Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson's fluid-mosaic model.

The recent rapid accumulation of knowledge on the dynamics and structure of the plasma membrane has prompted major modifications of the textbook fluid-mosaic model. However, because the new data have been obtained in a variety of research contexts using various biological paradigms, the impact of the critical conceptual modifications on biomedical research and development has been limited. In this review, we try to synthesize our current biological, chemical, and physical knowledge about the plasma membrane to provide new fundamental organizing principles of this structure that underlie every molecular mechanism that realizes its functions. Special attention is paid to signal transduction function and the dynamic aspect of the organizing principles. We propose that the cooperative action of the hierarchical three-tiered mesoscale (2-300 nm) domains--actin-membrane-skeleton induced compartments (40-300 nm), raft domains (2-20 nm), and dynamic protein complex domains (3-10 nm)--is critical for membrane function and distinguishes the plasma membrane from a classical Singer-Nicolson-type model.

A thermodynamic analysis of CLC transporter dimerization in lipid bilayers.

The CLC-ec1 chloride/proton antiporter is a membrane-embedded homodimer with subunits that can dissociate and associate, but the thermodynamic driving forces favor the assembled dimer at biological densities. Yet, the physical reasons for this stability are confounding as dimerization occurs via the burial of hydrophobic interfaces away from the lipid solvent. For binding of nonpolar surfaces in aqueous solution, the driving force is often attributed to the hydrophobic effect, but this should not apply in the membrane since there is very little water. To investigate this further, we quantified the thermodynamic changes associated with CLC dimerization in membranes by carrying out a van 't Hoff analysis of the temperature dependency of the free energy of dimerization, ΔG°. To ensure that the reaction reached equilibrium at different temperatures, we utilized a Förster resonance energy transfer assay to report on relaxation kinetics of subunit exchange as a function of temperature. Equilibration times were then applied to measure CLC-ec1 dimerization isotherms at different temperatures using the single-molecule subunit-capture photobleaching analysis approach. The results demonstrate that the dimerization free energy of CLC in Escherichia coli-like membranes exhibits a nonlinear temperature dependency corresponding to a large, negative change in heat capacity, a signature of solvent ordering effects such as the hydrophobic effect. Consolidating this with our previous molecular analyses suggests that the nonbilayer defect required to solvate the monomeric state is one source of the observed change in heat capacity and indicates the existence of a generalizable driving force for protein association in membranes.

Fluorescent human RPA to track assembly dynamics on DNA.

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. The fluorescent human RPA described here will enable high-resolution structure-function analysis of RPA-ssDNA interactions.

Mechanistic insight into AP-endonuclease 1 cleavage of abasic sites at stalled replication fork mimics.

Many types of damage, including abasic sites, block replicative DNA polymerases causing replication fork uncoupling and generating ssDNA. AP-Endonuclease 1 (APE1) has been shown to cleave abasic sites in ssDNA. Importantly, APE1 cleavage of ssDNA at a replication fork has significant biological implications by generating double strand breaks that could collapse the replication fork. Despite this, the molecular basis and efficiency of APE1 processing abasic sites at replication forks remain elusive. Here, we investigate APE1 cleavage of abasic substrates that mimic APE1 interactions at stalled replication forks or gaps. We determine that APE1 has robust activity on these substrates, like dsDNA, and report rates for cleavage and product release. X-ray structures visualize the APE1 active site, highlighting an analogous mechanism is used to process ssDNA substrates as canonical APE1 activity on dsDNA. However, mutational analysis reveals R177 to be uniquely critical for the APE1 ssDNA cleavage mechanism. Additionally, we investigate the interplay between APE1 and Replication Protein A (RPA), the major ssDNA-binding protein at replication forks, revealing that APE1 can cleave an abasic site while RPA is still bound to the DNA. Together, this work provides molecular level insights into abasic ssDNA processing by APE1, including the presence of RPA.

Nanoclusters of GPI-anchored proteins are formed by cortical actin-driven activity.

