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Meihua Ju, Sofia Ioannidou, Victoria Fry, Peter Munro, Eija Jokitalo, David T. Shima; Moesin and Annexin II Differentially Regulate Formation of the Actin-fodrin Cytoskeleton and Endothelial Fenestra Biogenesis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4080.
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Fenestrae are transcellular membrane pores that mediate blood-tissue exchange in highly specialized vascular endothelium such as in choroidal capillaries. Substances that traverse the pore never encounter the contents of the cytoplasm and are transported in a rapid and presumably energy-efficient manner. Fenestrae arise in attenuated regions of the endothelial cell periphery and are highly organized in clusters termed sieve plates. Here we provide evidence for a structural and biochemical link between actin binding proteins and the formation of a sub-membrane cytoskeleton involved in fenestra biogenesis.
Using an in vitro biogenesis model coupled with proteomic analysis we identified several proteins enriched in fenestrated plasma membranes. Localization of candidate proteins was accomplished by immunolabelling and both confocal and transmission electron microscopy. Functional roles of the candidate proteins in fenestra biogenesis were probed through gain and loss of function techniques.
We identified the ERM (ezrin/radixin/moesin) protein moesin as a component of fenestra sieve plates. Inhibition of moesin function by expression of a dominant negative mutant or by siRNA resulted in an inhibition of fenestra formation, whilst knockdown of another regulator of the actin cytoskeleton, annexin II, led to a robust increase in fenestra formation. Biochemical and structural analyses showed that these modulators controlled the formation of an actin-fodrin submembrane cytoskeleton that was essential for sieve plate and fenestra formation, and that this cytoskeleton was directly linked to the fenestra pore protein PV-1.
These findings provide a conceptual framework linking actin rearrangements to membrane remodeling during fenestra biogenesis and new molecular tools for probing fenestra structure and function.
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