Steven J. Winder

Functional plasticity of CH domains

With the refinement of algorithms for the identification of distinct motifs from sequence databases, especially those using secondary structure predictions, new protein modules have been determined in recent years. Calponin homology (CH) domains were identified in a variety of proteins ranging from actin cross‐linking to signaling and have been proposed to function either as autonomous actin binding motifs or serve a regulatory function. Despite the overall structural conservation of the unique CH domain fold, the individual modules display a quite striking functional variability. Analysis of the actopaxin/parvin protein family suggests the existence of novel (type 4 and type 5) CH domain families which require special attention, as they appear to be a good example for how CH domains may function as scaffolds for other functional motifs of different properties.

A role for the actin cytoskeleton in cell death and aging in yeast

Several determinants of aging, including metabolic capacity and genetic stability, are recognized in both yeast and humans. However, many aspects of the pathways leading to cell death remain to be elucidated. Here we report a role for the actin cytoskeleton both in cell death and in promoting longevity. We have analyzed yeast strains expressing mutants with either increased or decreased actin dynamics. We show that decreased actin dynamics causes depolarization of the mitochondrial membrane and an increase in reactive oxygen species (ROS) production, resulting in cell death. Important, however, is the demonstration that increasing actin dynamics, either by a specific actin allele or by deletion of a gene encoding the actin-bundling protein Scp1p, can increase lifespan by over 65%. Increased longevity appears to be due to these cells producing lower than wild-type levels of ROS. Homology between Scp1p and mammalian SM22/transgelin, which itself has been isolated in senescence screens, suggests a conserved mechanism linking aging to actin stability.

The complexities of dystroglycan.

The notion of dystroglycan as a simple laminin-binding receptor is increasingly being challenged. New roles and new binding partners are continually emerging. Recent structural advances have provided exciting new insights into the precise molecular interactions between dystroglycan and other key components of the dystroglycan complex. Coupled with an increasing understanding of dystroglycan function at the molecular level, we are finally beginning to probe the complexities of dystroglycan, not only in disease, but in development, adhesion and signalling.

Actin-binding proteins

Actin is an essential component of the cytoskeleton and plays a crucial role in eukaryotic cells. The actin cytoskeleton functions in the generation and maintenance of cell morphology and polarity, in endocytosis and intracellular trafficking, in contractility, motility and cell division. In cells,

The WW domain: Linking cell signalling to the membrane cytoskeleton

The WW domain is one of the smallest yet most versatile protein-protein interaction modules. The ability of this simple domain to interact with a number of proline-containing ligands has resulted in a great deal of functional diversity. Most recently it has been shown that WW domain interactions can also be differentially regulated by tyrosine phosphorylation. Here we briefly review WW domain ligands and structure in comparison to SH3 domain ligands and structure and discuss recent findings with regard to the regulation of WW domain interactions by phosphorylation. In particular we describe the potential for differential binding of the b-dystroglycan WW domain ligand by dystrophin or caveolin-3 in skeletal muscle and show how this could act as a switch to alter the relative affinity of the muscle dystroglycan complex for caveolin-3 or dystrophin and utrophin.

Dystroglycan, a scaffold for the ERK–MAP kinase cascade

Dystroglycan is an important cell adhesion receptor linking the actin cytoskeleton, via utrophin and dystrophin, to laminin in the extracellular matrix. To identify adhesion‐related signalling molecules associated with dystroglycan, we conducted a yeast two‐hybrid screen and identified mitogen‐activated protein (MAP) kinase kinase 2 (MEK2) as a β‐dystroglycan interactor. Pull‐down experiments and localization studies substantiated a physiological link between β‐dystroglycan and MEK and localized MEK with dystroglycan in membrane ruffles. Moreover, we also identified active extracellular signal‐regulated kinase (ERK), the downstream kinase from MEK, as another interacting partner for β‐dystroglycan and localized both active ERK and dystroglycan to focal adhesions in fibroblast cells. These studies suggest a role for dystroglycan as a multifunctional adaptor or scaffold capable of interacting with components of the ERK–MAP kinase cascade including MEK and ERK. These findings have important implications for our understanding of the role of dystroglycan in normal cellular processes and in disease states such as muscular dystrophy.

