Biology subjects

Wagenknecht-Wiesner, A.

Publications and source records attributed to Wagenknecht-Wiesner, A..

3 recordsLinked to original sources

bioRxiv2023

Alpha Synuclein Modulates Mitochondrial Ca2+ Uptake from ER During Cell Stimulation and Under Stress Conditions

Alpha synuclein (a-syn) is an intrinsically disordered protein prevalent in neurons, and aggregated forms are associated with synucleinopathies including Parkinson disease (PD). Despite the biomedical importance and extensive studies, the physiological role of a-syn and its participation in etiology of PD remain uncertain. We showed previously in model RBL cells that a-syn colocalizes with mitochondrial membranes, depending on formation of N-terminal helices and increasing with mitochondrial stress.1 We have now characterized this colocalization and functional correlates in RBL, HEK293, and N2a cells. We find that expression of a-syn enhances stimulated mitochondrial uptake of Ca2+ from the ER, depending on formation of its N-terminal helices but not on its disordered C-terminal tail. Our results are consistent with a-syn acting as a tether between mitochondria and ER, and we show increased contacts between these two organelles using structured illumination microscopy. We tested mitochondrial stress caused by toxins related to PD, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and found that a-syn prevents recovery of stimulated mitochondrial Ca2+ uptake. The C-terminal tail, and not N-terminal helices, is involved in this inhibitory activity, which is abrogated when phosphorylation site serine-129 is mutated (S129A). Correspondingly, we find that MPTP/MPP+ and CCCP stress is accompanied by both phosphorylation (pS129) and aggregation of a-syn. Overall, our results indicate that a-syn can participate as a tethering protein to modulate Ca2+ flux between ER and mitochondria, with potential physiological significance. A-syn can also prevent cellular recovery from toxin-induced mitochondrial dysfunction, which may represent a pathological role of a-syn in the etiology of PD.

cell biology↗

bioRxiv2022

Lipid Driven Inter-leaflet Coupling of Plasma Membrane Order Regulates FcεRI Signaling in Mast Cells

Engagement of high affinity immunoglobulin E (IgE) receptor Fc{varepsilon}RI with extracellular, multivalent antigen (Ag) stabilizes co-existing ordered and disordered phases in the inner leaflet of the plasma membrane. This optimally controls biochemical interactions between signaling components required for transmembrane (TM) signaling in mast cells. The biophysical organization of the resting inner leaflet is poised to respond appropriately to this extracellular stimulation. The resting inner leaflet is generally less ordered than the outer leaflet, with a lipid composition that does not spontaneously phase separate in model membranes. We proposed that coupling between the two leaflets mediates separation into different phase-like domains in the inner leaflet. To test this hypothesis in live cells, we first established a straightforward approach to evaluate changes in membrane order due to inter-leaflet coupling by measuring inner leaflet diffusion of phase-specific lipid probes with Imaging Fluorescence Correlation Spectroscopy (ImFCS) before and after methyl--cyclodextrin (mCD)-catalyzed exchange of outer leaflet lipids (LEX) with exogenous order- or disorder-promoting phospholipids. We examined the functional impact of LEX by monitoring two Ag-stimulated cellular responses, namely early-stage recruitment of Syk kinase to the inner leaflet and late-stage exocytosis of secretory granules (degranulation). Based on changes in probe diffusion, we observed global increase or decrease of inner leaflet order when outer leaflet is exchanged with order or disorder promoting lipids, respectively, in unstimulated cells. Furthermore, the degree of stimulated Syk recruitment and degranulation correlates with the inner leaflet order of the resting cells, which was varied using LEX. Overall, combined LEX and ImFCS platform provides strong evidence of lipid-based control of stimulated TM signaling in live mast cells. In addition, our functional results imply that resting-state lipid composition and ordering of the outer leaflet sets the ordering of the inner leaflet, likely via interleaflet coupling, and correspondingly modulates TM signaling initiated by antigen-activated IgE-Fc{varepsilon}RI. STATEMENT OF SIGNIFICANCECoupling between plasma membrane leaflets, which are biochemically and biophysically asymmetric, results in a steady-state membrane organization that is thought to play fundamental roles in cellular functions. Here, we present a straightforward assay built around mCD-catalyzed lipid exchange (LEX) and Imaging Fluorescence Correlation Spectroscopy (ImFCS) to quantitatively characterize a novel, lipid-driven, interleaflet coupling mechanism and its functional impact in live mast cells. We showed that elevation of outer leaflet lipid order induces ordering throughout the inner leaflet in resting cells. This ordering enhances protein-based reactions during Ag-stimulated Fc{varepsilon}RI signaling and consequent cellular response. Overall, we provide a compelling evidence of functional relevance of plasma membrane organizational heterogeneity driven by lipid-based interleaflet coupling.

biophysics↗

bioRxiv2020

Lipid-based, protein-based, and steric interactions synergize to facilitate transmembrane signaling stimulated by antigen-clustering of IgE receptors

Antigen (Ag) crosslinking of immunoglobulin E-receptor (IgE-Fc{varepsilon}RI) complexes in mast cells stimulates transmembrane (TM) signaling, requiring phosphorylation of the clustered Fc{varepsilon}RI by lipid-anchored Lyn tyrosine kinase. Previous studies showed that this stimulated coupling between Lyn and Fc{varepsilon}RI occurs in liquid ordered (Lo)-like nanodomains of the plasma membrane and that Lyn binds directly to cytosolic segments of Fc{varepsilon}RI that it initially phosphorylates for amplified activity. Net phosphorylation above a non-functional threshold is achieved in the stimulated state, but not in the resting state, and current evidence supports the hypothesis that this relies on disruption by Ag-crosslinking of a balance between Lyn and tyrosine phosphatase activities. However, the structural interactions that underlie the stimulation process remain poorly defined. This study evaluates the relative contributions and functional importance of different types of interactions leading to supra-threshold phosphorylation of Ag-crosslinked IgE-Fc{varepsilon}RI in live rat basophilic leukemia (RBL) mast cells. Our high-precision diffusion measurements by Imaging Fluorescence Correlation Spectroscopy (ImFCS) on multiple structural variants of Lyn and other lipid-anchored probes confirm subtle, stimulated stabilization of the Lo-like nanodomains and concomitant sharpening of segregation from liquid-disordered (Ld)-like regions. With other structural variants we determine that lipid-based interactions are essential for access by Lyn leading to phosphorylation of and protein-based binding to clustered Fc{varepsilon}RI. By contrast, TM tyrosine phosphatase, PTP, is excluded from these regions by steric repulsion of TM segments and preference for Ld-like regions. Overall, we establish a synergy of lipid-based, protein-based, and steric interactions underlying functional TM signaling in mast cells. SIGNIFICANCE STATEMENTLipid organization of the plasma membrane is known to be important for facilitating protein interactions in transmembrane signaling. However, the orchestration of these interactions in live cells has been elusive. We employed ImFCS to systemically investigate the interplay of lipids and proteins during signaling in mast cells, initiated as phosphorylation of Ag-crosslinked IgE-Fc{varepsilon}RI by lipid-anchored Lyn kinase. We find lipid-based interactions are first required for protein-based phosphorylation of the clustered Fc{varepsilon}RI within Lo-like nanodomains. Transmembrane phosphatases must be excluded from these regions, and we find this is mediated by their preference for Ld-like regions and by steric exclusion from the clustered Fc{varepsilon}RI proteins. ImFCS provides quantitative characterization of the functional link between features of plasma membrane organization and transmembrane signaling.

biophysics↗