No abstract

Local surface charge density of lipid membranes influences membrane–protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values.

Understanding the mechanics and dynamics of active matter at high density is indispensable to a range of physical and biological processes such as swarm dynamics, tissue formation and cancer metastasis. Here, we study the dynamics and mechanics of an MCF-10A epithelial cell monolayer on the multi-cellular and single-cell scales and over a wide density range. We show that the dynamics and Young's modulus of the monolayer are spatially heterogeneous on the multi-cellular scale. With increasing cell density, the monolayer approached kinetic arrest and the Young's modulus scaled critically. On the single-cell scale, as the cell density increased, cells were intermittently trapped in cages formed by their neighbors and their motion evolved from a ballistic motion to a sub-diffusive motion. Furthermore, the relaxation time and inverse self-diffusivity increased exponentially with the cell density. These findings provide a mechanism for long-ranged mechanical stress propagation, tissue remodeling and patterning at very high cell densities.

No abstract

The local surface charge density of the cell membrane influences regulation and localization of membrane proteins. The local surface charge density could, until recently, not be measured directly under physiological conditions, and it was largely a hypothetical yet very important parameter. Here we use unsaturated lipids of a distinct charge (DOTAP, DOPC, and DOPG) and a neutral fully saturated lipid (DPPC) to create model membranes with phase separating domains of a defined charge. We then apply quantitative surface charge microscopy (QSCM) to investigate the local surface charge density; this is a technique based on a scanning ion conductance microscope (SICM) capable of measuring surface charge density with nanoscale lateral resolution. We are able to clearly distinguish lipid domains from charge and topography in all three model membranes. The measured surface charge densities furthermore reveal that disordered domains formed by charged lipids are in fact not only impure, but also incorporate uncharged saturated lipids. We estimate that at least 30% of disordered domains in DOPG : DPPC and DOTAP : DPPC will be DPPC. These ratios could present a limit for the formation of charged domains in lipid membranes.

In the setting of ischemic stroke, the neurofilament subunit NF-L and the microtubule-associated protein MAP2 have proven to be exceptionally ischemia-sensitive elements of the neuronal cytoskeleton. Since alterations of the cytoskeleton have been linked to the transition from reversible to irreversible tissue damage, the present study investigates underlying time- and region-specific alterations of NF-L and MAP2 in different animal models of focal cerebral ischemia. Although NF-L is increasingly established as a clinical stroke biomarker, MAP2 serum measurements after stroke are still lacking. Therefore, the present study further compares serum levels of MAP2 with NF-L in stroke patients. In the applied animal models, MAP2-related immunofluorescence intensities were decreased in ischemic areas, whereas the abundance of NF-L degradation products accounted for an increase of NF-L-related immunofluorescence intensity. Accordingly, Western blot analyses of ischemic areas revealed decreased protein levels of both MAP2 and NF-L. The cytoskeletal alterations are further reflected at an ultrastructural level as indicated by a significant reduction of detectable neurofilaments in cortical axons of ischemia-affected areas. Moreover, atomic force microscopy measurements confirmed altered mechanical properties as indicated by a decreased elastic strength in ischemia-affected tissue. In addition to the results from the animal models, stroke patients exhibited significantly elevated serum levels of MAP2, which increased with infarct size, whereas serum levels of NF-L did not differ significantly. Thus, MAP2 appears to be a more sensitive stroke biomarker than NF-L, especially for early neuronal damage. This perspective is strengthened by the results from the animal models, showing MAP2-related alterations at earlier time points compared to NF-L. The profound ischemia-induced alterations further qualify both cytoskeletal elements as promising targets for neuroprotective therapies.

The lamellipodium, a thin veil-like structure at the leading edge of motile cells, is fundamental for cell migration and growth. Orchestrated activities of membrane components and an underlying biopolymer film result in a controlled movement of the whole system. Dynamics in two-dimensional cell motility are primarily driven by the actively moving protein film in the lamellipodium. Polymerization of actin filaments at the leading edge, back-transport of the actin network due to myosin motor activity, depolymerization in the back, and diffusive transport of actin monomers to the front control these dynamics. The same molecular prerequisites for lamellipodial motion are found in most eukaryotic cells and can function independently of the cell body. Here we show that lamellipodial dynamics differ strongly in different cell types according to their function. Path finding neuronal growth cones display strong stochastic fluctuations, wound healing fibroblasts that locally migrate in tissues exhibit reduced fluctuations while fish keratocytes move highly persistently. Nevertheless, experimental analysis and computer simulations show that changes in the parameters for actin polymerization and retrograde actin transport alone are sufficient for the cell to utilize the same, highly adaptive machinery to display this rich variety of behaviors.

Confronting motile cells with obstacles doubling as force sensors we tested the limits of the driving actin and myosin machinery. We could directly measure the force necessary to stop actin polymerization as well as the force present in the retrograde actin flow. Combined with detailed measurements of the retrograde flow velocity and specific manipulation of actin and myosin we found that actin polymerization and myosin contractility are not enough to explain the cells behavior. We show that ever-present depolymerization forces, a direct entropic consequence of actin filament recycling, are sufficient to fill this gap, even under heavy loads.