Showing papers by "Helge Ewers published in 2014"
TL;DR: It is shown that molecular pinning and interleaflet coupling between lipid tail domains on a nanoscopic scale suffice to induce transient immobilization and thereby anomalous subdiffusion on the millisecond time scale.
Abstract: The biological functions of the cell membrane are influenced by the mobility of its constituents, which are thought to be strongly affected by nanoscale structure and organization. Interactions with the actin cytoskeleton have been proposed as a potential mechanism with the control of mobility imparted through transmembrane “pickets” or GPI-anchored lipid nanodomains. This hypothesis is based on observations of molecular mobility using various methods, although many of these lack the spatiotemporal resolution required to fully capture all the details of the interaction dynamics. In addition, the validity of certain experimental approaches, particularly single-particle tracking, has been questioned due to a number of potential experimental artifacts. Here, we use interferometric scattering microscopy to track molecules labeled with 20–40 nm scattering gold beads with simultaneous <2 nm spatial and 20 μs temporal precision to investigate the existence and mechanistic origin of anomalous diffusion in bilayer...
102 citations
TL;DR: It is found that, during development, a marker of the septin complex, Septin7 (Sept7), becomes localized to the spine neck where it forms a stable structure underneath the plasma membrane, which indicates that Sept7 regulates membrane protein access to spines.
Abstract: Excitatory glutamatergic synapses at dendritic spines exchange and modulate their receptor content via lateral membrane diffusion. Several studies have shown that the thin spine neck impedes the access of membrane and solute molecules to the spine head. However, it is unclear whether the spine neck geometry alone restricts access to dendritic spines or if a physical barrier to the diffusion of molecules exists. Here, we investigated whether a complex of septin cytoskeletal GTPases localized at the base of the spine neck regulates diffusion across the spine neck. We found that, during development, a marker of the septin complex, Septin7 (Sept7), becomes localized to the spine neck where it forms a stable structure underneath the plasma membrane. We show that diffusion of receptors and bulk membrane, but not cytoplasmic proteins, is slower in spines bearing Sept7 at their neck. Finally, when Sept7 expression was suppressed by RNA interference, membrane molecules explored larger membrane areas. Our findings indicate that Sept7 regulates membrane protein access to spines.
85 citations
TL;DR: This work demonstrates the potential of the commonly used red fluorescent protein mCherry for single-molecule super-resolution imaging and shows the C-terminus of the nuclear pore protein POM121, which is on the inside of the pore and not readily accessible for external labeling.
Abstract: We demonstrate the potential of the commonly used red fluorescent protein mCherry for single-molecule super-resolution imaging. mCherry can be driven into a light-induced dark state in the presence of a thiol from which it can recover spontaneously or by irradiation with near UV light. We show imaging of subcellular protein structures such as microtubules and the nuclear pore complex with a resolution below 40 nm. We were able to image the C-terminus of the nuclear pore protein POM121, which is on the inside of the pore and not readily accessible for external labeling. The photon yield for mCherry is comparable to that of the latest optical highlighter fluorescent proteins. Our findings show that the widely used mCherry red fluorescent protein and the vast number of existing mCherry fusion proteins are readily amenable to super-resolution imaging. This obviates the need for generating novel protein fusions that may compromise function or the need for external fluorescent labeling.
27 citations