Effects of plasma membrane cholesterol level and cytoskeleton F-actin on cell protrusion mechanics.

Protrusions are deformations that form at the surface of living cells during biological activities such as cell migration. Using combined optical tweezers and fluorescent microscopy, we quantified the mechanical properties of protrusions in adherent human embryonic kidney cells in response to application of an external force at the cell surface. The mechanical properties of protrusions were analyzed by obtaining the associated force-length plots during protrusion formation, and force relaxation at constant length. Protrusion mechanics were interpretable by a standard linear solid (Kelvin) model, consisting of two stiffness parameters, k0 and k1 (with k0>k1), and a viscous coefficient. While both stiffness parameters contribute to the time-dependant mechanical behavior of the protrusions, k0 and k1 in particular dominated the early and late stages of the protrusion formation and elongation process, respectively. Lowering the membrane cholesterol content by 25% increased the k0 stiffness by 74%, and shortened the protrusion length by almost half. Enhancement of membrane cholesterol content by nearly two-fold increased the protrusion length by 30%, and decreased the k0 stiffness by nearly two-and-half-fold as compared with control cells. Cytoskeleton integrity was found to make a major contribution to protrusion mechanics as evidenced by the effects of F-actin disruption on the resulting mechanical parameters. Viscoelastic behavior of protrusions was further characterized by hysteresis and force relaxation after formation. The results of this study elucidate the coordination of plasma membrane composition and cytoskeleton during protrusion formation

Viscoelastic Properties of Plasma Membranes Varies with Cholesterol Level

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Optical tweezers escape forces

With suitable calibration, optical tweezers can be used to measure forces. If the maximum force that can be exerted is of interest, calibration can be performed using viscous drag to remove a particle from the trap, typically by moving the stage. The stage velocity required to remove the particle then gives the escape force. However, the escape force can vary by up to 30% or more, depending on the particle trajectory. This can have significant quantitative impact on measurements. We describe the variation of escape force and escape trajectory, using both experimental measurements and simulations, and discuss implications for experimental measurement of forces

Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015

Forouzanfar MH, Afshin A, Alexander LT, et al. Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. LANCET. 2016;388(10053):1659-1724.Background The Global Burden of Diseases, Injuries, and Risk Factors Study 2015 provides an up-to-date synthesis of the evidence for risk factor exposure and the attributable burden of disease. By providing national and subnational assessments spanning the past 25 years, this study can inform debates on the importance of addressing risks in context. Methods We used the comparative risk assessment framework developed for previous iterations of the Global Burden of Disease Study to estimate attributable deaths, disability-adjusted life-years (DALYs), and trends in exposure by age group, sex, year, and geography for 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks from 1990 to 2015. This study included 388 risk-outcome pairs that met World Cancer Research Fund-defined criteria for convincing or probable evidence. We extracted relative risk and exposure estimates from randomised controlled trials, cohorts, pooled cohorts, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. We developed a metric that allows comparisons of exposure across risk factors-the summary exposure value. Using the counterfactual scenario of theoretical minimum risk level, we estimated the portion of deaths and DALYs that could be attributed to a given risk. We decomposed trends in attributable burden into contributions from population growth, population age structure, risk exposure, and risk-deleted cause-specific DALY rates. We characterised risk exposure in relation to a Socio-demographic Index (SDI). Findings Between 1990 and 2015, global exposure to unsafe sanitation, household air pollution, childhood underweight, childhood stunting, and smoking each decreased by more than 25%. Global exposure for several occupational risks, high body-mass index (BMI), and drug use increased by more than 25% over the same period. All risks jointly evaluated in 2015 accounted for 57.8% (95% CI 56.6-58.8) of global deaths and 41.2% (39.8-42.8) of DALYs. In 2015, the ten largest contributors to global DALYs among Level 3 risks were high systolic blood pressure (211.8 million [192.7 million to 231.1 million] global DALYs), smoking (148.6 million [134.2 million to 163.1 million]), high fasting plasma glucose (143.1 million [125.1 million to 163.5 million]), high BMI (120.1 million [83.8 million to 158.4 million]), childhood undernutrition (113.3 million [103.9 million to 123.4 million]), ambient particulate matter (103.1 million [90.8 million to 115.1 million]), high total cholesterol (88.7 million [74.6 million to 105.7 million]), household air pollution (85.6 million [66.7 million to 106.1 million]), alcohol use (85.0 million [77.2 million to 93.0 million]), and diets high in sodium (83.0 million [49.3 million to 127.5 million]). From 1990 to 2015, attributable DALYs declined for micronutrient deficiencies, childhood undernutrition, unsafe sanitation and water, and household air pollution; reductions in risk-deleted DALY rates rather than reductions in exposure drove these declines. Rising exposure contributed to notable increases in attributable DALYs from high BMI, high fasting plasma glucose, occupational carcinogens, and drug use. Environmental risks and childhood undernutrition declined steadily with SDI; low physical activity, high BMI, and high fasting plasma glucose increased with SDI. In 119 countries, metabolic risks, such as high BMI and fasting plasma glucose, contributed the most attributable DALYs in 2015. Regionally, smoking still ranked among the leading five risk factors for attributable DALYs in 109 countries; childhood underweight and unsafe sex remained primary drivers of early death and disability in much of sub-Saharan Africa. Interpretation Declines in some key environmental risks have contributed to declines in critical infectious diseases. Some risks appear to be invariant to SDI. Increasing risks, including high BMI, high fasting plasma glucose, drug use, and some occupational exposures, contribute to rising burden from some conditions, but also provide opportunities for intervention. Some highly preventable risks, such as smoking, remain major causes of attributable DALYs, even as exposure is declining. Public policy makers need to pay attention to the risks that are increasingly major contributors to global burden. Copyright (C) The Author(s). Published by Elsevier Ltd

