A simple guide to biochemical approaches for analyzing protein-lipid interactions

Eukaryotic cells contain many different membrane compartments with characteristic shapes, lipid compositions, and dynamics. A large fraction of cytoplasmic proteins associate with these membrane compartments. Such protein-lipid interactions, which regulate the subcellular localizations and activities of peripheral membrane proteins, are fundamentally important for a variety of cell biological processes ranging from cytoskeletal dynamics and membrane trafficking to intracellular signaling. Reciprocally, many membrane-associated proteins can modulate the shape, lipid composition, and dynamics of cellular membranes. Determining the exact mechanisms by which these proteins interact with membranes will be essential to understanding their biological functions. In this Technical Perspective, we provide a brief introduction to selected biochemical methods that can be applied to study protein-lipid interactions. We also discuss how important it is to choose proper lipid composition, type of model membrane, and biochemical assay to obtain reliable and informative data from the lipid-interaction mechanism of a protein of interest.     

Phospholipids undergo hop diffusion in compartmentalized cell membrane

The diffusion rate of lipids in the cell membrane is reduced by a factor of 5-100 from that in artificial bilayers. This slowing mechanism has puzzled cell biologists for the last 25 yr. Here we address this issue by studying the movement of unsaturated phospholipids in rat kidney fibroblasts at the single molecule level at the temporal resolution of 25 micros. The cell membrane was found to be compartmentalized: phospholipids are confined within 230-nm-diameter (phi) compartments for 11 ms on average before hopping to adjacent compartments. These 230-nm compartments exist within greater 750-nm-phi compartments where these phospholipids are confined for 0.33 s on average. The diffusion rate within 230-nm compartments is 5.4 microm2/s, which is nearly as fast as that in large unilamellar vesicles, indicating that the diffusion in the cell membrane is reduced not because diffusion per se is slow, but because the cell membrane is compartmentalized with regard to lateral diffusion of phospholipids. Such compartmentalization depends on the actin-based membrane skeleton, but not on the extracellular matrix, extracellular domains of membrane proteins, or cholesterol-enriched rafts. We propose that various transmembrane proteins anchored to the actin-based membrane skeleton meshwork act as rows of pickets that temporarily confine phospholipids.     

Cell biology of viruses that assemble along the biosynthetic pathway

In this review we discuss five groups of viruses that bud into, or assemble from, different compartments along the biosynthetic pathway. These are herpes-, rota-, corona-, bunya- and poxviruses. Our main emphasis will be on the virally-encoded membrane glycoproteins that are responsible for determining the site of virus assembly. In a number of cases these proteins have been well characterized and appear to serve as resident markers of the budding compartments. The assembly and dissemination of these viruses raises many questions of cell biological interest.     

Rapid,combinatorial analysis of membrane compartments in intact plants with a multicolor marker set

Plant membrane compartments and trafficking pathways are highly complex, and are often distinct from those of animals and fungi. Progress has been made in defining trafficking in plants using transient expression systems. However, many processes require a precise understanding of plant membrane trafficking in a developmental context, and in diverse, specialized cell types. These include defense responses to pathogens, regulation of transporter accumulation in plant nutrition or polar auxin transport in development. In all of these cases a central role is played by the endosomal membrane system, which, however, is the most divergent and ill‐defined aspect of plant cell compartmentation. We have designed a new vector series, and have generated a large number of stably transformed plants expressing membrane protein fusions to spectrally distinct, fluorescent tags. We selected lines with distinct subcellular localization patterns, and stable, non‐toxic expression. We demonstrate the power of this multicolor ‘Wave’ marker set for rapid, combinatorial analysis of plant cell membrane compartments, both in live‐imaging and immunoelectron microscopy. Among other findings, our systematic co‐localization analysis revealed that a class of plant Rab1‐homologs has a much more extended localization than was previously assumed, and also localizes to trans‐Golgi/endosomal compartments. Constructs that can be transformed into any genetic background or species, as well as seeds from transgenic Arabidopsis plants, will be freely available, and will promote rapid progress in diverse areas of plant cell biology.     

