Aminoglycoside-Induced Phosphatidylserine Externalization in Sensory Hair Cells Is Regionally Restricted, Rapid, and Reversible

The aminophospholipid phosphatidylserine (PS) is normally restricted to the inner leaflet of the plasma membrane. During certain cellular processes, including apoptosis, PS translocates to the outer leaflet and can be labeled with externally applied annexin V, a calcium-dependent PS-binding protein. In mouse cochlear cultures, annexin V labeling reveals that the aminoglycoside antibiotic neomycin induces rapid PS externalization, specifically on the apical surface of hair cells. PS externalization is observed within ~75 s of neomycin perfusion, first on the hair bundle and then on membrane blebs forming around the apical surface. Whole-cell capacitance also increases significantly within minutes of neomycin application, indicating that blebbing is accompanied by membrane addition to the hair cell surface. PS externalization and membrane blebbing can, nonetheless, occur independently. Pretreating hair cells with calcium chelators, a procedure that blocks mechanotransduction, or overexpressing a phosphatidylinositol 4,5-biphosphate (PIP2)-binding pleckstrin homology domain, can reduce neomycin-induced PS externalization, suggesting that neomycin enters hair cells via transduction channels, clusters PIP2, and thereby activates lipid scrambling. The effects of short-term neomycin treatment are reversible. After neomycin washout, PS is no longer detected on the apical surface, apical membrane blebs disappear, and surface-bound annexin V is internalized, distributing throughout the supranuclear cytoplasm of the hair cell. Hair cells can therefore repair, and recover from, neomycin-induced surface damage. Hair cells lacking myosin VI, a minus-end directed actin-based motor implicated in endocytosis, can also recover from brief neomycin treatment. Internalized annexin V, however, remains below the apical surface, thereby pinpointing a critical role for myosin VI in the transport of endocytosed material away from the periphery of the hair cell

Simple Model of the Transduction of Cell-Penetrating Peptides

Cell-penetrating peptides (CPPs) such as HIV's trans-activating transcriptional activator (TAT) and polyarginine rapidly pass through the plasma membranes of mammalian cells by an unknown mechanism called transduction. They may be medically useful when fused to well-chosen chains of fewer than about 35 amino acids. I offer a simple model of transduction in which phosphatidylserines and CPPs effectively form two plates of a capacitor with a voltage sufficient to cause the formation of transient pores (electroporation). The model is consistent with experimental data on the transduction of oligoarginine into mouse C2-C12 myoblasts and makes three testable predictions.Comment: Seven pages. For a more complete version including the effects of counterions, see arXiv:0810.2358v3 [q-bio.BM

Antiphosphatidylserine antibody as a cause of multiple dural venous sinus thromboses and ST-elevation myocardial infarction

Objective: Rare disease Background: Antiphospholipid syndrome (APS) is an autoimmune disease characterized by antibodies directed against phos-pholipids on plasma membranes. Through unclear mechanisms, APS confers hypercoagulability. APS may cause recurrent thromboses in the arterial and venous vasculature. We report a case of primary APS resulting in cerebral venous thrombosis and ST-elevation myocardial infarction (STEMI) for which only antiphosphatidylserine (aPS) IgM antibody was positive after extensive investigation. Case Report: A 48-year-old male was admitted after a witnessed generalized seizure with subsequent confusion. Imaging demonstrated thrombosis of multiple central nervous system (CNS) sinuses, including the superior sagittal sinus and bilateral transverse sinuses. The patient was heparinized with aggressive hydration, which proved inadequate, prompting endovascular thrombectomy. Three months later, despite anticoagulation therapy, the patient developed a STEMI when International Normalized Ratio (INR) was 1.8. Echocardiogram (ECHO) and PAN CT scan were normal. Initial coagulation studies demonstrated normal anticardiolipin antibody, prothrombin time, partial thromboplastin time, and platelet count. Outpatient coagulation studies revealed normal an-tithrombin III, protein C/S, hemoglobin electrophoresis, homocysteine, anti-b2 glycoprotein 1 antibodies, and D-Dimer. Factor V Leiden, JAK 2 mutation, prothrombin gene mutation, and tests for paroxysmal nocturnal he-moglobinuria (PNH) were negative. A positive phosphatidylserine IgM was detected. The patient was continued on warfarin (10 mg daily) with a target INR of 3.0–3.5 and clopidogrel (75 mg daily). Conclusions: Despite extensive investigation, this patient only showed evidence of elevated aPS IgM antibodies, likely contributing to his CNS venous sinus thromboses and STEMI. It is important to screen for antiphosphatidylserine antibodies in cases of unprovoked thrombosis when standard thrombophilia analysis is unrevealing. This will assist in identifying pathogenicity and help prevent recurrence of subsequent thromboses. © Am J Case Rep, 2018

The immune reaction against allogeneic necrotic cells is reduced in Annexin A5 knock out mice whose macrophages display an anti-inflammatory phenotype

