Senthil Arumugam

ENDOPHILIN-A2 FUNCTIONS IN MEMBRANE SCISSION IN CLATHRIN-INDEPENDENT ENDOCYTOSIS

During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin. Clathrin-independent endocytic events are often less reliant on dynamin, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we show, in human and other mammalian cell lines, that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which are both clathrin-independent endocytic cargoes. In controlled in vitro systems, endoA2 reshapes membranes before scission. Furthermore, we demonstrate that endoA2, dynamin and actin contribute in parallel to the scission of Shiga-toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling-force-driven dynamic scission.

Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers.

Several cell surface molecules including signalling receptors are internalized by clathrin-independent endocytosis. How this process is initiated, how cargo proteins are sorted and membranes are bent remains unknown. Here, we found that a carbohydrate-binding protein, galectin-3 (Gal3), triggered the glycosphingolipid (GSL)-dependent biogenesis of a morphologically distinct class of endocytic structures, termed clathrin-independent carriers (CLICs). Super-resolution and reconstitution studies showed that Gal3 required GSLs for clustering and membrane bending. Gal3 interacted with a defined set of cargo proteins. Cellular uptake of the CLIC cargo CD44 was dependent on Gal3, GSLs and branched N-glycosylation. Endocytosis of β1-integrin was also reliant on Gal3. Analysis of different galectins revealed a distinct profile of cargoes and uptake structures, suggesting the existence of different CLIC populations. We conclude that Gal3 functionally integrates carbohydrate specificity on cargo proteins with the capacity of GSLs to drive clathrin-independent plasma membrane bending as a first step of CLIC biogenesis.

Surface Topology Engineering of Membranes for the Mechanical Investigation of the Tubulin Homologue FtsZ

In spite of their small size, bacteria display highly organized cytoskeletal structures like coils, helices, or rings. Extensive mechanical modeling has been done to explain the occurrence of such specific structures within the small volume of bacterial cells. As they are difficult to image within cells, in vitro reconstitution provides a valuable approach to quantitatively analyze their properties under defined conditions. A particularly interesting cytoskeletal feature is the Z-ring, which plays a key role in cell division for many bacteria. It is composed of FtsZ, a tubulin homologue, and other components and has been implicated in constriction force generation. Mechanisms localizing FtsZ to the center of the cell are known, but how it takes the form of a functional helical or ring-like structure remains unclear. 6] We hypothesized that intrinsically curved FtsZ filaments should initially respond to the native shape of bacteria and align using geometric cues. Thus, we devised a controlled biomimetic platform with membrane-coated glass substrates mimicking biologically relevant curvatures, to elucidate the mechanical properties of membrane-associated FtsZ. We found that E. coli FtsZ is assembled into inherently curved and twisted filaments supporting a helical geometry, which showed preferential orientations at the native bacterial cell-like curvatures. Strikingly, the FtsZ did not recognize smaller curvatures in the same way, but rather oriented themselves at an angle in higher curvatures, which does not support the idea that FtsZ alone is able to exert a constriction force. In recent studies involving high-resolution imaging and cryo-electron microscopy, the “Z-ring” has generally been described as a helical structure. Purified FtsZ has been studied extensively by electron microscopy and atomic force microscopy. Consistently, the EM and AFM images from these studies show curved filaments. Cryo-EM on reconstituted FtsZ filaments in vitro seems to contradict the presence of any local spontaneous curvature. However, in a recent study, Osawa et al. showed the ability of FtsZ filaments with an artificially introduced membrane targeting sequence (MTS) to bend membranes, with an influence of the MTS placement in FtsZ on the membrane bending direction. They used an MTS from MinD at the C-terminus of FtsZ to mimic the recruitment of FtsZ to the membrane by adaptor proteins ZipA or FtsA. Upon shifting the MTS to the Nterminus, they find that the filaments bend the membrane in opposite directions. They interpret this to be caused by a constriction force produced by partial Z-rings. A dividing bacterial cell initially has a curvature of about 2 mm , but proceeds towards much higher curvature values as the cell progresses through division. It is unknown whether a bacterial membrane, fortified with many structural proteins, osmotic pressure, and a cell wall, would be as easily deformed. The spontaneous structure of FtsZ filaments may enable them to organize into highly curved suprastructures by sensing the inner cell-membrane curvature, but they may have to recruit other mechanically active factors for cytokinesis. The distortions observed in previous studies 18] could simply be caused by a bundle of curved filaments bending the flexible membrane towards their own curvature. We first repeated the experiments with MTS-FtsZ on freestanding giant unilamellar vesicle (GUV) membranes, and quantitatively evaluated the induced radii of curvature. We found that the filaments did not bend the membranes when the unilamellar vesicles were isotonic. They aligned into filament networks similar to those on planar supported bilayers (Figure 1 b). Changing the osmotic gradient across the membranes by adding 10 mm glucose decreased intravesicular pressure and relaxed the membrane surface tension. This resulted in a curved topology of the membrane as well as the filaments (Figure 1a,b). Only when the membrane tension was low, under hypertonic conditions, could the filaments [*] Prof. Dr. P. Schwille Dept. Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry Am Klopferspitz 18, 82152 Martinsried (Germany) E-mail: schwille@biochem.mpg.de S. Arumugam, G. Chwastek, C. Ehrig Max Planck Institute for Cell Biology and Genetics Pfotenhauerstrasse 108, 01307 Dresden (Germany) and Biotechnology Center of the TU Dresden Tatzberg 47/51, 01307 Dresden (Germany)

