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1.
EMBO J ; 43(19): 4274-4297, 2024 Oct.
Article in English | MEDLINE | ID: mdl-39143238

ABSTRACT

Gasdermin D (GSDMD) executes the cell death program of pyroptosis by assembling into oligomers that permeabilize the plasma membrane. Here, by single-molecule imaging, we elucidate the yet unclear mechanism of Gasdermin D pore assembly and the role of cysteine residues in GSDMD oligomerization. We show that GSDMD preassembles at the membrane into dimeric and trimeric building blocks that can either be inserted into the membrane, or further assemble into higher-order oligomers prior to insertion into the membrane. The GSDMD residues Cys39, Cys57, and Cys192 are the only relevant cysteines involved in GSDMD oligomerization. S-palmitoylation of Cys192, combined with the presence of negatively-charged lipids, controls GSDMD membrane targeting. Simultaneous Cys39/57/192-to-alanine (Ala) mutations, but not Ala mutations of Cys192 or the Cys39/57 pair individually, completely abolish GSDMD insertion into artificial membranes as well as into the plasma membrane. Finally, either Cys192 or the Cys39/Cys57 pair are sufficient to enable formation of GSDMD dimers/trimers, but they are all required for functional higher-order oligomer formation. Overall, our study unveils a cooperative role of Cys192 palmitoylation-mediated membrane binding and Cys39/57/192-mediated oligomerization in GSDMD pore assembly. This study supports a model in which Gasdermin D oligomerization relies on a two-step mechanism mediated by specific cysteine residues.


Subject(s)
Cell Membrane , Cysteine , Lipoylation , Phosphate-Binding Proteins , Phosphate-Binding Proteins/metabolism , Phosphate-Binding Proteins/genetics , Cysteine/metabolism , Humans , Cell Membrane/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Protein Multimerization , HEK293 Cells , Animals , Gasdermins
2.
Biochim Biophys Acta ; 1857(8): 1290-1299, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27016377

ABSTRACT

The cell is metabolically highly compartmentalized. Especially, mitochondria host many vital reactions in their different microcompartments. However, due to their small size, these microcompartments are not accessible by conventional microscopy. Here, we demonstrate that time-correlated single-photon counting (TCSPC) fluorescence lifetime-imaging microscopy (FLIM) classifies not only mitochondria, but different microcompartments inside mitochondria. Sensor proteins in the matrix had a different lifetime than probes at membrane proteins. Localization in the outer and inner mitochondrial membrane could be distinguished by significant differences in the lifetime. The method was sensitive enough to monitor shifts in protein location within mitochondrial microcompartments. Macromolecular crowding induced by changes in the protein content significantly affected the lifetime, while oxidizing conditions or physiological pH changes had only marginal effects. We suggest that FLIM is a versatile and completive method to monitor spatiotemporal events in mitochondria. The sensitivity in the time domain allows for gaining substantial information about sub-mitochondrial localization overcoming diffraction limitation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.


Subject(s)
Electron Transport Complex III/genetics , Mitochondria/metabolism , Mitochondria/ultrastructure , Optical Imaging/methods , A Kinase Anchor Proteins/genetics , A Kinase Anchor Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Electron Transport Complex III/metabolism , Gene Expression , Genes, Reporter , Glycerol/pharmacology , HeLa Cells , Humans , Hydrogen Peroxide/pharmacology , Hydrogen-Ion Concentration , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mitochondria/drug effects , Mitochondrial Precursor Protein Import Complex Proteins , Optical Imaging/instrumentation , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
3.
J Cell Sci ; 126(Pt 1): 103-16, 2013 Jan 01.
Article in English | MEDLINE | ID: mdl-23038773

ABSTRACT

Mitochondria are involved in cellular energy supply, signaling and apoptosis. Their ability to fuse and divide provides functional and morphological flexibility and is a key feature in mitochondrial quality maintenance. To study the impact of mitochondrial fusion/fission on the reorganization of inner membrane proteins, oxidative phosphorylation (OXPHOS) complexes in mitochondria of different HeLa cells were tagged with fluorescent proteins (GFP and DsRed-HA), and cells were fused by polyethylene glycol treatment. Redistribution of the tagged OXPHOS complexes was then followed by means of immunoelectron microscopy, two color super-resolution fluorescence microscopy and single molecule tracking. In contrast to outer membrane and matrix proteins, which mix quickly and homogeneously upon mitochondrial fusion, the mixing of inner membrane proteins was decelerated. Our data suggest that the composition of cristae is preserved during fusion of mitochondria and that cristae with mixed OXPHOS complexes are only slowly and successively formed by restricted diffusion of inner membrane proteins into existing cristae. The resulting transitory mosaic composition of the inner mitochondrial membrane illuminates mitochondrial heterogeneity and potentially is linked to local differences in function and membrane potential.


