Cultured Xenopus Muscle Cells Research Articles

Directed motion of membrane proteins under an entropy-driven potential field generated by anchored proteins

Directed transport of proteins and other molecules in a crowded living cell is often carried out by diffusion at short distances and by motor-driven cargo transport over long distances. Here we demonstrate, by both experiments and theory, that anchored proteins inside the cell can generate a spatially varying and temporally stable potential (free-energy) landscape for intracellular or membrane transport in the mesoscale. By using a micropatterned substrate, we introduce a periodic array of anchored integrins on the basal membrane of cultured Xenopus muscle cells. This patterned array of anchored integrins imposes a periodic potential $U(x)$ to the lateral motion of nicotinic acetylcholine receptors (AChRs) on the cell membrane. From a thorough analysis of a large volume of AChR trajectories obtained over a wide range of sampling conditions and long durations from 385 cells, we find the trapping potential $U(x)$ and its effects on the drift velocity ${V}_{x}(x)$ and diffusion coefficient ${D}_{x}(x)$ of AChRs. Our findings suggest that anchored proteins may play an essential role in generating an effective potential landscape to guide molecular motion in the mesoscale ranging from protein trapping and directed motion to enhanced protein-protein interactions over a long range.

Topographic Modulation of Acetylcholine Receptors Diffusion Dynamics on Live Cell Membrane

In a recent single-molecule tracking experiment [1], the acetylcholine receptors (AChRs) on muscle cell membranes were found to display non-Gaussian statistics due to dynamic heterogeneity resulting from the complex cellular environment of the AChRs. In this study, we vary the cellular environment of AChRs by using the microgroove patterning techniques to control the spatial distribution of adhesion complexes on the basal membrane of live cells and study its effect on the diffusion dynamics of AChRs. Cultured Xenopus muscle cells are found to align along the microgroove patterns with their adhesion complexes, such as β1-integrins, predominantly anchored on the ridge region of the microgrooves, forming a stripe-like pattern. Interestingly, we find the movement of AChRs is largely restricted within the microgroove-aligned integrin stripes. Detailed statistical analysis of the AChR trajectories reveals that the AChRs are slowed down by the patterned integrins, and immobilization and clustering of the AChRs are correlated with the spatial distribution of integrins. The experiment demonstrates that the spatial distribution of integrins plays a crucial role in modulating the diffusion dynamics of AChRs. ∗The work was supported by the Research Grants Council of Hong Kong SAR. [1] W. He, H. Song, Y. Su, L. Geng, B. J. Ackerson, H. B. Peng, and P. Tong, Nature Communications, 7:11701 (2016).

Difffusion Dynamics of AChR Receptors on Live Muscle Cell Membrane

The dynamic structure of a cell membrane allows it to become an effective platform for various biological functions, such as signal transduction, molecule transportation and endocytosis. We report here a single-molecular tracking experiment on a quantum-dot-labeled transmembrane protein, acetylcholine receptor (AChR), in cultured Xenopus muscle cells. We carried out a complete statistical analysis on a large set of AChR trajectories with more than 500 cells examined. Various drug treatments were used to perturb F-actin and scaffold proteins and examine their roles in regulating the motion of the AChRs. The diffusion dynamics of AChRs was characterized by three quantities: the mean-square displacement 〈Δr2(τ)〉, the probability density function P(Dx) of instantaneous displacement Dx(τ) and the probability distribution f(δ) of instantaneous diffusion coefficient δ. After a careful analysis, we conclude that (1) AChRs show a hindered motion by the surrounding membrane molecules at short time and become diffusive at long time. (2) The mobile AChRs have a broad distribution in diffusion coefficient δ with a long exponential tail, which is universal and independent of different sample conditions. (3) The exponential distribution f(δ) leads to an exponential distribution P(Dx). Our measurements of membrane diffusion based on a large number of single molecular trajectories provide a complete statistical description of dynamic heterogeneity on live cell membrane. By combining all the experimental results available, we propose a dynamic picket-fence model of membrane organization involving slow active remodeling of the underlying cortical actin network to explain the observed non-Gaussian statistics and dynamic heterogeneity. (Work was supported by the Research Grants Council of Hong Kong SAR.)