Quantum dot-loaded monofunctionalized DNA icosahedra for single-particle tracking of endocytic pathways

Functionalization of quantum dots (QDs) with a single biomolecular tag using traditional approaches in bulk solution has met with limited success. DNA polyhedra consist of an internal void bounded by a well-defined three-dimensional structured surface. The void can house cargo and the surface can be functionalized with stoichiometric and spatial precision. Here, we show that monofunctionalized QDs can be achieved by encapsulating QDs inside DNA icosahedra and functionalizing the DNA shell with an endocytic ligand. We deployed the DNA-encapsulated QDs for real time imaging of three different endocytic ligands - folic acid, galectin-3 (Gal3) and the Shiga toxin B-subunit (STxB). Single particle tracking of Gal3 or STxB-functionalized, QD-loaded DNA icosahedra allows us to monitor compartmental dynamics along endocytic pathways. These DNA-encapsulated QDs that bear a unique stoichiometry of endocytic ligands represent a new class of molecular probes for quantitative imaging of endocytic receptor dynamics.

Three-photon microscopy shows that somatic release can be a quantitatively significant component of serotonergic neurotransmission in the mammalian brain

Recent experiments on monoaminergic neurons have shown that neurotransmission can originate from somatic release. However, little is known about the quantity of monoamine available to be released through this extrasynaptic pathway or about the intracellular dynamics that mediate such release. Using three‐photon microscopy, we directly imaged serotonin autofluorescence and investigated the total serotonin content, release competence, and release kinetics of somatic serotonergic vesicles in the dorsal raphe neurons of the rat. We found that the somata of primary cultured neurons contain a large number of serotonin‐filled vesicles arranged in a perinuclear fashion. A similar distribution is also observed in fresh tissue slice preparations obtained from the rat dorsal raphe. We estimate that the soma of a cultured neuron on an average contains about 9 fmoles of serotonin in about 450 vesicles (or vesicle clusters) of ≤370 nm average diameter. A substantial fraction (>30%) of this serotonin is released with a time scale of several minutes by K+‐induced depolarization or by para‐chloroamphetamine treatment. The amount of releasable serotonin stored in the somatic vesicles is comparable to the total serotonin content of all the synaptic vesicles in a raphe neuron, indicating that somatic release can potentially play a major role in serotonergic neurotransmission in the mammalian brain.

