Stefan Semrau

Accurate Determination of Elastic Parameters for Multicomponent Membranes

Heterogeneities in the cell membrane due to coexisting lipid phases have been conjectured to play a major functional role in cell signaling and membrane trafficking. Thereby the material properties of multiphase systems, such as the line tension and the bending moduli, are crucially involved in the kinetics and the asymptotic behavior of phase separation. In this Letter we present a combined analytical and experimental approach to determine the properties of phase-separated vesicle systems. First we develop an analytical model for the vesicle shape of weakly budded biphasic vesicles. Subsequently experimental data on vesicle shape and membrane fluctuations are taken and compared to the model. The parameters obtained set limits for the size and stability of nanodomains in the plasma membrane of living cells.

Membrane-Mediated Interactions Measured Using Membrane Domains

Cell membrane organization is the result of the collective effect of many driving forces. Several of these, such as electrostatic and van der Waals forces, have been identified and studied in detail. In this article, we investigate and quantify another force, the interaction between inclusions via deformations of the membrane shape. For electrically neutral systems, this interaction is the dominant organizing force. As a model system to study membrane-mediated interactions, we use phase-separated biomimetic vesicles that exhibit coexistence of liquid-ordered and liquid-disordered lipid domains. The membrane-mediated interactions between these domains lead to a rich variety of effects, including the creation of long-range order and the setting of a preferred domain size. Our findings also apply to the interaction of membrane protein patches, which induce similar membrane shape deformations and hence experience similar interactions.

Membrane heterogeneity : from lipid domains to curvature effects

The interest in membrane heterogeneity started with two biological questions: how is the plasma membrane organized on a microscopic scale and what is the influence of this structure on biological processes? The earliest model that set out to answer these questions was the homogeneous fluid mosaic (S. J. Singer and G. L. Nicolson, Science, 1972, 175, 720–731). This model was refined by including heterogeneity (Fig. 1), where either the lipid composition or the proteins were given the leading role. Both concepts are in the process of being reconciled in the light of new experiments on lipid–protein interactions. Those interactions range from specific chemical to unspecific and purely physical. The latter comprise membrane curvature mediated interactions which have recently been shown to influence a large number of biological processes. In parallel to the conception of refined models, new experimental techniques to determine membrane microstructure were developed. Single-molecule fluorescence has emerged as one of the leading technologies since it delivers the required spatial resolution and can be employed in living cells. In a complementary approach artificial model systems are used to study specific biophysical aspects of membranes in isolation and in a controllable way. Nowadays, artificial membranes have outgrown their initial status as simplistic mock cells: a rich spectrum of different phases and phase transitions and the unique possibility to study membrane material properties make them an exciting subject of research in their own right (U. Seifert, Adv. Phys., 1997, 46(1), 13–137, S. L. Veatch and S. L. Keller, Biochim. Biophys. Acta, 2005, 1746(3), 172–185). In this review we discuss state-of-the-art models for membrane microstructure on the basis of key experiments. We show how phase separated artificial membranes can be used to gain fundamental insight into lipid composition based heterogeneity and membrane mediated interactions. Finally, we review the basics of single-molecule tracking experiments in live cells and a new unbiased analysis method for single-molecule position data.

Differential Stoichiometry among Core Ribosomal Proteins.

Summary Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function.

Protein incorporation in giant lipid vesicles under physiological conditions.

One of the most essential components of a cell is the cell membrane. It forms both a protective boundary and a via for communication with the external environment. Because cell membranes are highly complex, simplified model systems in the form of giant vesicles (GVs) have been used extensively in vitro. GVs are attractive membrane models because they can be produced easily with the electroformation method, their sizes are comparable to natural cell sizes ranging from tens to hundreds of micrometers in diameter, and the choice for the types of lipids that can be used is broad. GVs are not only used to study lipid heterogeneity but also to examine the interaction of proteins with the membrane. However, the incorporation of proteins in GVs is difficult. Thus far only stable (small) membrane proteins have been successfully incorporated in GVs in a functional form. Compared to the gentle hydration method in which GVs are formed by spontaneous swelling, the classic electroformation method yields larger vesicles, which also have fewer defects. Despite its many advantages classic electroformation requires a low salt or saltless solution; 14] this severely limits the choice of proteins that can be studied. Applications of this method that require salt solutions rely on buffer exchange after electroformation is completed. Unfortunately, alternative methods to electroformation that can be applied in the presence of high salt solutions, such as spontaneous swelling or the freeze-thaw method, are not optimally adapted for incorporation of proteins. Swelling methods typically require higher temperatures that limit the lifetime of proteins in an experiment. Though freezethaw methods are useful for making vesicles in salt buffers, it is widely known that proteins degrade with each subsequent freeze-thaw cycle, so that the method is not ideal for studies with sensitive proteins. Moreover, the inverse emulsion method and microfluidic jetting are also not suited for membrane proteins. Here, we present two new applications of a specialized electroformation method, developed recently, that can produce GVs under physiologically relevant salt conditions by applying an electric field with a high frequency. 23] 1) We create GVs from native subcellular membranes containing transmembrane proteins. We produce, for the first time, GVs containing transmembrane proteins that require post-translational modifications. 2) We load GVs with biopolymer proteins as large as 110 kDa during electroformation at low temperatures, and show that the encapsulated proteins not only retain their function but also give shape to the GVs. First, we control the membrane composition of the GVs using native membrane material. These native membranes are acquired from eukaryotic cells by a mild extraction procedure, which retains intact subcellular structures from the endoplasmic reticulum (ER). Membrane proteins are incorporated in these membranes by a novel in vitro transcription–translation procedure. In this procedure, fully described in ref. [27] , properly folded and biologically active membrane proteins are synthesized in vitro in cell extracts. This approach extends upon earlier methods by using prokaryotic expression systems, which lack the ability to express proteins requiring post-translational modifications. Here, we incorporate a protein that requires such modification. Our in vitro translation reaction is comprised of the cell extract, purified mRNA encoding for the protein, complete amino acids, ATP and GTP. The mRNA codes for the membrane anchor of the small GTPase HRas (20–25 kDa), which contains the CAAX motif fused to eYFP (eYFP-CAAX). The formation of the membrane anchor of HRas requires post-translational modification in the form of covalent attachment of fatty acids (palmitoylation). By electroformation in saline buffer, GVs containing eYFPCAAX are formed. Figure 1 shows the cell extract on the surface of the electroformation chamber just prior to electroformation. Membrane material is colocalized with the cell extract