Quantifying immunogold labelling patterns of cellular compartments when they comprise mixtures of membranes (surface-occupying) and organelles (volume-occupying)

In quantitative immunoelectron microscopy, subcellular compartments that are preferentially labelled with colloidal gold particles can be identified by estimating labelling densities (LDs) and relative labelling indices (RLIs). Hitherto, this approach has been limited to compartments which are either surface occupying (membranes) or volume occupying (organelles) but not a mixture of both (membranes and organelles). However, some antigens are known to translocate between membrane and organelle compartments and the problem then arises of expressing gold particle LDs in a consistent manner (e.g., as number per compartment profile area). Here, we present one possible solution to tackle this problem. With this method, each membrane is treated as a volume-occupying compartment and this is achieved by creating an acceptance zone at a fixed distance on each side of membrane images. Gold signal intensity is then expressed as an LD within the membrane profile area so created and this LD can be compared to LDs found in volume-occupying compartments. Acceptance zone width is determined largely by the expected dispersion of gold labelling. In some cases, the zone can be applied to all visible membrane images but there is a potential problem when image loss occurs due to the fact that membranes are not cut orthogonal to their surface but are tilted within the section. The solution presented here is to select a subset of clear images representing orthogonally sectioned membranes (so-called local vertical windows, LVWs). The fraction of membrane images forming LVWs can be estimated in two ways: goniometrically (by determining the angle at which images become unclear) or stereologically (by counting intersections with test lines). The fraction obtained by either method can then be used to calculate a factor correcting for membrane image loss. In turn, this factor is used to estimate the total gold labelling associated with the acceptance zone of the entire (volume-occupying) membrane. However calculated, the LDs over the chosen (membrane and organelle) compartments are used to obtain observed and expected gold particle counts. The observed distribution is determined simply by counting gold particles associated with each compartment. Next, an expected distribution is created by randomly superimposing test points and counting those hitting each compartment. LDs of the chosen compartments are used to calculate RLI and chi-squared values and these serve to identify those compartments in which there is preferential labelling. The methods are illustrated by synthetic and real data.     

The plant secretory pathway seen through the lens of the cell wall

Secretion in plant cells is often studied by looking at well-characterised, evolutionarily conserved membrane proteins associated with particular endomembrane compartments. Studies using live cell microscopy and fluorescent proteins have illuminated the highly dynamic nature of trafficking, and electron microscopy studies have resolved the ultrastructure of many compartments. Biochemical and molecular analyses have further informed about the function of particular proteins and endomembrane compartments. In plants, there are over 40 cell types, each with highly specialised functions, and hence potential variations in cell biological processes and cell wall structure. As the primary function of secretion in plant cells is for the biosynthesis of cell wall polysaccharides and apoplastic transport complexes, it follows that utilising our knowledge of cell wall glycosyltransferases (GTs) and their polysaccharide products will inform us about secretion. Indeed, this knowledge has led to novel insights into the secretory pathway, including previously unseen post-TGN secretory compartments. Conversely, our knowledge of trafficking routes of secretion will inform us about polarised and localised deposition of cell walls and their constituent polysaccharides/glycoproteins. In this review, we look at what is known about cell wall biosynthesis and the secretory pathway and how the different approaches can be used in a complementary manner to study secretion and provide novel insights into these processes.     

Extracellular vesicles of blood plasma: content,origin, and properties

Extracellular vesicles (EVs) are bilayer membrane fragments that are released by different cell types upon activation or death. The most well studied EVs are those of blood plasma. Two types of EVs are usually distinguished: exosomes (formed by the membranes of intracellular compartments, 50–100 nm in diameter) and ectosomes (also called microparticles or microvesicles, formed from plasma membrane, 100–1000 nm in diameter). The real picture is much more complicated and is still poorly understood. EVs are enriched by various proteins, mRNA and miRNA, and the EV lipid and protein composition can substantially differ from that of the parental cells, from which EV originate. The blood concentration of EVs greatly increases in many diseases and conditions. EVs have a wide spectrum of biological activities, from pro-coagulant to immunomodulating ones. This activity can be physiologically important and is believed to be absolutely important pathophysiologically. In recent studies, EVs are considered to be important not only as objects of basic research, but also as potential biomarkers, drug candidates, drug carriers, or therapeutic targets.     