Proteins of the annexin family bind to phospholipids in a Ca2+ dependent manner. The exposure of phosphatidylserine (PS) by apoptotic as well as necrotic cells is one major eat-me-signal for macrophages. Annexin A5 (Anx A5) preferentially binds to PS. The availability of Anx A5 knock out (KO) mice allowed us to investigate for the first time if endogenous Anx A5 modulates the immune response towards allogeneic cells. Furthermore, the effect of Anx A5 gene deletion on the phagocytic process as well as on the inflammatory reaction of macrophages was explored. We found that Anx A5 KO mice have a strongly reduced allogeneic cellular immune reaction against primary as well as secondary necrotic cells. In vivo phagocytosis experiments revealed that macrophages of Anx A5 KO mice displayed an increased uptake of necrotic cells. Additionally, an increased secretion of the anti-inflammatory cytokine IL-10 of isolated macrophages of Anx A5 KO mice after contact with necrotic cells was observed. Furthermore, the promoter activity of the Anx A5 gene was enhanced after stimulation of macrophages. The tumour size of an allogeneic tumour regressed faster when endogenous Anx A5 was present. These data demonstrate that endogenous Anx A5 influences the phagocytosis of necrotic cells, modulates the immune response towards allogeneic cells and acts as an inflammatory protein

Phosphatidylserine polarization is required for proper Cdc42 localization and for development of cell polarity.

We used genetically-encoded fluorescent probes to visualize the distribution of phosphatidylserine (PS) in live S. cerevisiae. The majority of the PS was found to reside in the cytosolic leaflet of the plasma membrane. Remarkably, PS was polarized, accumulating in bud necks, the bud cortex and the tips of mating projections. Polarization required vectorial delivery of PS-enriched secretory and recycling vesicles. Rapid dissipation of the PS gradient is prevented by the slow diffusion of lipids along the plasmalemmal inner leaflet, estimated by photobleaching recovery measurements to be over an order of magnitude slower than in mammalian cells. In mutants lacking PS-synthase the absence of PS was associated with, and likely responsible for impaired polarization of the Cdc42 complex, leading to inhibition of bud emergence, diminished growth rate and abolishment of mating. The results indicate that PS polarization is required for optimal Cdc42 targeting and activation during cell division and mating

Role of Calcium in Phosphatidylserine Externalisation in Red Blood Cells from Sickle Cell Patients

Phosphatidylserine exposure occurs in red blood cells (RBCs) from sickle cell disease (SCD) patients and is increased by deoxygenation. The mechanisms responsible remain unclear. RBCs from SCD patients also have elevated cation permeability, and, in particular, a deoxygenation-induced cation conductance which mediates Ca2+ entry, providing an obvious link with phosphatidylserine exposure. The role of Ca2+ was investigated using FITC-labelled annexin. Results confirmed high phosphatidylserine exposure in RBCs from SCD patients increasing upon deoxygenation. When deoxygenated, phosphatidylserine exposure was further elevated as extracellular [Ca2+] was increased. This effect was inhibited by dipyridamole, intracellular Ca2+ chelation, and Gardos channel inhibition. Phosphatidylserine exposure was reduced in high K+ saline. Ca2+ levels required to elicit phosphatidylserine exposure were in the low micromolar range. Findings are consistent with Ca2+ entry through the deoxygenation-induced pathway (Psickle), activating the Gardos channel. [Ca2+] required for phosphatidylserine scrambling are in the range achievable in vivo

Loss of maternal annexin A5 increases the likelihood of placental platelet thrombosis and foetal loss

Antiphospholipid syndrome is associated with an increased risk of thrombosis and pregnancy loss. Annexin A5 (Anxa5) is a candidate autoantigen. It is not known, however, whether endogenous Anxa5 prevents foetal loss during normal pregnancy. We found significant reductions in litter size and foetal weight in Anxa5-null mice (Anxa5-KO). These changes occurred even when only the mother was Anxa5-KO. A small amount of placental fibrin deposition was observed in the decidual tissues, but did not noticeably differ between wild-type and Anxa5-KO mice. However, immunoreactivity for integrin beta 3/CD61, a platelet marker, was demonstrated within thrombi in the arterial canals only in Anxa5-KO mothers. Subcutaneous administration of the anticoagulant heparin to pregnant Anxa5-KO mice significantly reduced pregnancy loss, suggesting that maternal Anxa5 is crucial for maintaining intact placental circulation. Hence, the presence of maternal Anxa5 minimises the risk of thrombosis in the placental circulation and reduces the risk of foetal loss

Molecular Electroporation and the Transduction of Oligoarginines

Certain short polycations, such as TAT and polyarginine, rapidly pass through the plasma membranes of mammalian cells by an unknown mechanism called transduction as well as by endocytosis and macropinocytosis. These cell-penetrating peptides (CPPs) promise to be medically useful when fused to biologically active peptides. I offer a simple model in which one or more CPPs and the phosphatidylserines of the inner leaflet form a kind of capacitor with a voltage in excess of 180 mV, high enough to create a molecular electropore. The model is consistent with an empirical upper limit on the cargo peptide of 40--60 amino acids and with experimental data on how the transduction of a polyarginine-fluorophore into mouse C2C12 myoblasts depends on the number of arginines in the CPP and on the CPP concentration. The model makes three testable predictions.Comment: 15 pages, 5 figure