Jennifer L. Gallop

Membrane curvature and mechanisms of dynamic cell membrane remodelling

Membrane curvature is no longer seen as a passive consequence of cellular activity but an active means to create membrane domains and to organize centres for membrane trafficking. Curvature can be dynamically modulated by changes in lipid composition, the oligomerization of curvature scaffolding proteins and the reversible insertion of protein regions that act like wedges in membranes. There is an interplay between curvature-generating and curvature-sensing proteins during vesicle budding. This is seen during vesicle budding and in the formation of microenvironments. On a larger scale, membrane curvature is a prime player in growth, division and movement.

Mechanism of endophilin N-BAR domain-mediated membrane curvature.

Endophilin‐A1 is a BAR domain‐containing protein enriched at synapses and is implicated in synaptic vesicle endocytosis. It binds to dynamin and synaptojanin via a C‐terminal SH3 domain. We examine the mechanism by which the BAR domain and an N‐terminal amphipathic helix, which folds upon membrane binding, work as a functional unit (the N‐BAR domain) to promote dimerisation and membrane curvature generation. By electron paramagnetic resonance spectroscopy, we show that this amphipathic helix is peripherally bound in the plane of the membrane, with the midpoint of insertion aligned with the phosphate level of headgroups. This places the helix in an optimal position to effect membrane curvature generation. We solved the crystal structure of rat endophilin‐A1 BAR domain and examined a distinctive insert protruding from the membrane interaction face. This insert is predicted to form an additional amphipathic helix and is important for curvature generation. Its presence defines an endophilin/nadrin subclass of BAR domains. We propose that N‐BAR domains function as low‐affinity dimers regulating binding partner recruitment to areas of high membrane curvature.

Evolving nature of the AP2 α-appendage hub during clathrin-coated vesicle endocytosis

Clathrin‐mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoating. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein interaction hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to distinct subdomains on α‐appendages, called ‘top’ and side’ sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high‐affinity reversible interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered α(appendage)‐hubs have altered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated vesicle, most appendage binding partners are absent and thus the functional status of the appendage domain as an interaction hub is temporal and transitory giving directionality to vesicle assembly.

UBE2S drives elongation of K11-linked ubiquitin chains by the Anaphase-Promoting Complex

The Anaphase-Promoting Complex (APC) is an E3 ubiquitin ligase that regulates mitosis and G1 by sequentially targeting cell-cycle regulators for ubiquitination and proteasomal degradation. The mechanism of ubiquitin chain formation by APC and the resultant chain topology remains controversial. By using a single-lysine APC substrate to dissect the topology of ubiquitinated substrates, we find that APC-catalyzed ubiquitination has an intrinsic preference for the K11 linkage of ubiquitin that is essential for substrate degradation. K11 specificity is determined by an E2 enzyme, UBE2S/E2-EPF, that elongates ubiquitin chains after the substrates are pre-ubiquitinated by UbcH10 or UbcH5. UBE2S copurifies with APC; dominant-negative Ube2S slows down APC substrate degradation in functional cell-cycle extracts. We propose that Ube2S is a critical, unique component of the APC ubiquitination pathway.

Self-Assembly of Filopodia-Like Structures on Supported Lipid Bilayers

Pointing the Finger Filopodia are finger-like structures containing parallel bundles of actin filaments that are central to eukaryotic cell motility in a variety of contexts. K. Lee et al. (p. 1341) reconstituted filopodia-like structures that grow from supported lipid bilayers to explore filopodia assembly. A structural transition from actin networks to parallel bundles was observed that mediated self-assembly of filopodia-tip complexes on the membranes. Actin bundle structures formed on lipid bilayers give insight into formation of the finger-like structures involved in cell migration. Filopodia are finger-like protrusive structures, containing actin bundles. By incubating frog egg extracts with supported lipid bilayers containing phosphatidylinositol 4,5 bisphosphate, we have reconstituted the assembly of filopodia-like structures (FLSs). The actin assembles into parallel bundles, and known filopodial components localize to the tip and shaft. The filopodia tip complexes self-organize—they are not templated by preexisting membrane microdomains. The F-BAR domain protein toca-1 recruits N-WASP, followed by the Arp2/3 complex and actin. Elongation proteins, Diaphanous-related formin, VASP, and fascin are recruited subsequently. Although the Arp2/3 complex is required for FLS initiation, it is not essential for elongation, which involves formins. We propose that filopodia form via clustering of Arp2/3 complex activators, self-assembly of filopodial tip complexes on the membrane, and outgrowth of actin bundles.

Identification of factors associated with the diagnosis of delirium in elderly hospitalized patients.

