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Kathryn R Ayscough

Publications and source records attributed to Kathryn R Ayscough.

14 recordsLinked to original sources
PubMed2006

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.

Actins↗
PubMed2006

Apoptosis in yeast--mechanisms and benefits to a unicellular organism.

Initial observations that the budding yeast Saccharomyces cerevisiae can be induced to undergo a form of cell death exhibiting typical markers of apoptosis has led to the emergence of a thriving new field of research. Since this discovery, a number of conserved pro- and antiapoptotic proteins have been identified in yeast. Indeed, early experiments have successfully validated yeasts as a powerful genetic tool with which to investigate mechanisms of apoptosis. However, we still have little understanding as to why programmes of cell suicide exist in unicellular organisms and how they may be benefit such organisms. Recent research has begun to elucidate pathways that regulate yeast apoptosis in response to environmental stimuli. These reports strengthen the idea that physiologically relevant mechanisms of programmed cell death are present, and that these function as important regulators of yeast cell populations.

Apoptosis↗
PubMed2005

Defining protein modules for endocytosis.

Endocytosis is a complex process that controls the composition of the plasma membrane, nutrient uptake, and the regulation of cell signaling in eukaryotic cells. In this issue of Cell, Kaksonen et al. (2005) use real-time microscopy of yeast to reveal major insights, at the molecular level, into the spatial and temporal aspects of this critical process.

Actins↗
PubMed2005

The actin cytoskeleton in ageing and apoptosis.

Regulated cell death, or apoptosis, has evolved to fulfil a myriad of functions amongst multicellular organisms. It is now apparent that programmed cell death occurs in unicellular organisms such as yeast. In yeast, as in higher eukaryotes, the actin cytoskeleton is an essential component of a number of cellular activities, and many of the regulatory proteins involved are highly conserved. Recent evidence from diverse eukaryotic systems suggests that the actin cytoskeleton has a role in regulating apoptosis via interactions with the mitochondria. This interaction also appears to have a significant impact on the management of oxidative stress and so cellular ageing. In this mini-review we summarise some of the work, which suggests that actin is a key regulator of apoptosis and ageing in eukaryotic cells.

Actins↗
PubMed2005

Lsb5p interacts with actin regulators Sla1p and Las17p, ubiquitin and Arf3p to couple actin dynamics to membrane trafficking processes.

The importance of coupling the process of endocytosis to factors that regulate actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified. Sla1p is a well-characterized yeast protein that binds both to activators of actin dynamics, Las17p and Pan1p, and to cargo proteins, such as the pheromone receptor Ste2p. Previously, we reported that the Lsb5 protein plays a role in endocytosis in yeast and that it localizes to the plasma membrane. Lsb5p has a similar structure to the GGA [Golgi-localized, gamma-ear-containing, Arf (ADP-ribosylation factor)-binding] family of proteins with an N-terminal VHS [Vps27p (vacuolar protein sorting protein 27), Hrs, Stam] domain and a GAT (GGA and Tom1) domain. It does not, however, contain either a gamma-adaptin ear or a clathrin-binding motif. In the present study, we have further defined its interaction site with both Sla1p and with Las17p, two regulators of actin dynamics. The site of interaction with Sla1p involves the Sla1 HD1 (homology domain 1), which also was shown previously to interact with the pheromone receptor Ste2p. We also demonstrate hitherto unknown interactions between Lsb5p and the active form of the yeast Arf3 protein, and with ubiquitin. Finally, we demonstrate a requirement for Arf3p expression in order to localize Lsb5p to the correct cortical site in cells. Taken together, our data provide further evidence for the role of Lsb5p in membrane-trafficking events at the plasma membrane and also demonstrate for the first time an interaction of Arf3 with the endocytic machinery in yeast.

ADP-Ribosylation Factors↗
PubMed2005

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 Alzheimer's 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 deltaend3 cells. Our data suggest, for the first time, that a physiological link exists between actin regulation and cAMP signalling that regulates apoptosis in yeast.

Actins↗
PubMed2005

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.

Actins↗
PubMed2004

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.

Actins↗
PubMed2003

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.

Actins↗
PubMed2003

An interaction between Sla1p and Sla2p plays a role in regulating actin dynamics and endocytosis in budding yeast.

The importance of a dynamic actin cytoskeleton for facilitating endocytosis has been recognised for many years in budding yeast and is increasingly recognised in mammalian cells. However, the mechanism for actin recruitment and the role it plays in endocytosis is unclear. Here we show the importance of two yeast proteins in this process. We demonstrate that Sla1p and Sla2p interact in vitro and in vivo and that this interaction is mediated by the central domain of Sla2p, which includes its coiled-coil region, and by a domain of Sla1p between residues 118 and 361. Overexpression of the interacting fragment of Sla1p causes reduced fluid-phase endocytosis and, interestingly, defects in subsequent trafficking to vacuoles. We show that Sla2p is required for the polarised localisation of Sla1p in cells but not for its cortical localisation or for its overlapping localisation with actin. Generation of an Deltasla1Deltasla2 double mutant demonstrates that Sla2p is likely to act upstream of Sla1p in endocytosis, whereas sensitivity to latrunculin-A suggests that the proteins have opposite effects on actin dynamics. We propose that Sla2p recruits Sla1p to endocytic sites. Sla1p and its associated protein Pan1p then regulate actin assembly through interactions with Arp2/3 and Arp2/3-activating proteins Abp1p and Las17/Bee1p.

Actin Cytoskeleton↗
PubMed2003

The Ark1/Prk1 family of protein kinases. Regulators of endocytosis and the actin skeleton.

The Ark/Prk serine/threonine kinases initiate phosphorylation cycles that control the endocytic machinery in mammalian cells and in yeast, and the actin cytoskeleton in yeast. The members of this protein family are unified by homologies in their kinase domain, but are generally diverse in their other domains. The evolution of Ark/Prk family members in different organisms may have allowed the conserved role of the kinase domain, which is required for the phosphorylation of both endocytic and cytoskeletal components, to be coupled to other functional domains.

Actins↗
PubMed2002

Sla1p couples the yeast endocytic machinery to proteins regulating actin dynamics.

Sla1p is a protein required for cortical actin patch structure and organisation in budding yeast. Here we use a combination of immunofluorescence microscopy and biochemical approaches to demonstrate interactions of Sla1p both with proteins regulating actin dynamics and with proteins required for endocytosis. Using Sla1p-binding studies we reveal association of Sla1p with two proteins known to be important for activation of the Arp2/3 complex in yeast, Abp1p and the yeast WASP homologue Las17p/Bee1p. A recent report of Sla1p association with Pan1p puts Sla1p in the currently unique position of being the only yeast protein known to interact with all three known Arp2/3-activating proteins in yeast. Localisation of Sla1p at the cell cortex is, however, dependent on the EH-domain-containing protein End3p, which is part of the yeast endocytic machinery. Using spectral variants of GFP on Sla1p (YFP) and on Abp1p (CFP) we show for the first time that these proteins can exist in discrete complexes at the cell cortex. However, the detection of a significant FRET signal means that these proteins also come close together in a single complex, and it is in this larger complex that we propose that Sla1p binding to Abp1p and Las17p/Bee1p is able to link actin dynamics to the endocytic machinery. Finally, we demonstrate marked defects in both fluid-phase and receptor-mediated endocytosis in cells that do not express SLA1, indicating that Sla1p is central to the requirement in yeast to couple endocytosis with the actin cytoskeleton.

Actins↗