Using Xenopus tissue cultures for the study of myasthenia gravis pathogenesis

Myasthenia gravis (MG), the most common autoimmune disease of neuromuscular junction (NMJ), is heterogeneous in terms of pathophysiology, which is determined by the pathogenic antigen of autoantibodies targeting to synaptic proteins at the NMJs. Currently, patients suspected with MG are routinely screened for the presence of autoantibodies against acetylcholine receptor (AChR) or muscle-specific kinase (MuSK) using a cell-based assay (CBA) that involves the expression of target synaptic membrane protein in heterologous cell lines. However, some autoantibodies may only show reactivity for binding to densely clustered AChR in the physiological conformation, while AChR clustering is known to involve signaling events orchestrated by over a dozen of postsynaptic proteins. To improve the existing serological diagnosis of MG, this study explored the possibility of using the well-established Xenopus primary culture system as a novel CBA for MG. Here, by examining the pathogenic effects of four MG human plasma samples, we found that the samples from both seropositive and seronegative MG patients effectively induced the disassembly of aneural AChR clusters in cultured Xenopus muscle cells, as well as the nerve-induced AChR clusters in the nerve–muscle co-cultures. Importantly, the disassembly of AChR clusters was spatio-temporally correlated to the disappearance of actin depolymerizing factor (ADF)/cofilin, an actin regulator involved in AChR trafficking and clustering. Taken together, this study develops a reliable CBA using Xenopus primary cultures for screening the pathogenicity of human MG plasma samples, and providing a platform for investigating the pathogenic mechanisms underlying the endocytic trafficking and degradation of AChRs at NMJs in MG patients.

Mechanosensitivity of nicotinic receptors

Nicotinic acetylcholine receptors (nAChRs) are heteropentameric ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junction (NMJ) and other peripheral and central synapses. At the NMJ, acetylcholine receptors (AChRs) are constantly exposed to mechanical stress resulting from muscle contraction. It is therefore of interest to understand if their function is influenced by mechanical stimuli. In this study, patch-clamp recordings showed that AChR channel activity was enhanced upon membrane stretching in both cultured Xenopus muscle cells and C2C12 myotubes. To examine how this property is physiologically regulated, effects of membrane-intrinsic and membrane-extrinsic factors on AChRs expressed in HEK293T cells were studied. As in muscle cells, AChR single channel currents recorded under cell-attached configuration were significantly increased—without change in current amplitude—when negative pressure was applied through the patch pipette. GsMTx-4, a peptide toxin that blocks mechanically activated cation channels, inhibited this effect on AChRs. The mechanosensitivity decreased when cells were treated with MβCD, latrunculin A or cytochalasin D, but increased when exposed to lysophosphatidylcholine, indicating contributions from both membrane lipids and the cytoskeleton. Rapsyn, which binds to AChRs and mediates their cytoskeletal interaction in muscle, suppressed AChR mechanosensitivity when co-expressed in HEK293T cells, but this influence of rapsyn was impaired following the deletion of rapsyn’s AChR-binding domain or upon cytoskeletal disruption by cytochalasin D. These results suggest a mechanism for regulating AChR’s mechanosensitivity through its cytoskeletal linkage via rapsyn, which may serve to protect the receptors and sarcolemmal integrity under high mechanical stress encountered by the NMJ.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-012-1132-9) contains supplementary material, which is available to authorized users.

HGF induction of postsynaptic specializations at the neuromuscular junction

A critical event in the formation of vertebrate neuromuscular junctions (NMJs) is the postsynaptic clustering of acetylcholine receptors (AChRs) in muscle. AChR clustering is triggered by the activation of MuSK, a muscle-specific tyrosine kinase that is part of the functional receptor for agrin, a nerve-derived heparan sulfate proteoglycan (HSPG). At the NMJ, heparan sulfate (HS)-binding growth factors and their receptors are also localized but their involvement in postsynaptic signaling is poorly understood. In this study we found that hepatocyte growth factor (HGF), an HS-binding growth factor, surrounded muscle fibers and was localized at NMJs in rat muscle sections. In cultured Xenopus muscle cells, HGF was enriched at spontaneously occurring AChR clusters (hot spots), where HSPGs were also concentrated, and, following stimulation of muscle cells by agrin or cocultured neurons, HGF associated with newly formed AChR clusters. HGF presented locally to cultured muscle cells by latex beads induced new AChR clusters and dispersed AChR hot spots, and HGF beads also clustered phosphotyrosine, activated c-Met, and proteins of dystrophin complex; clustering of AChRs and associated proteins by HGF beads required actin polymerization. Lastly, although bath-applied HGF alone did not induce new AChR clusters, addition of HGF potentiated agrin-dependent AChR clustering in muscle. Our findings suggest that HGF promotes AChR clustering and synaptogenic signaling in muscle during NMJ development.

Localization of sodium and potassium currents at sites of nerve-muscle contact in embryonic Xenopus muscle cells in culture.

