The Movement of Goods
Around the Cell
A biologist and a physicist collaborate on a decade-long exploration of the physical parameters of membrane traffic
in eukaryotic cells.
In prokaryotic cells, simple diffusion is largely responsible for getting nutrients to where they need to be and for removing waste products. But eukaryotes, which are much more complex, require a specialized mass-transit system. This system consists of membrane-bound structures called transport carriers that ferry cargo into, out of, and around the cell. Over the past decade, our interest has centered on this system, particularly on the interplay between the biophysical properties of the membranes and the way in which these properties are exploited by specific biological molecules to construct and direct this transport system. It is an ideal topic for collaboration between a biologist and a physicist.
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The other of us (Patricia) was trained as an experimental physicist in soft matter and had worked initially on the physical aspects of liquid crystals. The intrinsic nonequilibrium nature of biological membranes captured Patricia’s interest, leading her to study, in collaboration with Jacques Prost, the fluctuations of model lipid membranes in the presence of membrane proteins. Patricia had already known of Bruno’s findings on Rab6-decorated membrane tubules before we met.
Almost immediately, we agreed on our joint goal: we would develop an in vitro assay that mimics the initial steps of intracellular transport. In particular, we would concentrate on the creation of the tubular carriers and the membrane deformation involved in their formation. We recruited a student, Aurélien Roux, to work with us. Aurélien, who now has his own lab in the biochemistry department of the University of Geneva, would generate tubular carriers by attaching biotinylated kinesin motor proteins to biotinylated lipid membranes using 100-nm polystyrene beads coated with streptavidin. The membranes known as giant unilamellar vesicles (GUVs) provide a simplified model of a cell membrane lipid bilayer. (See figure 1, below.) When incubated with microtubules and ATP in small chambers, GUVs did indeed give rise to membrane tubes and to complex tubular networks that could be visualized by confocal microscopy.2 (See photomicrograph on opposite page.) This experiment was the first demonstration that the force generated by kinesins was sufficient to pull a membrane tube from a membrane reservoir. Remarkably, as shown by transmission electron microscopy, the tubes that were pulled from GUVs made of egg phosphatidylcholine (EPC) had a constant diameter of 40±10 nm, a value close to that estimated for tubular transport carriers operating, for example, between the Golgi and the plasma membrane in vivo.
Using this minimal model, we also set out to investigate physical parameters involved in the early steps of intracellular transport, including membrane curvature, membrane bending rigidity, and membrane tension.
Because of the small diameter of actual transport carriers inside cells (typically 40–100 nm), they represent highly curved structures in comparison with the membrane from which they originate, which can be viewed as “flat.” During the early stages of vesicle formation from cell organelles, membrane proteins and lipids are sorted, ensuring efficient and accurate transport between cell compartments and the maintenance of homeostasis in organelle membranes. By 2004, the sorting of proteins had already been well described, but lipid sorting was much less clearly understood. To investigate constraints on lipid sorting, we pulled tubes from GUVs that were prepared from ternary mixtures of brain sphingomyelin, cholesterol, and dioleoylphosphatidylcholine (DOPC), representing the three major lipid components of the external leaflet of the plasma membrane. Depending on the relative proportion of the three lipids, they either mix to form a single homogeneous phase, or they demix and preferentially segregate in different phases. In the latter case, two phases coexist, a liquid disordered phase enriched in DOPC, and a liquid ordered phase enriched in cholesterol and sphingomyelin. The disordered phase is so called because the lipid tails in these patches of membrane have kinks and are disorganized so they do not pack together as closely as in the ordered phase.
The force required to pull a tube is proportional to the bending rigidity and the tension of the membrane.5 Using optical tweezers coupled to a micropipette system, we measured the bending rigidity of the ordered and disordered phases. (See figure 2.) Membranes in ordered phase are about twice as rigid as membranes in the more loosely packed disordered phase.6 Given this, we predicted that lipids of the ordered phase should be excluded from tube formation, to reduce the energy cost needed to bend the membrane into tubes. This is exactly what we observed. In phase-separated vesicles, tubes were preferentially pulled out from the disordered phase; when pulled from homogeneous vesicles, the tubes were enriched in lipids of the disordered phase (DOPC). These experiments provided the first direct demonstration that lipid sorting can occur during the formation of highly curved membrane tubes.6 There are two hypotheses to explain lipid sorting during vesicle formation: either the vesicle is formed from domains of the donor membrane where the lipids are already segregated, or lipid sorting occurs at the same time as the vesicle forms. Our in vitro experiments support the latter hypothesis, namely the dynamic sorting of lipids.
