Moderator: Fred Maxfield

Two outstanding and inter-related questions in membrane biology are: what are the organizational and dynamic properties of membranes at the molecular level?; and how are membrane molecules trafficked among the various organelles in mammalian cells?

Understanding membrane trafficking requires a description of the various cellular compartments, an analysis of the transport pathways that link these compartments, characterization of the molecules that determine their properties, and an understanding of how the properties of an individual cargo molecule determine its sorting at various branch points in the trafficking route. At present, we have only a partial understanding of these parameters. Much of what is known at the molecular level about how proteins are transported among membrane compartments is derived from studies of transport through the biosynthetic system and internalization at the plasma membrane. Several fundamental issues remain unresolved. For example, we know that a high fraction of endocytosis occurs by non-clathrin-mediated mechanisms, but our understanding of these mechanisms is rudimentary. For many steps of intracellular trafficking, we know the proteins that are involved, but we really don't know in detail how or where many of these molecules work. Resolving these issues will require a combined effort using many different techniques, including structural biology, quantitative morphological and kinetic studies, and the use of mutants and chemical and genomic inhibitors.

While it has long been understood that various organelle membranes have distinct lipid compositions, and that these differences are somehow tied to their specialized functions, the role of proteins in carrying out specialized functions has certainly been much better characterized than the role of lipids. In the past few years, however, the importance of the lipid components of biological membranes has become increasingly appreciated. This increase in our understanding of the role of lipids comes from several important areas. Biophysical studies of membrane systems have advanced significantly, with better understanding of increasingly complex model membranes and improved tools for examining the dynamic behaviour of lipids. These studies have carried over to studies in living cells, often using fluorescent molecules as lipid analogues. Although there is substantial evidence that there is lateral heterogeneity in the lipid composition and physical properties of membrane bilayers, there is considerable uncertainty about what the nature and effect of this heterogeneity might be.

An important area that links these two areas of research is enzymatic modification of lipids. These modifications are used in general signal transduction mechanisms and also to regulate specific processes in membrane trafficking.

Moderator: Ben de Kruijff

Biological membranes have unique and highly diverse compositions with respect to both their lipid and protein components. This session will address the outstanding questions related to this compositional diversity from a structural, functional and experimental perspective. The highly regulated and specific lipid composition of membranes suggests that lipids give rise to the general bilayer structure that is essential for function. But what effect does lipid composition have on membrane fluidity, order, thickness, shape, surface curvature, the interface, the pressure profile or the ability to form rafts? Membrane lipids are highly dynamic, not only as they move at considerable rates within the membrane and within the cell, but also as chemical entities that undergo rapid turnover. To understand the function of lipids will require their detection in space and time, both within the cell and in relation to function; but how are we going to do this? Lipids have both general and more specific functions in membranes; precisely what are these functions, and what more is there to learn, in particular for the vast number of minor lipids that remain to be identified? What is the potential to explore lipid-specific toxins and polypeptides? Functional membranes require the insertion of proteins into lipids, but what story do these proteins tell us about the lipid environment they function in, and what do we know about such proteins? We know that the genome specifies that one in three open reading frames code for a membrane protein, but how does this translate to the specific protein composition of membranes, and what tricks can we play to access the membrane proteome?

Key questions

Moderator: John Silvius

This session will explore how strongly, how broadly and in what ways the properties of membrane lipids influence the function and the biogenesis of membrane proteins in living cells. Lipids play a major role in defining the milieu that membrane-associated proteins operate within and, in some cases, on. It is therefore not surprising that membrane lipids can influence the function, trafficking and assembly of membrane proteins. However, defining the extent and biological consequences of such effects in vivo, particularly in higher organisms, remains a challenging experimental issue.

