Cell-substratum adhesives in diatoms : structural, nanomechanical and dynamic properties of adhesive nanofibers determined with AFM force spectroscopy
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2006
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University of Melbourne
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Science Collected Works
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PhD thesis
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Thesis (PhD)-- University of Melbourne, Faculty of Science, 2007
Includes bibliographical references.
Thesis (PhD)-- University of Melbourne, Faculty of Science, 2007
Includes bibliographical references.
Abstract
Chapter 2 reports on the adhesive and mechanical properties of a cell-substratum adhesive secreted by live diatom cells (Toxarium undulatum Bailey) examined in situ using Atomic Force Microscopy (AFM). The resulting force curves have a regular saw-tooth pattern, the characteristic fingerprint of modular proteins, and when bridged between tip and surface can repeatedly be stretched and relaxed resulting in precisely overlaying saw-tooth curves (up to ~600 successive cycles). The average rupture force of the peaks is 0.794 � 0.007 (SE) nN at a loading rate of 0.8 ?m/s and the average persistence length is 0.026 � <0.001 (SE) nm (fit using the Worm-Like Chain model). I propose that the force curves represent the unfolding of single adhesive nanofibers (ANFs), each a cohesive unit composed of a set number of modular proteins aligned in register. Furthermore, multi-modal distributions of force and persistence length provide a mechanism that allows the observation and differentiation of up to three bridged ANFs. The high force required for bond rupture, high extensibility (~1.2 ?m), and the accurate and rapid refolding upon relaxation, together provide strong and flexible properties ideally suited for the cell-substratum adhesion of this fouling diatom and provides an understanding of the mechanism responsible for the strength of adhesion.
Chapter 3 reports on further force curves recorded with a different AFM system that reveals additional adhesive components within the T. undulatum adhesive mucilage. The mucilage is a biocomposite containing four additional adhesive components, including single modular proteins that are likely to be the structural units from which the ANFs are assembled. Two further distinct supramolecular assemblies were observed to coexist with ANFs (ANFs II and III), along with a continuum of single modular proteins through oligomers made up of varying numbers of modular proteins arranged in parallel. All components of the adhesive biocomposite produce a characteristic force spectrum with the same interpeak distance (35.3 � 0.3 (mean � SE) nm) suggesting they are derived from discrete supramolecular assemblies of the same modular protein, but they are distinguishable from one another based on the rupture force, persistence length and interpeak force measured from their saw-tooth curves.
Chapter 4 reports on adhesive modular proteins recorded by AFM on live cells from the extracellular mucilage secreted from, and deposited around, the motile form of the pennate diatom Phaeodactylum tricornutum Bohlin. This is the first report of modular proteins and their supramolecular assemblies (ANFs) to be found on diatoms that use adhesives not only for substratum adhesion, but also as a conduit for cell motility. The permanent adhesive pads secreted by Toxarium undulatum, a sessile centric diatom, were previously shown to possess ANFs with a modular protein backbone. The results reported here suggest that modular proteins may be an important component of diatom adhesives in general, and that diatoms utilize the tensile strength, toughness and flexibility of ANFs for multiple functions. Significantly, the genome of P. tricornutum has recently been sequenced; this will allow directed searches of the genome to be made for genes with modular protein homologues, and subsequent detailed studies of their molecular structure and function.
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Keywords
Diatoms, Atomic force microscopy, Adhesives, Supramolecular chemistry, Nanostructures, Mucins.