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Title: Role of membrane cytoskeleton in fenestra biogenesis
Author: Ju, M.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2013
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Fenestrae are transcellular membrane pores that mediate blood-tissue exchange in highly specialized vascular endothelia 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. My PhD project was based on the identification of novel components of fenestrae and how these components contribute to mechanisms regulating fenestra formation. Using an in vitro biogenesis model coupled with proteomic analysis, we identified several proteins enriched in fenestrated plasma membranes. Localisation of candidate proteins was accomplished by immunolabelling, confocal microscopy, and transmission electron microscopy. Functional roles for the candidate proteins in fenestra biogenesis were probed through gain- and loss of function techniques. Coimmunoprecipitation was used to uncover protein-protein interactions, and biochemical reagents were applied to probe the signalling pathways involved in fenestra formation. Through extensive investigation, we identified the ERM (ezrin/radixin/moesin) protein moesin as a component of fenestral sieve plates. Inhibition of moesin function by expression of a dominant negative mutant or siRNA resulted in inhibition of fenestra formation, whereas 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 control the formation of an actin-fodrin submembrane cytoskeleton that is essential for sieve plate and fenestra formation, and that this cytoskeleton is directly linked to the fenestra pore protein PV-1. The transmembrane protein Na,K-ATPase is also a structural component of the submembrane complex, and functions as a regulator of fenestra formation in vitro and in vivo. These findings provide a conceptual framework linking the actin cytoskeleton to membrane remodeling during fenestra biogenesis and new molecular tools for probing fenestra structure and function.
Supervisor: Shima, D. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available