Pore-forming proteins as drivers of membrane permeabilization in cell death pathways.

Regulated cell death (RCD) relies on activation and recruitment of pore-forming proteins (PFPs) that function as executioners of specific cell death pathways: apoptosis regulator BAX (BAX), BCL-2 homologous antagonist/killer (BAK) and BCL-2-related ovarian killer protein (BOK) for apoptosis, gasdermins (GSDMs) for pyroptosis and mixed lineage kinase domain-like protein (MLKL) for necroptosis. Inactive precursors of PFPs are converted into pore-forming entities through activation, membrane recruitment, membrane insertion and oligomerization. These mechanisms involve protein-protein and protein-lipid interactions, proteolytic processing and phosphorylation. In this Review, we discuss the structural rearrangements incurred by RCD-related PFPs and describe the mechanisms that manifest conversion from autoinhibited to membrane-embedded molecular states. We further discuss the formation and maturation of membrane pores formed by BAX/BAK/BOK, GSDMs and MLKL, leading to diverse pore architectures. Lastly, we highlight commonalities and differences of PFP mechanisms involving BAX/BAK/BOK, GSDMs and MLKL and conclude with a discussion on how, in a population of challenged cells, the coexistence of cell death modalities may have profound physiological and pathophysiological implications.

Generation of nanoscopic membrane curvature for membrane trafficking.

Curved membranes are key features of intracellular organelles, and their generation involves dynamic protein complexes. Here we describe the fundamental mechanisms such as the hydrophobic insertion, scaffolding and crowding mechanisms these proteins use to produce membrane curvatures and complex shapes required to form intracellular organelles and vesicular structures involved in endocytosis and secretion. For each mechanism, we discuss its cellular functions as well as the underlying physical principles and the specific membrane properties required for the mechanism to be feasible. We propose that the integration of individual mechanisms into a highly controlled, robust process of curvature generation often relies on the assembly of proteins into coats. How cells unify and organize the curvature-generating factors at the nanoscale is presented for three ubiquitous coats central for membrane trafficking in eukaryotes: clathrin-coated pits, caveolae, and COPI and COPII coats. The emerging theme is that these coats arrange and coordinate curvature-generating factors in time and space to dynamically shape membranes to accomplish membrane trafficking within cells.

Membrane and organelle dynamics during cell division.

During division, eukaryotic cells undergo a dramatic, complex and coordinated remodelling of their cytoskeleton and membranes. For cell division to occur, chromosomes must be segregated and new cellular structures, such as the spindle apparatus, must be assembled. Pre-existing organelles, such as the nuclear envelope, endoplasmic reticulum and Golgi apparatus, must be disassembled or remodelled, distributed and reformed. Smaller organelles such as mitochondria as well as cytoplasmic content must also be properly distributed between daughter cells. This mixture of organelles and cytoplasm is bound by a plasma membrane that is itself subject to remodelling as division progresses. The lipids resident in these different membrane compartments play important roles in facilitating the division process. In recent years, we have begun to understand how membrane remodelling is coordinated during division; however, there is still much to learn. In this Review, we discuss recent insights into how these important cellular events are performed and regulated.

Structure, regulation, and functional diversity of microvilli on the apical domain of epithelial cells.

Microvilli are actin-based structures found on the apical aspect of many epithelial cells. In this review, we discuss different types of microvilli, as well as comparisons with actin-based sensory stereocilia and filopodia. Much is known about the actin-bundling proteins of these structures; we summarize recent studies that focus on the components of the microvillar membrane. We pay special attention to mechanisms of membrane microfilament attachment by the ezrin/radixin/moesin family and regulation of this protein family. We also discuss the NHERF family of scaffolding proteins that are found in microvilli and their role in microvilli regulation. Microvilli on cultured cells are not static structures, and their dynamics and those of their components are discussed. Finally, we mention diseases related to microvilli and outline questions that our current knowledge will allow the field to address in the near future.

Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca2+-sensor ALG-2.

Apoptosis linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining giant unilamellar vesicle-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.

Membrane localization accelerates association under conditions relevant to cellular signaling.

