Lipids and proteins in membranes: From in silico to in vivo

The cell can be called an elementary unit of the living world. In order to maintain its vital functions, metabolic, signalling and other important processes need to be efficient and robust. One mechanism how to achieve such characteristics is the precise localization/compartmentalization of these processes. Membranes, in general, are useful tools how to generate a semi-permeable barrier between two environments. The semi-permeability allows separation and, at the same time, fluent communication. In nature, membranes are cleverly used to compartmentalise living organisms into functional units such as cells, organelles or viruses. Semi-permeability of the membranes enables protection from the surrounding environment and, concurrently, intense but controlled exchange of material and information. Phenomena such as metabolism, signal transduction and trafficking, to name just few, are organized and maintained by membranes. The importance of biological membranes intrigued scientists for many decades. On the other hand, knowledge about membranes is limited (e.g., compared to some protein structures or chromosomes) largely due to the technical constraints. Current technological progress has enabled the uncovering of details of membrane structure and dynamics (e.g., Owen et al. 2010, Fitzpatrick and Lillemeier 2011, Digman and Gratton 2011).

Natural membranes are lipid bilayers containing a vast number of different proteins; both lipids and proteins can be glycosylated. These three chemical entities – lipids, proteins and carbohydrates – cooperatively define the structure and function of biological membranes. This Thematic Issue of Molecular Membrane Biology focuses on the proteins and lipids in membranes, keeping sugars for the future. Of note, the term ‘proteins' is used throughout this text even though many of the experiments were performed using peptides in lipid bilayers.

The organization of cellular membranes is defined by the kinetics of processes taking place therein. In other words, the mobility of molecules and their interactions are responsible for the function of membranes. In a two-dimensional (2D) platform such as a membrane, molecules can undergo three types of diffusion. First, lateral diffusion of lipids and proteins is well known and frequently explored by membrane biophysicists. It has been shown that there is a dramatic difference in the lateral diffusion of molecules in model and cellular membranes, the latter being substantially slower. The reduced mobility in cellular membranes is frequently called anomalous lateral diffusion. Cholesterol, an essential component of the eukaryotic plasma membrane, influences the diffusion of lipids (and proteins) but cannot be the only factor responsible for the difference. In addition, lateral diffusion of membrane proteins is cell and compartment-specific. Secondly, transverse diffusion (flip-flop) of lipids between the leaflets of the bilayer is in the focus of scientists studying metabolism of cells and genetics of metabolic diseases. The position of individual lipids influences the metabolism of various molecules. Sanderson in this issue reminds us of another type of mobility of lipids – rotational diffusion, rotation of lipids with regard to the 2D surface of the membrane. In his review, all three diffusion types of membrane molecules are thoroughly explained, suitable techniques (e.g., sum frequency generation vibrational and fluorescence correlation spectroscopy or single molecule imaging) and model membrane systems suggested and examples of the importance noted. The influence of peripheral and integral membrane peptides is discussed. In addition, a kinetic model of peptide interaction with the membrane is described and shown to add another level of complexity to membrane kinetic studies.

Sezgin and Schwille stress the importance of splitting the specific questions into individual experiments when analyzing protein-lipid relations in membranes. We are also reminded of five important membrane features influencing protein-lipid interactions: (i) Lipid composition, (ii) lipid packing, (iii) lateral pressure, (iv) hydrophobic (mis)match, and (v) membrane curvature. To study the impact of individual features on proteins and lipids in membranes may require a different model system and the appropriate technique. Sezgin and Schwille provide details on model membrane systems, their advantages and limitations and some technical issues related to the preparation. Interesting references to a number of protein-lipid oriented studies are always provided. These authors also introduce an advanced ‘model' system – giant plasma membrane-derived vesicles (GPMVs) – a hypothetical bridge between the complexity of membranes in living cells and the limiting composition of artificial membranes. Such vesicles mimic cellular membranes in terms of molecular complexity but at the same time provide a reasonably robust and equilibrium system for well-established biophysical approaches, e.g., by avoiding the high lipid turnover of membranes in living cells. On the other hand, simple model membranes such as supported planar bilayers, SPBs, and giant unilamellar vesicles, GUVs, by virtue of their simple lipid composition, allow interpretation of the observed results with regard to the individual chemical entities. Most of the studied model membranes to date, including GPMVs, suffer from symmetry of the leaflets in the bilayer and the absence of membrane potential. Sezgin and Schwille, together with Sanderson in the first review, emphasize the importance of the cell plasma membrane asymmetry and provide brief insight into potential techniques which may help to generate model asymmetric membranes. Could asymmetric, non-equilibrium membrane, a truly biomimetic model, be established in the future?

