Sutherland K. Maciver

The ADF/cofilin family: actin-remodeling proteins

SummaryThe ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined. Members of this family remodel the actin cytoskeleton, for example during cytokinesis, when the actin-rich contractile ring shrinks as it contracts through the interaction of ADF/cofilins with both monomeric and filamentous actin. The depolymerizing activity is twofold: ADF/cofilins sever actin filaments and also increase the rate at which monomers leave the filaments pointed end. The three-dimensional structure of ADF/cofilins is similar to a fold in members of the gelsolin family of actin-binding proteins in which this fold is typically repeated three or six times; although both families bind polyphosphoinositide lipids and actin in a pH-dependent manner, they share no obvious sequence similarity. Plants and animals have multiple ADF/cofilin genes, belonging in vertebrates to two types, ADF and cofilins. Other eukaryotes (such as yeast, Acanthamoeba and slime moulds) have a single ADF/cofilin gene. Phylogenetic analysis of the ADF/cofilins reveals that, with few exceptions, their relationships reflect conventional views of the relationships between the major groups of organisms.

Nemaline myopathy with minicores caused by mutation of the CFL2 gene encoding the skeletal muscle actin-binding protein, cofilin-2.

Nemaline myopathy (NM) is a congenital myopathy characterized by muscle weakness and nemaline bodies in affected myofibers. Five NM genes, all encoding components of the sarcomeric thin filament, are known. We report identification of a sixth gene, CFL2, encoding the actin-binding protein muscle cofilin-2, which is mutated in two siblings with congenital myopathy. The probands muscle contained characteristic nemaline bodies, as well as occasional fibers with minicores, concentric laminated bodies, and areas of F-actin accumulation. Her affected sisters muscle was reported to exhibit nonspecific myopathic changes. Cofilin-2 levels were significantly lower in the probands muscle, and the mutant protein was less soluble when expressed in Escherichia coli, suggesting that deficiency of cofilin-2 may result in reduced depolymerization of actin filaments, causing their accumulation in nemaline bodies, minicores, and, possibly, concentric laminated bodies.

F-actin capping proteins

Recent research on F-actin capping proteins has concentrated on three main areas. The discovery that controlled actin polymerization is the driving force for intracellular movement suggests an important role for capping proteins in regulating filament number and length. A capping protein from Dictyostelium (related to heat-shock protein HSP70) has been characterized that is activated by external stimuli. This provides a pivotal connection between extracellular signalling, cytoskeletal reorganization and locomotory behaviour. The roles of individual actin-binding sites in the gelsolin family of severing/capping proteins and binding sites for phosphatidylinositol 4,5-bisphosphate have been identified.

Actophorin preferentially binds monomeric ADP-Actin over ATP-bound actin: consequences for cell locomotion

Actophorin from Acanthamoeba castellanii severs actin filaments and sequesters actin monomers. Here we report that actophorin binds ADP‐bound monomers with higher affinity than ATP‐bound monomers. Actophorin is therefore much less efficient at severing actin filaments in the presence of ADP compared to ATP, particularly taking account of the higher critical concentration in ADP. Monomer binding is also reduced in the presence of 25 mM inorganic phosphate (which is assumed to form ADP · Pi‐actin). These findings are discussed in the light of observations on the nucleotide specificity of other monomer binding proteins and related to the role of actin in lamellar protrusion and cell locomotion.

Two actin binding proteins, actin depolymerizing factor and cofilin, are associated with Hirano bodies.

Hirano bodies are intracellular, paracrystalline, rod-like structures which contain actin, tropomyosin, vinculin, alpha-actinin, amyloid beta-protein precursor and several microtubule associated proteins (MAPs). These bodies are observed more frequently in the elderly and in a number of neurodegenerative diseases including Alzheimers disease. Many of the proteins known to be associated with Hirano bodies are actin binding proteins. We present immunohistological evidence that actin depolymerizing factor (ADF) and cofilin, two closely related proteins that bind and sever actin filaments, are also components of Hirano bodies. However, we could detect no difference in the levels of expression of either ADF or cofilin in the hippocampal tissue from normal individuals and Alzheimers disease patients.

Gelsolin Induces Colorectal Tumor Cell Invasion via Modulation of the Urokinase-Type Plasminogen Activator Cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin’s influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites.