Macro-patch current recordings were used to determine the spatial distribution of INa, inactivating (IIK) and non-inactivating (IK) outward potassium currents on nerve-contacted Xenopus muscle cells grown in culture. Sites of nerve contact were identified under 400x magnification and macroscopic currents recorded from cell-attached membrane patches ranging in diameter from 2 microm to 6 microm. Membrane patch (n=133) recordings revealed no detectable current, or INa and/or IIK, or just IK. More than 85% of the membrane patches from which INa (n=35) could be recorded occurred within 40 microm of the position of nerve contact. In addition, more than 90% of the membrane patches which contained INa also contained IIK. The density of IIK in these membrane patches that also contained INa was significantly higher than the density of IIK or IK in membrane patches without INa. Sites of ACh receptor localization at nerve-muscle contacts, identified by labelling with rhodamine-alpha-bungarotoxin, were also found to contain both INa (4/5 cells) and IIK (5/5 cells). These results suggest that in Xenopus muscle cells in culture the channels mediating IIK along with sodium channels are clustered at sites of nerve contact and synaptic specializations.

A role of tyrosine phosphatase in acetylcholine receptor cluster dispersal and formation.

Innervation of the skeletal muscle involves local signaling, leading to acetylcholine receptor (AChR) clustering, and global signaling, manifested by the dispersal of preexisting AChR clusters (hot spots). Receptor tyrosine kinase (RTK) activation has been shown to mediate AChR clustering. In this study, the role of tyrosine phosphatase (PTPase) in the dispersal of hot spots was examined. Hot spot dispersal in cultured Xenopus muscle cells was initiated immediately upon the presentation of growth factor-coated beads that induce both AChR cluster formation and dispersal. Whereas the density of AChRs decreased with time, the fine structure of the hot spot remained relatively constant. Although AChR, rapsyn, and phosphotyrosine disappeared, a large part of the original hot spot-associated cytoskeleton remained. This suggests that the dispersal involves the removal of a key linkage between the receptor and its cytoskeletal infrastructure. The rate of hot spot dispersal is inversely related to its distance from the site of synaptic stimulation, implicating the diffusible nature of the signal. PTPase inhibitors, such as pervanadate or phenylarsine oxide, inhibited hot spot dispersal. In addition, they also affected the formation of new clusters in such a way that AChR microclusters extended beyond the boundary set by the clustering stimuli. Furthermore, by introducing a constitutively active PTPase into cultured muscle cells, hot spots were dispersed in a stimulus- independent fashion. This effect of exogenous PTPase was also blocked by pervanadate. These results implicate a role of PTPase in AChR cluster dispersal and formation. In addition to RTK activation, synaptic stimulation may also activate PTPase which acts globally to destabilize preexisting AChR hot spots and locally to facilitate AChR clustering in a spatially discrete manner by countering the action of RTKs.

The Role of an Agrin–Growth Factor Interaction in ACh Receptor Clustering

The clustering of acetylcholine receptors (AChRs) at the neuromuscular junction is mediated in part by the heparan-sulfate proteoglycan agrin. However, our previous studies have also suggested the role of heparin-binding growth-associated molecule (HB-GAM) in AChR clustering. Here the role of an agrin–HB-GAM interaction in this process was examined using culturedXenopusmuscle cells. Agrin-coated beads were found to be weakly effective while agrin-coated beads further treated with HB-GAM were highly effective in AChR cluster induction. Protein overlay assays showed specific binding of HB-GAM to agrin. In addition, agrin-enriched neuritic tracks bound HB-GAM in a manner that showed a high degree of colocalization between the neural agrin and the applied factor. Finally, the introduction of exogenous HB-GAM together with soluble agrin resulted in the appearance of AChR clusters on the dorsal surface of cells in an agrin isoform-dependent manner; a dramatic change from the characteristic ventral AChR clustering seen in response to agrin alone. These results suggest that agrin may mediate AChR clustering by interacting with muscle-bound heparin-binding growth factors such as HB-GAM.

Full-Length Agrin Isoform Activities and Binding Site Distributions on CulturedXenopusMuscle Cells

Agrin is a large multidomain protein involved in the induction of postsynaptic differentiation of the neuromuscular junction. As a step toward further understanding the mechanisms by which agrin induces the aggregation of acetylcholine receptors (AChRs), we have characterized the activity of purified, full-length chick agrin isoforms onXenopusmuscle cells. Incubation with agrin isoforms led to the formation of numerous small AChR clusters primarily on the ventral surface of cells with differing efficacies: Y4Z8> Y4Z11= Y4Z19, with Y4B0being ineffective. Agrin activity appeared to be tyrosine phosphorylation-dependent as the kinase inhibitor tyrphostin RG50864 (80 μM) completely abolished the effect. Initial binding sites for all agrin isoforms were evenly distributed in a punctate manner on the muscle cell surface. After a 14-h incubation, the active isoforms induced AChR clustering, and agrin was enriched at these sites of clustering. Agrin binding to the cell surface and induction of AChR clustering were Ca2+-dependent, as previously shown in other systems. This is the first quantitative characterization of agrin's effects usingXenopuscell culture, providing a basis for further elucidating agrin's role in synaptogenesis using this elegant system.