Subject(s)
Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , HeLa Cells , Humans , Microscopy, Confocal , Microscopy, Immunoelectron , Mitochondria/ultrastructure , Mitochondrial Dynamics/genetics , Mitochondrial Dynamics/physiology , Mitochondrial Membranes/ultrastructure , Oxidative Phosphorylation
4.
Sci Rep ; 7: 46055, 2017 04 06.
Article in English | MEDLINE | ID: mdl-28383048

ABSTRACT

The assembly of respiratory complexes into macromolecular supercomplexes is currently a hot topic, especially in the context of newly available structural details. However, most work to date has been done with purified detergent-solubilized material and in situ confirmation is absent. We here set out to enable the recording of respiratory supercomplex formation in living cells. Fluorescent sensor proteins were placed at specific positions at cytochrome c oxidase suspected to either be at the surface of a CI1CIII2CIV1 supercomplex or buried within this supercomplex. In contrast to other loci, sensors at subunits CoxVIIIa and CoxVIIc reported a dense protein environment, as detected by significantly shortened fluorescence lifetimes. According to 3D modelling CoxVIIIa and CoxVIIc are buried in the CI1CIII2CIV1 supercomplex. Suppression of supercomplex scaffold proteins HIGD2A and CoxVIIa2l was accompanied by an increase in the lifetime of the CoxVIIIa-sensor in line with release of CIV from supercomplexes. Strikingly, our data provide strong evidence for defined stable supercomplex configuration in situ.


Subject(s)
Electron Transport Complex IV/metabolism , Green Fluorescent Proteins/metabolism , Imaging, Three-Dimensional , Multiprotein Complexes/metabolism , Cell Respiration , Cell Survival , Fluorescence Resonance Energy Transfer , HeLa Cells , Humans , Models, Molecular , Oxidative Phosphorylation , Protein Multimerization , Protein Subunits/metabolism
5.
PLoS One ; 5(7): e11910, 2010 Jul 30.
Article in English | MEDLINE | ID: mdl-20689601

ABSTRACT

BACKGROUND: Mitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism. METHODOLOGY/PRINCIPAL FINDINGS: The question discussed in this paper is whether fusion and fission of mitochondria in different cell lines result in re-localization of respiratory chain (RC) complexes and of the ATP synthase. This was addressed by fusing cells containing mitochondria with respiratory complexes labelled with different fluorescent proteins and resolving their time dependent re-localization in living cells. We found a complete reshuffling of RC complexes throughout the entire chondriome in single HeLa cells within 2-3 h by organelle fusion and fission. Polykaryons of fused cells completely re-mixed their RC complexes in 10-24 h in a progressive way. In contrast to the recently described homogeneous mixing of matrix-targeted proteins or outer membrane proteins, the distribution of RC complexes and ATP synthase in fused hybrid mitochondria, however, was not homogeneous but patterned. Thus, complete equilibration of respiratory chain complexes as integral inner mitochondrial membrane complexes is a slow process compared with matrix proteins probably limited by complete fusion. In co-expressing cells, complex II is more homogenously distributed than complex I and V, resp. Indeed, this result argues for higher mobility and less integration in supercomplexes. CONCLUSION/SIGNIFICANCE: Our results clearly demonstrate that mitochondrial fusion and fission dynamics favours the re-mixing of all RC complexes within the chondriome. This permanent mixing avoids a static situation with a fixed composition of RC complexes per mitochondrion.


Subject(s)
Mitochondria/metabolism , Electrophoresis , Electrophoresis, Gel, Two-Dimensional , HeLa Cells , Humans , Membrane Fusion/physiology , Microscopy, Electron , Microscopy, Fluorescence , Mitochondria/ultrastructure , Mitochondrial Membranes/metabolism , Mitochondrial Proton-Translocating ATPases/metabolism , Oxygen Consumption
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