Insights into the morphological pattern of erythrocytes' aging: Coupling quantitative AFM data to microcalorimetry and Raman spectroscopy.

Erythrocytes (RBCs) constitute a very interesting class of cells both for their physiological function and for a variety of peculiarities. Due to their exceptionally strong relationship with the environment, the morphology and nanoscale characteristics of these cells can reveal their biochemical status and structural integrity. Among the possible subjects of investigations, the RBCs' ageing is of the utmost importance. This is a fundamental phenomenon that, in physiological conditions, triggers the cell turnover and ensures the blood homeostasis. With these premises, in recent years, we have presented an atomic force microscopy-based methodology to characterize the patterns of RBC ageing from the morphological point of view. In the present work, we used an ageing protocol more similar to the physiological conditions and we used differential scanning calorimetry and atomic force microscopy to probe the cross correlation between important structural and functional proteins. We also assessed the role played by fundamental structural and membrane proteins in the development of the most relevant morphological intermediates observed along the ageing. Furthermore, we coupled the morphological ageing patterns to the (bio)chemical alterations detected by Raman spectroscopy. This allowed identifying the chronology of the ageing morphologies and the metabolic pathways most involved in their development. As a whole, the present study provides the base to correlate specific molecular alterations to the development of structural anomalies, and these latter to the functional status of blood cells.

Heat- and light-induced detachment of the light-harvesting antenna complexes of photosystem I in isolated stroma thylakoid membranes.

The multisubunit pigment-protein complex of photosystem I (PSI) consists of a core and peripheral light-harvesting antenna (LHCI). PSI is thought to be a rather rigid system and very little is known about its structural and functional flexibility. Recent data, however, suggest LHCI detachment from the PSI supercomplex upon heat and light treatments. Furthermore, it was suggested that the splitting off of LHCI acts as a safety valve for PSI core upon photoinhibition (Alboresi et al., 2009). In this work we analyzed the heat- and light-induced reorganizations in isolated PSI vesicles (stroma membrane vesicles enriched in PSI). Using differential scanning calorimetry we revealed a stepwise disassembly of PSI supercomplex above 50°C. Circular dichroism, sucrose gradient centrifugation and 77K fluorescence experiments identified the sequence of events of PSI destabilization: 3min heating at 60°C or 40min white light illumination at 25°C resulted in pronounced Lhca1/4 detachment from the PSI supercomplex, which is then followed by the degradation of Lhca2/3. The similarity of the main structural effects due to heat and light treatments supports the notion that thermo-optic mechanism, structural changes induced by ultrafast local thermal transients, which has earlier been shown to be responsible for structural changes in the antenna system of photosystem II, can also regulate the assembly and functioning of PSI antenna.

Rapid reversible formation of a metastable subgel phase in saturated diacylphosphatidylcholines.

Formation of well ordered lamellar subgel (SGII) phase in aqueous dispersions of L-dipalmitoylphosphatidylcholine upon cooling from the lamellar gel phase, without low-temperature equilibration, is observed in real time using synchrotron x-ray diffraction. It has the same lamellar repeat period as the gel phase from which it was formed but differs in its wide-angle diffraction pattern. The SGII phase forms at about 7 degrees C upon cooling at 2 degrees C/min. In temperature jump experiments at 1 degree C/s from 50 to -5 degrees C, the relaxation time of the lamellar gel-SGII transition is found to be approximately 15 s. The conversion between the lamellar gel and SGII phase is cooperative and rapidly reversible. Upon heating, it coincides in temperature with an endothermic event with a calorimetric enthalpy of 0.35 kcal/mol, the so-called sub-subtransition. Similar sub-subtransitions are also observed calorimetrically at temperatures approximately 10 degrees C below the subtransition, without low-temperature storage, in aqueous dispersions of L-dimyristoylphosphatidylcholine and L-distearoylphosphatidylcholine, but not in racemic DL-dipalmitoylphosphatidylcholine. The formation of the equilibrium lamellar crystalline Lc phase appears to take place only from within the SGII phase.