Translocation of cytoplasmic molecules to the plasma membrane is commonplace in cell signaling. Membrane localization has been hypothesized to increase intermolecular association rates; however, it has also been argued that association should be faster in the cytosol because membrane diffusion is slow. Here, we directly compare an identical association reaction, the binding of complementary DNA strands, in solution and on supported membranes. The measured rate constants show that for a 10-µm-radius spherical cell, association is 22- to 33-fold faster at the membrane than in the cytoplasm. The kinetic advantage depends on cell size and is essentially negligible for typical ~1 µm prokaryotic cells. The rate enhancement is attributable to a combination of higher encounter rates in two dimensions and a higher reaction probability per encounter.

Lipid scrambling is a general feature of protein insertases.

Glycerophospholipids are synthesized primarily in the cytosolic leaflet of the endoplasmic reticulum (ER) membrane and must be equilibrated between bilayer leaflets to allow the ER and membranes derived from it to grow. Lipid equilibration is facilitated by integral membrane proteins called "scramblases." These proteins feature a hydrophilic groove allowing the polar heads of lipids to traverse the hydrophobic membrane interior, similar to a credit card moving through a reader. Nevertheless, despite their fundamental role in membrane expansion and dynamics, the identity of most scramblases has remained elusive. Here, combining biochemical reconstitution and molecular dynamics simulations, we show that lipid scrambling is a general feature of protein insertases, integral membrane proteins which insert polypeptide chains into membranes of the ER and organelles disconnected from vesicle trafficking. Our data indicate that lipid scrambling occurs in the same hydrophilic channel through which protein insertion takes place and that scrambling is abolished in the presence of nascent polypeptide chains. We propose that protein insertases could have a so-far-overlooked role in membrane dynamics as scramblases.

RNA Crossing Membranes: Systems and Mechanisms Contextualizing Extracellular RNA and Cell Surface GlycoRNAs.

The subcellular localization of a biopolymer often informs its function. RNA is traditionally confined to the cytosolic and nuclear spaces, where it plays critical and conserved roles across nearly all biochemical processes. Our recent observation of cell surface glycoRNAs may further explain the extracellular role of RNA. While cellular membranes are efficient gatekeepers of charged polymers such as RNAs, a large body of research has demonstrated the accumulation of specific RNA species outside of the cell, termed extracellular RNAs (exRNAs). Across various species and forms of life, protein pores have evolved to transport RNA across membranes, thus providing a mechanistic path for exRNAs to achieve their extracellular topology. Here, we review types of exRNAs and the pores capable of RNA transport to provide a logical and testable path toward understanding the biogenesis and regulation of cell surface glycoRNAs.

Fluidity is the way to life: lipid phase separation in bacterial membranes.

A hallmark of biological membranes is the dynamic localization of lipids and proteins. Lipids respond to temperature reduction below a critical point with phase separation, and poikilothermic animals and also bacteria adapt their lipid content to prevent gel phase formation in membranes. In a new study, Gohrbandt et al (2022) show that reduced membrane fluidity in bacterial cells causes reversible phase separation without membrane rupture in vivo, highlighting the physical robustness of biological membranes.

On the role of phase separation in the biogenesis of membraneless compartments.

Molecular mechanistic biology has ushered us into the world of life's building blocks, revealing their interactions in macromolecular complexes and inspiring strategies for detailed functional interrogations. The biogenesis of membraneless cellular compartments, functional mesoscale subcellular locales devoid of strong internal order and delimiting membranes, is among mechanistic biology's most demanding current challenges. A developing paradigm, biomolecular phase separation, emphasizes solvation of the building blocks through low-affinity, weakly adhesive unspecific interactions as the driver of biogenesis of membraneless compartments. Here, I discuss the molecular underpinnings of the phase separation paradigm and demonstrate that validating its assumptions is much more challenging than hitherto appreciated. I also discuss that highly specific interactions, rather than unspecific ones, appear to be the main driver of biogenesis of subcellular compartments, while phase separation may be harnessed locally in selected instances to generate material properties tailored for specific functions, as exemplified by nucleocytoplasmic transport.

Cryo-EM structures of perforin-2 in isolation and assembled on a membrane suggest a mechanism for pore formation.