MinCDE exploits the dynamic nature of FtsZ filaments for its spatial regulation

Significance Although the mechanisms of microtubule depolymerization are relatively well understood, those of the tubulin homologue FtsZ have been difficult to understand owing to differences in its filament architecture and dynamics compared with those of microtubules. MinC, an important negative regulator of FtsZ and a component of the Min oscillatory system in Escherichia coli, positions the Z-ring to the midcell. With single-molecule fluorescence imaging in a cell-free minimal system on supported lipid bilayers, in which a network of FtsZ bundles assemble in a chemically well-defined system, the dynamic nature of the FtsZ bundles and the mechanism of disassembly by MinC is elucidated. In Escherichia coli, a contractile ring (Z-ring) is formed at midcell before cytokinesis. This ring consists primarily of FtsZ, a tubulin-like GTPase, that assembles into protofilaments similar to those in microtubules but different in their suprastructures. The Min proteins MinC, MinD, and MinE are determinants of Z-ring positioning in E. coli. MinD and MinE oscillate from pole to pole, and genetic and biochemical evidence concludes that MinC positions the Z-ring by coupling its assembly to the oscillations by direct inhibitory interaction. The mechanism of inhibition of FtsZ polymerization and, thus, positioning by MinC, however, is not understood completely. Our in vitro reconstitution experiments suggest that the Z-ring consists of dynamic protofilament bundles in which monomers constantly are exchanged throughout, stochastically creating protofilament ends along the length of the filament. From the coreconstitution of FtsZ with MinCDE, we propose that MinC acts on the filaments in two ways: by increasing the detachment rate of FtsZ-GDP within the filaments and by reducing the attachment rate of FtsZ monomers to filaments by occupying binding sites on the FtsZ filament lattice. Furthermore, our data show that the MinCDE system indeed is sufficient to cause spatial regulation of FtsZ, required for Z-ring positioning.

Mechanism of Shiga Toxin Clustering on Membranes

The bacterial Shiga toxin interacts with its cellular receptor, the glycosphingolipid globotriaosylceramide (Gb3 or CD77), as a first step to entering target cells. Previous studies have shown that toxin molecules cluster on the plasma membrane, despite the apparent lack of direct interactions between them. The precise mechanism by which this clustering occurs remains poorly defined. Here, we used vesicle and cell systems and computer simulations to show that line tension due to curvature, height, or compositional mismatch, and lipid or solvent depletion cannot drive the clustering of Shiga toxin molecules. By contrast, in coarse-grained computer simulations, a correlation was found between clustering and toxin nanoparticle-driven suppression of membrane fluctuations, and experimentally we observed that clustering required the toxin molecules to be tightly bound to the membrane surface. The most likely interpretation of these findings is that a membrane fluctuation-induced force generates an effective attraction between toxin molecules. Such force would be of similar strength to the electrostatic force at separations around 1 nm, remain strong at distances up to the size of toxin molecules (several nanometers), and persist even beyond. This force is predicted to operate between manufactured nanoparticles providing they are sufficiently rigid and tightly bound to the plasma membrane, thereby suggesting a route for the targeting of nanoparticles to cells for biomedical applications.

Protein-membrane interactions: the virtue of minimal systems in systems biology

The plasma membrane of cells can be viewed as a highly dynamic, regulated, heterogeneous environment with multiple functions. It constitutes the boundary of the cell, encapsulating all its components. Proteins interact with the membrane in many ways to accommodate essential processes, such as membrane trafficking, membrane protrusions, cytokinesis, signaling, and cell–cell communication. A vast amount of literature has already fostered our current understanding of membrane–protein interactions. However, many phenomena still remain to be understood, e.g., the exact mechanisms of how certain proteins cause or assist membrane transformations. Systems biology aims to predict biological processes on the basis of the set of molecules involved. Many key processes arise from interactions with the lipid membrane. Protein interactome maps do not consider such specific interactions, and thus cannot predict precise outcomes of the interactions of the involved proteins. These can only be inferred from experimental approaches. We describe examples of how an emergent behavior of protein–membrane interactions has been demonstrated by the use of minimal systems. These studies contribute to a deeper understanding of protein interactomes involving membranes and complement other approaches of systems biology. WIREs Syst Biol Med 2011 3 269–280 DOI: 10.1002/wsbm.119

