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Dynamics of emerging actin networks

Deshpande, Siddharth. Dynamics of emerging actin networks. 2013, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_10573

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Abstract

Life is an ensemble of countless emerging properties arising through self-assembly and self-organization phenomena, manifesting at the cellular, the tissue and the organismal level. The mechanical integrity of a cell is orchestrated by the cytoskeleton, a dynamic system comprised of three biopolymers, actin, microtubules and intermediate filaments, acting in symphony, facilitated by a plethora of accessory proteins. Understanding the cytoskeletal functionality and its relation to other cellular components and properties is a prominent question in biophysics. Actin, a dynamic and polymorphic component, forms a variety of structures such as filaments, bundles, and their networks. The unique viscoelastic properties shown by actin-based structures have been extensively probed via rheological means. On the contrary, the underlying microstructural dynamics remain mostly uncovered. Actin bundles are crucial for eukaryotic cells; they are involved in the intracellular transport, contractive forces, mechanical stability, cell motility and environment exploration. This thesis takes a step forward to fathom the rich dynamics and emergent properties exhibited by actin bundles within flow-free confinements, a prerequisite for the study.
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.
Advisors:Pfohl, Thomas
Committee Members:Lim, Roderick
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie > Biophysikalische Chemie (Pfohl)
Item Type:Thesis
Thesis no:10573
Bibsysno:Link to catalogue
Number of Pages:169 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:54
Deposited On:12 Nov 2013 13:45

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