Showing papers by "Ana J. García-Sáez published in 2013"
TL;DR: The results demonstrate that Bax and BakΔC21 follow similar mechanisms of membrane permeabilization characterized by the formation of protein-permeable pores of dynamic size, in agreement with the proteolipidic nature of these apoptotic pores.
132 citations
TL;DR: It is found that proapoptotic Bax forms large, stable pores via an all-or-none mechanism that can release cytochrome c, and antiap optotic Bcl-xL induces transient permeability alterations in pure lipid membranes that have no consequences for the mitochondrial outer membrane but inhibit Bax membrane insertion.
70 citations
TL;DR: Light is shed on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria, for caspase-8 activation and Bid binding and cleavage in type II cells.
Abstract: Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria.
25 citations
TL;DR: The theoretical basis of point FCS as well as the scanning FCS (SFCS) approach are described, which is a practical way to address the challenges of FCS with membranes.
Abstract: 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.
3 citations
18 Sep 2013
TL;DR: In this article, the authors 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.
Abstract: 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.
Keywords:
Diffusion, triplet state;
Membrane dynamics;
Fluorescence correlation spectroscopy;
Single particle tracking;
Laurdan;
Fluorescence recovery after photobleaching
1 citations