Translocation of Phosphatidylserine

One of the early events of the apoptotic process involves the translocation of phosphatidylserine on the surface of cell membranes annexin V binding and propidium iodide uptake reveals various cellular states. After treatment with organotin(IV) compounds the cells could be categorized into populations vital cells (annexin V /P ), early apoptotic cells (annexin V /P ), late apoptotic cells (annexin V /P ), and necrotic cells (annexin V /P" ). Cells are observed with a fluorescence microscope and it is possible to observe translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer one and to see a green stain for annexin V FLUOS bound to PS, and a red stain for propidium iodide. 

Changes occur at the cell surface and plasma membrane in the early stages of apoptosis. One of the major plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer for external exposure (FI). This change of exposure requires the activation of caspase-3, a Ca flux over the plasma membrane, and a change in Bcl-2 family (B8,B13, M6). 

The second phase, proposed by Moghimi et al., was said to occur 24 h after exposure, and consisted of a loss of mitochondrial membrane potential which was revealed by translocation of phosphatidylserine as a consequence of PEI-induced channel formation. This led to release of the pro-apoptotic cytochrome c and subsequent activation of caspases-3 triggering apoptosis. Mitochondrial-mediated apoptotic events induced by polycations have also been reported in cell lines treated with high MW PLL in free form, DNA polyplexes and PAMAM dendrimers. In the study by Lee et al., PAMAM dendrimers around 45 nm in size exhibited mitochondrial co-localization, decreased expression of mitochondrial genes and mitochondria membrane... 

Phosphatidylserine is a membrane phospholipid, which is normally restricted to the inner leaflet of the plasma membrane. The translocation of phosphatidylserine from the inner to the... 

Phosphatidylserine is a membrane phospholipid, which is normally restricted to the inner leaflet of the plasma membrane. The translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane is an early event in apoptosis. Annexin V, an endogenous human protein with a high affinity for membrane-bound phosphatidylserine, can be used in vitro to detect apoptosis. An annexin V assay in a microfluidic system has been developed for flow cytometric analysis of apoptosis using a minimal number of cells. 

Methods used to demonstrate the existence of membrane phospholipid asymmetry, such as chemical labelling and susceptibility to hydrolysis or modification by phospholipases and other enzymes, are rmsuitable for dynamic studies because the rates of chemical and biochemical reactions are of a different order compared to the transmembrane translocahon of the phospholipids. Indirect methods have therefore been developed to measure the translocation rate which are consequent on the loss of membrane phospholipid asymmetry. Thus time scales appropriate to rates of lipid scrambling under resting conditions or when the forces preserving the asymmetric phospholipid distribution are disturbed can be monitored. Generally the methods rely on detecting the appearance of phosphatidylserine on the surface of cells. Methods of demonstrating Upid translocation in mammalian cells has been the subject of a recent review (Bevers etal., 1999). 

The control of the processes of phosphohpid translocation and scrambling by cytoplasmic calcium concentration is known to be one factor in regulating the appearance of phosphatidylserine on the cell surface. There is now evidence that other factors are involved in modulating membrane phospholipid distribution and for preserving phospholipid homeostasis. Current research is directed to clarify the role of these agents and to establish any connections with the initiation of apoptosis. 

Translocation of the phosphatidylserine from the inner to the outer leaflet of the plasma membrane is an initial event related to the apoptotic process and possibly serves as a signal for the removal of apoptotic bodies by phagocytic cells (Martin et al., 1995). The exposure of this phospholipid has been largely used as a specific apoptosis marker. 

Voelker, D.R. 1990. Characterization of phosphatidylserine synthesis and translocation in permeabilized animal cells. J. Biol. Chem. 265 14340-14346. 

The classic example of this targeting mechanism is the translocation of PKC. Binding of Ca + or diacyl-glycerol to PKC in the presence of phosphatidylserine causes a conformational change in the molecule that results in exposure of the pseudosubstrate domain (Orr el al., 1992 Bosca and Moran, 1993) and presumably increases hydrophohicity of the molecule, which facilitates binding of the enzyme to membrane lipids. 

Persistent membrane anchorage - newly translocated cPKC binds DAG in the presence of phosphatidylserine and calcium, and becomes fuUy active in the proximity of substrates cPKC is maintained at the plasma membrane until the DAG signal is cleared (1-2 minutes). 

PKC is one member of a femily of serine and threonine kinases - so-called because they catalyse the phosphorylation fix)m ATP of serine and threonine amino acid bases found in many different proteins. PKC is distinguished from other protein kinases because it is dependent on Ca and also the phospholipid phosphatidylserine (PS) for activity. When DAG appears free in the plasma membrane, quiescent PKC translocates from the cytosol to the plasma membrane, binding at sites rich in PS. It is this binding of PKC to membrane PS, in the presence of DAG, which confers increased Ca sensitivity to the enzyme and allows release of the catalytic subunit of PKC into the cytosol where target protein phosphorylation... 

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