Lattices, rafts, and scaffolds: domain regulation of receptor signaling at the plasma membrane

The plasma membrane is organized into various subdomains of clustered macromolecules. Such domains include adhesive structures (cellular synapses, substrate adhesions, and cell–cell junctions) and membrane invaginations (clathrin-coated pits and caveolae), as well as less well-defined domains such as lipid rafts and lectin-glycoprotein lattices. Domains are organized by specialized scaffold proteins including the intramembranous caveolins, which stabilize lipid raft domains, and the galectins, a family of animal lectins that cross-link glycoproteins forming molecular lattices. We review evidence that these heterogeneous microdomains interact to regulate substratum adhesion and cytokine receptor dynamics at the cell surface

Proteomic Analysis of the Dysferlin Protein Complex Unveils Its Importance for Sarcolemmal Maintenance and Integrity

Dysferlin is critical for repair of muscle membranes after damage. Mutations in dysferlin lead to a progressive muscular dystrophy. Recent studies suggest additional roles for dysferlin. We set out to study dysferlin's protein-protein interactions to obtain comprehensive knowledge of dysferlin functionalities in a myogenic context. We developed a robust and reproducible method to isolate dysferlin protein complexes from cells and tissue. We analyzed the composition of these complexes in cultured myoblasts, myotubes and skeletal muscle tissue by mass spectrometry and subsequently inferred potential protein functions through bioinformatics analyses. Our data confirm previously reported interactions and support a function for dysferlin as a vesicle trafficking protein. In addition novel potential functionalities were uncovered, including phagocytosis and focal adhesion. Our data reveal that the dysferlin protein complex has a dynamic composition as a function of myogenic differentiation. We provide additional experimental evidence and show dysferlin localization to, and interaction with the focal adhesion protein vinculin at the sarcolemma. Finally, our studies reveal evidence for cross-talk between dysferlin and its protein family member myoferlin. Together our analyses show that dysferlin is not only a membrane repair protein but also important for muscle membrane maintenance and integrity

MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes

Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer–based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein

Caveolin regulates endocytosis of the muscle repair protein, dysferlin

Dysferlin and Caveolin-3 are plasma membrane proteins associated wtih muscular dystrophy. Patients with mutations in the CAV3 gene show dysferlin mislocalization in muscle cells

C9ORF72 interaction with cofilin modulates actin dynamics in motor neurons.

Intronic hexanucleotide expansions in C9ORF72 are common in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non-ATG-initiated translation are responsible for the pathophysiology. We determined the interactome of C9ORF72 in motor neurons and found that C9ORF72 was present in a complex with cofilin and other actin binding proteins. Phosphorylation of cofilin was enhanced in C9ORF72-depleted motor neurons, in patient-derived lymphoblastoid cells, induced pluripotent stem cell-derived motor neurons and post-mortem brain samples from ALS patients. C9ORF72 modulates the activity of the small GTPases Arf6 and Rac1, resulting in enhanced activity of LIM-kinases 1 and 2 (LIMK1/2). This results in reduced axonal actin dynamics in C9ORF72-depleted motor neurons. Dominant negative Arf6 rescues this defect, suggesting that C9ORF72 acts as a modulator of small GTPases in a pathway that regulates axonal actin dynamics

Defective Membrane Remodeling in Neuromuscular Diseases: Insights from Animal Models

Proteins involved in membrane remodeling play an essential role in a plethora of cell functions including endocytosis and intracellular transport. Defects in several of them lead to human diseases. Myotubularins, amphiphysins, and dynamins are all proteins implicated in membrane trafficking and/or remodeling. Mutations in myotubularin, amphiphysin 2 (BIN1), and dynamin 2 lead to different forms of centronuclear myopathy, while mutations in myotubularin-related proteins cause Charcot-Marie-Tooth neuropathies. In addition to centronuclear myopathy, dynamin 2 is also mutated in a dominant form of Charcot-Marie-Tooth neuropathy. While several proteins from these different families are implicated in similar diseases, mutations in close homologues or in the same protein in the case of dynamin 2 lead to diseases affecting different tissues. This suggests (1) a common molecular pathway underlying these different neuromuscular diseases, and (2) tissue-specific regulation of these proteins. This review discusses the pathophysiology of the related neuromuscular diseases on the basis of animal models developed for proteins of the myotubularin, amphiphysin, and dynamin families. A better understanding of the common mechanisms between these neuromuscular disorders will lead to more specific health care and therapeutic approaches

The ARF-GEF ARNO and ARF6 regulate dendritic and axonal development in cultured rat hippocampal neurons

The unique morphology of nerve cells is a key feature of the complex organization of the nervous system. Neurite extension and branching are dynamic processes that take place throughout the life of a neuron. The development of dendritic and axonal processes requires the coordination of membrane-cytoskeletal rearrangements to ensure the establishment of proper neural connections. These events are thought to be largely regulated by signaling pathways involving the Rho-family of small guanine triphosphatases (GTPases). Another family of small GTP-binding proteins, the ARF (ADP-ribosylation factor) family, has been implicated in the regulation of membrane transport and actin filament reorganization in non-neuronal cells. However, it is unknown what role ARF family members play in the modulation of these events during neurite extension and branching. Therefore, we have analyzed the role of the ARF-guanine exchange factor (GEF) ARNO (ARF-nucleotide binding site opener) in dendritic arbor development and axonal elongation and branching by overexpressing wild-type and mutant forms of ARNO and ARF6 in cultured rat hippocampal neurons. The overexpression of catalytically inactive ARNO and dominant-negative ARF6 result in increased dendritic branching as well as enhanced axonal extension and branching. Expression of inactive ARNO caused a redistribution of a subset of endosomes to the axonal plasma membrane and displacement of the profilin-binding protein Mena from the growth cone plasma membrane. These results suggest that ARNO through ARF6 negatively regulates dendritic branching and axonal elongation and branching during neural development