Because the tool set available to biologists to study cellular function have been predominantly biochemical techniques, the story of how cells work is dominated by protein interactions. Recently, researchers have begun to appreciate that physical properties play a much bigger role in cellular activities than was previously suspected. In fact, it is the ways in which a cell takes advantage of physical and biochemical properties together that has interested us most.
Aurélien had also observed that when phase separation of lipids occurs in the tubes, fission events take place at the boundary between ordered and disordered domains.6 It turns out that these observations are consistent with a theoretical analysis in which membrane rupture was predicted to originate from the difference in surface energy between the two phases, caused by their different composition. Much as nonmiscible liquids minimize their surface of contact, the lipids in bidimensional lipid domains minimize the length of their contact, resulting in a constricting force called line tension.8 Since the lipids in cell membranes are likely close to phase separation, these results raised the interesting prospect that the role of the numerous proteins implicated in sorting and fission events in vivo could be to trigger phase separation in membrane lipids, either by clustering specific lipids or by inducing membrane tubulation.9
Mechanoenzymes, including dynamin, are known to contribute to membrane fission. Dynamin is a large GTPase that polymerizes into a helical collar at the neck of endocytic buds, and induces the formation of endocytic vesicles through neck fission. Our work on line tension–induced membrane fission motivated us to explore the role of membrane curvature in the helical assembly of dynamin. Using a combination of confocal microscopy and optical tweezers, we discovered that membrane curvature triggers dynamin assembly, and thus the precise timing of the detachment of endocytic vesicles from the membrane.10
The functions of proteins that sense or induce membrane curvature have received considerable attention recently because of the importance of these phenomena during the formation of vesicles and tubular carriers involved in intracellular transport. During formation, vesicles and tubules are surrounded by coat proteins, such as the COPI coatomer, which are recruited to the site by activated coat-recruitment proteins such as Arf1 (ADP-ribosylation factor), a small G protein that binds to Golgi membranes as the first step in coat assembly. Several proteins involved in vesicle formation, including the ArfGAP1 protein, contain a lipid-binding structural motif, named ALPS, that senses membrane curvature.
Our assay system was ideal for studying the spatial distribution of proteins between curved and noncurved membrane regions. Ernesto Ambroggio, a postdoc, worked with Benoît to compare the sensitivity to curvature of Arf1 and ArfGAP1. Arf1 bound almost equally well to the GUV membrane and to a tube pulled with kinesin motors or optical tweezers. Thus, Arf1 binding is, at most, only weakly sensitive to membrane curvature. In contrast, ArfGAP1 did not bind to the GUV at all. A curvature threshold was found for its binding to the membrane tubes: almost no binding was detected on tubes with a radius above 35±5 nm, while below this critical radius, ArfGAP1 density on the membrane increased linearly.13
Recently, Benoît has used a similar approach to study amphiphysin, a protein with a crescent-shaped binding domain that is involved in the generation of clathrin-coated vesicles. He showed that this protein has a dual behavior: at low concentration, its levels in membranes depend on membrane curvature—reminiscent of ArfGAP1—but it cannot deform the membrane. At high concentration, amphiphysin constricts a membrane tube, independently of the membrane tension (Sorre et al., submitted).
Our collaboration, combining biophysics and cell biology, and illuminated by our interactions with theoretical physicists, has been particularly fruitful and gratifying over the past 10 years. Right now we are planning to deepen our partnership still further with an ambitious project aimed at understanding how different classes of actin-based motors of the myosin family function in membrane trafficking and membrane dynamics. This project will exploit the minimal in vitro system developed in our laboratories.
Over the last decade we have challenged one another and generated reciprocal interests: Bruno has become more receptive to and interested in physics concepts, and Patricia continues to explore projects more related to cell biology. Based on the results of our cross-disciplinary collaboration, we advise others to embrace the approach. The challenges and rewards of considering alternative perspectives will add exciting new dimensions to your research design and experimentation.
Patricia Bassereau and F1000 Member References:
|This article is adapted from a review in F1000 Biology Reports, DOI:10.3410/B3-7 (open access at http://bit.ly/hwBycR). For citation purposes, please refer to that version.|