Actin is a highly ubiquitous and evolutionarily conserved protein capable of polymerizing and forming filamentous polymers which play a central role in cell mechanics and motility. Here, we study the in vitro regulation of actin mechanics and dynamics by calponin and caldesmon, two actin binding proteins believed to be involved in regulating cytoskeletal mechanics and structure through mechanisms not currently well understood.
Chapters 1 and 2 introduce the reader to actin and its roles in the cell, as well as to the methods and theoretical foundations used in this work.
In Chapter 3, we use total internal reflection and confocal fluorescence microscopy to investigate the polymerization dynamics of actin in the presence of a caldesmon C terminal fragment, H32K. We show that H32K stabilizes a nascent structural state of actin without altering the polymerization dynamics of the filament. We also show that H32K stabilized nascent actin has increased affinity for the actin branching protein complex Arp2/3 involved in driving membrane protrusions during cell motility, and propose the nascent state of actin as a possible transient differentiator targeting certain actin binding proteins to actin in vivo. This is to our knowledge the first reported direct functional effect of nascent actin.
In Chapter 4, we use fluorescence microscopy to quantify actin bending mechanics in the presence of the binding protein calponin and show that calponin reduces the persistence length of actin. We compare our results to the literature and compare the mechanical change to electron microscopy reconstructions, which suggest that calponin affects actin intermonomer contacts through interactions with actin subdomain 2.
In Chapter 5, we expand on the results from Chapter 4 using bulk rheology and show that calponin increases the tensile strength of reconstituted actin networks, similar to the effect seen in whole cells and tissues. We discuss these data within an affine network model and show that the results can be entirely explained in terms of the reduced actin persistence length. We use this to propose a novel physical mechanism for calponin function in vivo.
This work elucidates the physical mechanisms of calponin and caldesmon function and their role in regulating the cellular cytoskeleton. / 2031-01-01T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/13663 |
| Date | 24 September 2015 |
| Creators | Jensen, Mikkel Herholdt |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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