Using molecular tethering to analyze the role of nuclear compartmentalization in the regulation of mammalian gene activity

The mammalian nucleus has a complex structural organization that dynamically interacts with the genome. Chromatin is organized into discrete domains by association with distinct nuclear compartments enriched in structural and regulatory proteins. Growing evidence suggests that gene activity is modulated by interactions with these sub-nuclear compartments. Therefore, analyzing how nuclear architecture controls genome activity will be necessary to fully understand complex biological processes such as development and disease. In this article we describe a molecular methodology involving inducible tethering that can be used to position genes at the inner nuclear membrane (INM)-lamina compartment. The consequences of such directed re-positioning on gene activity or other DNA transactions can then be analyzed. This approach can be generalized and extended to position genes or chromosomal domains within other nuclear compartments thereby greatly facilitating the analysis of nuclear structure and its impact on genome activity.     

Lipid rafts as viral entry routes and immune platforms: A double-edged sword in SARS-CoV-2 infection?

Lipid rafts are nanoscopic compartments of cell membranes that serve a variety of biological functions. They play a crucial role in viral infections, as enveloped viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can exploit rafts to enter or quit target cells. On the other hand, lipid rafts contribute to the formation of immune synapses and their proper functioning is a prerequisite for adequate immune response and viral clearance. In this narrative review we dissect the panorama focusing on this singular aspect of cell biology in the context of SARS-CoV-2 infection and therapy. A lipid raft-mediated mechanism can be hypothesized for many drugs recommended or considered for the treatment of SARS-CoV-2 infection, such as glucocorticoids, antimalarials, immunosuppressants and antiviral agents. Furthermore, the additional use of lipid-lowering agents, like statins, may affect the lipid composition of membrane rafts and thus influence the processes occurring in these compartments. The combination of drugs acting on lipid rafts may be successful in the treatment of more severe forms of the disease and should be reserved for further investigation.     

Exocytosis of late endosomes does not directly contribute membrane to the formation of phagocytic cups or pseudopods in Dictyostelium

Exocytosis of late endocytic compartments in Dictyostelium has mostly been studied by live microscopy. Here we show that this exocytosis is accompanied by a complete fusion of late endosomes with the plasma membrane resulting in the transient formation of membrane microdomains that can be visualized by immunofluorescence in fixed cells. This permitted to demonstrate that fusion of late endocytic compartments with the cell surface does not occur in regions of the plasma membrane engaged in the formation of pseudopods, macropinosomes or phagosomes. Our results propose that exocytosis of late endosomes and actin-driven membrane remodeling are mutually exclusive processes.     

Microdosimetric Study for Nanosecond Pulsed Electric Fields on a Cell Circuit Model with Nucleus

Recently, scientific interest in electric pulses, always more intense and shorter and able to induce biological effects on both plasma and nuclear membranes, has greatly increased. Hence, microdosimetric models that include internal organelles like the nucleus have assumed increasing importance. In this work, a circuit model of the cell including the nucleus is proposed, which accounts for the dielectric dispersion of all cell compartments. The setup of the dielectric model of the nucleus is of fundamental importance in determining the transmembrane potential (TMP) induced on the nuclear membrane; here, this is demonstrated by comparing results for three different sets of nuclear dielectric properties present in the literature. The results have been compared, even including or disregarding the dielectric dispersion of the nucleus. The main differences have been found when using pulses shorter than 10 ns. This is due to the fact that the high spectral components of the shortest pulses are differently taken into account by the nuclear membrane transfer functions computed with and without nuclear dielectric dispersion. The shortest pulses are also the most effective in porating the intracellular structures, as confirmed by the time courses of the TMP calculated across the plasma and nuclear membranes. We show how dispersive nucleus models are unavoidable when dealing with pulses shorter than 10 ns because of the large spectral contents arriving above 100 MHz, i.e., over the typical relaxation frequencies of the dipolar mechanism of the molecules constituting the nuclear membrane and the subcellular cell compartments.     

