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Title: Role of the actin cytoskeleton in regulating LYVE-1 lateral diffusion and function
Author: Stanly, Tess Archana
ISNI:       0000 0004 6498 1346
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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The lymphatic endothelial receptor LYVE-1 investigated here is a weak binder to its ligand hyaluronan (HA) and uses receptor clustering to achieve the avidity required for stable interaction to HA. These clustering based interactions are highly dynamic and are often controlled by several molecular players that contribute to receptor functional changes. One such contributor is the cortical actin cytoskeleton. As the actin cytoskeleton lies immediately below the membrane surface, it creates compartments on the membrane that control the receptor organisation, dynamics and allows physical interaction of the receptors with actin, which either hinder or enhance their functions. In this study, I investigate the role of the actin cytoskeleton in regulating the dynamics and function of LYVE-1. At first, the native organisation of LYVE-1 and cross-linked clustered state of LYVE-1 (that are known to cause avidity induced increase in HA binding), on primary human dermal lymphatic endothelial cells (HDLECs), were identified by confocal and super-resolution STED microscopy. Following which the effects of actin disassembly on LYVE-1:HA binding function and cross-linked clustering of LYVE-1 were investigated using FACS and STED microscopy. The dynamics of LYVE-1 during actin steady state or disassembly was studied using diffusion measurements on primary and cytoplasmic tail truncated hLYVE-1 transfected HDLECs. The results from the diffusion experiments suggest that LYVE-1 mobility decreases after actin depletion and correlates with the increase in HA interaction as determined by FACS during actin disassembly, although there was no change in the surface organisation of LYVE-1. Further diffusion measurements using STED-FCS showed that LYVE-1 was 'trapped' or confined on the membrane surface of HDLECs in native conditions. This data along with biochemical assays show a hitherto unknown direct link of the actin cytoskeleton regulating LYVE-1:HA binding and dynamics, giving an indication of how LYVE-1 in vivo may modulate binding to HA.
Supervisor: Jackson, David ; Eggeling, Christian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available