Showing papers by "Ana J. García-Sáez published in 2015"
TL;DR: By analysing the stoichiometry of Bax oligomers at the single-molecule level, it is found that Bax binds to the membrane in a monomersic state and then self-assembles in <1 min, and active Bax does not exist in a unique oligomeric state, but as several different species based on dimer units.
Abstract: 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.
146 citations
TL;DR: A single viral protein can mediate all events necessary for membrane budding and abscission, and pUL34 recruits pUL31 to the membrane, which, on its own, deforms membranes to produce nuclear envelope vesicles.
70 citations
TL;DR: How PFPs and lipids cooperate and remodel each other to achieve pore formation are discussed, and new evidences of protein-lipid pore structures are explored.
Abstract: Pore-forming proteins (PFPs) punch holes in their target cell membrane to alter their permeability. Permeabilization of lipid membranes by PFPs has received special attention to study the basic molecular mechanisms of protein insertion into membranes and the development of biotechnological tools. PFPs act through a general multi-step mechanism that involves (i) membrane partitioning, (ii) insertion into the hydrophobic core of the bilayer, (iii) oligomerization, and (iv) pore formation. Interestingly, PFPs and membranes show a dynamic interplay. As PFPs are usually produced as soluble proteins, they require a large conformational change for membrane insertion. Moreover, membrane structure is modified upon PFPs insertion. In this context, the toroidal pore model has been proposed to describe a pore architecture in which not only protein molecules but also lipids are directly involved in the structure. Here, we discuss how PFPs and lipids cooperate and remodel each other to achieve pore formation, and explore new evidences of protein-lipid pore structures.
65 citations
TL;DR: It is shown that phosphatidylinositol 4,5-bisphosphate-dependent FGF2 oligomerization concomitant with the generation of membrane pores depends on F GF2 surface cysteines as either chemical alkylation or substitution with alanines impairs these processes.
51 citations
TL;DR: Surprisingly, it was found that on the cell surface EqtII did not organize into a unique oligomeric form, Instead, it existed as a mixture of oligomersic species mostly including monomers, dimers, tetramers, and hexamers.
50 citations
TL;DR: A comparison of StI, StII, EqtII, and FraC activities with that of their respective N-terminal synthetic peptides shows that a higher hydrophobicity contributes to increase the activity, reinforcing the notion that this property must be taken into account to design new potent membranotropic agents.
34 citations
TL;DR: This protocol demonstrates how to prepare supported bilayers and characterize their structure and mechanical properties using AFM, which play an important role in cellular processes such as maintaining cell hemostasis from environmental stress and stabilizing protein complexes.
Abstract: 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.
25 citations
TL;DR: Three distinct apoptosis-related factors all promote membrane association of BCL2-like structural folds belonging to both MCL1 and BAK, however, at the same time, the two proteins exhibited distinguishing features in their membrane association modes under apoptotic-like conditions.
11 citations
TL;DR: This book chapter explains the theoretical framework of point FCS and Scanning FCS (SFCS), which is a variation especially suitable for membrane studies, and presents a list of materials necessary for SFCS studies on Giant Unilamellar Vesicles (GUVs).
Abstract: Fluorescence correlation spectroscopy (FCS) is a powerful quantitative method to study dynamical properties of biophysical systems. It exploits the temporal autocorrelation of fluorescence intensity fluctuations originating from a tiny volume (~fL). A theoretical model function can be then fitted to the measured auto-correlation curve to obtain physical parameters such as local concentration and diffusion time. However, the application of FCS on membranes is coupled to several difficulties like accurate positioning and stability of the set-up. In this book chapter, we explain the theoretical framework of point FCS and Scanning FCS (SFCS), which is a variation especially suitable for membrane studies. We present a list of materials necessary for SFCS studies on Giant Unilamellar Vesicles (GUVs). Finally, we provide simple protocols for the preparation of GUVs, calibration of the microscope setup, and acquisition and analysis of SFCS data to determine diffusion coefficients and concentrations of fluorescent particles embedded in lipid membranes.
9 citations
TL;DR: How microscopy approaches applied to model membranes are used to understand the intrinsic apoptotic pathway involving MOMP is discussed, which reveals interesting features like the role of membrane in altering the affinity of Bcl-2 proteins, Bax and Bak proAPoptotic activity, mechanistic differences between pro- and antiapoptotic members, and the critical helices involved in pore formation by Bax.
Abstract: Apoptosis is a form of programmed cell death that plays an important role in key biological processes like development of organisms, the correct functioning of the immune system, and the maintenance of the cellular homeostasis. Dysregulation in the apoptotic pathway leads to diseases like cancer or neurodegenerative disorders. The proteins of the B-cell lymphoma 2 (Bcl-2) family are key regulators of mitochondrial outer membrane permeabilization (MOMP) during apoptosis, which is a critical step in the cell's commitment to death. However, their mechanism of action is still under intense investigation. Here, we discuss how microscopy approaches applied to model membranes are used to understand the intrinsic apoptotic pathway involving MOMP. We describe how model membranes mimicking the outer mitochondrial membrane (LUVs, GUVs, SLB) are used to understand the mechanism of Bcl-2-mediated apoptosis using state-of-the-art techniques like atomic force microscopy and fluorescence correlation spectroscopy. These studies have revealed interesting features like the role of membrane in altering the affinity of Bcl-2 proteins, Bax and Bak proapoptotic activity, mechanistic differences between pro- and antiapoptotic members, and the critical helices involved in pore formation by Bax.
4 citations
15 Sep 2015