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Mechanosensitive Biological Function...

Hsu, Yen-Yu.

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Mechanosensitive Biological Functions: From Membrane Tension to Synthetic Exocytosis.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Mechanosensitive Biological Functions: From Membrane Tension to Synthetic Exocytosis./
Author:	Hsu, Yen-Yu.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2024,
Description:	220 p.
Notes:	Source: Dissertations Abstracts International, Volume: 85-12, Section: B.
Contained By:	Dissertations Abstracts International85-12B.
Subject:	Biology. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=31349137
ISBN:	9798382741260

Mechanosensitive Biological Functions: From Membrane Tension to Synthetic Exocytosis.
Hsu, Yen-Yu.

Mechanosensitive Biological Functions: From Membrane Tension to Synthetic Exocytosis. - Ann Arbor : ProQuest Dissertations & Theses, 2024 - 220 p.

Source: Dissertations Abstracts International, Volume: 85-12, Section: B.

Thesis (Ph.D.)--University of Michigan, 2024.

Cell communication is crucial for coordinating development and facilitating environmental adaptations in biological processes. Cells receive and transmit messages through various chemical or physical signals, including mechanical force, which is transmitted via mechanosensitive (MS) ion channels. These channels convert mechanical inputs into biochemical or electrical signals, regulating cellular behaviors. Despite the presence of MS channels as natural membrane tension sensors, a lack of suitable measurement tools has hindered the characterization of membrane tension changes in living cells. In Chapter 2, I developed an optical membrane tension reporter using a genetically modified MS channel, MscL, incorporating circularly permuted green fluorescence protein (cpGFP). This reporter serves as an OFF sensor in living cells, with fluorescence negatively correlated to membrane stretching and substrate stiffness, offering a valuable approach for detailed investigations in cellular membrane biophysics. While many MS channels have been investigated in living cells, studying their molecular mechanisms in response to mechanical force remains challenging due to the complexity of their protein structures, interactions with other cellular components, low expression levels, and toxicity issues in conventional cell culture systems. Therefore, in Chapter 3, I describe the use of cell-free expression (CFE) systems encapsulated inside lipid bilayers vesicles to reconstitute a putative MS channel found in fission yeast, Pkd2, and delved into its function without the involvement of complex cell signaling pathways. By co-encapsulating a cell-free expressed calcium indicator G-GECO with Pkd2 inside vesicles, I demonstrated that Pkd2 became calcium permeable when vesicle membranes were being stretched by hypo-osmotic shock. Moreover, the peak fluorescence intensities increased proportionally to the strength and duration of hypo-osmotic pressure. Our findings present the potential of using synthetic cells as a useful reconstitution platform for in vitro study of MS channels and other complicated membrane proteins. Realizing synthetic cells provide a powerful platform to replicate cellular functions, the final goal of my dissertation is to construct synthetic cells capable of intercellular communication, based on mimicking calcium-triggered exocytosis in living cells. It is well known that membrane fusion is an essential process for exocytosis. In Chapter 4, I describe the development of a DNA-mediated membrane fusion system that can be triggered by calcium ions by using surface-bound PEG (polyethylene glycol) chains which are cleavable by calpain, a calcium-activated protease. I demonstrated that membrane interactions and fusions are only observed in the presence of calcium ions, mimicking in vivo SNARE-mediated membrane fusion by recapitulating its calcium-dependent nature. I further showed that this strategy can be integrated into a vesicle-in-vesicle system reconstituted with MS channels for generating force-activated synthetic exocytosis which could enable communication between synthetic cells and natural cells in the future. In summary, my dissertation established the application of MS channel as an optical membrane tension reporter in living cells. I also advanced knowledge by leveraging encapsulated CFE systems inside synthetic cells for in vitro study of Pkd2p channels. To explore the use of the synthetic cell platform for controlled cell communications, a calcium-dependent membrane fusion strategy was developed and used in vesicle systems for the reconstitution of synthetic exocytosis, which can be integrated with mechanosensing mechanisms using MS channels. By improving the understanding of the biological functions of MS channels, I hope this research can help decipher the sophisticated mechanism of force-dependent signaling pathways and explore their potential biomedical applications.

ISBN: 9798382741260Subjects--Topical Terms:

522710
Biology.
Subjects--Index Terms:

Mechanosensitive proteins

Mechanosensitive Biological Functions: From Membrane Tension to Synthetic Exocytosis.
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520 $a Cell communication is crucial for coordinating development and facilitating environmental adaptations in biological processes. Cells receive and transmit messages through various chemical or physical signals, including mechanical force, which is transmitted via mechanosensitive (MS) ion channels. These channels convert mechanical inputs into biochemical or electrical signals, regulating cellular behaviors. Despite the presence of MS channels as natural membrane tension sensors, a lack of suitable measurement tools has hindered the characterization of membrane tension changes in living cells. In Chapter 2, I developed an optical membrane tension reporter using a genetically modified MS channel, MscL, incorporating circularly permuted green fluorescence protein (cpGFP). This reporter serves as an OFF sensor in living cells, with fluorescence negatively correlated to membrane stretching and substrate stiffness, offering a valuable approach for detailed investigations in cellular membrane biophysics. While many MS channels have been investigated in living cells, studying their molecular mechanisms in response to mechanical force remains challenging due to the complexity of their protein structures, interactions with other cellular components, low expression levels, and toxicity issues in conventional cell culture systems. Therefore, in Chapter 3, I describe the use of cell-free expression (CFE) systems encapsulated inside lipid bilayers vesicles to reconstitute a putative MS channel found in fission yeast, Pkd2, and delved into its function without the involvement of complex cell signaling pathways. By co-encapsulating a cell-free expressed calcium indicator G-GECO with Pkd2 inside vesicles, I demonstrated that Pkd2 became calcium permeable when vesicle membranes were being stretched by hypo-osmotic shock. Moreover, the peak fluorescence intensities increased proportionally to the strength and duration of hypo-osmotic pressure. Our findings present the potential of using synthetic cells as a useful reconstitution platform for in vitro study of MS channels and other complicated membrane proteins. Realizing synthetic cells provide a powerful platform to replicate cellular functions, the final goal of my dissertation is to construct synthetic cells capable of intercellular communication, based on mimicking calcium-triggered exocytosis in living cells. It is well known that membrane fusion is an essential process for exocytosis. In Chapter 4, I describe the development of a DNA-mediated membrane fusion system that can be triggered by calcium ions by using surface-bound PEG (polyethylene glycol) chains which are cleavable by calpain, a calcium-activated protease. I demonstrated that membrane interactions and fusions are only observed in the presence of calcium ions, mimicking in vivo SNARE-mediated membrane fusion by recapitulating its calcium-dependent nature. I further showed that this strategy can be integrated into a vesicle-in-vesicle system reconstituted with MS channels for generating force-activated synthetic exocytosis which could enable communication between synthetic cells and natural cells in the future. In summary, my dissertation established the application of MS channel as an optical membrane tension reporter in living cells. I also advanced knowledge by leveraging encapsulated CFE systems inside synthetic cells for in vitro study of Pkd2p channels. To explore the use of the synthetic cell platform for controlled cell communications, a calcium-dependent membrane fusion strategy was developed and used in vesicle systems for the reconstitution of synthetic exocytosis, which can be integrated with mechanosensing mechanisms using MS channels. By improving the understanding of the biological functions of MS channels, I hope this research can help decipher the sophisticated mechanism of force-dependent signaling pathways and explore their potential biomedical applications.
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650 4 $a Biomechanics. $3 548685
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