Lipid rafts serve as anchoring platforms for membrane proteins. Thus far they escaped direct observation by light microscopy due to their small size. Here we used differently colored dyes as reporters for the registration of both ordered and disordered lipids from the two leaves of a freestanding bilayer. Photoswitchable lipids dissolved or reformed the domains. Measurements of domain mobility indicated the presence of 120 nm wide ordered and 40 nm wide disordered domains. These sizes are in line with the predicted roles of line tension and membrane undulation as driving forces for alignment.

The polyphosphate glucokinases can phosphorylate glucose to glucose 6-phosphate using polyphosphate as the substrate. ORF all1371 encodes a putative polyphosphate glucokinase in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Here, ORF all1371 was heterologously expressed in Escherichia coli, and its purified product was characterized. Enzyme activity assays revealed that All1371 is an active polyphosphate glucokinase that can phosphorylate both glucose and mannose in the presence of divalent cations in vitro. Unlike many other polyphosphate glucokinases, for which nucleoside triphosphates (e.g. ATP or GTP) act as phosphoryl group donors, All1371 required polyphosphate to confer its enzymic activity. The enzymic reaction catalysed by All1371 followed classical Michaelis–Menten kinetics, with kcat = 48.2 s−1 at pH 7.5 and 28 °C and KM = 1.76 µM and 0.118 mM for polyphosphate and glucose, respectively. Its reaction mechanism was identified as a particular multi-substrate mechanism called the ‘bi-bi ping-pong mechanism’. Bioinformatic analyses revealed numerous polyphosphate-dependent glucokinases in heterocyst-forming cyanobacteria. Viability of an Anabaena sp. PCC 7120 mutant strain lacking all1371 was impaired under nitrogen-fixing conditions. GFP promoter studies indicate expression of all1371 under combined nitrogen deprivation. All1371 might play a substantial role in Anabaena sp. PCC 7120 under these conditions.

Lipid domains less than 200 nm in size may form a scaffold, enabling the concerted function of plasma membrane proteins. The size-regulating mechanism is under debate. We tested the hypotheses that large values of spontaneous monolayer curvature are incompatible with micrometer-sized domains. Here, we used the transition of photoswitchable lipids from their cylindrical conformation to a conical conformation to increase the negative curvature of a bilayer-forming lipid mixture. In contrast to the hypothesis, pre-existing micrometer-sized domains did not dissipate in our planar bilayers, as indicated by fluorescence images and domain mobility measurements. Elasticity theory supports the observation by predicting the zero free energy gain for splitting large domains into smaller ones. It also indicates an alternative size-determining mechanism: The cone-shaped photolipids reduce the line tension associated with lipid deformations at the phase boundary and thus slow down the kinetics of domain fusion. The competing influence of two approaching domains on the deformation of the intervening lipids is responsible for the kinetic fusion trap. Our experiments indicate that the resulting local energy barrier may restrict the domain size in a dynamic system.

Fluorescence correlation spectroscopy (FCS) is one of the prominent tools to elucidate these dynamics in living cells but can only report on the dynamics at one given spatial position at a time. Using scanning fluorescence correlation spectroscopy (sFCS), we obtain a multitude of FCS measurements at different spatial locations. Here, we present a statistical analysis pipeline for sFCS data which allows for the accurate determination of the diffusion dynamics and the differentiation of free (Brownian) from hindered (non-Brownian) diffusion modes. We show free diffusion for phospholipids in model membranes and cells but reveal hindered diffusion of sphingolipids and GPI-anchored proteins in cells. Notably, these measurements can be performed using standard fluorescent dyes or proteins on a conventional confocal laser scanning microscope. To further investigate the dynamics on single cell level, we combine sFCS with stimulated emission depletion (STED) microscopy and by alternating conventional and super-resolved excitation we introduce line interleaved excitation scanning STED-FCS (LIESS-FCS). With LIESS-FCS the diffusion modes can be directly determined at multiple spots within the cellular plasma membrane providing detailed insights into organisation and function. Overall we are presenting a novel toolkit to investigate nano-scale molecular diffusion dynamics for shedding a new light on membrane organisation and heterogeneity. Ordered lipid domains (OLD) are thought to serve as signaling platforms. Here we show that their assembly does not involve intermediates in terms of nonregistered domains in the individual leaflets. The addition of photoswitchable lipids (ceramide PhoDAG-1) to the membrane forming lipid mixture allowed solubilizing and re-assembling OLDs by illuminating freestanding planar lipid bilayers at 365 nm and 475 nm, respectively. Differently colored fluorescent labels adopted a matching distribution in the two monolayers indicating that OLDs from the two leaflets were always in register. Tracing domain diffusion revealed perfect agreement with the Saffman-Delbr€ uck equation. The registration of domains as small as 10 nm in diameter is in line with the predicted role of line tension as a driving force for alignment (1). Registration of the observed micrometer sized domains is mainly driven by membrane undulations (2). 1. Galimzyanov, et al. Phys. Rev. Lett. 2015, 115:088101. 2. Galimzyanov, et al. Biophys. J. 2017. 1617-Plat Asymmetric Membranes and the Study of Lipid Movement across SingleThe great variety of lipid molecules in the cell membrane suggests their complex andunique role in cell function. The cell has further established unique lipid composition indifferent membranes within the cell for directed functionality. In addition, in membranes likethe plasma membrane (PM), there is an asymmetric distribution of lipids between the outer orexoplasmic and the inner or cytoplasmic leaflets and the physiological fate of cells depends onthe strict maintenance of this asymmetry. However, the exact ...

Stable asymmetric membranes with DOPC on one side and DOPC/DOPG mixtures on the other were created with transmembrane potentials ranging from 10 to 25 mV. increasing membrane charge asymmetry increases the offset voltage and also increases the stiffness of the membrane. These initial successes demonstrate a viable pathway to quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membrane processes in the future.