Analysis of sphingolipid-signaling at the plasma membrane of Saccharomyces cerevisiae

The protein and lipid composition of eukaryotic plasma membranes is highly dynamic and regulated according to need. Despite its great plasticity, the plasma membrane retains some organizational features, such as its lateral organization into distinct domains. In the yeast, Saccharomyces cerevisiae, large immobile protein clusters, termed eisosomes, are important for plasma membrane organization. Eisosomes help to sort proteins into discrete domains, function in endocytosis and are implicated in cellular signaling. The major eisosome components Pil1 and Lsp1 were first identified as in vitro targets of the sphingolipid long chain base-regulated Pkhkinases. However, it is not known if eisosomes are targets of Pkh-mediated sphingolipid signaling in vivo. In this thesis, I show that Pkh-kinases regulate eisosome formation in response to alterations of complex sphingolipid levels in the plasma membrane. I found that Pkh-kinase-dependent phosphorylation of Pil1 controls the assembly state of eisosomes. The combination of different unbiased, global analysis methods, such as proteomics and high content screening enabled me to identify Nce102 as a negative regulator of Pkh-kinases. Nce102 relocalizes between MCC domains, overlaying eisosomes, and the remainder of the plasma membrane in response to alterations in sphingolipid levels. Together with its regulatory function on Pkh-kinases that localize at eisosomes, this relocalization suggests that it is part of a sphingolipid sensor. Furthermore, I identified Rom2, a Rho1 GTPase exchange factor, as a novel regulator of sphingolipid metabolism. My data reveal several new insights into regulation of sphingolipid metabolism and plasma membrane organization. I provide a model how a homeostatic feedback loop may control sphingolipid levels. This likely will help in understanding how cells adjust processes, such as eisosome driven domain organization, endocytosis and actin organization to altered conditions. Furthermore, I anticipate that the datasets created in this thesis will serve as a resource for future studies on plasma membrane function

Comparing cellular proteomes by mass spectrometry

Mass spectrometry and cryo-electron tomography together can enable absolute protein abundance to be determined

On the semiclassical limit of 4d spin foam models

We study the semiclassical properties of the Riemannian spin foam models with Immirzi parameter that are constructed via coherent states. We show that in the semiclassical limit the quantum spin foam amplitudes of an arbitrary triangulation are exponentially suppressed, if the face spins do not correspond to a discrete geometry. When they do arise from a geometry, the amplitudes reduce to the exponential of i times the Regge action. Remarkably, the dependence on the Immirzi parameter disappears in this limit.Comment: 32 pages, 5 figure

Seg1 Controls Eisosome Assembly and Shape

Eisosomes are stable domains at the plasma membrane of the budding yeast Saccharomyces cerevisiae and have been proposed to function in endocytosis. Eisosomes are composed of two main cytoplasmic proteins, Pil1 and Lsp1, that form a scaffold around furrow-like plasma membrane invaginations. We show here that the poorly characterized eisosome protein Seg1/Ymr086w is important for eisosome biogenesis and architecture. Seg1 was required for efficient incorporation of Pil1 into eisosomes and the generation of normal plasma membrane furrows. Seg1 preceded Pil1 during eisosome formation and established a platform for the assembly of other eisosome components. This platform was further shaped and stabilized upon the arrival of Pil1 and Lsp1. Moreover, Seg1 abundance controlled the shape of eisosomes by determining their length. Similarly, the Schizosaccharomyces pombe Seg1-like protein Sle1 was necessary to generate the filamentous eisosomes present in fission yeast. The function of Seg1 in the stepwise biogenesis of eisosomes reveals striking architectural similarities between eisosomes in yeast and caveolae in mammals

Effect of a 25 ingredient sport drink on exercise performance and muscle oxygen extraction: a randomized controlled cross-over trial

Many sport drinks contain a mixture of potential ergogenic substances. Recently, a new sport drink with 25 different ingredients was introduced to the market. Various athletes reported beneficial performance effects from the supplement, though without scientific evidence. The aim of this study was to investigate the effects of the sport drink on exercise performance. Nine sport students performed 3 test sessions including a cycle exercise tests to exhaustion, a leg strength test and a jump test. Each session was separated by 1 week. The first session was performed as a familiarization trial. In a random order, half of the participants performed the second session after consumption of the multi ingredient sport drink (MISD intake of 40g, 24 and 1h before each test) and half after placebo ingestion (same amount). During test session 3 the conditions were reversed (cross-over setting). Near infrared spectroscopy analyses were performed on the vastus lateralis during the MISD and placebo cycling test. The sport drink compared to placebo, improved maximal power output (7 watts, 95% CI 1.1-13.4), increased maximal lactate concentration (2.5 mmol/l, 95% CI 1.6-3.4), and power output at the individual threshold (Dmax) (6.1 watts, 95% CI 1.9-10.3). Power output at the 4 mmol/l threshold was reduced (9.0 watts, 95% CI -17.4 to -0.6) during the MISD trial. Additionally, the sport drink led to a steeper tissue oxygenation index decrease (TOI, slope: -0.0182±0.0084 vs. -0.0256±0.0073, p<0.005) during the test. Leg strength and jump ability was not affected by the supplement. The sport drink slightly increased power output during an incremental exercise test. Due to the broad range of substances in the supplement and their different effects, the factors involved in the performance enhancement are speculative. Data show that factors other than muscle oxygen extraction (represented by TOI) are involved in the improved maximal power output