Biophysical and biochemical studies have shown that the composition and physical properties of membrane lipids influence the behaviour of a variety of membrane proteins in vitro. Biological studies have shown that major perturbations of membrane lipid composition or physical properties can alter the behaviour of various membrane proteins in living cells, and that cells regulate their lipid compositions through numerous mechanisms, presumably to maintain optimal membrane function. Nonetheless, various microorganisms have been found to tolerate a surprising extent of variation in membrane lipid composition and properties without loss of viability. Such findings highlight some still-unanswered questions concerning the effects of membrane lipids on membrane protein function. Is a strong sensitivity to lipid properties characteristic of the majority of membrane proteins or of a small (if interesting) minority? What specific physical properties of bilayer lipids 'matter' most strongly, and to the greatest proportion of, membrane proteins? Can we adequately define, quantitate and measure these key properties? How stringently must cells regulate the properties of membrane lipids in order to maintain optimal membrane protein function, and how closely must membrane proteins and lipids therefore co-evolve?

Some proteins interact with the membrane as a substrate, mediating either chemical or physical/morphological remodelling of the lipid bilayer. The composition and physical properties of the bilayer can affect the function of these proteins in a particularly direct manner, by determining the susceptibility of the bilayer to undergo the transformations that these proteins are designed to mediate. However, we still have much to learn about how these proteins and the bilayers they act on are mutually tailored to support such biological processes. To what extent do lipid bilayer properties promote, regulate or, in the 'wrong' context, resist the action of proteins that mediate processes such as membrane fusion and fission, or the formation of tubular or vesicular membrane structures? How do proteins 'read', exploit and where necessary modify the mechanical properties of bilayers to sculpt them into organelles, vesicles and so on, with well-defined yet dynamic morphologies?

A particularly fascinating question in the area of lipid-protein interactions is how integral membrane proteins assemble and insert into the hydrophobic interior of the membrane. Biophysical studies of proteins that insert spontaneously into lipid bilayers, high-resolution imaging of integral membrane protein structure and novel findings concerning translocon-mediated integration of proteins into membranes have stimulated much recent ferment in this field. It remains, however, to elucidate the mechanisms by which the information encoded in the sequences of nascent membrane proteins is deciphered by the translocon, and by combining such knowledge with other physical and structural data, to move closer to the goal of predicting with high accuracy the structure and topology of membrane proteins on the basis of their primary sequence.

Key questions

Moderator: Marino Zerial

A fundamental feature of eukaryotic cells is their compartmentalization into membrane-bound structures or organelles. Despite the dynamic exchange of material in and out of these compartments, they are able to retain their structural integrity and functionality. To understand the molecular mechanisms responsible for transport between these cellular compartments, researchers have examined individual processes, such as vesicle formation or membrane fusion; identifying their regulatory components, analysing their biological effects and, ultimately, reconstituting the processes from purified components in artificial systems. This strategy has succeeded in elucidating the mechanisms that underlie intracellular transport in terms of understanding the behaviour of molecules within an individual transport step. However, to further our understanding requires a change in strategy - we need to go beyond studying single molecules or even molecular complexes (for example, vesicular coats), to examining the function of entire machineries (for example, the entire complement of molecules necessary for membrane tethering, fusion and motility) in time and space, and explaining how, collectively, they assemble into cellular organelles.

This task will not be easy. However, the notion that cellular organelles are built as a modular system, in which each module carries out a specific set of functions and different modules are functionally connected to each other, provides a general framework for compartmental organization. A functional module can be, for example, a membrane domain in which various components participate in a transport reaction (for example, vesicle tethering and fusion) and are coordinated via their spatial distribution and temporal regulation. The existence of such modules is supported by morphological as well as biochemical data.