Ig alpha/Ig beta complexes generate signals for B cell development independent of selective plasma membrane compartmentalization

Ligand-induced BCR association with detergent-resistant plasma membrane compartments (lipid rafts) has been argued to be essential for initiating and/or sustaining Igalpha/Igbeta-dependent BCR signaling. Because a fraction of the BCR and an even larger fraction of the preBCR associates with lipid rafts in the apparent absence of ligand stimulation, it has been proposed that raft-associated receptor complexes mediate the ligand-independent basal signaling events observed in resting B lineage cells. However, there is no direct evidence that localization of Igalpha/Igbeta-containing complexes to detergent-resistant membrane compartments is absolutely required for the signaling events that drive B cell development. To address these issues we have designed surrogate preBCR/Igalpha/Igbeta complexes that are incapable of ligand-induced aggregation and that are preferentially targeted to either raft or nonraft compartments. An analysis of their ability to promote the preBCR-dependent proB-->preB cell transition of murine B cell progenitors revealed that expression of these surrogate receptor complexes at levels that approximate that of the conventional preBCR can drive B cell development in a manner independent of both aggregation and lipid raft localization.     

Membrane lysis during biological membrane fusion: collateral damage by misregulated fusion machines

In the canonical model of membrane fusion, the integrity of the fusing membranes is never compromised, preserving the identity of fusing compartments. However, recent molecular simulations provided evidence for a pathway to fusion in which holes in the membrane evolve into a fusion pore. Additionally, two biological membrane fusion models—yeast cell mating and in vitro vacuole fusion—have shown that modifying the composition or altering the relative expression levels of membrane fusion complexes can result in membrane lysis. The convergence of these findings showing membrane integrity loss during biological membrane fusion suggests new mechanistic models for membrane fusion and the role of membrane fusion complexes.     

Computer Simulations of Protein Diffusion in Compartmentalized Cell Membranes

The diffusion of proteins in the cell membrane is investigated using computer simulations of a two-dimensional model. The membrane is assumed to be divided into compartments, with adjacent compartments separated by a barrier of stationary obstacles. Each compartment contains traps represented by stationary attractive disks. Depending on their size, these traps are intended to model either smaller compartments or binding sites. The simulations are intended to model the double-compartment model, which has been used to interpret single molecule experiments in normal rat kidney cells, where five regimes of transport are observed. The simulations show, however, that five regimes are observed only when there is a large separation between the sizes of the traps and large compartments, casting doubt on the double compartment model for the membrane. The diffusive behavior is sensitive to the concentration and size of traps and the strength of the barrier between compartments suggesting that the diffusion of proteins can be effectively used to characterize the structure of the membrane.     

Theoretical analysis of an iron mineral-based magnetoreceptor model in birds

Sensing the magnetic field has been established as an essential part of navigation and orientation of various animals for many years. Only recently has the first detailed receptor concept for magnetoreception been published based on histological and physical results. The considered mechanism involves two types of iron minerals (magnetite and maghemite) that were found in subcellular compartments within sensory dendrites of the upper beak of several bird species. But so far a quantitative evaluation of the proposed receptor is missing. In this article, we develop a theoretical model to quantitatively and qualitatively describe the magnetic field effects among particles containing iron minerals. The analysis of forces acting between these subcellular compartments shows a particular dependence on the orientation of the external magnetic field. The iron minerals in the beak are found in the form of crystalline maghemite platelets and assemblies of magnetite nanoparticles. We demonstrate that the pull or push to the magnetite assemblies, which are connected to the cell membrane, may reach a value of 0.2 pN -- sufficient to excite specific mechanoreceptive membrane channels in the nerve cell. The theoretical analysis of the assumed magnetoreceptor system in the avian beak skin clearly shows that it might indeed be a sensitive biological magnetometer providing an essential part of the magnetic map for navigation.     

