Vikram Kjøller Bhatia

How curved membranes recruit amphipathic helices and protein anchoring motifs

Lipids and several specialized proteins are thought to be able to sense the curvature of membranes (MC). Here we used quantitative fluorescence microscopy to measure curvature-selective binding of amphipathic motifs on single liposomes 50-700 nm in diameter. Our results revealed that sensing is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity. We proposed a model based on curvature-induced defects in lipid packing that related these findings to lipid sorting and accurately predicted the existence of a new ubiquitous class of curvature sensors: membrane-anchored proteins. The fact that unrelated structural motifs such as alpha-helices and alkyl chains sense MC led us to propose that MC sensing is a generic property of curved membranes rather than a property of the anchoring molecules. We therefore anticipate that MC will promote the redistribution of proteins that are anchored in membranes through other types of hydrophobic moieties.

Amphipathic motifs in BAR domains are essential for membrane curvature sensing

BAR (Bin/Amphiphysin/Rvs) domains and amphipathic α‐helices (AHs) are believed to be sensors of membrane curvature thus facilitating the assembly of protein complexes on curved membranes. Here, we used quantitative fluorescence microscopy to compare the binding of both motifs on single nanosized liposomes of different diameters and therefore membrane curvature. Characterization of members of the three BAR domain families showed surprisingly that the crescent‐shaped BAR dimer with its positively charged concave face is not able to sense membrane curvature. Mutagenesis on BAR domains showed that membrane curvature sensing critically depends on the N‐terminal AH and furthermore that BAR domains sense membrane curvature through hydrophobic insertion in lipid packing defects and not through electrostatics. Consequently, amphipathic motifs, such as AHs, that are often associated with BAR domains emerge as an important means for a protein to sense membrane curvature. Measurements on single liposomes allowed us to document heterogeneous binding behaviour within the ensemble and quantify the influence of liposome polydispersity on bulk membrane curvature sensing experiments. The latter results suggest that bulk liposome‐binding experiments should be interpreted with great caution.

Membrane Curvature Induction and Tubulation Are Common Features of Synucleins and Apolipoproteins

Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, β-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.

Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules.

Sorting nexins (SNXs) are regulators of endosomal sorting. For the SNX‐BAR subgroup, a Bin/Amphiphysin/Rvs (BAR) domain is vital for formation/stabilization of tubular subdomains that mediate cargo recycling. Here, by analysing the in vitro membrane remodelling properties of all 12 human SNX‐BARs, we report that some, but not all, can elicit the formation of tubules with diameters that resemble sorting tubules observed in cells. We reveal that SNX‐BARs display a restricted pattern of BAR domain‐mediated dimerization, and by resolving a 2.8 Å structure of a SNX1‐BAR domain homodimer, establish that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR‐dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX‐BARs, and the formation of high order SNX‐BAR oligomers through selective ‘tip–loop’ interactions. Overall, the restricted and selective nature of these interactions provide a molecular explanation for how distinct SNX‐BAR‐decorated tubules are nucleated from the same endosomal vacuole, as observed in living cells. Our data provide insight into the molecular mechanism that generates and organizes the tubular endosomal network.

BAR domains, amphipathic helices and membrane-anchored proteins use the same mechanism to sense membrane curvature

The internal membranes of eukaryotic cells are all twists and bends characterized by high curvature. During recent years it has become clear that specific proteins sustain these curvatures while others simply recognize membrane shape and use it as “molecular information” to organize cellular processes in space and time. Here we discuss this new important recognition process termed membrane curvature sensing (MCS). First, we review a new fluorescence‐based experimental method that allows characterization of MCS using measurements on single vesicles and compare it to sensing assays that use bulk/ensemble liposome samples of different mean diameter. Next, we describe two different MCS protein motifs (amphipathic helices and BAR domains) and suggest that in both cases curvature sensitive membrane binding results from asymmetric insertion of hydrophobic amino acids in the lipid membrane. This mechanism can be extended to include the insertion of alkyl chain in the lipid membrane and consequently palmitoylated and myristoylated proteins are predicted to display similar curvature sensitive binding. Surprisingly, in all the aforementioned cases, MCS is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity as assumed so far. Finally, we integrate these new insights into the debate about which motifs are involved in sensing versus induction of membrane curvature and what role MCS proteins may play in biology.

Membrane Curvature Sensing by Amphipathic Helices A SINGLE LIPOSOME STUDY USING α-SYNUCLEIN AND ANNEXIN B12

