Showing papers by "Ana J. García-Sáez published in 2023"
TL;DR: The Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease as mentioned in this paper .
Abstract: Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.
4 citations
TL;DR: A review of the structure and molecular mechanisms of mitochondria pore formation at the interface between cell death and inflammatory signaling to regulate cellular outcomes is presented in this paper , where the authors discuss the current understanding of the mitochondrial pore structure.
1 citations
TL;DR: In this article , the authors used advanced microscopy methods to provide a holistic view of the macromolecular assembly of the key effectors BAX and BAK that uncovers key aspects of the dynamic architecture of the apoptotic pore.
TL;DR: In this article , the effect of membrane fluidity on the permeabilizing activity of sticholysin I (St I), a toxin that belongs to the actinoporins family of α-PFTs, was studied.
Abstract: Actinoporins have emerged as archetypal α-pore-forming toxins (PFTs) that promote the formation of pores in membranes upon oligomerization and insertion of an α-helix pore-forming domain in the bilayer. These proteins have been used as active components of immunotoxins, therefore, understanding their lytic mechanism is crucial for developing this and other applications. However, the mechanism of how the biophysical properties of the membrane modulate the properties of pores generated by actinoporins remains unclear. Here we studied the effect of membrane fluidity on the permeabilizing activity of sticholysin I (St I), a toxin that belongs to the actinoporins family of α-PFTs. To modulate membrane fluidity we used vesicles made of an equimolar mixture of phosphatidylcholine (PC) and egg sphingomyelin (eggSM), in which PC contained fatty acids of different acyl chain lengths and degrees of unsaturation. Our detailed single-vesicle analysis revealed that when membrane fluidity is high, most of the vesicles are partially permeabilized in a graded manner. In contrast, more rigid membranes can be either completely permeabilized or not, indicating an all-or-none mechanism. Altogether, our results reveal that St I pores can be heterogeneous in size and stability, and that these properties depend on the fluid state of the lipid bilayer. We propose that membrane fluidity at different regions of cellular membranes is a key factor to modulate the activity of the actinoporins, which has implications for the design of different therapeutic strategies based on their lytic action.
TL;DR: In this paper , the authors present an automated analysis software to accurately measure the real-time kinetics of assembly of individual high-order oligomer complexes in cellular membranes, which is suitable for the analysis of intracellular protein oligomers, whose stoichiometry is usually difficult to quantify due to variability in signal detection in different areas of the cell.
Abstract: Single molecule fluorescence microscopy has the unique advantage to provide real-time information on the spatiotemporal assembly of individual protein complexes in cellular membranes. This includes the assembly of proteins into oligomer species of numerous copy numbers. However, there is a need for improved tracing analysis of the real-time growth kinetics of these assemblies in cells with single molecule resolution. Here, we present an automated analysis software to accurately measure the real-time kinetics of assembly of individual high-order oligomer complexes. Our software comes with a simple Graphical User Interface (GUI), is available as a source code and an executable, and can analyze a full data set of several hundred to thousand molecules in less than 2 minutes. Importantly, this software is suitable for the analysis of intracellular protein oligomers, whose stoichiometry is usually more difficult to quantify due to variability in signal detection in the different areas of the cell. We validated our method with simulated ground-truth data and time-lapse images of diffraction-limited oligomeric assemblies of BAX and BAK proteins on mitochondria of cells undergoing apoptosis. Our approach provides the broad community of biologists with a fast, user-friendly tool to trace the compositional evolution of macromolecular assemblies, and potentially model their growth for a deeper understanding of the structural and biophysical mechanisms underlying their functions.
TL;DR: In this article , two variants of HaloTag7 with restored dehalogenase activity were engineered for reversible labeling, which enabled controlled and stable labeling density over extended time periods by combining with structured illumination.
Abstract: Self-labeling enzymes (SLE) such as the HaloTag have emerged as powerful tools in high and super-resolution fluorescence microscopy. Newly developed fluorogenic SLE substrates enable imaging in the presence of excess dye. To exploit this feature for reversible labeling, we engineered two variants of HaloTag7 with restored dehalogenase activity. Kinetic studies in vitro showed different turnover kinetics for reHaloTagS (~0.006 s-1) and reHaloTagF (~0.055 s-1). Imaging by confocal and stimulated emission depletion microscopy yielded 3-time enhanced photostability of reHaloTag labeling. Prominently, single molecule imaging with reHaloTags enabled controlled and stable labeling density over extended time periods. By combination with structured illumination, simultaneous visualization of single molecule diffusion and organellar dynamics was achieved. These applications highlight the potential of reHaloTag labeling for pushing the limits of advanced fluorescence microscopy techniques.
TL;DR: In this paper , the authors reviewed calcium signaling in the context of RN pathways, with a focus on ferroptosis, a type of RN in which plasma membrane damage is elicited by the accumulation of oxidized lipids.
TL;DR: In this paper , the authors investigated the influence of polymer concentration and temperature on the nanodisc structure and concluded that the transition from a gel toward a liquid-crystalline lipid phase proceeds over a broad T-range compared to a continuous lipid bilayer.
Abstract: Membrane proteins are an essential part of signaling and transport processes and are targeted by multiple drugs. To isolate and investigate them in their native state, polymer-bounded nanodiscs have become valuable tools. In this study, we investigate the lipid model system dimyristoyl-phosphocholine (DMPC) with the nanodisc-forming copolymers styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA). Using small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS), we studied the influence of polymer concentration and temperature on the nanodisc structure. In Tris buffer, the size of nanodiscs formed with SMA is smaller compared to DIBMA at the same polymer ratio. In both cases, the size decreases monotonically with increasing polymer concentration, and this effect is more pronounced when using SMA. Measurements at temperatures (T) between 5 and 30 °C in phosphate buffer showed an incomplete solubilization at high T even at polymer/lipid ratios above that required for complete lipid solubilization. For DIBMA, the nanodiscs developed at lower temperatures are stable and the net repulsion increases, while for SMA, the individual nanodiscs possess smaller sizes and are less affected by T. However, using DLS, one can observe SMA agglomerates at low T. Interestingly, for both polymers, no drastic changes of the observable parameters (radius and bilayer thickness) are seen upon cooling, which would indicate a sharp (first-order) phase transition from liquid-crystalline to gel, but only gradual changes. Hence, we conclude that the transition from a gel toward a liquid-crystalline lipid phase proceeds over a broad T-range compared to a continuous lipid bilayer. These results can pave the way toward the development of better protocols for studying membrane proteins stabilized in this type of membrane mimics.
TL;DR: In this article , the authors developed a new optogenetic system, Opto-GPX4Deg, for light-induced degradation of the lipid reducing protein GPX4, which allows controlled ferroptosis induction with high precision in time and space.
Abstract: Ferroptosis is an iron-dependent form of regulated cell death characterized by accumulation of peroxidized lipids and plasma membrane disruption, whose molecular mechanism of execution remains poorly understood. Here, we developed a new optogenetic system, Opto-GPX4Deg, for light-induced degradation of the lipid reducing protein GPX4, which allows controlled ferroptosis induction with high precision in time and space. By using Opto-GPX4Deg to study cell death dynamics within the cellular population, we found that lipid peroxidation, followed by ferroptotic death, spread to neighboring cells in a distance-dependent manner. Remarkably, ferroptosis propagation showed a strong dependency on cell confluence and preferentially affected adjacent cells. Our findings establish cell death propagation as a feature of ferroptosis and provide new understanding of the mechanism involved.