Several cell-surface lipid-tethered proteins exhibit a concentration-independent, cholesterol-sensitive organization of nanoscale clusters and monomers. To understand the mechanism of formation of these clusters, we investigate the spatial distribution and steady-state dynamics of fluorescently tagged GPI-anchored protein nanoclusters using high-spatial and temporal resolution FRET microscopy. These studies reveal a nonrandom spatial distribution of nanoclusters, concentrated in optically resolvable domains. Monitoring the dynamics of recovery of fluorescence intensity and anisotropy, we find that nanoclusters are immobile, and the dynamics of interconversion between nanoclusters and monomers, over a range of temperatures, is spatially heterogeneous and non-Arrhenius, with a sharp crossover coinciding with a reduction in the activity of cortical actin. Cholesterol depletion perturbs cortical actin and the spatial scale and interconversion dynamics of nanoclusters. Direct perturbations of cortical actin activity also affect the construction, dynamics, and spatial organization of nanoclusters. These results suggest a unique mechanism of complexation of cell-surface molecules regulated by cortical actin activity.

The FtsLB subcomplex of the bacterial divisome is a tetramer with an uninterrupted FtsL helix linking the transmembrane and periplasmic regions.

In Escherichia coli, FtsLB plays a central role in the initiation of cell division, possibly transducing a signal that will eventually lead to the activation of peptidoglycan remodeling at the forming septum. The molecular mechanisms by which FtsLB operates in the divisome, however, are not understood. Here, we present a structural analysis of the FtsLB complex, performed with biophysical, computational, and in vivo methods, that establishes the organization of the transmembrane region and proximal coiled coil of the complex. FRET analysis in vitro is consistent with formation of a tetramer composed of two FtsL and two FtsB subunits. We predicted subunit contacts through co-evolutionary analysis and used them to compute a structural model of the complex. The transmembrane region of FtsLB is stabilized by hydrophobic packing and by a complex network of hydrogen bonds. The coiled coil domain probably terminates near the critical constriction control domain, which might correspond to a structural transition. The presence of strongly polar amino acids within the core of the tetrameric coiled coil suggests that the coil may split into two independent FtsQ-binding domains. The helix of FtsB is interrupted between the transmembrane and coiled coil regions by a flexible Gly-rich linker. Conversely, the data suggest that FtsL forms an uninterrupted helix across the two regions and that the integrity of this helix is indispensable for the function of the complex. The FtsL helix is thus a candidate for acting as a potential mechanical connection to communicate conformational changes between periplasmic, membrane, and cytoplasmic regions.

Raft-based interactions of gangliosides with a GPI-anchored receptor.

Gangliosides, glycosphingolipids containing one or more sialic acid(s) in the glyco-chain, are involved in various important physiological and pathological processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or preparations. Single-fluorescent-molecule imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59's transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside molecules were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities.

Membrane mechanisms for signal transduction: the coupling of the meso-scale raft domains to membrane-skeleton-induced compartments and dynamic protein complexes.

Virtually all biological membranes on earth share the basic structure of a two-dimensional liquid. Such universality and peculiarity are comparable to those of the double helical structure of DNA, strongly suggesting the possibility that the fundamental mechanisms for the various functions of the plasma membrane could essentially be understood by a set of simple organizing principles, developed during the course of evolution. As an initial effort toward the development of such understanding, in this review, we present the concept of the cooperative action of the hierarchical three-tiered meso-scale (2-300 nm) domains in the plasma membrane: (1) actin membrane-skeleton-induced compartments (40-300 nm), (2) raft domains (2-20 nm), and (3) dynamic protein complex domains (3-10nm). Special attention is paid to the concept of meso-scale domains, where both thermal fluctuations and weak cooperativity play critical roles, and the coupling of the raft domains to the membrane-skeleton-induced compartments as well as dynamic protein complexes. The three-tiered meso-domain architecture of the plasma membrane provides an excellent perspective for understanding the membrane mechanisms of signal transduction.