Health problems occurring in national-level female soccer players are different between leagues and throughout the season: a 6-month prospective cohort study

OBJECTIVES This study investigated the prevalence and severity of health problems in national-level female soccer players with respect to league and seasonality. METHODS In a prospective cohort study, 46 female soccer players aged 22.8±3.9 years playing in the three highest leagues in Switzerland were surveyed biweekly using the Oslo Sports Trauma Research Centre health problem (OSTRC-H) questionnaire. All definitions and measures followed the OSTRC-H-specific recommendations. The 6-month observation period included parts of the off-season and one half of the match season. RESULTS The average 2-weekly health problem prevalence was 37.3% (illnesses: 8.8%; sudden onset injuries-both acute and repetitive mechanisms: 19.7%; repetitive gradual onset injuries: 12.4%) and 25.1% for substantial problems as defined in the OSTRC-H context (7.3%; 12.0% and 7.3%, respectively). The absolute injury rates amounted to 148 injuries per 100 players per half season, of which 96 injuries per 100 players per half season were substantial. Female players in the 2nd and 3rd highest national leagues showed more gradual onset injuries (p<0.001) and fewer illnesses than those in the top league (p<0.05). At the same time, there were no league-specific differences in sudden onset injuries. Such injuries had a higher cumulative severity score than gradual onset injuries. Among sudden onset injuries, the ankle was the most affected body part, while the thigh was affected by for gradual onset injuries. The average 2-weekly health problem prevalence values steadily increased during the match season. CONCLUSION Among national-level female soccer players, the risk of health problems is relatively high and differs between leagues and across seasons

A role for eisosomes in maintenance of plasma membrane phosphoinositide levels

The plasma membrane delineates the cell and mediates its communication and material exchange with the environment. Many processes of the plasma membrane occur through interactions of proteins with phosphatidylinositol(4,5)-bisphosphate (PI(4,5)P2), which is highly enriched in this membrane and is a key determinant of its identity. Eisosomes function in lateral organization of the plasma membrane, but the molecular function of their major protein subunits, the BAR domain–containing proteins Pil1 and Lsp1, is poorly understood. Here we show that eisosomes interact with the PI(4,5)P2 phosphatase Inp51/Sjl1, thereby recruiting it to the plasma membrane. Pil1 is essential for plasma membrane localization and function of Inp51 but not for the homologous phosphatidylinositol bisphosphate phosphatases Inp52/Sjl2 and Inp53/Sjl3. Consistent with this, absence of Pil1 increases total and available PI(4,5)P2 levels at the plasma membrane. On the basis of these findings, we propose a model in which the eisosomes function in maintaining PI(4,5)P2 levels by Inp51/Sjl1 recruitment

Breaking new grounds in injury risk screening in soccer by deploying unsupervised learning with a special focus on sex and fatigue effects

In injury prevention, a vertical drop jump (DJ) is often used for screening athletes at risk for injury; however, the large variation in individual movement patterns might mask potentially relevant strategies when analysed on a group-based level. Two movement strategies are commonly discussed as predisposing athletes to ACL injuries: a deficient leg axis and increased leg stiffness during landing. This study investigated the individual movement pattern of 39 female and male competitive soccer players performing DJs at rest and after being fatigued. The joint angles were used to train a Kohonen self-organising map. Out of 19,596 input vectors, the SOM identified 700 unique postures. Visualising the movement trajectories and adding the latent parameters contact time, medial knee displacement (MKD) and knee abduction moment allow identification of zones with presumably increased injury risk and whether the individual movement patterns pass these zones. This information can be used, e.g., for individual screening and for feedback purposes. Additionally, an athlete's reaction to fatigue can be explored by comparing the rested and fatigued movement trajectories. The results highlight the ability of unsupervised learning to visualise movement patterns and to give further insight into an individual athlete's status without the necessity of a priori assumptions