Force Mapping Study of Actinoporin Effect in Membranes Presenting Phase Domains.

Equinatoxin II (EqtII) and Fragaceatoxin C (FraC) are pore-forming toxins (PFTs) from the actinoporin family that have enhanced membrane affinity in the presence of sphingomyelin (SM) and phase coexistence in the membrane. However, little is known about the effect of these proteins on the nanoscopic properties of membrane domains. Here, we used combined confocal microscopy and force mapping by atomic force microscopy to study the effect of EqtII and FraC on the organization of phase-separated phosphatidylcholine/SM/cholesterol membranes. To this aim, we developed a fast, high-throughput processing tool to correlate structural and nano-mechanical information from force mapping. We found that both proteins changed the lipid domain shape. Strikingly, they induced a reduction in the domain area and circularity, suggesting a decrease in the line tension due to a lipid phase height mismatch, which correlated with proteins binding to the domain interfaces. Moreover, force mapping suggested that the proteins affected the mechanical properties at the edge, but not in the bulk, of the domains. This effect could not be revealed by ensemble force spectroscopy measurements supporting the suitability of force mapping to study local membrane topographical and mechanical alterations by membranotropic proteins.

LGBTQ+ in STEM: Visibility and Beyond.

Lesbian, gay, bisexual, trans, queer (LGBTQ+, "+"=plus other sexes, gender identities, and sexual orientation) people in the STEM fields (STEM=science, technology, engineering, and mathematics) continue to experience discrimination in their workplace. In the last decade, the clamor for safe and diverse spaces continue to rise. Indeed, social media plays a huge part on increasing visibility of the LGBTQ+ community in STEM. LGBTQ+ scientists have not only promoted discourse and raised concerns to the online community, but more importantly, supported each other in platforms such as Twitter. Beyond social media, work at the institutes by LGBTQ+ individuals are also bearing fruit. This piece explores the grassroots work of scientists as they work hand-in-hand with their institute staff and leadership to promote diversity and inclusivity within and beyond their community.

AFM to Study Pore-Forming Proteins.

Atomic force microscopy (AFM) is a form of contact microscopy that uses a very sharp tip to scan the surface of a sample. It provides a 3D image of the surface structure and in the force mode it can also be used to test the mechanical properties of the sample. AFM has been successfully applied to study the molecular mechanism of pore-forming proteins on model membranes. It gives information about both the structural reorganization of the membrane surface and the changes in the force required for membrane piercing upon incubation with this special type of proteins. Here we describe robust protocols to investigate the effect of pore-forming proteins in supported lipid bilayers .

Scanning Fluorescence Correlation Spectroscopy for Quantification of the Dynamics and Interactions in Tube Organelles of Living Cells.

Single-molecule spectroscopic quantification of protein-protein interactions directly in the organelles of living cells is highly desirable but remains challenging. Bulk methods, such as Förster resonance energy transfer (FRET), currently only give a relative quantification of the strength of protein-protein interactions. Here, we introduce tube scanning fluorescence cross-correlation spectroscopy (tubeSFCCS) for the absolute quantification of diffusion and complex formation of fluorescently labeled molecules in the mitochondrial compartments. We determined the extent of association between the apoptosis regulators Bcl-xL and tBid at the mitochondrial outer membrane of living cells and discovered that practically all mitochondria-bound Bcl-xL and tBid are associated with each other, in contrast to undetectable association in the cytosol. Furthermore, we show further applicability of our method to other mitochondrial proteins, as well as to proteins in the endoplasmic reticulum (ER) membrane.

Pro-apoptotic cBid and Bax exhibit distinct membrane remodeling activities: An AFM study.

Bcl-2 proteins are key regulators of the mitochondrial outer membrane (MOM) permeabilization that mediates apoptosis. During apoptosis, Bid is cleaved (cBid) and translocates to the MOM, where it activates Bax. Bax then oligomerizes and induces MOM permeabilization. However, little is known about how these proteins affect membrane organization aside from pore formation. In previous studies, we have shown that both cBid and Bax are able to remodel membranes and stabilize curvature. Here, we dissected the independent effects of Bax and cBid on supported lipid structures mimicking the mitochondrial composition by means of atomic force spectroscopy. We show that cBid did not permeabilize the membrane but lowered the membrane breakthrough force. On the other hand, Bax effects were dependent on its oligomeric state. Monomeric Bax did not affect the membrane properties. In contrast, oligomeric Bax lowered the breakthrough force of the membrane, which in the context of pore formation, implies a lowering of the line tension at the edge of the pore.

Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores.

Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual-color single-molecule localization-based super-resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc-shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane.

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers.

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

A single herpesvirus protein can mediate vesicle formation in the nuclear envelope.

Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane, delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor, pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles, we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be bypassed by membrane tethering of pUL31, demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodeling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds that constrict to vesicles. Therefore, a single viral protein can mediate all events necessary for membrane budding and abscission.

Scanning fluorescence correlation spectroscopy in model membrane systems.

Fluorescence correlation spectroscopy (FCS) is an emerging technique employed in biophysical studies that exploits the temporal autocorrelation of fluorescence intensity fluctuations measured in a tiny volume (in the order of fL). The autocorrelation curve derived from the fluctuations can then be fitted with diffusion models to obtain parameters such as diffusion time and number of particles in the diffusion volume/area. Application of FCS to membranes allows studying membrane component dynamics, which includes mobility and interactions between the components. However, FCS encounters several difficulties like accurate positioning and stability of the setup when applied to membranes. Here, we describe the theoretical basis of point FCS as well as the scanning FCS (SFCS) approach, which is a practical way to address the challenges of FCS with membranes. We also list materials necessary for FCS experiments on two model membrane systems: (1) supported lipid bilayers and (2) giant unilamellar vesicles. Finally, we present simple protocols for the preparation of these model membrane systems, calibration of the microscope setup for FCS, and acquisition and analysis of point FCS and SFCS data so that diffusion coefficients and concentrations of fluorescent probes within lipid membranes can be calculated.

Cardiolipin effects on membrane structure and dynamics.

Cardiolipin (CL) is a lipid with unique properties solely found in membranes generating electrochemical potential. It contains four acyl chains and tends to form nonlamellar structures, which are believed to play a key role in membrane structure and function. Indeed, CL alterations have been linked to disorders such as Barth syndrome and Parkinson's disease. However, the molecular effects of CL on membrane organization remain poorly understood. Here, we investigated the structure and physical properties of CL-containing membranes using confocal microscopy, fluorescence correlation spectroscopy, and atomic force microscopy. We found that the fluidity of the lipid bilayer increased and its mechanical stability decreased with CL concentration, indicating that CL decreases the packing of the membrane. Although the presence of up to 20% CL gave rise to flat, stable bilayers, the inclusion of 5% CL promoted the formation of flowerlike domains that grew with time. Surprisingly, we often observed two membrane-piercing events in atomic force spectroscopy experiments with CL-containing membranes. Similar behavior was observed with a lipid mixture mimicking the mitochondrial outer membrane composition. This suggests that CL promotes the formation of membrane areas with apposed double bilayers or nonlamellar structures, similar to those proposed for mitochondrial contact sites. All together, we show that CL induces membrane alterations that support the role of CL in facilitating bilayer structure remodeling, deformation, and permeabilization.