The existence of such organizational principles raises a number of questions, however. In the first instance, if a functional module is a discrete membrane entity, what molecular mechanisms account for the compartmentalization of membranes, and what are the physical properties of each membrane module? The principles of membrane compartmentalization need to be defined more rigorously and adapted to the requirements of modular organization. For example, we need to understand how different modules are linked together within a compartment, how different functions are coordinated, and how cargo is sorted within each compartment and delivered to its next destination. Is cargo a passive passenger that needs to be transferred by the means of membrane carriers, or does it play a more fundamental role in determining the identity and trafficking fate of a compartment? In addition, what is the role of the cytoskeleton in membrane organization, and is (intracellular) positional information exploited to regulate compartmental identity in addition to structure and function? The question of compartmental organization needs to be addressed, not only in terms of membrane phase separation and spatial constraints, but also in relation to temporal regulation. Can compartments be formed de novo and undergo transformations, or are they maintained unaltered over long periods of time? Must they be inherited after cell division? And finally, how has compartmental organization been modified during evolution; have modules increased in complexity or have new modules appeared? Answering these questions will require molecular and cell biological methods; combining functional genomics with mathematical and biophysical approaches. Such integration will enable us to make quantitative predictions of metabolic pathways and cell behaviour in response to various stimuli, rewarding us with basic knowledge as well biotechnological potential.

Key questions

Moderator: Jennifer Lippincott-Schwartz

Proteins associated with lipid bilayers, either peripherally or integrally, have a profound capacity to alter membrane fluidity, chemistry and phase behaviour. In the environment of a living cell, this can give rise to microdomain formation, protein sorting, vesicle/tubule budding, translocation through the cytoplasm, membrane fusion or compartmentalization. The challenge facing cell biologists interested in membrane organization, therefore, is to gain a detailed understanding of how lipid bilayers are affected by lipid and protein interactions, and how these interactions yield particular membrane behaviours, such as membrane sorting, budding and fusion events. In this session, we will discuss techniques (and their shortcomings) that are available for determining these interactions and their effects.

Among the most promising approaches for investigating membrane dynamics and organization is molecular imaging, which can show when and where genetically or biochemically modified molecules, signals or processes are formed, transformed and then consumed in space and time. The discovery of genetically encoded fluorescent tags has revolutionized this approach by making it possible to analyse the dynamics of proteins and organelles in living cells and to probe interactions between molecules in vivo. By combining in vivo microscopy with innovative imaging techniques, it is now possible to extract quantitative information about the morphological and biophysical properties of membranes. For example, until recently, the manner in which proteins are transported between compartments of the secretory or endocytic pathways was inferred largely from electron-microscopy studies of fixed cells and biochemical characterization of isolated organelles. However, recent imaging of the transport of green fluorescent protein (GFP)-tagged cargo proteins has shown that the carriers are larger and more pleiomorphic than previously thought, and that cytoskeletal components are important for determining a carrier's direction of motion. Furthermore, such imaging has revealed that the coat complexes responsible for budding off transport carriers, once thought of as rigid assemblies similar to viral capsid structures, undergo rapid exchange with pools of these complexes in the cytoplasm.

Various classic techniques, including fluorescence recovery after photobleaching (FRAP), fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS), are now experiencing a resurgence of interest as in vivo tools to probe cellular binding interactions, intracellular trafficking pathways and the existence of membrane microdomains. This renaissance is occurring because commercial instruments are now available for performing these techniques, and readily generated GFP fusion proteins allow the methods to be applied to numerous important problems. The first question posed in this session asks how these and other molecular imaging techniques can be used to address specific questions related to membrane organization, including the size and dynamics of rafts, protein-lipid and protein-protein interactions within membranes, and other features of membrane protein behaviour within cells (such as whether a protein or lipid is undergoing diffusion, flow, binding/dissociation reactions or percolation within a membrane).

Within this area, discussion will also focus on the limitations of these techniques. For example, there is currently no consensus on how to analyse and interpret FRAP data when binding contributes to recovery. How can we foster more accurate interpretations of FRAP in the presence of binding interactions or partitioning in and out of membranes? Would a combination of imaging strategies, such as FRAP in combination with total internal reflection microscopy (TIF-FM) for studying events at the cell surface, or photoactivation combined with FRET for studying protein-protein interactions, be helpful? We will also consider potential new methods for analysing molecular composition changes in membranes in the context of living cells, for detecting changes in the functional states of proteins on membranes, and for sensing physiologically relevant stimuli on membranes (chemical, electrical or mechanical). Such tools include the use of GFP variants engineered to respond to pH, halides, free Ca⁺² or redox potentials, or to undergo photochemical colour conversion. Alternatives to fluorescent proteins, including tetracysteine-biarsenical labelling, which have minimal steric bulk and are suitable for staining in electron microscopy, will also be discussed.

