Biology subjects

Czogalla, A.

Publications and source records attributed to Czogalla, A..

4 recordsLinked to original sources

bioRxiv2022

A combined biochemical and cellular approach reveals Zn2+-dependent hetero- and homodimeric CD4 and Lck assemblies in T cells

The CD4 or CD8 co-receptors interaction with the protein-tyrosine kinase Lck is widely accepted as the initiator of the tyrosine phosphorylation cascade leading to T-cell activation. These co-receptors potentially enhance T-cell antigen sensitivity, but how they function is still debated. A critical question is: to what extent are co-receptors and signal-initiating Lck coupled? Our contribution concerns the small - but indispensable for CD4- and CD8-Lck formation - element Zn2+. The intracellular Zn2+ pool is strictly buffered but undergoes dynamic changes, also reported during T-cell activation. Furthermore, the identical Zn2+-binding cysteinyl residues may alter co-receptor dimerization or heterodimerization with Lck. Following initial research demonstrating a significant difference in the affinity of Zn2+ to CD4 and CD4-Lck in solution, we combined biochemical and cellular approaches to show that fluctuations of buffered Zn2+ in physiological ranges indeed influence Zn(CD4)2 and Zn(CD4)(Lck). This conclusion was supported by the simulation of complexes equilibria, demonstrating that Zn2+ changes can alter the molar ratio between those complexes. In T cells, increased intracellular free Zn2+ concentration causes higher CD4 partitioning in the plasma membrane by a still unknown mechanism. We additionally found that CD4 palmitoylation decreases the specificity of CD4-Lck formation in the reconstituted membrane model, suggesting that this reversible modification may also be involved. Our findings help elucidate co-receptor-Lck coupling stoichiometry and demonstrate that intracellular free Zn2+ has a major role in the interplay between CD4 dimers and CD4-Lck assembly.

biophysics↗

bioRxiv2022

Spherocytosis-related L1340P mutation in ankyrin affects its interactions with spectrin

Previously, we reported a new missense mutation in the ANK1 gene correlated with the HS phenotype. This mutation, resulting in L1340P substitution (HGMD CM149731), likely leads to the changes in the conformation of the ankyrin ZZUD domain important for ankyrin binding to spectrin. In this report, we have shown the molecular and physiological effects of this mutation. First, we assessed the binding activity of human {beta}-spectrin to the mutated ZZUDL1340P domain of ankyrin using two different experimental approaches - the study of association and dissociation responses of spectrin ankyrin binding domain and sedimentation assay. In addition, we demonstrated changes in morphology caused by the overexpressed ankyrin ZZUD domain in human cell models. Our results prove the key role of L1340 aa residue in the UPA domain for the correct alignment of the ZZUD domain of ankyrin, which results in binding the latter with spectrin within the erythrocyte membrane. Replacing the L1340 with a proline residue disrupts the spectrin binding activity of ankyrin.

biochemistry↗

bioRxiv2022

Effect of leaflet asymmetry on mechanical properties of lipid bilayers with phosphatidic acid

The asymmetry of membranes has a significant impact on their biophysical characteristics and behavior. This study investigates the composition and mechanical properties of symmetric and asymmetric membranes in giant unilamellar vesicles (GUVs) made of phosphatidylcholine (POPC) and phosphatidic acid (POPA). A combination of fluorescence quantification, zeta potential measurements, micropipette aspiration and bilayer molecular dynamics simulations are used to characterize these membranes. The outer leaflet composition in vesicles is found consistent across the two preparation methods we employed, namely electroformation and inverted emulsion transfer. However, characterizing the inner leaflet poses challenges. Micropipette aspiration of GUVs show that oil residues do not substantially alter membrane elasticity, but simulations reveal increased membrane thickness and decreased interleaflet coupling in the presence of oil. Asymmetric membranes with a POPC:POPA mixture in the outer leaflet and POPC in the inner leaflet display similar stretching elasticity values to symmetric POPC:POPA membranes, suggesting potential POPA insertion into the inner leaflet during vesicle formation and suppressed asymmetry. The inverse compositional asymmetry, with POPC in the outer leaflet and POPC:POPA in the inner yield less stretchable membranes with higher compressibility modulus compared to their symmetric counterparts. Challenges in achieving and predicting compositional correspondence highlight the limitations of phase-transfer-based methods. Additionally, caution is advised when using fluorescently labeled lipids (even at low fractions of 0.5 mol%), as unexpected gel-like domains in symmetric POPC:POPA membranes were observed only with a specific type of labeled DOPE (dioleoylphosphatidylethanolamine) and the same fraction of unlabeled DOPE. The latter suggest that such phase separation may result from interactions between lipids and membrane fluorescent probes. Overall, this study underscores the complexity of factors influencing GUV membrane asymmetry, emphasizing the need for further research and improvement of characterization techniques. SIGNIFICANCEAsymmetrically charged lipid bilayer models are superior to commonly used symmetrical ones, exhibiting naturally present asymmetry, thereby exhibiting a more adequate range of biophysical membrane characteristics better reflecting biological membranes. This study focuses on the mechanical properties of phosphatidic acid (PA)-enriched membranes, a crucial lipid for cellular lipid metabolism, e.g. glycerophospholipid synthesis, and for signal transduction. Micropipette aspiration, fluorescent PA-sensor, and zeta potential studies demonstrate that asymmetric membranes are less stretchable than symmetric ones. Accompanying in silico studies on the symmetric membranes confirm that oil impurities do not influence the membrane stretching elasticity but increase its thickness and decrease the coupling of the two leaflets, which sheds light on the elastic behavior of experimental models of asymmetric lipid bilayers.

biophysics↗

bioRxiv2022

Investigation of nano- and micro-domains formed by ceramide-1-phosphate in freestanding lipid bilayers

Biological membranes are known for their complex nature and formation of domains is crucial for proper execution of multiple cellular processes. Such domains, mostly due to their nanoscale character, are rarely studied since implementation of techniques for their quantitative investigation is difficult. In this article we introduce spot-variation z-scan FCS implemented for the first time for artificial lipid vesicles of defined lipid composition. We used this approach to investigate behaviour of different species of ceramide-1-phosphate within membranes. We were able to provide quantitative description of lipid domains generated by ceramide-1-phosphate in nano and microscale. Aligning these results with in silico studies helped us to validate the approach and draw conclusions on complex behaviour of signalling lipids within biological membranes.

biophysics↗