Lipoprotein lipase stored in adipocytes and muscle cells is a cryptic enzyme

The status of lipoprotein lipase (LPL) has been examined in different cell types (adipose, skeletal muscle, and heart muscle cells) and different tissues (adipose, muscle, and cardiac tissues) from mouse, rat, and human. Cell and secreted activities were compared in cycloheximide-, heparin-treated cells present in culture. A gross underestimation of cell LPL activity was found; excess of LPL over substrate and/or apolipoprotein C-II was excluded as well as inhibition by cell component(s) or detergent molecules used to disrupt membrane structures in the cell lysates. Unmasking of LPL activity occurred upon dilution: the higher the concentration of LPL, the higher were the dilution factor and the concentration of heparin required to reach a plateau of activity. This maximal value was found to be identical to that determined in the secretion medium, indicating that the cell LPL activity can be determined in toto. The unmasking effect of dilution upon LPL activity was extended to adipose, muscle, and cardiac tissues from rat and to adipose tissues from mouse and human. In agreement with previous results (Vannier et al., 1989, J. Biol. 264: 13199-13205), our results are in favor of LPL as being cryptic within the cell. A model is proposed, in which potentially active LPL molecules are present as aggregates in various membrane compartments. It is concluded that the determination of the pool size of catalytically active cell LPL has to be estimated in vitro under the appropriate conditions described herein.     

Diffusion signal in magnetic resonance imaging: origin and interpretation in neurosciences

Diffusion Magnetic Resonance Imaging provides images of unquestionable diagnostic value. It is commonly used in the assessment of stroke and in white matter fiber tracking, among other applications. The diffusion coefficient has been shown to depend on cell concentration, membrane permeability, and cell orientation in the case of white matter or muscle fiber tracking; yet a clear relation between diffusion measurements and known physiological parameters is not established. The aim of this paper is to review hypotheses and actual knowledge on diffusion signal origin to provide assistance in the interpretation of diffusion MR images. Focus will be set on brain images, as most common applications of diffusion MRI are found in neuroradiology. Diffusion signal does not come from two intra- or extracellular compartments, as was first assumed. Restriction of water displacement due to membranes, hindrance in the extracellular space, and tissue heterogeneity are important factors. Unanswered questions remain on how to deal with tissue heterogeneity, and how to retrieve parameters less troublesome to work with from biological and clinical points of view. Diffusion quantification should be done with care, as many variables can lead to variation in measurements.     

A new method to visualize the Helicobacter pylori-associated Lewis(b)-binding adhesin utilizing SDS-digested freeze-fracture replica labeling.

Freeze-fracture replica labeling has become a versatile tool to visualize both membrane components and other cell structures using SDS-replica cleaning before specific immunogold labeling of proteins or lipids. We report here for the first time the adoption and optimization of the method to studies of bacterial envelopes, as applied to structural analysis of the distribution of the unique BabA-adhesin of the gastric pathogen Helicobacter pylori. BabA is important for bacterial adherence to the human epithelial cell lining of the stomach. The adhesin was found to be distributed all over the bacterial cell surfaces. Our results suggest that the SDS-replica labeling allows assessment of protein localization to distinct cell compartments and analysis of co-localization with neighboring membrane structures.     

The Salmonella effector PipB2 affects late endosome/lysosome distribution to mediate Sif extension

After internalization into mammalian cells, the bacterial pathogen Salmonella enterica resides within a membrane-bound compartment, the Salmonella-containing vacuole (SCV). During its maturation process, the SCV interacts extensively with host cell endocytic compartments, especially late endosomes/lysosomes (LE/Lys) at later stages. These interactions are mediated by the activities of multiple bacterial and host cell proteins. Here, we show that the Salmonella type III effector PipB2 reorganizes LE/Lys compartments in mammalian cells. This activity results in the centrifugal extension of lysosomal glycoprotein-rich membrane tubules, known as Salmonella-induced filaments, away from the SCV along microtubules. Salmonella overexpressing pipB2 induce the peripheral accumulation of LE/Lys compartments, reducing the frequency of LE/Lys tubulation. Furthermore, ectopic expression of pipB2 redistributes LE/Lys, but not other cellular organelles, to the cell periphery. In coexpression studies, PipB2 can overcome the effects of dominant-active Rab7 or Rab34 on LE/Lys positioning. Deletion of a C-terminal pentapeptide motif of PipB2, LFNEF, prevents its peripheral targeting and effect on organelle positioning. The PipB2 homologue PipB does not possess this motif or the same biological activity as PipB2. Therefore, it seems that a divergence in the biological functions of these two effectors can be accounted for by sequence divergence in their C termini.