REVIEW: The membrane--cytoskeleton interface: the role of dystrophin and utrophin

Recent studies with transgenic animals have consid erably advanced our knowledge of the roles of dystrophin and utrophin in both muscle and non-muscle tissues. Rigorous analyses of the roles of the various mdxmutations in mice, as well as the use of artificial transgenes in an mdxbackground, are beginning to define the functional importance of various regions of the dystrophin protein in normal muscle. Furthermore, recent biochemical analyses have revealed new insights into the role and organization of dystrophin at the membrane--cytoskeleton interface. Transgenic approaches have also revealed surprising and encouraging results with respect to utrophin. Against expectations, the long-awaited utrophin knockout mice have a remarkably mild phenotype with only subtle changes in neuromuscular junction architecture. On the other hand, mdxmice transgenic for a mini-utrophin construct showed rescue of the muscular dystrophy phenotype, clearly an encouraging finding with obvious therapeutic possibilities. These and other recent findings are discussed in the context of the structure and function of dystrophin and utrophin at the membrane--cytoskeleton interface

Structural insights into actin-binding, branching and bundling proteins

Structural advances in our understanding of the functions of the actin cytoskeleton have come from diverse sources. On the one hand, the determination of the structure of a bacterial actin-like protein MreB reveals the prokaryotic origins of the actin cytoskeleton, whereas on the other, cryo-electron microscopy and crystallography have yielded reconstructions of many actin crosslinking, regulatory and binding proteins in complex with F-actin. Not least, a high-resolution structure of the Arp2/3 complex and a reconstruction with F-actin provides considerable insight into the eukaryotic machinery, vital for the formation of new F-actin barbed ends, a prerequisite for rapid actin polymerisation involved in cell shape change and motility.

Direct interaction of beta-dystroglycan with F-actin.

Dystroglycans are essential transmembrane adhesion receptors for laminin. Alpha-dystroglycan is a highly glycosylated extracellular protein that interacts with laminin in the extracellular matrix and the transmembrane region of beta-dystroglycan. Beta-dystroglycan, via its cytoplasmic tail, interacts with dystrophin and utrophin and also with the actin cytoskeleton. As a part of the dystrophin-glycoprotein complex of muscles, dystroglycan is also important in maintaining sarcolemmal integrity. Mutations in dystrophin that lead to Duchenne muscular dystrophy also lead to a loss of dystroglycan from the sarcolemma, and chimaeric mice lacking muscle dystroglycan exhibit a severe muscular dystrophy phenotype. Using yeast two-hybrid analysis and biochemical and cell biological studies, we show, in the present study, that the cytoplasmic tail of beta-dystroglycan interacts directly with F-actin and, furthermore, that it bundles actin filaments and induces an aberrant actin phenotype when overexpressed in cells.

SCP1 encodes an actin-bundling protein in yeast.

The association of F-actin (filamentous actin) with a large number of binding proteins is essential for cellular function. Actin-binding proteins control the dynamics of actin filaments, nucleate new filaments and facilitate formation of higher-order structures such as actin bundles. The yeast gene SCP1 encodes a small protein with significant homology to mammalian SM22/transgelin. We have investigated the role of Scp1p in budding yeast to probe the fundamental role of this family of proteins. Here, we demonstrate that Scp1p binds to F-actin and induces the formation of tight F-actin bundles in vitro. Deletion of SCP1 in yeast lacking the actin-bundling protein, fimbrin (Sac6p), exacerbates the disrupted actin phenotype and enhances latrunculin-A sensitivity. Furthermore, Scp1p co-localizes with actin in cortical patches and its localization is lost in the presence of latrunculin-A. Our data support a role for Scp1p in bundling actin filaments and, in concert with Sac6p, acting as a second actin-bundling activity crucial to the stability of the yeast actin cytoskeleton.