Quantification of Biological Interactions with Particle Image Cross-Correlation Spectroscopy (PICCS)

A multitude of biological processes that involve multiple interaction partners are observed by two-color microscopy. Here we describe an analysis method for the robust quantification of correlation between signals in different color channels: particle image cross-correlation spectroscopy (PICCS). The method, which exploits the superior positional accuracy obtained in single-object and single-molecule microscopy, can extract the correlation fraction and length scale. We applied PICCS to correlation measurements in living tissues. The morphogen Decapentaplegic (Dpp) was imaged in wing imaginal disks of fruit fly larvae and we quantified what fraction of early endosomes contained Dpp.

Transcriptional profiling of cells sorted by RNA abundance

We have developed a quantitative technique for sorting cells on the basis of endogenous RNA abundance, with a molecular resolution of 10–20 transcripts. We demonstrate efficient and unbiased RNA extraction from transcriptionally sorted cells and report a high-fidelity transcriptome measurement of mouse induced pluripotent stem cells (iPSCs) isolated from a heterogeneous reprogramming culture. This method is broadly applicable to profiling transcriptionally distinct cellular states without requiring antibodies or transgenic fluorescent proteins.

Studying Lineage Decision-Making In Vitro: Emerging Concepts and Novel Tools

Correct and timely lineage decisions are critical for normal embryonic development and homeostasis of adult tissues. Therefore, the search for fundamental principles that underlie lineage decision-making lies at the heart of developmental biology. Here, we review attempts to understand lineage decision-making as the interplay of single-cell heterogeneity and gene regulation. Fluctuations at the single-cell level are an important driving force behind cell-state transitions and the creation of cell-type diversity. Gene regulatory networks amplify such fluctuations and define stable cell types. They also mediate the influence of signaling inputs on the lineage decision. In this review, we focus on insights gleaned from in vitro differentiation of embryonic stem cells. We discuss emerging concepts, with an emphasis on transcriptional regulation, dynamical aspects of differentiation, and functional single-cell heterogeneity. We also highlight some novel tools to study lineage decision-making in vitro.

Robust assessment of protein complex formation in vivo via single-molecule intensity distributions of autofluorescent proteins

The formation of protein complexes or clusters in the plasma membrane is essential for many biological processes, such as signaling. We develop a tool, based on single-molecule microscopy, for following cluster formation in vivo. Detection and tracing of single autofluorescent proteins have become standard biophysical techniques. The determination of the number of proteins in a cluster, however, remains challenging. The reasons are (i) the poor photophysical stability and complex photophysics of fluorescent proteins and (ii) noise and autofluorescent background in live cell recordings. We show that, despite those obstacles, the accurate fraction of signals in which a certain (or set) number of labeled proteins reside, can be determined in an accurate an robust way in vivo. We define experimental conditions under which fluorescent proteins exhibit predictable distributions of intensity and quantify the influence of noise. Finally, we confirm our theoretical predictions by measurements of the intensities of individual enhanced yellow fluorescent protein (EYFP) molecules in living cells. Quantification of the average number of EYFP-C10HRAS chimeras in diffraction-limited spots finally confirm that the membrane anchor of human Harvey rat sarcoma (HRAS) heterogeneously distributes in the plasma membrane of living Chinese hamster ovary cells.

Membrane lysis by gramicidin S visualized in red blood cells and giant vesicles

The cationic amphiphilic antimicrobial peptide gramicidin S (GS) is an effective antibiotic. Its applicability is however restricted to topical infections due to its hemolytic activity. In this study, the process of GS induced hemolysis was investigated in detail for the first time. The morphological changes of red blood cells (RBCs) inflicted by GS were visualized and explained in terms of a physical model. The observed fast rupture events were further investigated with giant unilamellar vesicles (GUVs) as model systems for RBCs. Measurements of membrane fluctuations in GUVs revealed that the membrane surface tension was increased after incubation with GS. These findings are in agreement with the hypothesis that amphiphilic peptides induce membrane rupture by an increase in membrane tension.