Campbell W. Gourlay

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.

A role for actin in regulating apoptosis/programmed cell death : evidence spanning yeast, plants and animals

Achieving an understanding of how apoptosis/PCD (programmed cell death) is integrated within cellular responses to environmental and intracellular signals is a daunting task. From the sensation of a stimulus to the point of no return, a programme of cell death must engage specific pro-death components, whose effects can in turn be enhanced or repressed by downstream regulatory factors. In recent years, considerable progress has been made in our understanding of how components involved in these processes function. We now know that some of the factors involved in PCD networks have ancient origins that pre-date multicellularity and, indeed, eukaryotes themselves. A subject attracting much attention is the role that the actin cytoskeleton, itself a cellular component with ancient origins, plays in cell death regulation. Actin, a key cellular component, has an established role as a cellular sensor, with reorganization and alterations in actin dynamics being a well known consequence of signalling. A range of studies have revealed that actin also plays a key role in apoptosis/PCD regulation. Evidence implicating actin as a regulator of eukaryotic cell death has emerged from studies from the Animal, Plant and Fungal Kingdoms. Here we review recent data that provide evidence for an active, functional role for actin in determining whether PCD is triggered and executed, and discuss these findings within the context of regulation of actin dynamics.

Fumarate Hydratase Deficiency in Renal Cancer Induces Glycolytic Addiction and Hypoxia-Inducible Transcription Factor 1α Stabilization by Glucose-Dependent Generation of Reactive Oxygen Species

ABSTRACT Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an inherited cancer syndrome linked to biallelic inactivation of the gene encoding the tricarboxylic acid cycle enzyme fumarate hydratase (FH). Individuals with HLRCC are at risk to develop cutaneous and uterine leiomyomas and an aggressive form of kidney cancer. Pseudohypoxic drive—the aberrant activation of cellular hypoxia response pathways despite normal oxygen tension—is considered to be a likely mechanism underlying the etiology of this tumor. Pseudohypoxia requires the oxygen-independent stabilization of the α subunit of the hypoxia-inducible transcription factor (HIF-1α). Under normoxic conditions, proline hydroxylation of HIF-1α permits VHL recognition and subsequent targeting for proteasomal degradation. Here, we demonstrate that inactivating mutations of FH in an HLRCC-derived cell line result in glucose-mediated generation of cellular reactive oxygen species (ROS) and ROS-dependent HIF-1α stabilization. Additionally, we demonstrate that stable knockdown of FH in immortalized renal epithelial cells results in ROS-dependent HIF-1α stabilization. These data reveal that the obligate glycolytic switch present in HLRCC is critical to HIF stabilization via ROS generation.

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.

Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, COCHLEATA, AFILA, and TENDRIL-LESS.

The compound leaf primordium of pea represents a marginal blastozone that initiates organ primordia, in an acropetal manner, from its growing distal region. The UNIFOLIATA (UNI) gene is important in marginal blastozone maintenance because loss or reduction of its function results in uni mutant leaves of reduced complexity. In this study, we show that UNI is expressed in the leaf blastozone over the period in which organ primordia are initiated and is downregulated at the time of leaf primordium determination. Prolonged UNI expression was associated with increased blastozone activity in the complex leaves of afila (af), cochleata (coch), and afila tendril-less (af tl) mutant plants. Our analysis suggests that UNI expression is negatively regulated by COCH in stipule primordia, by AF in proximal leaflet primordia, and by AF and TL in distal and terminal tendril primordia. We propose that the control of UNI expression by AF, TL, and COCH is important in the regulation of blastozone activity and pattern formation in the compound leaf primordium of the pea.

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.

Loss of Cytochrome c Oxidase Promotes RAS-Dependent ROS Production from the ER Resident NADPH Oxidase, Yno1p, in Yeast

Many disease states, including the aging process, are associated with the accumulation of mitochondria harboring respiratory dysfunction. Mitochondrial dysfunction is often accompanied by increased ROS levels that can contribute to cellular dysfunction and disease etiology. Here we use the model eukaryote S. cerevisiae to investigate whether reduced cytochrome c oxidase (COX) activity, commonly reported in aging organisms and associated with neurodegenerative disorders, leads to ROS production from mitochondria. We provide evidence that although reduced COX complex activity correlates with ROS accumulation, mitochondria are not the major production center. Instead we show that COX-deficient mitochondria activate Ras upon their outer membrane that establishes a pro-ROS accumulation environment by suppressing antioxidant defenses and the ERAD-mediated turnover of the ER-localized NADPH oxidase Yno1p. Our data suggest that dysfunctional mitochondria can serve as a signaling platform to promote the loss of redox homeostasis, ROS accumulation, and accelerate aging in yeast.

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 Δsla1Δsla2 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.

Yno1p/Aim14p, a NADPH-oxidase ortholog, controls extramitochondrial reactive oxygen species generation, apoptosis, and actin cable formation in yeast

The large protein superfamily of NADPH oxidases (NOX enzymes) is found in members of all eukaryotic kingdoms: animals, plants, fungi, and protists. The physiological functions of these NOX enzymes range from defense to specialized oxidative biosynthesis and to signaling. In filamentous fungi, NOX enzymes are involved in signaling cell differentiation, in particular in the formation of fruiting bodies. On the basis of bioinformatics analysis, until now it was believed that the genomes of unicellular fungi like Saccharomyces cerevisiae and Schizosaccharomyces pombe do not harbor genes coding for NOX enzymes. Nevertheless, the genome of S. cerevisiae contains nine ORFs showing sequence similarity to the catalytic subunits of mammalian NOX enzymes, only some of which have been functionally assigned as ferric reductases involved in iron ion transport. Here we show that one of the nine ORFs (YGL160W, AIM14) encodes a genuine NADPH oxidase, which is located in the endoplasmic reticulum (ER) and produces superoxide in a NADPH-dependent fashion. We renamed this ORF YNO1 (yeast NADPH oxidase 1). Overexpression of YNO1 causes YCA1-dependent apoptosis, whereas deletion of the gene makes cells less sensitive to apoptotic stimuli. Several independent lines of evidence point to regulation of the actin cytoskeleton by reactive oxygen species (ROS) produced by Yno1p.