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Molecular DNA Sensors to Measure Distribution of Cytoskeletal Forces

Christina Jayachandran
Doctoral thesis
Date of Examination: 2019-09-27
Date of issue: 2020-08-24
Advisor: Schmidt, Christoph F. Prof. Dr.
Advisor: Rehfeldt, Florian Dr.
Referee: Schmidt, Christoph F. Prof. Dr.
Referee: Wouters, Fred Prof. Dr.
Referee: Rehfeldt, Florian Dr.
Referee: Wardetzky, Max Prof. Dr.
Referee: Neef, Andreas Dr.
Referee: Klumpp, Stefan Prof. Dr.

Persistent Address: http://hdl.handle.net/21.11130/00-1735-0000-0005-1463-5

 

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Abstract

English

Actin, a major cytoskeletal biopolymer in eukaryotic cells, is crosslinked into networks of filaments and bundles. These networks are largely responsible for the maintenance of cellular shape, rigidity, and mechanical stability. Other assemblies of actin are involved in a myriad of cellular processes, such as cell migration, division, intracellular transport, and morphogenesis. In these processes, the spatial and temporal regulation of the network structure, their dynamics, and force generation due to myosin motors are crucial. Experimentally, one of the challenges is to measure force transmission across such networks, which is vital to properly understand the function, failure, and repair mechanisms beyond the linear regime. To measure forces across the cytoskeletal network, we have developed a FRET-based, reversible DNA force sensor. We employ these DNA constructs as flexible crosslinkers across semiflexible actin, thereby reconstituting model networks of cytoskeletal structures. Characterization of the rheology and frequency response of these model actin-DNA sensor networks is performed via a macrorheometer and also by utilizing a large bandwidth, high-resolution microrheology set up. DNA force sensors are crosslinked in vitro with actin filaments in order to map force distributions and stress relaxations in the resulting network. We characterize the DNA force sensor in solution and across actin networks through fluorescence lifetime imaging microscopy (FLIM) measurements. From these results, we estimate the FRET efficiency of our DNA sensor. We also test DNA sensors in a cellular environment and describe its preliminary results.
Keywords: DNA based molecular force sensors; FRET; FLIM; Frequency response of networks; Bulk fluorescence measurements; Actin-DNA sensor networks
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