpression of Schip1 in vitro causes accumulation of cortical F-actin with dissolution of transversal stress fibers and promotes cell migration in response to PDGF-BB stimulation. Upon actin disassembly by latrunculin A treatment, Schip1 remains associated with the residual F-actin-containing structures, suggesting a functional connection with actin cytoskeleton possibly via its interaction partners. A similar assay with cytochalasin D points to stabilization of cortical actin cytoskeleton in Schip1 overexpressing cells by attenuation of actin depolymerisation.ConclusionsSchip1 is a novel glomerular protein predominantly expressed in podocytes, necessary for the zebrafish pronephros development and function. Schip1 associates with the cortical actin cytoskeleton network and modulates its dynamics in response to PDGF signaling via interaction with the Nherf2/ezrin complex. Its implication in proteinuric diseases remains to be further investigated.

Effect of hypoxia on TRPV1 and TRPV4 channels in rat pulmonary arterial smooth muscle cells.

Transient receptor potential (TRP) channels of the vanilloid subfamily, mainly TRPV1 and TRPV4, are expressed in pulmonary artery smooth muscle cells (PASMC) and implicated in the remodeling of pulmonary artery, a landmark of pulmonary hypertension (PH). Among a variety of PH subtypes, PH of group 3 are mostly related to a prolonged hypoxia exposure occurring in a variety of chronic lung diseases. In the present study, we thus investigated the role of hypoxia on TRPV1 and TRPV4 channels independently of the increased pulmonary arterial pressure that occurs during PH. We isolated PASMC from normoxic rat and cultured these cells under in vitro hypoxia. Using microspectrofluorimetry and the patch-clamp technique, we showed that hypoxia (1% O2 for 48h) significantly increased stretch- and TRPV4-induced calcium responses. qRT-PCR, Western blotting, and immunostaining experiments revealed that the expression of TRPV1 and TRPV4 was not enhanced under hypoxic conditions, but we observed a membrane translocation of TRPV1. Furthermore, hypoxia induced a reorganization of the F-actin cytoskeleton, the tubulin, and intermediate filament networks (immunostaining experiments), associated with an enhanced TRPV1- and TRPV4-induced migratory response (wound-healing assay). Finally, as assessed by immunostaining, exposure to in vitro hypoxia elicited a significant increase in NFATc4 nuclear localization. Cyclosporin A and BAPTA-AM inhibited NFATc4 translocation, indicating the activation of the Ca(2+)/calcineurin/NFAT pathway. In conclusion, these data point out the effect of hypoxia on TRPV1 and TRPV4 channels in rat PASMC, suggesting that these channels can act as direct signal transducers in the pathophysiology of PH.

The role of multifilament structures and lateral interactions in dynamics of cytoskeleton proteins and assemblies.

Microtubules and actin filaments are biopolymer molecules that are major components of cytoskeleton networks in biological cells. They play important roles in supporting fundamental cellular processes such as cell division, signaling, locomotion, and intracellular transport. In cells, cytoskeleton proteins function under nonequilibrium conditions that are powered by hydrolysis of adenosine triphosphate (ATP) or guanosine triphosphate (GTP) molecules attached to them. Although these biopolymers are critically important for all cellular processes, the mechanisms that govern their complex dynamics and force generation remain not well explained. One of the most difficult fundamental issues is to understand how different components of cytoskeleton proteins interact together. We develop an approximate theoretical approach for analyzing complex processes in cytoskeleton proteins that takes into account the multifilament structure, lateral interactions between parallel protofilaments, and the most relevant biochemical transitions during the biopolymer growth. It allows us to fully evaluate collective dynamic properties of cytoskeleton filaments as well as the effect of external forces on them. It is found that for the case of strong lateral interactions the stall force of the multifilament protein is a linear function of the number of protofilaments. However, for weak lateral interactions, deviations from the linearity are observed. We also show that stall forces, mean velocities, and dispersions are increasing functions of the lateral interactions. Physical-chemical explanations of these phenomena are presented. Our theoretical predictions are supported by extensive Monte Carlo computer simulations.

