Ubiquitous among eukaryotes, the ADF/cofilins are essential proteins responsible for the high turnover rates of actin filaments in vivo. In vertebrates, ADF and cofilin are products of different genes. Both bind to F-actin cooperatively and induce a twist in the actin filament that results in the loss of the phalloidin-binding site. This conformational change may be responsible for the enhancement of the off rate of subunits at the minus end of ADF/cofilin-decorated filaments and for the weak filament-severing activity. Binding of ADF/cofilin is competitive with tropomyosin. Other regulatory mechanisms in animal cells include binding of phosphoinositides, phosphorylation by LIM kinases on a single serine, and changes in pH. Although vertebrate ADF/cofilins contain a nuclear localization sequence, they are usually concentrated in regions containing dynamic actin pools, such as the leading edge of migrating cells and neuronal growth cones. ADF/cofilins are essential for cytokinesis, phagocytosis, fluid phase endocytosis, and other cellular processes dependent upon actin dynamics.

Proteins of the ADF/Cofilin Family: Essential Regulators of Actin Dynamics

https://ressources.unisciel.fr/biocell/chap4/res/04_04_cell_migration_review_rottner.pdf

The lamellipodium: where motility begins

Actin polymerization often occurs at the plasma membrane to drive the protrusion of lamellipodia and filopodia at the leading edge of migrating cells. A role for actin polymerization in another cellular process that involves the reshaping of the plasma membrane--namely endocytosis--has recently been established. Live-cell imaging studies are shedding light on the order and timing of the molecular events and mechanisms of actin function during endocytosis.

Harnessing actin dynamics for clathrin-mediated endocytosis

A pathway for association of receptors, adaptors, and actin during endocytic internalization.

There has recently been considerable progress in understanding the regulation of clathrin-coated vesicle (CCV) formation and function. These advances are due to the determination of the structure of a number of CCV coat components at molecular resolution and the identification of novel regulatory proteins that control CCV formation in the cell. In addition, pathways of (a) phosphorylation, (b) receptor signaling, and (c) lipid modification that influence CCV formation, as well as the interaction between the cytoskeleton and CCV transport pathways are becoming better defined. It is evident that although clathrin coat assembly drives CCV formation, this fundamental reaction is modified by different regulatory proteins, depending on where CCVs are forming in the cell. This regulatory difference likely reflects the distinct biological roles of CCVs at the plasma membrane and trans-Golgi network, as well as the distinct properties of these membranes themselves. Tissue-specific functions of CCVs require even more-specialized regulation and defects in these pathways can now be correlated with human diseases.

Biological basket weaving: formation and function of clathrin-coated vesicles.

Genome sequencing projects have dramatically increased the amount of sequence data available in public databases, providing an opportunity to identify new protein families. The definition of new protein families allows knowledge from studies of one or a few members of a protein family to be applied to many other proteins in the family. The analysis presented here defines an actin-binding module, the actin-depolymerizing factor homology (ADF-H) domain, which is present in each member of a newly identified, extensive protein family. This family consists of three phylogenetically distinct classes: the ADF/cofilins, the twinfilins, and the drebrin/Abp1s. Each class has been used by eukaryotic organisms for more than 1 billion years, since before the divergence of fungi and animals. The ADF-H domain can be considered a functional building block that is arranged differently in each of the three protein classes in the family. In addition to being evolutionarily distinguishable, proteins in each class seem to possess distinct biochemical properties. Thus, ancient duplication of the ADF-H domain allowed the development of functional diversity.