Wrapping of single-stranded DNA by Replication Protein A and modulation through phosphorylation.

Single-stranded DNA (ssDNA) intermediates, which emerge during DNA metabolic processes are shielded by Replication Protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper, directing the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent specificity requires a comprehensive understanding of the structural conformation of ssDNA when bound to RPA. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and Double Electron-Electron Resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

Structural basis of pH-dependent activation in a CLC transporter.

CLCs are dimeric chloride channels and anion/proton exchangers that regulate processes such as muscle contraction and endo-lysosome acidification. Common gating controls their activity; its closure simultaneously silences both protomers, and its opening allows them to independently transport ions. Mutations affecting common gating in human CLCs cause dominant genetic disorders. The structural rearrangements underlying common gating are unknown. Here, using single-particle cryo-electron microscopy, we show that the prototypical Escherichia coli CLC-ec1 undergoes large-scale rearrangements in activating conditions. The slow, pH-dependent remodeling of the dimer interface leads to the concerted opening of the intracellular H+ pathways and is required for transport. The more frequent formation of short water wires in the open H+ pathway enables Cl- pore openings. Mutations at disease-causing sites favor CLC-ec1 activation and accelerate common gate opening in the human CLC-7 exchanger. We suggest that the pH activation mechanism of CLC-ec1 is related to the common gating of CLC-7.

A thermodynamic analysis of CLC transporter dimerization in lipid bilayers.

The CLC-ec1 chloride/proton antiporter is a membrane embedded homodimer where subunits can dissociate and associate, but the thermodynamic driving forces favor the assembled form at biological densities. Yet, the physical reasons for this stability are confounding since binding occurs via the burial of hydrophobic protein interfaces yet the hydrophobic effect should not apply since there is little water within the membrane. To investigate this further, we quantified the thermodynamic changes associated with CLC dimerization in membranes by carrying out a van 't Hoff analysis of the temperature dependency of the free energy of dimerization, ΔG° . To ensure that the reaction reached equilibrium under changing conditions, we utilized a Förster Resonance Energy Transfer based assay to report on the relaxation kinetics of subunit exchange as a function of temperature. These equilibration times were then applied to measure CLC-ec1 dimerization isotherms as a function of temperature using the single-molecule subunit-capture photobleaching analysis approach. The results demonstrate that the dimerization free energy of CLC in E. coli membranes exhibits a non-linear temperature dependency corresponding to a large, negative change in heat capacity, a signature of solvent ordering effects including the hydrophobic effect. Consolidating this with our previous molecular analyses suggests that the non-bilayer defect required to solvate the monomeric state is the molecular source of this large change in heat capacity and is a major and generalizable driving force for protein association in membranes.

Fluorescent human RPA to track assembly dynamics on DNA.

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described.

An Aurora B-RPA signaling axis secures chromosome segregation fidelity.

Errors in chromosome segregation underlie genomic instability associated with cancers. Resolution of replication and recombination intermediates and protection of vulnerable single-stranded DNA (ssDNA) intermediates during mitotic progression requires the ssDNA binding protein Replication Protein A (RPA). However, the mechanisms that regulate RPA specifically during unperturbed mitotic progression are poorly resolved. RPA is a heterotrimer composed of RPA70, RPA32 and RPA14 subunits and is predominantly regulated through hyperphosphorylation of RPA32 in response to DNA damage. Here, we have uncovered a mitosis-specific regulation of RPA by Aurora B kinase. Aurora B phosphorylates Ser-384 in the DNA binding domain B of the large RPA70 subunit and highlights a mode of regulation distinct from RPA32. Disruption of Ser-384 phosphorylation in RPA70 leads to defects in chromosome segregation with loss of viability and a feedback modulation of Aurora B activity. Phosphorylation at Ser-384 remodels the protein interaction domains of RPA. Furthermore, phosphorylation impairs RPA binding to DSS1 that likely suppresses homologous recombination during mitosis by preventing recruitment of DSS1-BRCA2 to exposed ssDNA. We showcase a critical Aurora B-RPA signaling axis in mitosis that is essential for maintaining genomic integrity.