If the first two articles in this Thematic Issue focus on the model systems and techniques generally used to study membrane organization and dynamics, the following brings light to the experimental procedures investigating specific lipid-protein interactions within membranes. Lena Mäler introduces solution-state NMR and its advantages in lipid-protein interaction and integral membrane protein structure studies. NMR and its variants have limitations in the samples which can be investigated using these techniques. Mäler describes in detail which model membrane systems are compatible with solution-state NMR, the limitations of the used systems and, especially, provides expert insight into how NMR experiments can profit from the growing experience with the formation and use of bicelles and nanodiscs/macrodiscs. Personally, it was exciting to read how complex dynamic studies of membranes and interacting peptides can be performed using solution-state NMR. For example, the importance of lipid headgroups and the internal molecular dynamics for the interaction of peptide with membrane was resolved at almost atomic level using this technique. Frequently, the peptide orientation in the bilayer is unknown but essential for diffusion studies. Solution-state NMR provides high resolution localization information of peptides in membranes and the dynamics of peptide and the surrounding lipids.

The last two reviews of this Thematic Issue emphasize the importance of cooperativity between proteins and lipids in membranes to maintain vital functions of cells and organisms. Two phenomena crucial for biological processes are discussed: Multi-domain organization of the membrane and protein sorting. Highly probably, similar physical processes are responsible for protein sorting and partitioning into membrane domains. Hanulová and Weiss highlight the active role of lipids in these processes even though short sorting motifs have been discovered in the amino acid sequence of proteins (e.g., KDEL for the endoplasmic reticulum targeting). Not just direct lipid-protein interactions but the hydrophobic mismatch can influence the transport and organization of proteins in membranes. The clustering of proteins due to the negative hydrophobic mismatch is mentioned. In other words, proteins in the lipid bilayer with poorly matched thickness due to long (or short) lipid acyl chains can form clusters or migrate increasingly into the areas with more favourable structure. On the other hand, membrane proteins do not actively search for the optimal environment but rather stochastically reach the area with favourable structure. Hanulová and Weiss do not over-exaggerate the impact of hydrophobic mismatch and refer to protein acylation as another significant factor governing the localization of proteins in cellular membranes.

Models proposed for the multi-domain organization of cellular membranes are summarized by Mueller, Wedlich-Söldner and Spira in the last review of this Thematic Issue. The authors sympathetically remind us of the communication barrier between biophysicists and biologists (biochemists) dividing the lateral membrane organization theories into lipidocentric and proteocentric, respectively. A unifying model of membrane organization called the ‘patchwork membrane' is here suggested. This model combines the characteristics of the three main models previously proposed to describe the membrane heterogeneities: Hydrophobic mismatch, lipid rafts and picket-and-fence models. Spira and co-workers argue that current progress in methodological options (especially imaging techniques) has enabled the fusion of lipidocentric and proteocentric models into a more general model applicable to membranes of living cells. The ‘patchwork membrane' model is based on the interdependence of different levels of membrane organization. Two levels of clustering and segregation are highlighted: (i) Self-organization through weak interactions, and (ii) formation of larger assemblies through higher affinity intermolecular interactions (e.g., protein-protein or protein-lipid).

All the presented articles underline the beauty of membrane science in its complexity and resistance to uncover its details. We read about the fast diffusion of molecules still inaccessible by current techniques, sizes smaller than the resolution of light microscopy capable to visualise structures of living cells, and other hurdles preventing rapid disclosure of membrane secrets. It is the intense dialogue between highly diverse scientific fields which enabled another big step in the understanding of cellular walls. It was my pleasure to work with the people who contributed to this Thematic Issue and I hope you will enjoy reading these diverse articles describing the complex character of cellular membranes.

Acknowledgements

Dr Marek Cebecauer would like to acknowledge Purkyne Fellowship of the Academy of Sciences of the Czech Republic and funding from the Czech Science Foundation (P305/11/0459).