Kathryn R. Ayscough

Latrunculin alters the actin-monomer subunit interface to prevent polymerization

atrunculin-A is a drug that is capable of rapidly, reversibly and specifically disrupting the actin cytoskeleton. The efficacy of its action has made it a compound of choice in many cell-biology laboratories, supplanting the classic actin-depolymerizing drug cytochalasin-D. One reason for this is that the mode of action of latrunculin seems to be less complex than that of cytochalasin. Whereas the latter affects the kinetics of actin-filament polymerization at both the barbed and pointed ends, latrunculin-A seems to associate only with actin monomers, thereby preventing them from repolymerizing into filaments. The association of latrunculin with monomeric, rather than filamentous, actin gave us the opportunity to further our understanding of this interaction by detailed structural analysis of actin monomers using crystallographic techniques. Here we show the first high-resolution structure of an actin-disrupting drug in association with actin and discuss how its interactions with actin, and the conformational changes that its binding causes, may explain its mode of action within the cell. Latrunculin (Fig. 1a) is purified from Latrunculia magnificans, a Red Sea sponge that exudes a noxious, red fluid that kills fish within minutes. Two related compounds, latrunculin-A and latrunculinB, isolated from the fluid were shown to depolymerize actin structures both in vitro and in vivo. The in vitro studies showed that latrunculin binds only to the actin monomer and that the kinetics of this interaction are consistent with the complex being unable to polymerize. Unlike cytochalasin, latrunculin can disrupt the actin cytoskeleton in yeast cells. This has enabled genetic studies to be carried out that have facilitated the identification of point mutations in the actin gene that cause cells to become resistant to the effects of the drug (Fig. 1b). The mutations that give rise to latrunculin resistance were found to be clustered around a distinct site, close to the nucleotide-binding site, which indicated that they might identify a potential binding site for latrunculin. However, as this site is not close to recognized subunit contacts in the filament, or to known binding sites for other proteins that associate with actin, the mechanism by which latrunculin exerts its effects has remained unclear. Actin has never been known to crystallize in the absence of a binding protein that keeps it in a monodispersed state. Of the three known examples of such binding proteins, profilin is inappropriate as it promotes nucleotide exchange, whereas deoxyribonuclease1 binds to domains that have been implicated, in studies of yeast genetics, in latrunculin binding. In contrast, gelsolin domain 1 in complex with actin leaves these domains free and also reduces nucleotide exchange, as does latrunculin. We therefore soaked latrunculin-A L

The actin cytoskeleton: a key regulator of apoptosis and ageing?

Evidence from many organisms has shown that the accumulation of reactive oxygen species (ROS) has a detrimental effect on cell well-being. High levels of ROS have been linked to programmed cell death pathways and to ageing. Recent reports have implicated changes to the dynamics of the actin cytoskeleton in the release of ROS from mitochondria and subsequent cell death.

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.

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,

In vivo functions of actin-binding proteins.

Actin and actin-binding proteins have been identified in eukaryotic organisms across the evolutionary spectrum. Although many actin-binding proteins have been purified and studied in vitro, our understanding of the in vivo functions of these proteins has, until recently, lagged behind. In the past year, in vivo studies, especially those using genetic approaches, have led to significant advances in our understanding of how actin-binding proteins function in a cellular environment.

Actin regulation in endocytosis

Increasing evidence from a variety of cell types has highlighted the importance of the actin cytoskeleton during endocytosis. No longer is actin viewed as a passive barrier that must be removed to allow endocytosis to proceed. Rather, actin structures are dynamically organised to assist the remodelling of the cell surface to allow inward movement of vesicles. The majority of our mechanistic insight into the role of actin in endocytosis has come from studies in budding yeast. Although endocytosis in mammalian cells is clearly more complex and subject to a greater array of regulatory signals, recent advances have revealed actin, and actin-regulatory proteins, to be present at endocytic sites. Furthermore, live cell imaging indicates that spatiotemporal aspects of actin recruitment and vesicle formation are likely to be conserved across eukaryotic evolution.

Differential requirements for actin during yeast and mammalian endocytosis

Key features of clathrin-mediated endocytosis have been conserved across evolution. However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis. We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure. By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis. Conversely, increased turgor pressure in specific yeast mutants correlated with a decreased rate of endocytic patch invagination.

Actin-induced hyperactivation of the Ras signaling pathway leads to apoptosis in Saccharomyces cerevisiae

ABSTRACT Recent research has revealed a conserved role for the actin cytoskeleton in the regulation of aging and apoptosis among eukaryotes. Here we show that the stabilization of the actin cytoskeleton caused by deletion of Sla1p or End3p leads to hyperactivation of the Ras signaling pathway. The consequent rise in cyclic AMP (cAMP) levels leads to the loss of mitochondrial membrane potential, accumulation of reactive oxygen species (ROS), and cell death. We have established a mechanistic link between Ras signaling and actin by demonstrating that ROS production in actin-stabilized cells is dependent on the G-actin binding region of the cyclase-associated protein Srv2p/CAP. Furthermore, the artificial elevation of cAMP directly mimics the apoptotic phenotypes displayed by actin-stabilized cells. The effect of cAMP elevation in inducing actin-mediated apoptosis functions primarily through the Tpk3p subunit of protein kinase A. This pathway represents the first defined link between environmental sensing, actin remodeling, and apoptosis in Saccharomyces cerevisiae.

Identification of an upstream regulatory pathway controlling actin-mediated apoptosis in yeast

The build up of reactive oxygen species (ROS) is known to contribute to a reduction in the lifespan of a cell and to their degeneration in diseases such as Alzheimers and tissue ischaemia. It is therefore important to elucidate pathways that regulate cellular oxidative stress. We have previously shown that actin dynamics can affect the oxidative-stress burden on a yeast cell and thereby its potential lifespan. To elucidate further the connection between actin dynamics and oxidative stress, we sought to identify regulators of this process. The actin regulatory proteins Sla1p and End3p are important in maintaining a rapid turnover of F-actin in cortical patches. We show that cells expressing a mutated form of Sla1p or lacking End3p display markers of apoptosis such as depolarized mitochondrial membranes and elevated levels of reactive oxygen species. Overexpression of the ubiquitin ligase RSP5 can alleviate the oxidative-stress phenotype observed in cells lacking End3p by targeting Sla1p to the cortex and restoring actin remodelling capability. We also demonstrate that overexpression of PDE2, a negative regulator of the Ras/cAMP pathway rescues actin dynamics, reduces oxidative stress sensitivity and restores viability in Δend3 cells. Our data suggest, for the first time, that a physiological link exists between actin regulation and cAMP signalling that regulates apoptosis in yeast.

Functions of actin in endocytosis.

Endocytosis is a fundamental eukaryotic process required for remodelling plasma-membrane lipids and protein to ensure appropriate membrane composition. Increasing evidence from a number of cell types reveals that actin plays an active, and often essential, role at key endocytic stages. Much of our current mechanistic understanding of the endocytic process has come from studies in budding yeast and has been facilitated by yeast’s genetic amenability and by technological advances in live cell imaging. While endocytosis in metazoans is likely to be subject to a greater array of regulatory signals, recent reports indicate that spatiotemporal aspects of vesicle formation requiring actin are likely to be conserved across eukaryotic evolution. In this review we focus on the ‘modular’ model of endocytosis in yeast before highlighting comparisons with other cell types. Our discussion is limited to endocytosis involving clathrin as other types of endocytosis have not been demonstrated in yeast.