Active and Passive Microrheology of F-Actin Membrane Composites

Nöding, Helen

From Minimal Cortex Model Systems to Living Cells

dc.contributor.advisor	Janshoff, Andreas Prof. Dr.
dc.contributor.author	Nöding, Helen
dc.date.accessioned	2018-07-19T09:34:39Z
dc.date.available	2018-07-19T09:34:39Z
dc.date.issued	2018-07-19
dc.identifier.uri	http://hdl.handle.net/11858/00-1735-0000-002E-E457-0
dc.identifier.uri	http://dx.doi.org/10.53846/goediss-6970
dc.language.iso	eng	de
dc.rights.uri	http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc	571.4	de
dc.title	Active and Passive Microrheology of F-Actin Membrane Composites	de
dc.title.alternative	From Minimal Cortex Model Systems to Living Cells	de
dc.type	doctoralThesis	de
dc.contributor.referee	Janshoff, Andreas Prof. Dr.
dc.date.examination	2017-10-20
dc.description.abstracteng	The complex mechanical properties of a living cell are not only a function of its structural components but also of the organizational super-structure and the dynamic interconnection of filaments, proteins and the plasma membrane. Especially, the F-actin cortex plays a central role in cell adhesion, migration, division, growth and differentiation. Abnormalities in these essential cellular processes are tightly connected to diseases such as cancer and malaria. It is thus of pivotal interest to study the determinants of cellular mechanics. The main part of this thesis is dedicated to the investigation of a minimal cortex model of the F-actin cortex. A pre-polymerized network of semi-flexible F-actin polymers is attached to a lipid bilayer by the physiological cross-linker ezrin. A pseudo-phosphorylated mutant of ezrin, T567D, is used to study the impact of transient membrane linkage on the frequency dependent viscoelastic properties. Here, passive as well as active microrheological measurements are established on these thin composite materials and frequency spectra ranging from 10^-3-10^2 Hz are measured. In an intermediate frequency regime (10^-2-10^1 Hz) the elastic properties dominate the force response of the model system. The stiffness of the system is dominated by the mesh size of the self-organized model cortex, which is in turn a function of the availability of pinning-points in the membrane. These findings suggest the formation of an affine network. The low frequency regime (10^-3-10^-2 Hz) of the shear modulus is dominated by the transient binding kinetic of the membrane cross-link ezrin. An apparent unbinding rate constant is determined from the microrheological data and the transient nature of the membrane attachment is supported by a low energy barrier for the unbinding. For the high frequency regime (10^1-10^2 Hz) deviations from the typically found power law scaling of ¾ are observed and discussed in the context of increased inertia upon F-actin attachment to a solid supported model membrane. The minimal actin cortex model is compared to entangled networks of the semi-dilute F-actin filament as well as F-actin membrane composites isolated from the apical cortex of living cells by the sandwich cleavage method. In the second part of this thesis, the focus is set on mechanotransduction and rigidity sensing in epithelial monolayers. Cell mechanics in response to surface elasticity are studied by active atomic force microscopy based microrheology and interpreted in terms of (active) soft glassy rheology. Epithelial cells from the kidney (MDCK II) and mammary gland (MCF-10A) are studied as a function of substrate elasticity (E=1-100 kPa). Cells cultured on soft substrates (1 kPa) exhibit similar frequency dependent viscoelastic properties as cells, which are F-actin depleted by latrunculin A. Both cell lines behave stiffer when cultured on surfaces of higher elasticity. Above a certain threshold of substrate stiffness no further changes can be observed upon increase in surface stiffness. This final cortical stiffness can only be increased for a short time by the F actin reinforcing drug jasplakinolide. Additionally, the impact of cell size on the frequency dependent viscoelastic properties is elucidated. Altogether, the essential contribution of the membrane linkage to the viscoelastic properties of the F-actin cortex is shown in a minimal model system and the importance of extracellular mechanical cues, such as substrate elasticity, on the organization and mechanics of the F-actin cytoskeleton in living cells is emphasized.	de
dc.contributor.coReferee	Rehfeldt, Florian Dr.
dc.contributor.thirdReferee	Köster, Sarah Prof. Dr.
dc.contributor.thirdReferee	Enderlein, Jörg Prof. Dr.
dc.contributor.thirdReferee	Meinecke, Michael Prof. Dr.
dc.contributor.thirdReferee	Rizzoli, Silvio O. Prof. Dr.
dc.subject.eng	Minimal Actin Cortex	de
dc.subject.eng	Microrheology	de
dc.subject.eng	Cell Cortex	de
dc.subject.eng	F-Actin	de
dc.subject.eng	Mechanotransduction	de
dc.subject.eng	Latrunculin Jasplakinolid	de
dc.subject.eng	Video Particle Tracking	de
dc.identifier.urn	urn:nbn:de:gbv:7-11858/00-1735-0000-002E-E457-0-5
dc.affiliation.institute	Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)	de
dc.subject.gokfull	Biologie (PPN619462639)	de
dc.identifier.ppn	1027326676

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GGNB - Göttinger Graduiertenzentrum für Neurowissenschaften, Biophysik und molekulare Biowissenschaften [1116]
GGNB - Göttingen Graduate Center for Neurosciences, Biophysics and Molecular Biosciences

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