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The Resource Cell mechanics studied using atomic force microscopy, by Mingzhai Sun, (electronic resource)

Cell mechanics studied using atomic force microscopy, by Mingzhai Sun, (electronic resource)

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Contributor
Summary
Cholesterol plays an indispensable role in regulating the properties of the cell membrane. In particular, lipid rafts, specific membrane domains, which are thought to be required for a number of cell functions, such as receptor mediated signaling and membrane trafficking, are dispersed when cell cholesterol is extracted. There is also evidence showing that cholesterol affects the cells' deformability, an important factor in the development of atherosclerosis. In this study, we investigated the effect of cellular cholesterol on the mechanical properties of bovine aortic endothelial cells (BAECs) and their correlation with the development of atherosclerosis. To compare the mechanical properties of cells with different cholesterol content, we have developed a method to measure the forces needed to extract nanotubes (tethers) from their membranes, using atomic force microscopy (AFM). Our observations show that cholesterol depletion of BAECs resulted in significant increase of membrane-cytoskeleton adhesion. An increase in cellular cholesterol to a level higher than that in normal cells caused decrease of the membrane cytoskeleton adhesion and dramatic decrease of the effective surface viscosity of their membranes. While cholesterol depletion and enrichment had no apparent effect on the intensity of F-actin specific fluorescence, disrupting F-actin with latrunculin A abrogated the observed effects. Fluorescence recovery after photobleaching experiments were also performed to measure the lateral mobility of a lipid probe (DiIC12) at different cholesterol contents. The results are consistent with the AFM measurement.To investigate the molecular bases of the phenomena, we focussed on the regulatory phospholipid, phosphatidylinositol 4,5-biophosphate (PIP2), which is involved in a variety of cell functions, especially the regulation of cytoskeleon, and membrane-cytoskeleton adhesion. In the plasma membrane, PIP2 accumulates in cholesterol-rich domains, and its concentration decreases upon cholesterol depletion.By culturing BAECs with neomycin or by transfecting them to express the GFP-tagged PH domain from phospholipase C [delta], we sequester PIP2 to mimic the effect induced by cholesterol depletion. Interestingly, PIP2 sequestering by either approach decreases cell membrane deformability as cholesterol depletion does. This result suggests that cholesterol depletion affects cell mechanical properties by altering the concentration/distribution of PIP2, which may further change the cortical F-actin network. Furthermore, our studies demonstrate that AFM can be used to relate and correlate biomolecular and biophysical properties
Language
eng
Work
Publication
Note
  • The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file
  • Title from title screen of research.pdf file (viewed on June 17, 2009)
  • Vita
Instance
Label
Cell mechanics studied using atomic force microscopy
Title
Cell mechanics studied using atomic force microscopy
Statement of responsibility
by Mingzhai Sun
Creator
Contributor
Thesis advisor
Subject
Genre
Language
eng
Summary
Cholesterol plays an indispensable role in regulating the properties of the cell membrane. In particular, lipid rafts, specific membrane domains, which are thought to be required for a number of cell functions, such as receptor mediated signaling and membrane trafficking, are dispersed when cell cholesterol is extracted. There is also evidence showing that cholesterol affects the cells' deformability, an important factor in the development of atherosclerosis. In this study, we investigated the effect of cellular cholesterol on the mechanical properties of bovine aortic endothelial cells (BAECs) and their correlation with the development of atherosclerosis. To compare the mechanical properties of cells with different cholesterol content, we have developed a method to measure the forces needed to extract nanotubes (tethers) from their membranes, using atomic force microscopy (AFM). Our observations show that cholesterol depletion of BAECs resulted in significant increase of membrane-cytoskeleton adhesion. An increase in cellular cholesterol to a level higher than that in normal cells caused decrease of the membrane cytoskeleton adhesion and dramatic decrease of the effective surface viscosity of their membranes. While cholesterol depletion and enrichment had no apparent effect on the intensity of F-actin specific fluorescence, disrupting F-actin with latrunculin A abrogated the observed effects. Fluorescence recovery after photobleaching experiments were also performed to measure the lateral mobility of a lipid probe (DiIC12) at different cholesterol contents. The results are consistent with the AFM measurement.To investigate the molecular bases of the phenomena, we focussed on the regulatory phospholipid, phosphatidylinositol 4,5-biophosphate (PIP2), which is involved in a variety of cell functions, especially the regulation of cytoskeleon, and membrane-cytoskeleton adhesion. In the plasma membrane, PIP2 accumulates in cholesterol-rich domains, and its concentration decreases upon cholesterol depletion.By culturing BAECs with neomycin or by transfecting them to express the GFP-tagged PH domain from phospholipase C [delta], we sequester PIP2 to mimic the effect induced by cholesterol depletion. Interestingly, PIP2 sequestering by either approach decreases cell membrane deformability as cholesterol depletion does. This result suggests that cholesterol depletion affects cell mechanical properties by altering the concentration/distribution of PIP2, which may further change the cortical F-actin network. Furthermore, our studies demonstrate that AFM can be used to relate and correlate biomolecular and biophysical properties
Cataloging source
MUU
http://library.link/vocab/creatorDate
1979-
http://library.link/vocab/creatorName
Sun, Mingzhai
Degree
Ph. D.
Dissertation year
2008.
Granting institution
University of Missouri-Columbia
Illustrations
illustrations
Index
no index present
Literary form
non fiction
Nature of contents
  • dictionaries
  • bibliography
  • theses
http://library.link/vocab/relatedWorkOrContributorName
Forgács, G.
http://library.link/vocab/subjectName
  • Atherosclerosis
  • Phospholipids
  • Cholesterol
  • Cell membranes
  • Atomic force microscopy
Target audience
specialized
Label
Cell mechanics studied using atomic force microscopy, by Mingzhai Sun, (electronic resource)
Instantiates
Publication
Note
  • The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file
  • Title from title screen of research.pdf file (viewed on June 17, 2009)
  • Vita
Bibliography note
Includes bibliographical references
Carrier category
online resource
Carrier category code
  • cr
Carrier MARC source
rdacarrier
Color
mixed
Content category
text
Content type code
  • txt
Content type MARC source
rdacontent
Control code
401748548
Dimensions
unknown
Form of item
electronic
Media category
computer
Media MARC source
rdamedia
Media type code
  • c
Specific material designation
remote
System control number
(OCoLC)401748548
System details
Mode of access: World Wide Web
Label
Cell mechanics studied using atomic force microscopy, by Mingzhai Sun, (electronic resource)
Publication
Note
  • The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file
  • Title from title screen of research.pdf file (viewed on June 17, 2009)
  • Vita
Bibliography note
Includes bibliographical references
Carrier category
online resource
Carrier category code
  • cr
Carrier MARC source
rdacarrier
Color
mixed
Content category
text
Content type code
  • txt
Content type MARC source
rdacontent
Control code
401748548
Dimensions
unknown
Form of item
electronic
Media category
computer
Media MARC source
rdamedia
Media type code
  • c
Specific material designation
remote
System control number
(OCoLC)401748548
System details
Mode of access: World Wide Web

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