Experimental Investigation of Cell Membrane Nano-mechanics and Plasma Membrane-Cytoskeletal Interactions Using Optical Tweezers

The mechanical properties of the cell components, cell plasma membrane and cytoskeleton, as well as membrane-cytoskeleton associations, determine the mechanical properties of the whole cell which is important in cellular shape change behavior and mechanical signal transduction in living cells. Examples of biologically important processes involving cellular shape changes are deformation of erythrocytes in capillaries, cell division, phagocytosis, pseudopodium and dendritic spine formation, and electromotility of the outer hair cells. This dissertation focuses on investigating the mechanical properties of the living cell plasma membrane and the local mechanical associations of the plasma membrane with the underlying cytoskeleton. Mechanical properties of the living cell plasma membrane are investigated by forming membrane nanotubes (tethers) from human embryonic kidney cells using optical tweezers technique. In order to analyze the role of membrane composition on its nano-mechanical properties, the membrane cholesterol content, the major lipid component of the plasma membrane, is manipulated and the obtained membrane mechanical properties are correlated to the membrane cholesterol content. The results reveal significant effects of membrane cholesterol in specific, and membrane composition in general, on membrane nano-mechanical properties. Specifically, decreases in membrane cholesterol content were associated with increased plasma membrane equilibrium force, plasma membrane tether stiffness and plasma membrane-cytoskeleton adhesion energy per unit area. Elevation of the membrane cholesterol content was followed by lower membrane tether equilibrium force, lower stiffness values, and lower membrane-cytoskeleton adhesion energy. The membrane bending modulus was almost unchanged upon membrane cholesterol manipulations. In order to discern the role of cell cytoskeleton on membrane mechanical properties the experiments were repeated after F-actin disruption and in control cell and cholesterol manipulated cells. The disruption of F-actin filaments showed a noticeable impact on the membrane mechanical properties and diminished the observed disparity in membrane mechanical properties upon cholesterol depletion and cholesterol enrichment. This dissertation also focuses on local cell deformations (protrusions). These deformations occur at an intermediate region between deformations at cell plasma membrane level and whole cell deformation, and are biologically important in formation of pseudopodia and filopodia, deformation of macrophages to engulf particles, and the surface protrusions on the cells preceding formation of membrane tethers. We used a combined optical tweezers-fluorescent microscopy technique to study cellular protrusions in adherent living cells. The mechanical properties of the protrusions were analyzed by obtaining the associated force-length plots and a Maxwell viscoelastic model is used to fit the force-length plots a. The experiments are performed on adherent human embryonic kidney cells, under cholesterol depleted and cholesterol enriched conditions to examine the effects of membrane cholesterol on protrusions. The experiments indicated greater maximum protrusion forces and shorter protrusion length under cholesterol depleted conditions in addition to greater values of the protrusion stiffness. Cholesterol enrichment experiments were associated with lower values of maximum protrusion force and protrusion stiffness, and formation of longer protrusions The observations suggest a significant contribution of the cytoskeletal F-actin filaments on the observed mechanical properties of protrusion regardless of membrane cholesterol content