Homodecameric Rad52 promotes single-position Rad51 nucleation in homologous recombination.

Homologous recombination (HR) is a pathway for the accurate repair of double-stranded DNA breaks. These breaks are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Saccharomyces cerevisiae Rad52 is a mediator protein that promotes HR by facilitating formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Canonically, Rad52 has been described to function by displacing RPA to promote Rad51 binding. However, in vitro, Rad51 readily forms a filament by displacing RPA in the absence of Rad52. Yet, in vivo, Rad52 is essential for HR. Here, we resolve how Rad52 functions as a mediator using single-particle cryo-electron microscopy and biophysical approaches. We show that Rad52 functions as a homodecamer and catalyzes single-position nucleation of Rad51. The N-terminal half of Rad52 is a well-ordered ring, while the C-terminal half is disordered. An intrinsic asymmetry within Rad52 is observed, where one or a few of the C-terminal halves interact with the ordered N-terminal ring. Within the C-terminal half, we identify two conserved charged patches that harbor the Rad51 and RPA interacting motifs. Interactions between these two charged patches regulate a ssDNA binding. These features drive Rad51 binding to a single position on the Rad52 decameric ring. We propose a Rad52 catalyzed single-position nucleation model for the formation of pre-synaptic Rad51 filaments in HR.

Yeast Rad52 is a homodecamer and possesses BRCA2-like bipartite Rad51 binding modes.

Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.

Rtt105 regulates RPA function by configurationally stapling the flexible domains.

Replication Protein A (RPA) is a heterotrimeric complex that binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins. When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting the facilitated exchange activity of RPA. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely binding to RPA-interacting proteins, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states.

Over two-thirds of integral membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study, we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid a thinned-membrane defect formed by hydrophobic mismatch at their exposed dimerization interfaces. In this defect, lipids are strongly tilted and less densely packed than in the bulk, with a larger degree of entanglement between opposing leaflets and greater water penetration into the bilayer interior. Dimerization restores the membrane to a near-native state and therefore, appears to be driven by the larger free-energy cost of lipid solvation of the dissociated protomers. Supporting this theory, we demonstrate that addition of short-chain lipids strongly shifts the dimerization equilibrium toward the monomeric state, and show that the cause of this effect is that these lipids preferentially solvate the defect. Importantly, we show that this shift requires only minimal quantities of short-chain lipids, with no measurable impact on either the macroscopic physical state of the membrane or the protein's biological function. Based on these observations, we posit that free-energy differentials for local lipid solvation define membrane-protein association equilibria. With this, we argue that preferential lipid solvation is a plausible cellular mechanism for lipid regulation of oligomerization processes, as it can occur at low concentrations and does not require global changes in membrane properties.

A cell's outer membrane is made of molecules called lipids, which band together to form a flexible thin film, just two molecules thick. This membrane is dotted with proteins that transport materials in to and out of cells. Most of these membrane proteins join with other proteins to form structures known as oligomers. Except, how membrane-bound proteins assemble into oligomers ­ the physical forces driving these molecules to take shape ­ remains unclear. This is partly because the structural, physical and chemical properties of fat-like lipid membranes are radically different to the cell's watery interior. Consequently, the conditions under which membrane oligomers form are distinct from those surrounding proteins inside cells. Membrane proteins are also more difficult to study and characterize than water-soluble proteins inside the cell, and yet many therapeutic drugs such as antibiotics specifically target membrane proteins. Overall, our understanding of how the unique properties of lipid membranes affect the formation of protein structures embedded within, is lacking and warrants further investigation. Now, Chadda, Bernhardt et al. focused on one membrane protein, known as CLC, which tends to exist in pairs ­ or dimers. To understand why these proteins form dimers (a process called dimerization) Chadda, Bernhardt et al. first used computer simulations, and then validated the findings in experimental tests. These complementary approaches demonstrated that the main reason CLC proteins 'dimerize' lies in their interaction with the lipid membrane, and not the attraction of one protein to its partner. When CLC proteins are on their own, they deform the surrounding membrane and create structural defects that put the membrane under strain. But when two CLC proteins join as a dimer, this membrane strain disappears ­ making dimerization the more stable and energetically favorable option. Chadda, Bernhardt et al. also showed that with the addition of a few certain lipids, specifically smaller lipids, cell membranes become more tolerant of protein-induced structural changes. This might explain how cells could use various lipids to fine-tune the activity of membrane proteins by controlling how oligomers form. However, the theory needs to be examined further. Altogether, this work has provided fundamental insights into the physical forces shaping membrane-bound proteins, relevant to researchers studying cell biology and pharmacology alike.

Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles.

Using quantitative light microscopy and a modified immunoelectron microscopic technique, we have characterized the entry pathway of the cholera toxin binding subunit (CTB) in primary embryonic fibroblasts. CTB trafficking to the Golgi complex was identical in caveolin-1null (Cav1-/-) mouse embryonic fibroblasts (MEFs) and wild-type (WT) MEFs. CTB entry in the Cav1-/- MEFs was predominantly clathrin and dynamin independent but relatively cholesterol dependent. Immunoelectron microscopy was used to quantify budded and surface-connected caveolae and to identify noncaveolar endocytic vehicles. In WT MEFs, a small fraction of the total Cav1-positive structures were shown to bud from the plasma membrane (2% per minute), and budding increased upon okadaic acid or lactosyl ceramide treatment. However, the major carriers involved in initial entry of CTB were identified as uncoated tubular or ring-shaped structures. These carriers contained GPI-anchored proteins and fluid phase markers and represented the major vehicles mediating CTB uptake in both WT and caveolae-null cells.

Occupancy distributions of membrane proteins in heterogeneous liposome populations.

Studies of membrane protein structure and function often rely on reconstituting the protein into lipid bilayers through the formation of liposomes. Many measurements conducted in proteoliposomes, e.g. transport rates, single-molecule dynamics, monomer-oligomer equilibrium, require some understanding of the occupancy statistics of the liposome population for correct interpretation of the results. In homogenous liposomes, this is easy to calculate as the act of protein incorporation can be described by the Poisson distribution. However, in reality, liposomes are heterogeneous, which alters the statistics of occupancy in several ways. Here, we determine the liposome occupancy distribution for membrane protein reconstitution while considering liposome size heterogeneity. We calculate the protein occupancy for a homogenous population of liposomes with radius r = 200 nm, representing an idealization of vesicles extruded through 400 nm pores and compare it to the right-skewed distribution of 400 nm 2:1 POPE:POPG vesicles. As is the case for E. coli polar lipids, this synthetic composition yields a sub-population of small liposomes, 25-30 nm in radius with a long tail of larger vesicles. Previously published microscopy data of the co-localization of the CLC-ec1 Cl-/H+ transporter with liposomes, and vesicle occupancy measurements using functional transport assays, shows agreement with the heterogeneous 2:1 POPE:POPG population. Next, distributions of 100 nm and 30 nm extruded 2:1 POPE:POPG liposomes are measured by cryo-electron microscopy, demonstrating that extrusion through smaller pores does not shift the peak, but reduces polydispersity arising from large liposomes. Single-molecule photobleaching analysis of CLC-ec1-Cy5 shows the 30 nm extruded population increases the 'Poisson-dilution' range, reducing the probability of vesicles with more than one protein at higher protein/lipid densities. These results demonstrate that the occupancy distributions of membrane proteins into vesicles can be accurately predicted in heterogeneous populations with experimental knowledge of the liposome size distribution. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3'-kinase-dependent machinery.

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3'-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.