Bax is a key regulator of apoptosis that mediates the release of cytochrome c to the cytosol via oligomerization in the outer mitochondrial membrane before pore formation. However, the molecular mechanism of Bax assembly and regulation by other Bcl-2 members remains obscure. Here, by analysing the stoichiometry of Bax oligomers at the single-molecule level, we find that Bax binds to the membrane in a monomeric state and then self-assembles in <1 min. Strikingly, active Bax does not exist in a unique oligomeric state, but as several different species based on dimer units. Moreover, we show that cBid activates Bax without affecting its assembly, while Bcl-xL induces the dissociation of Bax oligomers. On the basis of our experimental data and theoretical modelling, we propose a new mechanism for the molecular pathway of Bax assembly to form the apoptotic pore.

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.

Background:Equinatoxin II is a model ␣-pore-forming toxin that kills cells by porating the host plasma membrane. Results: On the membrane, equinatoxin II does not adopt a unique oligomeric state, but assembles into multiple coexisting species related to toxicity. Conclusion: Toxicity of Equinatoxin II depends on its assembly mechanism. Significance: A new molecular mechanism is proposed for ␣-pore-forming toxins action.

Abstract:In the present investigation we studied the molecular mechanisms of the monodesmosidic saponin digitonin on natural and artificial membranes. We measured the hemolytic activity of digitonin on red blood cells (RBCs). Also different lipid membrane models (large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs) in the presence and absence of cholesterol were employed. The stability and permeability of the different vesicle systems were studied by using calcein release assay, GUVs membrane permeability assay using confocal microscopy (CM) and fluorescence correlation spectroscopy (FCS) and vesicle size measurement by dynamic light scattering (DLS). The results support the essential role of cholesterol in explaining how digitonin can disintegrate biological and artificial membranes. Digitonin induces membrane permeability or causes membrane rupturing only in the presence of cholesterol in an all-or-none mechanism. This effect depends on the concentrations of both digitonin and cholesterol. At low concentrations, digitonin induces membrane permeability while keeping the membrane intact. When digitonin is combined with other drugs, a synergistic potentiation can be observed because it facilitates their uptake.

The complete software is implemented in MATLAB (The MathWorks, Inc., USA) and available as a standalone as well as the full source code at http://www.ifib.uni-tuebingen.de/research/garcia-saez/guv-software.html.

The startling increase in the occurrence of rifampicin (Rif) resistance in the clinical isolates of Mycobacterium tuberculosis worldwide is posing a serious concern to tuberculosis management. The majority of Rif resistance in bacteria arises from mutations in the RpoB subunit of the RNA polymerase. We isolated M. smegmatis strains harbouring either an insertion (6 aa) or a deletion (10 aa) in their RpoB proteins. Although these strains showed a compromised fitness for growth in 7H9 Middlebrook medium, their resistance to Rif was remarkably high. The attenuated growth of the strains correlated with decreased specific activities of the RNA polymerases from the mutants. While the RNA polymerases from the parent or a mutant strain (harbouring a frequently occurring mutation, H442Y, in RpoB) were susceptible to Rif-mediated inhibition of transcription from calf thymus DNA, those from the insertion and deletion mutants were essentially refractory to such inhibition. Three-dimensional structure modelling revealed that the RpoB amino acids that interact with Rif are either deleted or unable to interact with Rif due to their unsuitable spatial positioning in these mutants. We discuss possible uses of the RpoB mutants in studying transcriptional regulation in mycobacteria and as potential targets for drug design.

BackgroundInteraction of non-structural protein 5A (NS5A) of Hepatitis C virus (HCV) with human kinases namely, casein kinase 1α (ck1α) and protein kinase R (PKR) have different functional implications such as regulation of viral replication and evasion of interferon induced immune response respectively. Understanding the structural and molecular basis of interactions of the viral protein with two different human kinases can be useful in developing strategies for treatment against HCV.ResultsSerine 232 of NS5A is known to be phosphorylated by human ck1α. A structural model of NS5A peptide containing phosphoacceptor residue Serine 232 bound to ck1α has been generated using the known 3-D structures of kinase-peptide complexes. The substrate interacting residues in ck1α has been identified from the model and these are found to be conserved well in the ck1 family. ck1α – substrate peptide complex has also been used to understand the structural basis of association between ck1α and its other viral stress induced substrate, tumour suppressor p53 transactivation domain which has a crystal structure available.Interaction of NS5A with another human kinase PKR is primarily genotype specific. NS5A from genotype 1b has been shown to interact and inhibit PKR whereas NS5A from genotype 2a/3a are unable to bind and inhibit PKR efficiently. This is one of the main reasons for the varied response to interferon therapy in HCV patients across different genotypes. Using PKR crystal structure, sequence alignment and evolutionary trace analysis some of the critical residues responsible for the interaction of NS5A 1b with PKR have been identified.ConclusionsThe substrate interacting residues in ck1α have been identified using the structural model of kinase - substrate peptide. The PKR interacting NS5A 1b residues have also been predicted using PKR crystal structure, NS5A sequence analysis along with known experimental results. Functional significance and nature of interaction of interferon sensitivity determining region and variable region 3 of NS5A in different genotypes with PKR which was experimentally shown are also supported by the findings of evolutionary trace analysis. Designing inhibitors to prevent this interaction could enable the HCV genotype 1 infected patients respond well to interferon therapy.

Membranes are dynamic lipophilic structures that constitute a selective barrier essential for the smooth functioning of cellular machinery. Their complex and highly heterogeneous composition, as well as the intricate diffusion patterns that they display, have been intensely studied in the past decades. Several innovative microscopy techniques have recently been developed to gain insight into the basis of membrane dynamics, interactions, and molecular organization. In this article, we introduce four pioneer fluorescence‐based approaches that have substantially broadened our overall understanding of membranes: fluorescence correlation spectroscopy (FCS), single particle tracking (SPT), fluorescence recovery after photobleaching (FRAP), and 6‐lauroyl‐2‐(dimethylamino)‐naphtalene (LAURDAN) microscopy. We discuss each technique in detail, explaining their principles, methodology, and applications in the research of the dynamic processes controlled by membranes.