Role of autophagy in response to titanium dioxide nanoparticles

The development and the increasing production of manufactured nanoparticles (NP) are raising safety concerns because of the potential effects of NP on human health, particularly at the respiratory level. Several studies have shown that exposure to manufactured NP can induce pathogenic biological effects, with lung remodelling (fibrosis, emphysema…), depending on the physicochemical characteristics of the NP. Currently, oxidative stress and inflammation are the most widely accepted paradigms of NP toxicity; however, the exact underlying mechanisms in the biological effects of NP still remain unknown. Autophagy is a physiological process that allows the autodigestion of the subcellar components and which is also involved in the elimination of intracellular pathogens. It has been shown that this process can negatively regulate inflammation and oxidative stress. Thus, the hypothesis of this study is that a defective autophagy could be a new mechanism explaining, at least in part, NP effects. We choose to focus on TiO2 NP since it is one of the most abundantly produced and widely used NP. We used seven TiO2 NPs presenting different physicochemicals properties (size, crystal phase, surface embedding) and compared their effects to those of micron-size TiO2 and carbon black (CB - for chemical composition effects). NPs were characterized by electron microscopy, dynamic light scattering and X-ray diffraction. Murine macrophages (RAW cell line) were exposed to 50 μg/mL TiO2 and CB NP for 6 hours. Effects of NP on the autophagy process were analysed by looking at the expression of autophagy markers (LC3-II and p62) and lysosomal proteins (LAMPs and cathepsins). We also analysed the cytoskeleton network by fluorescence microscopy. Moreover, expression of inflammatory cytokines was determined in macrophages exposed to NP. All particles, except the micrometric one, induced an increase of LC3-II protein expression, indicating an accumulation of autophagosomes. These particles also increased p62 protein level, suggesting an autophagy blockade. This perturbation of the autophagy process by NP does not seem to result from a disruption of the cytoskeleton but from a defect in the lysosome function as suggested by a decrease of mature cathepsins expression. Moreover, preliminary results showed that exposure to TiO2 NP induce inflammation, at different levels depending on their physicochemicals characteristics. These results suggest that some TiO2 NP, depending on their physicochemical properties, can block the autophagy process. The future work will be to understand the mechanisms explaining this autophagy dysfunction and to determine its consequence on the toxicity induced by these NP.

Zyxin Antagonizes the FERM Protein Expanded to Couple F-Actin and Yorkie-Dependent Organ Growth

Coordinated multicellular growth during development is achieved by the sensing of spatial and nutritional boundaries. The conserved Hippo (Hpo) signaling pathway has been proposed to restrict tissue growth by perceiving mechanical constraints through actin cytoskeleton networks. The actin-associated LIM proteins Zyxin (Zyx) and Ajuba (Jub) have been linked to the control of tissue growth via regulation of Hpo signaling, but the study of Zyx has been hampered by a lack of genetic tools. We generated a zyx mutant in Drosophila using TALEN endonucleases and used this to show that Zyx antagonizes the FERM-domain protein Expanded (Ex) to control tissue growth, eye differentiation, and F-actin accumulation. Zyx membrane targeting promotes the interaction between the transcriptional co-activator Yorkie (Yki) and the transcription factor Scalloped (Sd), leading to activation of Yki target gene expression and promoting tissue growth. Finally, we show that Zyx's growth-promoting function is dependent on its interaction with the actin-associated protein Enabled (Ena) via a conserved LPPPP motif and is antagonized by Capping Protein (CP). Our results show that Zyx is a functional antagonist of Ex in growth control and establish a link between actin filament polymerization and Yki activity.

Epidermal Growth Factor Activates the Rho GTPase-activating Protein (GAP) Deleted in Liver Cancer 1 via Focal Adhesion Kinase and Protein Phosphatase 2A

Deleted in Liver Cancer 1 (DLC1) is a RHO GTPase-activating protein (GAP) that negatively regulates RHO. Through its GAP activity, it modulates the actin cytoskeleton network and focal adhesion dynamics, ultimately leading to suppression of cell invasion and metastasis. Despite its presence in various structural and signaling components, little is known about how the activity of DLC1 is regulated at focal adhesions. Here we show that EGF stimulation activates the GAP activity of DLC1 through a concerted mechanism involving DLC1 phosphorylation by MEK/ERK and its subsequent dephosphorylation by protein phosphatase 2A (PP2A) and inhibition of focal adhesion kinase by MEK/ERK to allow the binding between DLC1 and PP2A. Phosphoproteomics and mutation studies revealed that threonine 301 and serine 308 on DLC1, known previously to be mutated in certain cancers, are required for DLC1-PP2A interaction and the subsequent activation of DLC1 upon their dephosphorylation. The intricate interplay of this "MEK/ERK-focal adhesion kinase-DLC1-PP2A" quartet provides a novel checkpoint in the spatiotemporal control of cell spreading and cell motility.

