Alf Honigmann

A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins

The ideal fluorescent probe for bioimaging is bright, absorbs at long wavelengths and can be implemented flexibly in living cells and in vivo. However, the design of synthetic fluorophores that combine all of these properties has proved to be extremely difficult. Here, we introduce a biocompatible near-infrared silicon-rhodamine probe that can be coupled specifically to proteins using different labelling techniques. Importantly, its high permeability and fluorogenic character permit the imaging of proteins in living cells and tissues, and its brightness and photostability make it ideally suited for live-cell super-resolution microscopy. The excellent spectroscopic properties of the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging.

Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution.

We report on a fiber laser-based stimulated emission-depletion microscope providing down to ∼20 nm resolution in raw data images as well as 15-19 nm diameter probing areas in fluorescence correlation spectroscopy. Stimulated emission depletion pulses of nanosecond duration and 775 nm wavelength are used to silence two fluorophores simultaneously, ensuring offset-free colocalization analysis. The versatility of this superresolution method is exemplified by revealing the octameric arrangement of Xenopus nuclear pore complexes and by quantifying the diffusion of labeled lipid molecules in artificial and living cell membranes.

Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment

Synaptic-vesicle exocytosis is mediated by the vesicular Ca2+ sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca2+ through PIP2. This interaction allows both Ca2+-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca2+ triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca2+ influx bringing the vesicle membrane close enough for membrane fusion.

Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells.

The interaction of lipids and proteins plays an important role in plasma membrane bioactivity, and much can be learned from their diffusion characteristics. Here we present the combination of super-resolution STED microscopy with scanning fluorescence correlation spectroscopy (scanning STED-FCS, sSTED-FCS) to characterize the spatial and temporal heterogeneity of lipid interactions. sSTED-FCS reveals transient molecular interaction hotspots for a fluorescent sphingolipid analogue. The interaction sites are smaller than 80 nm in diameter and lipids are transiently trapped for several milliseconds in these areas. In comparison, newly developed fluorescent phospholipid and cholesterol analogues with improved phase-partitioning properties show more homogenous diffusion, independent of the preference for liquid-ordered or disordered membrane environments. Our results do not support the presence of nanodomains based on lipid-phase separation in the basal membrane of our cultured nonstimulated cells, and show that alternative interactions are responsible for the strong local trapping of our sphingolipid analogue.

The 2015 super-resolution microscopy roadmap.

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio) physical and biomedical research, particularly with respect to ...

Multi-protein assemblies underlie the mesoscale organization of the plasma membrane

Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. The latter hypothesis has been difficult to test in the past. Here, we introduce several approaches based on click chemistry, through which we study the distribution of membrane proteins in living cells, as well as in membrane sheets. We found that the plasma membrane proteins form multi-protein assemblies that are long lived (minutes), and in which protein diffusion is restricted. The formation of the assemblies is dependent on cholesterol. They are separated and anchored by the actin cytoskeleton. Specific proteins are preferentially located in different regions of the assemblies, from their cores to their edges. We conclude that the assemblies constitute a basic mesoscale feature of the membrane, which affects the patterning of most membrane proteins, and possibly also their activity.

A lipid bound actin meshwork organizes liquid phase separation in model membranes

The eukaryotic cell membrane is connected to a dense actin rich cortex. We present FCS and STED experiments showing that dense membrane bound actin networks have severe influence on lipid phase separation. A minimal actin cortex was bound to a supported lipid bilayer via biotinylated lipid streptavidin complexes (pinning sites). In general, actin binding to ternary membranes prevented macroscopic liquid-ordered and liquid-disordered domain formation, even at low temperature. Instead, depending on the type of pinning lipid, an actin correlated multi-domain pattern was observed. FCS measurements revealed hindered diffusion of lipids in the presence of an actin network. To explain our experimental findings, a new simulation model is proposed, in which the membrane composition, the membrane curvature, and the actin pinning sites are all coupled. Our results reveal a mechanism how cells may prevent macroscopic demixing of their membrane components, while at the same time regulate the local membrane composition. DOI: http://dx.doi.org/10.7554/eLife.01671.001

STED microscopy detects and quantifies liquid phase separation in lipid membranes using a new far-red emitting fluorescent phosphoglycerolipid analogue

We have developed a bright, photostable, and far-red emitting fluorescent phosphoglycerolipid analogue to probe diffusion characteristics of lipids in membranes. The lipid analogue consists of a saturated (C18) phosphoethanolamine and a hydrophilic far-red emitting fluorescent dye (KK114) that is tethered to the head group by a long polyethylenglycol linker. In contrast to reported far-red emitting fluorescent lipid analogues, this one partitions predominantly into liquid ordered domains of phase-separated ternary bilayers. We performed fluorescence correlation spectroscopy with a super-resolution STED microscope (STED-FCS) to measure the lateral diffusion of the new lipid analogue in the liquid ordered (Lo) and disordered (Ld) phase. On a mica support, we observed micrometer large phases and found that the lipid analogue diffuses freely on all tested spatial scales (40-250 nm) in both the Ld and Lo phase with diffusion coefficients of 1.8 microm2 s(-1) and 0.7 microm2 s(-1) respectively. This indicates that the tight molecular packing of the Lo phase mainly slows down the diffusion rather than causing anomalous sub-diffusion. The same ternary mixture deposited on acid-cleaned glass forms Lo nanodomains of < 40 nm to 300 nm in diameter as only revealed by STED microscopy, which demonstrates the severe influence of interactions with the substrate on the sizes of domains in membranes. When averaging over different positions, STEd-FCS measurements on such glass supported membranes displayed anomalous sub-diffusion. This anomaly can be attributed to a transient partitioning of the lipid analogue into the nano-domains, where diffusion is slowed down. Our results suggest that STED-FCS in combination with a Lo-partitioning fluorescent lipid analogue can directly probe the presence of Lo nano-domains, which in the future should allow the study of potential lipid rafts in live-cell membranes.

Interaction of calmodulin with Sec61α limits Ca2+ leakage from the endoplasmic reticulum

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein‐conducting channel, the Sec61 complex. The presence of large, water‐filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)‐binding motif in the cytosolic N‐terminus of mammalian Sec61α that bound CaM but not Ca2+‐free apocalmodulin with nanomolar affinity and sequence specificity. In single‐channel measurements, CaM potently mediated Sec61‐channel closure in Ca2+‐dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+‐CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.

GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane

The organization of proteins and lipids in the plasma membrane has been subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here, we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase, nor result in any enrichment of nanoscopic ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane.