Cell culture-based analysis of postsynaptic membrane assembly in muscle cells

We report a method for studying postsynaptic membrane assembly utilizing the replating of aneural cultures of differentiated skeletal muscle cells onto laminin-coated surfaces. A significant limitation to the current cell culturebased approaches has been their inability to recapitulate the multistage surface acetylcholine receptor (AChR) redistribution events that produce complex AChR clusters found at the intact neuromuscular junction (NMJ). By taking advantage of the ability of substrate laminin to induce advanced maturation of AChR aggregates on the surface of myotubes, we have developed a secondary-plating method that allows more precise analysis of the signaling events connecting substrate laminin stimulation to complex AChR cluster formation. We validate the utility of this method for biochemical and microscopy studies by demonstrating the roles of RhoGTPases in substrate laminin-induced complex cluster assembly

Mid-Left Ventricular Ballooning Variant Takotsubo Syndrome Induced by Treadmill Exercise Stress Testing

Stress-induced cardiomyopathy, also known as takotsubo cardiomyopathy, presents similar to a myocardial infarction after a physical or emotional stressor but without any evidence of obstructive coronary artery disease. Different patterns of myocardial involvement and several triggering events have been reported, but classically this condition is characterized by a stress-induced transient left ventricular apical systolic dysfunction. We describe a case of treadmill exercise stress testing-triggered variant of takotsubo cardiomyopathy with mid-left ventricular hypokinesis

Agrin and laminin induce acetylcholine receptor clustering by convergent, Rho GTPase-dependent signaling pathways.

During neuromuscular junction formation, extracellular matrix-mediated signals cause muscle surface acetylcholine receptors (AChRs) to aggregate at synaptic sites. Two extracellular matrix proteins, agrin and laminin, have each been shown to initiate signaling pathways that culminate in AChR clustering in cultured muscle cells. Here we present evidence that laminin-induced AChR clustering is mediated by the activation of the Rho GTPases Cdc42, Rac and Rho. Clustering in response to laminin is blocked by the dominant negative mutants Cdc42N17, RacN17 and RhoN19, as well as by the Rho inhibitor C3 transferase. Moreover, laminin-induced AChR clustering is impaired by the Rho kinase inhibitor Y-27632. Agrin-induced AChR clustering has previously been shown to require activation of Cdc42, Rac and Rho. Therefore, although agrin and laminin use distinct transmembrane receptors to initiate AChR clustering, their signaling pathways converge at the level of Rho GTPase activation

Cooperative regulation by Rac and Rho of agrin-induced acetylcholine receptor clustering in muscle cells.

A key aspect of neuromuscular synapse formation is the clustering of muscle acetylcholine receptors (AChR) at synaptic sites in response to neurally secreted agrin. Agrin-induced AChR clustering in cultured myotubes proceeds via the initial formation of small microclusters, which then aggregate to form AChR clusters. Here we show that the coupling of agrin signaling to AChR clustering is dependent on the coordinated activities of Rac and Rho GTPases. The addition of agrin induces the sequential activation of Rac and Rho in C2 muscle cells. The activation of Rac is rapid and transient and constitutes a prerequisite for the subsequent activation of Rho. This temporal pattern of agrin-induced Rac and Rho activation reflects their respective roles in AChR cluster formation. Whereas agrin-induced activation of Rac is necessary for the initial phase of AChR cluster formation, which involves the aggregation of diffuse AChR into microclusters, Rho activation is crucial for the subsequent condensation of these microclusters into full-size AChR clusters. Co-expression of constitutively active forms of Rac and Rho is sufficient to induce the formation of mature AChR clusters in the absence of agrin. These results establish that Rac and Rho play distinct but complementary roles in the mechanism of agrin-induced AChR clustering