Myosin Steps Symmetrically along Actin

Myosin 5a is a homodimer belonging to the family of processive molecular motors that transform chemical energy into directional motion along the cytoskeleton network. Despite a well understood kinetic cycle, the type of hand-over-hand motion describing each step remains unknown due to the intrinsic short lifetime of the unbound head state. Here, we differentially label the two motor domains of myosin 5a, one with a fluorescent quantum dot and the other with a small gold nanoparticle and simultaneously trace the three dimensional motion of each domain by combining single molecule fluorescence with interferometric scattering microscopy (iSCAT).Within individual trajectories, we observed an intermediate state for each labelled unbound head that is located to one and the same side of the actin filament from which myosin repeatedly sampled the desired actin site until strong binding occurred. Analysis over several hundred molecules showed no preference in the relative position of the transient state with respect to actin. The observation that the transient state orientation can change for the same molecule by binding to another actin filament shows that the directionality of the transient is not an intrinsic property of the motor. Instead the decision is made upon binding to actin, with both cases equally probable. Our work provides critical insight on the highly efficient mechano-chemical cycle of myosin 5a and provides strong evidence for a symmetric hand-over-hand model in which the trailing head swings by the leading head on the same side repeatedly.

Microrheology of In-Vitro Acto-Myosin Networks in Steady State

We show that complex fluids such as actin networks respond differently to deformations at intermediate length scales than they do at large distances as a bulk material. This intermediate response regime is characterized by a 1/r3 decay with distance. When characterizing passive entangled F-actin networks, we observed this intermediate response at surprisingly large distances of 2-6 µm, which are comparable to the size of a cell, and are over ten times larger than the mesh size of the actin network. We generalize the framework of microrheology to include and characterize this intermediate response, which in turn allows extracting the material's structural properties.We use this newfound understanding to extract structural information of active in-vitro reconstituted cytoskeleton networks, in which such analysis can be done in a controlled fashion.

AFM-Based Study of Fullerenol (C60(OH)24)-Induced Changes of Elasticity in Living SMCC-7721 Cells

In this study, the alterations of the morphology and biomechanical properties of living SMCC-7721 cancer cells treated with fullerenol (C60(OH)24) for 24, 48, and 72h were investigated using an atomic force microscope (AFM). Comparative analyses show that the elastic moduli of the SMCC-7721 cells exposed to fullerenol decrease significantly with the increase of the treatment periods. Furthermore, in different phases of the treatment, a global decrease in elasticity is accompanied by cellular morphological changes, and the time-dependent effect of the fullerenol can be observed using AFM and optical microscope. In addition, as the treatment duration increases, the indentation force and depth penetrated into the cell membrane by the AFM tip are in a declining trend. The reduction in the stiffness of the cells exposed to fullerenol could be associated with the disruption of the cellular cytoskeleton network. The investigation indicates that the elastic modulus of single living cells can be a useful biomarker to evaluate the effects of fullerenol or other anticancer agents on the cells and reveal instructive information for cellular dynamic behaviors.

An adhesome comprising laminin, dystroglycan and myosin IIA is required during notochord development in Xenopus laevis

Dystroglycan (Dg) is a transmembrane receptor for laminin that must be expressed at the right time and place in order to be involved in notochord morphogenesis. The function of Dg was examined in Xenopus laevis embryos by knockdown of Dg and overexpression and replacement of the endogenous Dg with a mutated form of the protein. This analysis revealed that Dg is required for correct laminin assembly, for cell polarization during mediolateral intercalation and for proper differentiation of vacuoles. Using mutations in the cytoplasmic domain, we identified two sites that are involved in cell polarization and are required for mediolateral cell intercalation, and a site that is required for vacuolation. Furthermore, using a proteomic analysis, the cytoskeletal non-muscle myosin IIA has been identified for the first time as a molecular link between the Dg-cytoplasmic domain and cortical actin. The data allowed us to identify the adhesome laminin-Dg-myosin IIA as being required to maintain the cortical actin cytoskeleton network during vacuolation, which is crucial to maintain the shape of notochordal cells.

Actin dynamics and the evolution of the memory trace

The goal of this essay is to link the regulation of actin dynamics to the idea that the synaptic changes that support long-term potentiation and memory evolve in temporally overlapping stages—generation, stabilization, and consolidation. Different cellular/molecular processes operate at each stage to change the spine cytoarchitecture and, in doing so, alter its function. Calcium-dependent processes that degrade the actin cytoskeleton network promote a rapid insertion of AMPA receptors into the post synaptic density, which increases a spine׳s capacity to express a potentiated response to glutamate. Other post-translation events then begin to stabilize and expand the actin cytoskeleton by increasing the filament actin content of the spine and reorganizing it to be resistant to depolymerizing events. Disrupting actin polymerization during this stabilization period is a terminal event—the actin cytoskeleton shrinks and potentiated synapses de-potentiate and memories are lost. Late-arriving, new proteins may consolidate changes in the actin cytoskeleton. However, to do so requires a stabilized actin cytoskeleton. The now enlarged spine has properties that enable it to capture other newly transcribed mRNAs or their protein products and thus enable the synaptic changes that support LTP and memory to be consolidated and maintained.This article is part of a Special Issue entitled SI: Brain and Memory.