Isolation of Balamuthia mandrillaris from urban dust, free of known infectious involvement

The free-living amoeba Balamuthia mandrillaris can cause fatal encephalitis in humans and other mammals. The organism is associated with soils, and soil exposure has been identified as a risk factor for this pathogen. However, B. mandrillaris has been isolated only once from soils believed to be the source of the infection in child from California, USA who died of Balamuthia amoebic encephalitis and once from another unrelated soil source. We report for a third time the isolation of B. mandrillaris from the environment and for the second time its isolation from a sample not known to be involved with pathogenicity. We have established the new clonal B. mandrillaris strain (ID-19) in axenic media. The identity of our isolate was originally by morphology using a light microscope and this has been confirmed by 16S rRNA gene PCR. The new strain ID-19 groups with others of the species. The fact that our isolate came from dust particles deposited on surfaces from the air in an urban environment may suggest that it is not just soil exposure that constitutes a risk factor for Balamuthia infection. This is the first report of this organism from Iran.

Arabidopsis CAP1 – a key regulator of actin organisation and development

Maintenance of F-actin turnover is essential for plant cell morphogenesis. Actin-binding protein mutants reveal that plants place emphasis on particular aspects of actin biochemistry distinct from animals and fungi. Here we show that mutants in CAP1, an A. thaliana member of the cyclase-associated protein family, display a phenotype that establishes CAP1 as a fundamental facilitator of actin dynamics over a wide range of plant tissues. Plants homozygous for cap1 alleles show a reduction in stature and morphogenetic disruption of multiple cell types. Pollen grains exhibit reduced germination efficiency, and cap1 pollen tubes and root hairs grow at a decreased rate and to a reduced length. Live cell imaging of growing root hairs reveals actin filament disruption and cytoplasmic disorganisation in the tip growth zone. Mutant cap1 alleles also show synthetic phenotypes when combined with mutants of the Arp2/3 complex pathway, which further suggests a contribution of CAP1 to in planta actin dynamics. In yeast, CAP interacts with adenylate cyclase in a Ras signalling cascade; but plants do not have Ras. Surprisingly, cap1 plants show disruption in plant signalling pathways required for co-ordinated organ expansion suggesting that plant CAP has evolved to attain plant-specific signalling functions.

A systematic analysis of Acanthamoeba genotype frequency correlated with source and pathogenicity: T4 is confirmed as a pathogen-rich genotype

Acanthamoeba is a genus of facultative human parasites that is currently classified into 17 genotypes (T1-T17) each of which arguably represents a species. These amoebae cause Acanthamoeba Keratitis (AK) a disease of the eye, and a rare but usually fatal Granulatomous Acanthamoeba Encephalitis (GAE). A database of strains derived from the literature and a number of fresh isolates has been constructed to detect trends of pathogenic and other associations with these genotypes. One genotype in particular, T4, was found to be over represented in human disease. The prevalence of this genotype has been commented upon previously, however T4 is also the most common type isolated from the environment. Our statistical analysis of the database allows us to claim that T4 is in fact the genotype most often associated with human disease, even after its abundance in the general environment is taken into account. T3 and T11 are closest relatives to T4 and they are the second and third most often associated with AK. A number of other more subtle correlations also emerge from this analysis.

The identification of a second cofilin binding site on actin suggests a novel, intercalated arrangement of F-actin binding.

The cofilins are members of a protein family that binds monomeric and filamentous actin, severs actin filaments, and increases monomer off-rate from the pointed end. Here, we characterize the cofilin-actin interface. We confirm earlier work suggesting the importance of the lower region of subdomain 1 encompassing the N and C termini (site 1) in cofilin binding. In addition, we report the discovery of a new cofilin binding site (site 2) from residues 112–125 that form a helix toward the upper, rear surface of subdomain 1 in the standard actin orientation (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Nature 347, 37–44). We propose that cofilin binds “behind” one monomer and “in front” of the other longitudinally associated monomer, accounting for the fact that cofilin alters the twist in the actin (McGough, A., Pope, B., Chiu, W., and Weeds, A. (1997) J. Cell Biol. 138, 771–781). The characterization of the cofilin-actin interface will facilitate an understanding of how cofilin severs and depolymerizes filaments and may shed light on the mechanism of the gelsolin family because they share a similar fold with the cofilins (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagiki, F. (1996) Cell 85, 1047–1055).