Perforin-2 (PFN2, MPEG1) is a key pore-forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane-bound pre-pore complex that converts to a pore-forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre-pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre-assembled complete pre-pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre-pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 ß-hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre-pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.

Functional and structural asymmetry suggest a unifying principle for catalysis in membrane-bound pyrophosphatases.

Membrane-bound pyrophosphatases (M-PPases) are homodimeric primary ion pumps that couple the transport of Na+- and/or H+ across membranes to the hydrolysis of pyrophosphate. Their role in the virulence of protist pathogens like Plasmodium falciparum makes them an intriguing target for structural and functional studies. Here, we show the first structure of a K+-independent M-PPase, asymmetric and time-dependent substrate binding in time-resolved structures of a K+-dependent M-PPase and demonstrate pumping-before-hydrolysis by electrometric studies. We suggest how key residues in helix 12, 13, and the exit channel loops affect ion selectivity and K+-activation due to a complex interplay of residues that are involved in subunit-subunit communication. Our findings not only explain ion selectivity in M-PPases but also why they display half-of-the-sites reactivity. Based on this, we propose, for the first time, a unified model for ion-pumping, hydrolysis, and energy coupling in all M-PPases, including those that pump both Na+ and H+.

The Chemical Reactivity of Membrane Lipids.

It is well-known that aqueous dispersions of phospholipids spontaneously assemble into bilayer structures. These structures have numerous applications across chemistry and materials science and form the fundamental structural unit of the biological membrane. The particular environment of the lipid bilayer, with a water-poor low dielectric core surrounded by a more polar and better hydrated interfacial region, gives the membrane particular biophysical and physicochemical properties and presents a unique environment for chemical reactions to occur. Many different types of molecule spanning a range of sizes, from dissolved gases through small organics to proteins, are able to interact with membranes and promote chemical changes to lipids that subsequently affect the physicochemical properties of the bilayer. This Review describes the chemical reactivity exhibited by lipids in their membrane form, with an emphasis on conditions where the lipids are well hydrated in the form of bilayers. Key topics include the following: lytic reactions of glyceryl esters, including hydrolysis, aminolysis, and transesterification; oxidation reactions of alkenes in unsaturated fatty acids and sterols, including autoxidation and oxidation by singlet oxygen; reactivity of headgroups, particularly with reactive carbonyl species; and E/Z isomerization of alkenes. The consequences of reactivity for biological activity and biophysical properties are also discussed.

Activation energy for pore opening in lipid membranes under an electric field.

The standard model of pore formation was introduced more than fifty years ago, and it has been since, despite some refinements, the cornerstone for interpreting experiments related to pores in membranes. A central prediction of the model concerning pore opening under an electric field is that the activation barrier for pore formation is lowered proportionally to the square of the electric potential. However, this has only been scarcely and inconclusively confronted to experiments. In this paper, we study the electropermeability of model lipid membranes composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) containing different fractions of POPC-OOH, the hydroperoxidized form of POPC, in the range 0 to 100 mol %. By measuring ion currents across a 50-µm-diameter black lipid membrane (BLM) with picoampere and millisecond resolution, we detect hydroperoxidation-induced changes to the intrinsic bilayer electropermeability and to the probability of opening angstrom-size or larger pores. Our results over the full range of lipid compositions show that the energy barrier to pore formation is lowered linearly by the absolute value of the electric field, in contradiction with the predictions of the standard model.

Optogenetic clustering and membrane translocation of the BcLOV4 photoreceptor.

Optogenetic tools respond to light through one of a small number of behaviors including allosteric changes, dimerization, clustering, or membrane translocation. Here, we describe a new class of optogenetic actuator that simultaneously clusters and translocates to the plasma membrane in response to blue light. We demonstrate that dual translocation and clustering of the BcLOV4 photoreceptor can be harnessed for novel single-component optogenetic tools, including for control of the entire family of epidermal growth factor receptor (ErbB1-4) tyrosine kinases. We further find that clustering and membrane translocation are mechanistically linked. Stronger clustering increased the magnitude of translocation and downstream signaling, increased sensitivity to light by ~threefold-to-fourfold, and decreased the expression levels needed for strong signal activation. Thus light-induced clustering of BcLOV4 provides a strategy to generate a new class of optogenetic tools and to enhance existing ones.