Deshpande, Siddharth. Dynamics of emerging actin networks. 2013, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_10573
To study a reversible reaction sequence in a step-by-step manner, one needs an open system. As a result, there have been relatively few studies in this direction, as most of the experimental systems are closed, for instance, sealed coverslips or liposomes. We created a straightforward microfluidic system, consisting of quasi two-dimensional, cell-sized compartments, enclosing sub-picolitre volumes. These `microchambers' are connected to the controlling channel (the reservoir) via narrow connecting channels, allowing exclusive diffusive transport into and out of the microchambers. The system represents an ideal environment to form an entangled network of actin filaments in a steady-state and is manipulable in a step-by-step fashion.
We induce bundling of actin filaments in three ways: counterion condensation aided by magnesium ions, depletion interactions mimicked by polyethylene glycol, acting as a crowding agent, and specific interactions with actin exhibited by filamin, an actin binding protein. Above the critical concentration of bundling agents, actin filaments transform into an emerging network of actin bundles, a process associated with percolation, leading to a single connected entity. Sharing of filaments is an important parameter for the observed behaviour, as reducing the actin filament length exclusively forms bundles without percolation. We encounter a hierarchical process of bundling: filaments coalesce into small bundles that further fuse to form bigger bundles. Disassembly involves a similar hierarchy, additionally involving peeling-off of single filaments. We explore the reactions using time-lapse image analyses and apply kinetic models.
Counterion condensation forms a network comprising of straight, rigid bundles facilitated by a zipping process (v ~ 12 µm/s), generating tension within the network. Disassembly leads to the release of the stored energy, utilized in the buckling of bundles, enabling us to estimate ~ 100 - 200 kT of stored energy. Crowding agents force the actin filaments to form an intriguing spindle-like structure, consisting of poles with sets of aligned filaments shared and stretched between them, which further transforms into a network of bundles. The disassembly constitutes the reversal of the process. Filamin forms ring-like networks, containing intrinsically curved bundles. Owing to the highly specific interactions, the network does not disassemble, even after 12 hours.
In essence, using a bottom-up approach, we explore the emerging properties of actin bundles, with an emphasis on their dynamics.
|Committee Members:||Lim, Roderick|
|Faculties and Departments:||05 Faculty of Science > Departement Chemie > Chemie > Biophysikalische Chemie (Pfohl)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||169 S.|
|Last Modified:||30 Jun 2016 10:54|
|Deposited On:||12 Nov 2013 13:45|
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