Induction of acetylcholine receptor cluster formation by local application of growth factors in cultured Xenopus muscle cells

A survey of several growth factors was carried out to determine their effect on the formation of acetylcholine receptor (AChR) clusters in cultured Xenopus muscle cells. Factors were locally applied via polystyrene beads and AChR clustering at bead-muscle contacts was assessed. Among the factors tested, basic fibroblast growth factor, insulin and insulin-like growth factor-I, as well as several polycations, were found to be effective in inducing AChR clusters. The action of these molecules appears to be mediated by tyrosine kinase receptors, since it can be blocked by a tyrosine kinase inhibitor. These results suggest that local ligand-mediated tyrosine phosphorylation is involved in the formation of AChR clusters.

Modulation of the nicotinic acetylcholine receptor channels by spermine in Xenopus muscle cell culture

The modulation by endogenous polyamine spermine on acetylcholine (ACh)-induced currents was evaluated using a outside-out patch and whole-cell voltage-clamp recordings from cultured Xenopus muscle cells. Spermine potentiated the ACh-induced whole-cell currents at micromolar concentrations but is less effective at millimolar concentrations at a holding potential of −60 mV. It produced a voltage-dependent potentiation of ACh-induced whole-cell currents; the degree of potentiation by 100 μM spermine was 18.3 ± 1.7% at a holding potential of −60 mV while 108.2 ± 2.9% increase at +60 mV. Arcaine, a putative competitive antagonist at the polyamine-binding site on the N- methyl- d-aspartate (NMDA) receptor-channel complex, reduced the spermine enhancement of ACh-induced whole-cell currents. Glycine (10 μM) had no effect on the potentiation of ACh-induced whole-cell currents by spermine. In single-channel recordings, spermine (1 μM to 1 mM) increased the opening frequency of both low- and high-conductance ACh channels and at higher concentrations (≥ 100 μM) it produced, in addition, a voltage-dependent decrease in channel amplitude and average open time of both types of ACh channels that limits its enhancing action. It is likely that these two actions of spermine, due to differences in concentration- and voltage-dependence, are mediated by different binding sites on the nicotinic ACh receptor-channel complex of the muscle cells.

Clustered acetylcholine receptors have two levels of organization in Xenopus muscle cells.

We studied the organization of acetylcholine receptor (AChR) clusters by shearing cultured Xenopus muscle cells with a stream of buffer, and preparing rotary replicas of the exposed cytoplasmic surface of the sarcolemma. AChR clusters contained numerous particles that protruded from the sarcolemma and formed an irregular array composed of discrete aggregates. AChR were located within these particle aggregates, as shown by comparison of the replicas to labeling by fluorescent alpha-bungarotoxin, and by immunogold cytochemistry with antibodies specific for the receptor. The aggregates were cross-linked by a dense network of 7 nm filaments that replicated with the banded pattern characteristic of actin microfilaments. The organization of receptors into the small aggregates was independent of the organization of these aggregates into clusters, as alkaline extraction removed the microfilament network and disrupted the irregular array of particle aggregates, but did not disperse individual receptors from the aggregates. We conclude that two levels of interactions organize AChR clusters in Xenopus muscle cells: short-range interactions that assemble individual AChR into small aggregates, and long-range interactions, perhaps mediated by actin microfilaments, that anchor the aggregates into larger clusters.

A role of tyrosine phosphorylation in the formation of acetylcholine receptor clusters induced by electric fields in cultured Xenopus muscle cells.