In addition to molecular imaging in vivo, the use of simple model membrane systems, such as giant vesicles, nanotubes or supported bilayers, are likely to be crucial for testing in a controlled environment the physical and biochemical parameters thought to control membrane organization and dynamics. The second question to be addressed in this session therefore considers how nanodevices and biomimetic systems can be used for testing and developing models of biological membranes. We will also discuss how these techniques can be used to define and analyse the combinations and conditions for complex relationships between different membrane components. Such information will be important for developing more refined hypotheses related to protein sorting, transport and fusion events of membranes. Biomimetic systems also have the potential to be introduced into living cells (as artificial membranes or organelles), allowing specific compartments or membrane trafficking pathways to be dissected and/or perturbed. This opens up the possibility of using biomimetic membrane complexes/structures to treat diseased or pathological states of cells, or to induce cell differentiation or dedifferentiation.

The session will conclude by considering the question of how to resolve the behaviour of specific proteins and complexes in membranes that are below the spatial resolution of fluorescence microscopy. Included in this discussion will be a consideration of the approaches for tracking specific molecules by single particle tracking, and how such an analysis can be used to better understand the organization, function and activity of membrane domains. It is possible that the analysis of membrane protein behaviour at this nanoscale will yield data that is inconsistent with larger, micrometre-scale observations obtained using FRAP, which samples large protein populations within membranes. How should we reconcile such potential differences?

Key questions

Moderator: Kai Simons

Membranes play a central role in cellular organization. How membranes perform their different cellular functions is an area of research still in its infancy, despite many years of work on the problem. About 30% of the proteome is located in the various cellular membranes, and many other proteins spend part of their lifetime in membranes. There are more than 500 individual lipid species in membranes, and their roles are poorly understood. Membranes not only surround cellular compartments; they are also able to compartmentalize themselves by forming dynamic microdomains that function in membrane trafficking and signal transduction. Membranes also play a very important role in cell polarization. A characteristic feature of polarized cells is the division of their cell surface into functionally distinct membrane domains. This requires intricate sorting machinery to deliver proteins and lipids to the right membrane domains. This sorting machinery is therefore fundamental to the generation of cellular polarity, as well as its maintenance in the face of continuous plasma membrane turnover by endocytosis.

Epithelial cells are an interesting example of membrane polarization. Their cell surface is divided into apical and basolateral membrane domains; the apical membrane forms a shield to protect the epithelium to hostilities of the outside environment. The unusual robustness of the apical membrane is largely due to its special lipid composition. It is strongly enriched in raft lipids, which have a propensity to form tightly packed membrane microdomains. The biogenesis of the apical membrane plays a key role in tube formation during organ development. It is also important to realize that the lipid bilayer can fold into different physical states - for example, different cubic symmetries - and this propensity is bound to be important for cell morphogenesis.

In this session, we will address the function of microdomains in membrane traffic and membrane interactions, and discuss the Golgi complex as an unusual example of membrane organization.

Key questions

Moderator: Lew Cantley

During the past 20 years, considerable progress has been made in understanding the complex interactions that occur between membrane lipids and proteins during receptor-dependent signalling to the cell interior. We have a greater knowledge of how receptor activation can lead to acute local stimulation of enzymes that convert lipid precursors, such as phosphatidylcholine and phosphatidylinositol, into lipid second messengers, such as diacylglycerol, phosphatidic acid or phosphatidylinositol-3,4,5-trisphosphate. However, this knowledge raises many new questions: How rapidly do the second messengers diffuse away from sites of generation? Can architectural elements of the membrane or local enzymatic activities that degrade the lipids limit the diffusion of the signalling lipids and thereby provide compartmentalized signalling? Does binding of signalling lipids to cytoskeleton-anchored proteins limit diffusion?

