Bending of a lipid membrane edge by annexin A5 trimers.

Plasma membrane damage occurs in healthy cells and more frequently in cancer cells where high growth rates and metastasis result in frequent membrane damage. The annexin family of proteins plays a key role in membrane repair. Annexins are recruited at the membrane injury site by Ca+2 and repair the damaged membrane in concert with several other proteins. Annexin A4 (ANXA4) and ANXA5 form trimers at the bilayer surface, and previous simulations show that the trimers induce high local negative membrane curvature on a flat bilayer. The membrane-curvature-inducing property of ANXA5 is presumed to be vital to the membrane repair mechanism. A previously proposed descriptive model hypothesizes that ANXA5-mediated curvature force is utilized at the free edge of the membrane at a wound site to pull the wound edges together, resulting in the formation of a "neck"-shaped structure, which, when combined with a constriction force exerted by ANXA6, leads to membrane repair. The molecular details and mechanisms of repair remain unknown, in part because the membrane edge is a transient structure that is difficult to investigate both experimentally and computationally. For the first time, we investigate the impact of ANXA5 near a membrane edge, which is modeled by a bicelle under periodic boundary conditions. ANXA5 trimers induce local curvature on the membrane leading to global bending of the bicelle. The global curvature depends on the density of annexins on the bicelle, and the curvature increases with the ANXA5 concentration until it reaches a plateau. The simulations suggest that not only do annexins induce local membrane curvature, but they can change the overall shape of a free-standing membrane. We also demonstrate that ANXA5 trimers reduce the rate of phosphatidylserine lipid diffusion from the cytoplasmic to the exoplasmic leaflet along the edge of the bicelle. In this way, membrane-bound annexins can potentially delay the apoptotic signal triggered by the presence of phosphatidylserine lipids in the outer leaflet, thus biding time for repair of the membrane hole. Our findings provide new insights into the role of ANXA5 at the edges of the membrane (the injury site) and support the curvature-constriction model of membrane repair.

Interplay of membrane crosslinking and curvature induction by annexins.

Efficient plasma membrane repair (PMR) is required to repair damage sustained in the cellular life cycle. The annexin family of proteins, involved in PMR, are activated by Ca2+ influx from extracellular media at the site of injury. Mechanistic studies of the annexins have been overwhelmingly performed using a single annexin, despite the recruitment of multiple annexins to membrane damage sites in living cells. Hence, we investigate the effect of the presence of the crosslinking annexins, annexin A1, A2 and A6 (ANXA1, ANXA2 and ANXA6) on the membrane curvature induction of annexin A4 (ANXA4) in model membrane systems. Our data support a mechanistic model of PMR where ANXA4 induced membrane curvature and ANXA6 crosslinking promotes wound closure. The model now can be expanded to include ANXA1 and ANXA2 as specialist free edge membrane crosslinkers that act in concert with ANXA4 induced curvature and ANXA6 crosslinking.

Molecular Mechanism of Hydrotropic Properties of GTP and ATP.

Hydrotropes are small amphiphilic compounds that increase the aqueous solubility of hydrophobic molecules. Recent evidence suggests that adenosine triphosphate (ATP), which is the primary energy carrier in cells, also assumes hydrotropic properties to prevent the aggregation of hydrophobic proteins, but the mechanism of hydrotropy is unknown. Here, we compare the hydrotropic behavior of all four biological nucleoside triphosphates (NTPs) using molecular dynamics (MD) simulations. We launch all atom MD simulations of aqueous solutions of NTPs [ATP, guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP)] with pyrene, which acts both as a model hydrophobic compound and as a spectroscopic reporter for aggregation. GTP prevents pyrene aggregation effectively. Dissolution is not achieved in the presence of CTP and UTP. The higher stability of the base stacking in guanine is responsible for the higher hydrotropic efficiency of GTP. Consistent with the simulations, spectroscopic measurements also suggest that the hydrotropic activity of GTP is higher than ATP. Stacking of aromatic pyrene with the aromatic base of NTPs is a characteristic feature of this hydrotropic property. Both ATP and GTP also dissolve clusters of di- and tripeptides containing tryptophan but with equal potency. Importantly, the presence of aromatic amino acids is a necessary condition for the hydrotropic potency of ATP and GTP. Our results can have broad implications for hydrotrope design in the pharmaceutical industry, as well as the possibility of cells employing GTP as a hydrotrope to regulate the hydrophobic protein aggregation in membrane-less biological condensates.

Interdisciplinary Synergy to Reveal Mechanisms of Annexin-Mediated Plasma Membrane Shaping and Repair.

The plasma membrane surrounds every single cell and essentially shapes cell life by separating the interior from the external environment. Thus, maintenance of cell membrane integrity is essential to prevent death caused by disruption of the plasma membrane. To counteract plasma membrane injuries, eukaryotic cells have developed efficient repair tools that depend on Ca2+- and phospholipid-binding annexin proteins. Upon membrane damage, annexin family members are activated by a Ca2+ influx, enabling them to quickly bind at the damaged membrane and facilitate wound healing. Our recent studies, based on interdisciplinary research synergy across molecular cell biology, experimental membrane physics, and computational simulations show that annexins have additional biophysical functions in the repair response besides enabling membrane fusion. Annexins possess different membrane-shaping properties, allowing for a tailored response that involves rapid bending, constriction, and fusion of membrane edges for resealing. Moreover, some annexins have high affinity for highly curved membranes that appear at free edges near rupture sites, a property that might accelerate their recruitment for rapid repair. Here, we discuss the mechanisms of annexin-mediated membrane shaping and curvature sensing in the light of our interdisciplinary approach to study plasma membrane repair.