Tracking Assembly Kinetics of Intermediate Filaments

Tracking Assembly Kinetics of Intermediate Filaments

Oliva Saldanha

Doctoral thesis

Date of Examination: 2016-04-22

Date of issue: 2017-04-13

Advisor: Köster, Sarah Prof. Dr.

Referee: Köster, Sarah Prof. Dr.

Referee: Hub, Jochen Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0023-3E14-3

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Abstract

English

Vimentin is an intermediate filament (IF) protein in eukaryotic cells and together with other IFs, actin filament and microtubules plays an instrumental role in providing stability and mechanical properties to the cell. From in vitro experiments it is known that the rod-like IF monomers self-assemble to first form tetramers. In a subsequent step, unit length filaments (ULFs) of 65 nm length form by lateral association of typically eight tetramers which then longitudinally anneal to form long filaments with a diameter of about 10 nm. Upon the addition of divalent salts, the long filamentous vimentin bundle and form networks. An in-depth understanding of the intricate assembly mechanisms and aggregation forming processes of vimentin is important to realize its biophysical role in cells. Hence, our study investigates the structural changes in vimentin by small angle X-ray scattering (SAXS) employing microfluidics and by time resolved dynamic and static light scattering (DLS and SLS). Dynamic tracking of the formation of μm-long filaments from tetrameric vimentin is studied in microflow. The initial vimentin assembly steps upon the addition of monovalent ions are accessible by SAXS and occur on ms time scales. Aggregation mechanism of vimentin filaments due to the addition of divalent ions can also be detected by the method of SAXS and occurs on the s time scales. These processes are investigated both in hydrodynamically focused continuous microflow as well as in droplets. In combination with SAXS, the microfluidic approaches ensure decreased radiation damage and the advantage of subsequent mixing of different aqueous components. However, the intermediary zone of elongation into thin-rod like vimentin filaments is inaccessible by X-rays (due to the length scales involved) and complementary to SAXS, time resolved DLS and SLS techniques are employed where time evolution of elongation can be measured from minutes to hours. The advantage of time resolved DLS and SLS is the tracking of the elongation process with access to growing mass per unit length. With the complementary approaches and the given sensitivity of SAXS and DLS/SLS, we can infer the spatial and temporal evolution of fundamental structural changes in vimentin.

Keywords: intermediate filaments; vimentin; biophysics; small angle x-ray scattering; light scattering; microfluidics; self-assembly