Apoptosis and the yeast actin cytoskeleton.

Actin represents one of the most abundant and extensively studied proteins found in eukaryotic cells. It has been identified as a major target for destruction during the process of apoptosis. Recent research has also highlighted a role for cytoskeletal components in the initiation and inhibition of apoptotic processes. The high degree of conservation that exists between actins from divergent eukaryotes, particularly with respect to those that contribute to the cytoskeleton, has meant that functional studies from the model yeast Saccharomyces cerevisiae have proven useful in elucidating its cellular roles. Within the context of apoptosis in yeasts, actin seems to function as part of the signalling mechanisms that link nutritional sensing to a mitochondrial-dependent commitment to cell death. Studies in yeasts have also shown that oxidative damage accrued by the actin cytoskeleton is closely monitored and is tethered to an apoptotic response. Strong, but as yet, undefined links between the actin cytoskeleton and apoptosis have also been described in studies from plant and animal systems. The widespread involvement of actin in apoptotic mechanisms from diverse eukaryotic organisms raises the possibility of conserved regulatory pathways, further strengthening the relevance of yeast research in this area.

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

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.

A role for actin in aging and apoptosis.

The actin cytoskeleton is central to many cell processes including membrane trafficking and generation of cell polarity. We have identified a role for actin in cell death and in promoting longevity of the budding yeast, Saccharomyces cerevisiae. Aging in yeast appears to occur via an apoptotic-like pathway with changes including DNA fragmentation, loss of mitochondrial membrane permeability, increase in levels of ROS (reactive oxygen species) and exposure of phosphatidylserine in the outer leaflet of the plasma membrane. This pathway can be induced by alterations in actin dynamics, such that reduced dynamics correlates with increased levels of ROS and decreased viability. Conversely, increased actin dynamics correlates with low ROS levels and increased survival. Our current studies have focused on identifying pathways which couple changes in actin dynamics to cell death.

Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, cochleata, afila, and tendril-lessn.

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.

Analytical performance of the Genzyme LipoPro Lp(a) kit for plasma lipoprotein(a)-cholesterol assay.

The analytical performance of a new assay for plasma lipoprotein(a)-cholesterol (Lp[a]-C) was compared with that of our existing Lp(a) protein assay. The Lp(a)-C assay utilises lectin affinity chromatography to isolate intact Lp(a) particles. The effect of apo(a) isoform size on this system was assessed and found to be negligible. Plasma Lp(a) concentrations measured by both assays were in excellent accord in 24 subjects with Lp(a) protein concentrations ranging from 1-65 mg/dL (r2 = 0.916). Linearity of the Lp(a)-C assay system was excellent (r2 = 0.997) and within-run precision was 6.9% at an Lp(a)-C concentration of 0.3 mmol/L. Between-calibration precision was checked and proved to be 7.9%. The lectin-binding reagent used in the assay bound different sized apo(a) isoforms equally, and the recovery of Lp(a) from the reagent was, on average, 64%. We conclude that the Lp(a)-C assay system performs well but that further information is required on what new information, if any, the assay provides over traditional Lp(a) protein measurements by enzyme-linked immunosorbent assay (ELISA) or immunoturbidimetry.