ABSTRACT
DIVALENT CATIONINDUCED REGULATION OF á5â1FIBRONECTIN INTERACTION FORCE ASSESSED USING ATOMIC FORCE MICROSCOPY
Nicolas Andres Perrusquia, Ph.D.
University of Pittsburgh, 2007
Cellular attachment to the extracellular matrix (ECM) via cell surface receptors is essential for signaling of the most basic of biological function such as differentiation and motility (Hynes, 1992). The integrin á5â1 binds its sole ECM ligand, fibronectin, through recognition of the RGD and (synergy) sequences; establishing a bi-directional signaling pathway between the cytoplasm and the ECM (Leahy, 1996; Redick, 2000; Krammer, 2001). During motility, forward cellular motion results in a rearward pull on the ECM, which physically loads the binding interface between á5â1 and fibronectin, forcing molecular separations at various speeds.
Divalent cations play a critical role in the á5â1fibronectin interaction as evidenced by (1) regulation of the affinity of interaction by cations [e.g., Ca2+ down regulates and Mg2+ or Mn2+ up regulates á5â1 binding affinity (Gailit, 1988; Mould, 1995)] and (2) loss of molecular interaction between á5â1 and fibronectin upon chelation using EDTA (Mould, 1995; Li, 2003).
The primary goal of the present study was to investigate the mechanisms underlying the cation-induced changes in á5â1fibronectin interaction. We used atomic force microscopy (AFM) to directly examine the á5â1-fibronectin interaction in the presence of affinity regulating divalent cations (i.e. either Ca2+, Mg2+, Mn2+, CaMg or CaMn) and at load rates that encompassed the known range of cellular motility speeds. Complimentary biochemical analyses were performed to examine the competitive binding of various cations to á5â1.
The rupture force was linearly proportional to load rate and two distinct patterns for this relationship were observed. There was only one linear region for the down regulated state of á5â1 for all load rates. In contrast, the up regulated state resulted in two piecewise linear segments; one segment was associated with low load rates (< ~ 10,000 pN/s) and the other with high load rates (> ~10,000 pN/s). Further, the data pattern associated with down regulated á5â1 results from a single (outer) energy barrier, while the up regulated data pattern results from two energy barriers; an outer barrier for low load rates and an inner energy barrier for high load rates.
No significant difference in bond rupture force (P = 0.68) existed at low load rates between the down and up regulated forms of á5â1, since each condition encountered the same outer energy barrier. However, the up regulated form of á5â1 encountered an additional energy barrier (i.e., the inner barrier) at high load rates, resulting in a sharp increase in rupture force (i.e. the second piecewise linear segment).
Although both Mg2+ and Mn2+ up regulated á5â1 (i.e., both inner and outer barriers present), the addition of Ca2+ down regulated á5â1 (i.e., eliminated the inner barrier) only for Mg2+; it was unable to do so for Mn2+. The complimentary biochemical assays showed that (45Ca2+) preferentially labelled á5â1 in the presence of Mg2+ (but not Mn2+) indicating that Mg2+ is displaced by Ca2+.
Overall, these results support the premise that a cation related mechanism is responsible for both down and up regulation of á5â1 binding affinity to fibronectin. Furthermore, this cation-induced regulation is related to the changes in the energy landscape (single vs. double energy barriers).
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07102007-181046 |
| Date | 25 September 2007 |
| Creators | Perrusquia, Nicolas |
| Contributors | Hai Lin, Harvey S. Borovetz, Scott X. Mao, Sanjeev G. Shroff |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-07102007-181046/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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