Optimization of cell unroofing and in vitro reconstitution of reticular adhesion

Reticular adhesions (RAs) are a novel type of cell-ECM adhesion, characterized by the presence of integrin αVβ5 and colocalized with flat clathrin lattices (FCLs) and lack many focal adhesion (FA) components. A distinctive feature of RAs is their persistence during the cytoskeletal changes that occu...

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Main Author: Quadir, Mohammed Mostafa Al
Other Authors: Matemaattis-luonnontieteellinen tiedekunta, Faculty of Sciences, Bio- ja ympäristötieteiden laitos, Department of Biological and Environmental Science, Jyväskylän yliopisto, University of Jyväskylä
Format: Master's thesis
Language:eng
Published: 2024
Subjects:
Online Access: https://jyx.jyu.fi/handle/123456789/96065
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author Quadir, Mohammed Mostafa Al
author2 Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science Jyväskylän yliopisto University of Jyväskylä
author_facet Quadir, Mohammed Mostafa Al Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science Jyväskylän yliopisto University of Jyväskylä Quadir, Mohammed Mostafa Al Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science Jyväskylän yliopisto University of Jyväskylä
author_sort Quadir, Mohammed Mostafa Al
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description Reticular adhesions (RAs) are a novel type of cell-ECM adhesion, characterized by the presence of integrin αVβ5 and colocalized with flat clathrin lattices (FCLs) and lack many focal adhesion (FA) components. A distinctive feature of RAs is their persistence during the cytoskeletal changes that occur in cell division, unlike other cell adhesions that typically disassemble. The formation of RAs involves the clustering of integrin αVβ5 and the formation of FCLs, which have been found to be interdependent events. A deeper understanding of why this occurs and what physiological factors trigger RA formation is needed. The in vitro reconstitution of RAs is crucial for studying these questions in a controlled manner. This thesis aimed to optimize a method for isolating membrane sheets attached to a solid substrate. It compared two cell unroofing methods, which remove the top membrane and cytosol, leaving the bottom membrane on a solid surface: squashing-peeling and ultrasonication. It also attempted to reconstitute RAs on these membrane sheets and visualize them using TIRF microscopy. This experiment was successful in producing high-quality membrane sheets and in reconstituting RAs on these sheets. Among the two methods employed for generating the membrane sheets, ultrasonication was found to be more effective than the squashing-peeling method for isolating high-quality membrane sheets. The research also indicated that RA formation is temperature-sensitive, with 37°C being the optimal temperature. In vitro reconstitution of reticular adhesions presents a powerful approach to studying these novel structures.
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spellingShingle Quadir, Mohammed Mostafa Al Optimization of cell unroofing and in vitro reconstitution of reticular adhesion reticular adhesion extracellular matrix Solu- ja molekyylibiologia Cell and molecular biology 4013 solubiologia integriinit adheesio solukalvot solut mikroskopia cell biology integrins adhesion cell membranes cells microscopy
title Optimization of cell unroofing and in vitro reconstitution of reticular adhesion
title_full Optimization of cell unroofing and in vitro reconstitution of reticular adhesion
title_fullStr Optimization of cell unroofing and in vitro reconstitution of reticular adhesion Optimization of cell unroofing and in vitro reconstitution of reticular adhesion
title_full_unstemmed Optimization of cell unroofing and in vitro reconstitution of reticular adhesion Optimization of cell unroofing and in vitro reconstitution of reticular adhesion
title_short Optimization of cell unroofing and in vitro reconstitution of reticular adhesion
title_sort optimization of cell unroofing and in vitro reconstitution of reticular adhesion
title_txtP Optimization of cell unroofing and in vitro reconstitution of reticular adhesion
topic reticular adhesion extracellular matrix Solu- ja molekyylibiologia Cell and molecular biology 4013 solubiologia integriinit adheesio solukalvot solut mikroskopia cell biology integrins adhesion cell membranes cells microscopy
topic_facet 4013 Cell and molecular biology Solu- ja molekyylibiologia adheesio adhesion cell biology cell membranes cells extracellular matrix integriinit integrins microscopy mikroskopia reticular adhesion solubiologia solukalvot solut
url https://jyx.jyu.fi/handle/123456789/96065 http://www.urn.fi/URN:NBN:fi:jyu-202406204829
work_keys_str_mv AT quadirmohammedmostafaal optimizationofcellunroofingandinvitroreconstitutionofreticularadhesion