Cell-surface receptors typically undergo internalization following stimulation. This process is generally thought to be a mechanism of down-regulation. However, there is growing evidence that internalized receptors can continue to generate signals or even acquire new pathways for signalling after internalization. How might the distinct membrane environment in transport vesicles and early or late endosomes influence signalling by receptors?

Finally, as cell-surface membrane receptors are in intimate contact with the lipids of the plasma membrane bilayer, the local lipid environment of the plasma membrane is likely to play a crucial role in regulation of receptor function. For example, it is now clear that different membrane compartments are enriched in different subsets of phosphoinositides, which are known to influence the function of both intrinsic and extrinsic membrane proteins. In addition, acute hydrolysis of phosphatidylcholine to phosphatidic acid, hydrolysis of phosphatidylinositol-4,5-bisphosphate to diacylglycerol or phosphorylation of phosphatidylinositol-4,5-bisphosphate to produce phosphatidylinositol-3,4,5-trisphosphate, can potentially cause large local effects on receptor function. This question is intimately related to the questions raised above.

The development of new tools for monitoring local changes in lipids and proteins in live single cells is likely to provide new insight into these important questions.

Key questions

Moderator: Richard Anderson

Lipid domains are coherent regions of membrane formed by the lateral association of specific lipids that compartmentalize membrane functions. The most studied of these domains are the caveolae and their close relatives, lipid rafts, which depend on cholesterol for their structure and function. This session is focused on a discussion of the pathophysiology of caveolae and rafts.

The crucial role of cholesterol in maintaining the structure and function of caveolae/rafts raises important questions about the impact of damaged or modified cholesterol on the function of these domains. The principal altered form of cholesterol to be considered in this discussion is oxidized cholesterol. Oxysterols can arise through natural processes in the cell or be delivered to the cell from extracellular sources. Experiments in tissue-culture cells indicate that the introduction of oxidized cholesterol into cells has a major impact on signal transduction from caveolae as well as on membrane traffic. So, what are the sources of oxidized cholesterol, how do oxysterols affect the behaviour of lipid bilayers, why do they inhibit signal transduction from lipid domains and what mechanisms do cells use to deal with oxidized cholesterol?

There is considerable morphological and biochemical evidence that both normal and misfolded prions concentrate in caveolae lipid domains. Moreover, both cholesterol depletion of cells and elimination of the glycosylphosphatidylinositol anchor on the prion prevents the conversion of wild-type prion to the scrapie isoform. This suggests that caveolae are sites of scrapie prion formation. An important topic of discussion, therefore, is the function of caveolae, as well as other lipid domains, in the pathogenesis of amyloid diseases. Is it the ability of caveolae to internalize molecules, their ability to compartmentalize processing machinery or simply their ability to concentrate essential substrates that is important for the pathogenesis?

Caveolae were originally described in endothelial cells as membrane invaginations that were involved in endocytosis. The presence of flask-shaped membrane invaginations on both surfaces of these cells, and the sequential appearance of tracers injected into the blood, first in lumenal and then ablumenal invaginations, suggested that caveolae were able to transport molecules across endothelial cells. We have recently learned that viruses such as SV40 are able to commandeer this machinery to infect cells. Moreover, there are credible reports that bacteria can activate an endocytic pathway that involves membranes rich in caveolae. Virtually nothing is known about the molecular mechanisms of caveolae invagination, the mechanism of caveolae vesicle formation, the role of tyrosine kinases and protein kinase C in regulating this process or the function of caveolin-1. The endocytic machinery that drives uptake through these lipid domains has clear clinical relevancy but remains a complete mystery.

Key questions