Cytoskeletal Pinning Controls Phase Separation in Multicomponent Lipid Membranes

We study the effect of a minimal cytoskeletal network formed on the surface of giant unilamellar vesicles by the prokaryotic tubulin homolog, FtsZ, on phase separation in freestanding lipid membranes. FtsZ has been modified to interact with the membrane through a membrane targeting sequence from the prokaryotic protein MinD. FtsZ with the attached membrane targeting sequence efficiently forms a highly interconnected network on membranes with a concentration-dependent mesh size, much similar to the eukaryotic cytoskeletal network underlying the plasma membrane. Using giant unilamellar vesicles formed from a quaternary lipid mixture, we demonstrate that the artificial membrane-associated cytoskeleton, on the one hand, suppresses large-scale phase separation below the phase transition temperature, and, on the other hand, preserves phase separation above the transition temperature. Our experimental observations support the ideas put forward in our previous simulation study: In particular, the picket fence effect on phase separation may explain why micrometer-scale membrane domains are observed in isolated, cytoskeleton-free giant plasma membrane vesicles, but not in intact cell membranes. The experimentally observed suppression of large-scale phase separation much below the transition temperatures also serves as an argument in favor of the cryoprotective role of the cytoskeleton.

MinC, MinD, and MinE Drive Counter-oscillation of Early-Cell-Division Proteins Prior to Escherichia coli Septum Formation

ABSTRACT Bacterial cell division initiates with the formation of a ring-like structure at the cell center composed of the tubulin homolog FtsZ (the Z-ring), which acts as a scaffold for the assembly of the cell division complex, the divisome. Previous studies have suggested that the divisome is initially composed of FtsZ polymers stabilized by membrane anchors FtsA and ZipA, which then recruit the remaining division proteins. The MinCDE proteins prevent the formation of the Z-ring at poles by oscillating from pole to pole, thereby ensuring that the concentration of the Z-ring inhibitor, MinC, is lowest at the cell center. We show that prior to septum formation, the early-division proteins ZipA, ZapA, and ZapB, along with FtsZ, assemble into complexes that counter-oscillate with respect to MinC, and with the same period. We propose that FtsZ molecules distal from high concentrations of MinC form relatively slowly diffusing filaments that are bound by ZapAB and targeted to the inner membrane by ZipA or FtsA. These complexes may facilitate the early stages of divisome assembly at midcell. As MinC oscillates toward these complexes, FtsZ oligomerization and bundling are inhibited, leading to shorter or monomeric FtsZ complexes, which become less visible by epifluorescence microscopy because of their rapid diffusion. Reconstitution of FtsZ-Min waves on lipid bilayers shows that FtsZ bundles partition away from high concentrations of MinC and that ZapA appears to protect FtsZ from MinC by inhibiting FtsZ turnover. IMPORTANCE A big issue in biology for the past 100 years has been that of how a cell finds its middle. In Escherichia coli, over 20 proteins assemble at the cell center at the time of division. We show that the MinCDE proteins, which prevent the formation of septa at the cell pole by inhibiting FtsZ, drive the counter-oscillation of early-cell-division proteins ZapA, ZapB, and ZipA, along with FtsZ. We propose that FtsZ forms filaments at the pole where the MinC concentration is the lowest and acts as a scaffold for binding of ZapA, ZapB, and ZipA: such complexes are disassembled by MinC and reform within the MinC oscillation period before accumulating at the cell center at the time of division. The ability of FtsZ to be targeted to the cell center in the form of oligomers bound by ZipA and ZapAB may facilitate the early stages of divisome assembly. A big issue in biology for the past 100 years has been that of how a cell finds its middle. In Escherichia coli, over 20 proteins assemble at the cell center at the time of division. We show that the MinCDE proteins, which prevent the formation of septa at the cell pole by inhibiting FtsZ, drive the counter-oscillation of early-cell-division proteins ZapA, ZapB, and ZipA, along with FtsZ. We propose that FtsZ forms filaments at the pole where the MinC concentration is the lowest and acts as a scaffold for binding of ZapA, ZapB, and ZipA: such complexes are disassembled by MinC and reform within the MinC oscillation period before accumulating at the cell center at the time of division. The ability of FtsZ to be targeted to the cell center in the form of oligomers bound by ZipA and ZapAB may facilitate the early stages of divisome assembly.