We analyzed factors associated with the discharge diagnosis of delirium among 1,285 patients admitted to a major teaching hospital during a 2‐year period, developed a model to classify the risk of developing delirium on the basis of clinical and diagnostic data, and tested the model on 471 patients admitted during the subsequent year. Using the multivariate technique of recursive partitioning, we identified four factors that distinguished 80% of all cases of delirium: 1) a urinary tract infection at any time during the hospital stay (odds ratio = 3.1; 95% confidence interval = 2.02–4.58); 2) no urinary tract infection, but low serum albumin on admission (odds ratio = 2.4; 95% confidence interval = 1.43–3.99); 3) neither urinary tract infection nor low serum albumin, but elevated white blood cell count on admission (odds ratio = 1.99; 95% confidence interval = 1.18‐3.37); 4) none of these risk factors, but proteinuria on admission (odds ratio = 1.82; 95% confidence interval = 2.25–2.66). Patients without any of these four risk factors had the lowest probability of developing delirium during their hospital stay. Among individuals with delirium, in‐hospital mortality and hospital charges were higher. The model developed accurately characterized the risk of delirium when it was tested on patients admitted to the same hospital during the subsequent year.

Endophilin and CtBP/BARS are not acyl transferases in endocytosis or Golgi fission

Endophilins have been proposed to have an enzymatic activity (a lysophosphatidic acid acyl transferase or LPAAT activity) that can make phosphatidic acid in membranes. This activity is thought to change the bilayer asymmetry in such a way that negative membrane curvature at the neck of a budding vesicle will be stabilized. An LPAAT activity has also been proposed for CtBP/BARS (carboxy-terminal binding protein/brefeldin A-ribosylated substrate), a transcription co-repressor that is implicated in dynamin-independent endocytosis and fission of the Golgi in mitosis. Here we show that the LPAAT activity associated with endophilin is a contaminant of the purification procedure and can be also found associated with the pleckstrin homology domain of dynamin. Likewise, the LPAAT activity associated with CtBP/BARS is also a co-purification artefact. The proposed locus of activity in endophilins includes the BAR domain, which has no catalytic site but instead senses positive membrane curvature. These data will prompt a re-evaluation of the molecular details of membrane budding.

Roles of Amphipathic Helices and the Bin/Amphiphysin/Rvs (BAR) Domain of Endophilin in Membrane Curvature Generation

Control of membrane curvature is required in many important cellular processes, including endocytosis and vesicular trafficking. Endophilin is a bin/amphiphysin/rvs (BAR) domain protein that induces vesicle formation by promotion of membrane curvature through membrane binding as a dimer. Using site-directed spin labeling and EPR spectroscopy, we show that the overall BAR domain structure of the rat endophilin A1 dimer determined crystallographically is maintained under predominantly vesiculating conditions. Spin-labeled side chains on the concave surface of the BAR domain do not penetrate into the acyl chain interior, indicating that the BAR domain interacts only peripherally with the surface of a curved bilayer. Using a combination of EPR data and computational refinement, we determined the structure of residues 63–86, a region that is disordered in the crystal structure of rat endophilin A1. Upon membrane binding, residues 63–75 in each subunit of the endophilin dimer form a slightly tilted, amphipathic α-helix that directly interacts with the membrane. In their predominant conformation, these helices are located orthogonal to the long axis of the BAR domain. In this conformation, the amphipathic helices are positioned to act as molecular wedges that induce membrane curvature along the concave surface of the BAR domain.

Membrane curvature in cell biology: An integration of molecular mechanisms

Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists.

Phosphoinositides and membrane curvature switch the mode of actin polymerization via selective recruitment of toca-1 and Snx9

The membrane–cytosol interface is the major locus of control of actin polymerization. At this interface, phosphoinositides act as second messengers to recruit membrane-binding proteins. We show that curved membranes, but not flat ones, can use phosphatidylinositol 3-phosphate [PI(3)P] along with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to stimulate actin polymerization. In this case, actin polymerization requires the small GTPase cell cycle division 42 (Cdc42), the nucleation-promoting factor neural Wiskott–Aldrich syndrome protein (N-WASP) and the actin nucleator the actin-related protein (Arp) 2/3 complex. In liposomes containing PI(4,5)P2 as the sole phosphoinositide, actin polymerization requires transducer of Cdc42 activation-1 (toca-1). In the presence of phosphatidylinositol 3-phosphate, polymerization is both more efficient and independent of toca-1. Under these conditions, sorting nexin 9 (Snx9) can be implicated as a specific adaptor that replaces toca-1 to mobilize neural Wiskott–Aldrich syndrome protein and the Arp2/3 complex. This switch in phosphoinositide and adaptor specificity for actin polymerization from membranes has implications for how different types of actin structures are generated at precise times and locations in the cell.