During the development of the neuromuscular junction, acetylcholine receptors (AChRs) become clustered in the postsynaptic membrane in response to innervation. In vitro, several non-neuronal stimuli can also induce the formation of AChR clusters. DC electric field (E field) is one of them. When cultured Xenopus muscle cells are exposed to an E field of 5-10 V/cm, AChRs become clustered along the cathode-facing edge of the cells within 2 h. Recent studies have suggested the involvement of tyrosine kinase activation in the action of several AChR clustering stimuli, including nerve, polymer beads, and agrin. We thus examined the role of tyrosine phosphorylation in E field-induced AChR clustering. An antibody against phosphotyrosine (PY) was used to examine the localization of PY-containing proteins in E field-treated muscle cells. We found that anti-PY staining was colocalized with AChR clusters along the cathodal edge of the cells. In fact, cathodal PY staining could be detected before the first appearance of AChR clusters. When cultures were subjected to E fields in the presence of a tyrosine kinase inhibitor, tyrphostin RG-50864, cathodal AChR clustering was abolished with a half maximal inhibitory dosage of 50 microM. An inactive form of tyrphostin (RG-50862) had no effect on the field-induced clustering. These data suggest that the activation of tyrosine kinases is an essential step in E field-induced AChR clustering. Thus, the actions of several disparate stimuli for AChR clustering seem to converge to a common signal transduction mechanism based on tyrosine phosphorylation at the molecular level.

Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells.

Aggregation of the nicotinic acetylcholine receptor (AChR) at sites of nerve-muscle contact is one of the earliest events to occur during the development of the neuromuscular junction. The stimulus presented to the muscle by nerve and the mechanisms underlying postsynaptic differentiation are not known. The purpose of this study was to examine the distribution of phosphotyrosine (PY)-containing proteins in cultured Xenopus muscle cells in response to AChR clustering stimuli. Results demonstrated a distinct accumulation of PY at AChR clusters induced by several stimuli, including nerve, the culture substratum, and polystyrene microbeads. AChR microclusters formed by external cross-linking did not show PY colocalization, implying that the accumulation of PY in response to clustering stimuli was not due to the aggregation of basally phosphorylated AChRs. A semi-quantitative determination of the time course for development of PY labeling at bead contacts revealed early PY accumulation within 15 min of contact before significant AChR aggregation. At later stages (within 15 h), the AChR signal came to approximate the PY signal. We have reported the inhibition of bead-induced AChR clustering in response to beads by a tyrphostin tyrosine kinase inhibitor (RG50864) (Peng, H. B., L. P. Baker, and Q. Chen. 1991. Neuron. 6:237-246). RG50864 also inhibited PY accumulation at bead contacts, providing evidence for tyrosine kinase activation in response to the bead stimulus. These results suggest that tyrosine phosphorylation may play an important role in the generative stages of cluster formation, and may involve protein(s) other than or in addition to AChRs.

Inhibition of nerve- and agrin-induced acetylcholine receptor clustering on Xenopus muscle cells in culture

During an early stage of neuromuscular junction formation the nerve induces acetylcholine (ACh) receptors to accumulate at the contact area. To elucidate the induction process we tested various glycosaminoglycans for their ability to inhibit nerve-induced receptor accumulation. The potency sequence was found as follows: fucyoidin > dextran sulfate > heparin = heparan sulfate > chondroitin sulfate type A and B. This sequence is similar to that for agrin-induced receptor clustering in chick myotubes [ J. Neurosci., 10 (1990) 3576–3582], suggesting that agrin-like molecules are involved in nerve-induced receptor accumulation in the Xenopus system. We further tested whether agrin in the culture medium competes with the endogenous inducing substance and found that agrin partially inhibited nerve-induced receptor accumulation. We compared nerve- and agrin-induced receptor accumulation under various experimental conditions. Generally, they behaved similarly except in the presence of heparin. Heparin in the culture medium partially blocked nerve-induced receptor accumulation, whereas it totally inhibited agrin-induced receptor clustering. Our observations are consistent with the hypothesis that an agrin-like molecule released by the nerve is the induction signal for receptor accumulation in the Xenopus system.

A role of tyrosine phosphorylation in the formation of acetylcholine receptor clusters induced by electric fields in cultured Xenopus muscle cells

A role of tyrosine phosphorylation in the formation of acetylcholine receptor clusters induced by electric fields in cultured Xenopus muscle cells

Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells

Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells

A K+ current in Xenopus muscle cells which shows inactivation

The purpose of this study was to describe an inactivating potassium current that is present in Xenopus muscle cells in culture. Whole cell currents were recorded from muscle cells grown in culture for 16-30 h. The majority of muscle cells had an outward current which inactivated during a 100 ms maintained depolarization. The outward current was reduced by tetraethylammonium or the aminopyridines, but not completely eliminated by either agent. Depolarizing prepulses reduced both the peak current and the late component of the current. The results suggest that by one day in culture these skeletal muscle cells develop an inactivating outward potassium current.

Induction of dystrophin localization in cultured Xenopus muscle cells by latex beads

Induction of dystrophin localization in cultured Xenopus muscle cells by latex beads