Background: Amphipathic helices preferentially bind highly curved lipid membranes, providing a method of protein sorting. Results: Curvature sensing requires the insertion of hydrophobic residues and is modulated by electrostatic interactions. Conclusion: The relative strength of hydrophobic and electrostatic membrane interactions determines whether helix-containing proteins sense curvature. Significance: Sensing cannot be described through simple physicochemical properties but depends on the total sum of membrane interactions. Preferential binding of proteins on curved membranes (membrane curvature sensing) is increasingly emerging as a general mechanism whereby cells may effect protein localization and trafficking. Here we use a novel single liposome fluorescence microscopy assay to examine a common sensing motif, the amphipathic helix (AH), and provide quantitative measures describing and distinguishing membrane binding and sensing behavior. By studying two AH-containing proteins, α-synuclein and annexin B12, as well as a range of AH peptide mutants, we reveal that both the hydrophobic and hydrophilic faces of the helix greatly influence binding and sensing. Although increased hydrophobic and electrostatic interactions with the membrane both lead to greater densities of bound protein, the former yields membrane curvature-sensitive binding, whereas the latter is not curvature-dependent. However, the relative contributions of both components determine the sensing of AHs. In contrast, charge density in the lipid membrane seems important primarily in attracting AHs to the membrane but does not significantly influence sensing. These observations were made possible by the ability of our assay to distinguish within our samples liposomes with and without bound protein as well as the density of bound protein. Our findings suggest that the description of membrane curvature-sensing requires consideration of several factors such as short and long range electrostatic interactions, hydrogen bonding, and the volume and structure of inserted hydrophobic residues.

A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins.

The discovery of proteins that recognize membrane curvature created a paradigm shift by suggesting that membrane shape may act as a cue for protein localization that is independent of lipid or protein composition. Here we review recent data on membrane curvature sensing by three structurally unrelated motifs: BAR domains, amphipathic helices and membrane-anchored proteins. We discuss the conclusion that the curvature of the BAR dimer is not responsible for sensing and that the sensing properties of all three motifs can be rationalized by the physicochemical properties of the curved membrane itself. We thus anticipate that membrane curvature will promote the redistribution of proteins that are anchored in membranes through any type of hydrophobic moiety, a thesis that broadens tremendously the implications of membrane curvature for protein sorting, trafficking and signaling in cell biology.

PICK1 Deficiency Impairs Secretory Vesicle Biogenesis and Leads to Growth Retardation and Decreased Glucose Tolerance

Two lipid membrane sculpting BAR domain proteins, PICK1 and ICA69, play a key role early in the biogenesis of peptide hormone secretory vesicles and are critical for normal growth and metabolic homeostasis.

Delineation of the GPRC6A Receptor Signaling Pathways Using a Mammalian Cell Line Stably Expressing the Receptor

The GPRC6A receptor is a recently “deorphanized” class C G protein–coupled receptor. We and others have shown that this receptor is coactivated by basic l-α-amino acids and divalent cations, whereas other groups have also suggested osteocalcin and testosterone to be agonists. Likewise, the GPRC6A receptor has been suggested to couple to multiple G protein classes albeit via indirect methods. Thus, the exact ligand preferences and signaling pathways are yet to be elucidated. In the present study, we generated a Chinese hamster ovary (CHO) cell line that stably expresses mouse GPRC6A. In an effort to establish fully the signaling properties of the receptor, we tested representatives of four previously reported GPRC6A agonist classes for activity in the Gq, Gs, Gi, and extracellular-signal regulated kinase signaling pathways. Our results confirm that GPRC6A is activated by basic l-α-amino acids and divalent cations, and for the first time, we conclusively show that these responses are mediated through the Gq pathway. We were not able to confirm previously published data demonstrating Gi- and Gs-mediated signaling; neither could we detect agonistic activity of testosterone and osteocalcin. Generation of the stable CHO cell line with robust receptor responsiveness and optimization of the highly sensitive homogeneous time resolved fluorescence technology allow fast assessment of Gq activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for GPRC6A is thus useful in high-throughput screening for novel pharmacological tool compounds, which are necessary to unravel the physiologic function of the receptor.

Membrane Localization is Critical for Activation of the PICK1 BAR Domain

The PSD‐95/Discs‐large/ZO‐1 homology (PDZ) domain protein, protein interacting with C kinase 1 (PICK1) contains a C‐terminal Bin/amphiphysin/Rvs (BAR) domain mediating recognition of curved membranes; however, the molecular mechanisms controlling the activity of this domain are poorly understood. In agreement with negative regulation of the BAR domain by the N‐terminal PDZ domain, PICK1 distributed evenly in the cytoplasm, whereas truncation of the PDZ domain caused BAR domain‐dependent redistribution to clusters colocalizing with markers of recycling endosomal compartments. A similar clustering was observed both upon truncation of a short putative α‐helical segment in the linker between the PDZ and the BAR domains and upon coexpression of PICK1 with a transmembrane PDZ ligand, including the alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor GluR2 subunit, the GluR2 C‐terminus transferred to the single transmembrane protein Tac or the dopamine transporter C‐terminus transferred to Tac. In contrast, transfer of the GluR2 C‐terminus to cyan fluorescent protein, a cytosolic protein, did not elicit BAR domain‐dependent clustering. Instead, localizing PICK1 to the membrane by introducing an N‐terminal myristoylation site produced BAR domain‐dependent, but ligand‐independent, PICK1 clustering. The data support that in the absence of PDZ ligand, the PICK1 BAR domain is inhibited through a PDZ domain‐dependent and linker‐dependent mechanism. Moreover, they suggest that unmasking of the BAR domain’s membrane‐binding capacity is not a consequence of ligand binding to the PDZ domain per se but results from, and coincides with, recruitment of PICK1 to a membrane compartment.