The ADF Homology (ADF-H) Domain: A Highly Exploited Actin-binding Module

The actin cytoskeleton has been implicated in endocytosis, yet few molecules that link these systems have been identified. Here, we have cloned and characterized mHip1R, a protein that is closely related to huntingtin interacting protein 1 (Hip1). These two proteins are mammalian homologues of Sla2p, an actin binding protein important for actin organization and endocytosis in yeast. Sequence alignments and secondary structure predictions verified that mHip1R belongs to the Sla2 protein family. Thus, mHip1R contains an NH2-terminal domain homologous to that implicated in Sla2p's endocytic function, three predicted coiled–coils, a leucine zipper, and a talin-like actin-binding domain at the COOH terminus. The talin-like domain of mHip1R binds to F-actin in vitro and colocalizes with F-actin in vivo, indicating that this activity has been conserved from yeast to mammals. mHip1R shows a punctate immunolocalization and is enriched at the cell cortex and in the perinuclear region. We concluded that the cortical localization represents endocytic compartments, because mHip1R colocalizes with clathrin, AP-2, and endocytosed transferrin, and because mHip1R fractionates biochemically with clathrin-coated vesicles. Time-lapse video microscopy of mHip1R–green fluorescence protein (GFP) revealed a blinking behavior similar to that reported for GFP-clathrin, and an actin-dependent inward movement of punctate structures from the cell periphery. These data show that mHip1R is a component of clathrin-coated pits and vesicles and suggest that it might link the endocytic machinery to the actin cytoskeleton.

/pdf/an-actin-binding-protein-of-the-sla2-huntingtin-interacting-3n5i5k5pdk.pdf

An Actin-Binding Protein of the Sla2/Huntingtin Interacting Protein 1 Family Is a Novel Component of Clathrin-Coated Pits and Vesicles

Yeast Abp1p is a cortical actin cytoskeleton protein implicated in cytoskeletal regulation, endocytosis, and cAMP-signaling. We have identified a gene encoding a mouse homologue of Abp1p, and it is identical to SH3P7, a protein shown recently to be a target of Src tyrosine kinases. Yeast and mouse Abp1p display the same domain structure including an N-terminal actin-depolymerizing factor homology domain and a C-terminal Src homology 3 domain. Using two independent actin-binding domains, mAbp1 binds to actin filaments with a 1:5 saturation stoichiometry. In stationary cells, mAbp1 colocalizes with cortical F-actin in fibroblast protrusions that represent sites of cellular growth. mAbp1 appears at the actin-rich leading edge of migrating cells. Growth factors cause mAbp1 to rapidly accumulate in lamellipodia. This response can be mimicked by expression of dominant-positive Rac1. mAbp1 recruitment appears to be dependent on de novo actin polymerization and occurs specifically at sites enriched for the Arp2/3 complex. mAbp1 is a newly identified cytoskeletal protein in mice and may serve as a signal-responsive link between the dynamic cortical actin cytoskeleton and regions of membrane dynamics.

/pdf/association-of-mouse-actin-binding-protein-1-mabp1-sh3p7-an-4g74kg2mo8.pdf

Association of mouse actin-binding protein 1 (mAbp1/SH3P7), an Src kinase target, with dynamic regions of the cortical actin cytoskeleton in response to Rac1 activation.

A modified molecular-replacement method is described that makes use of six-dimensional searches and the phased translation function, providing a systematic examination of all possible search-model orientations in an experimental electron-density map. As an example, the structure solution of the cofilin-homology domain of the Saccharomyces cerevisiae actin-binding protein 1 (ABP1) is presented in detail. Additional examples are presented in which these tools have significantly aided structure solutions in a variety of contexts. These results suggest that this approach might be of widespread utility for challenging structures involving weak phase information, complex asymmetric units and search models with weak structural homology. Furthermore, this approach supports an exhaustive molecular-replacement strategy in cases where an appropriate search model cannot readily be identified on the basis of sequence homology. The fully automated web-based implementation of this phased translation function is described.

Phased translation function revisited: structure solution of the cofilin-homology domain from yeast actin-binding protein 1 using six-dimensional searches.

Michael M. Kessels

Papers

The ADF Homology (ADF-H) Domain: A Highly Exploited Actin-binding Module

An Actin-Binding Protein of the Sla2/Huntingtin Interacting Protein 1 Family Is a Novel Component of Clathrin-Coated Pits and Vesicles

Association of mouse actin-binding protein 1 (mAbp1/SH3P7), an Src kinase target, with dynamic regions of the cortical actin cytoskeleton in response to Rac1 activation.

Phased translation function revisited: structure solution of the cofilin-homology domain from yeast actin-binding protein 1 using six-dimensional searches.