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Perrusquia, Nicolas (2007) DIVALENT CATION-INDUCED REGULATION OF á5â1-FIBRONECTIN INTERACTION FORCE ASSESSED USING ATOMIC FORCE MICROSCOPY. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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ABSTRACTDIVALENT CATION-INDUCED REGULATION OF á5â1-FIBRONECTIN INTERACTION FORCE ASSESSED USING ATOMIC FORCE MICROSCOPYNicolas Andres Perrusquia, Ph.D.University of Pittsburgh, 2007Cellular 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 á5â1-fibronectin 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 á5â1-fibronectin 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 (&lt ~ 10,000 pN/s) and the other with high load rates (&gt ~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).


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairShroff, Sanjeev Gsshroff@pitt.eduSSHROFF
Committee MemberLin,
Committee MemberBorovetz, Harvey Sborovetzhs@upmc.eduBOROVETZ
Committee MemberMao, Scott Xsmao@engr.pitt.eduSXM2
Date: 25 September 2007
Date Type: Completion
Defense Date: 23 July 2007
Approval Date: 25 September 2007
Submission Date: 10 July 2007
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Swanson School of Engineering > Bioengineering
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Other ID:, etd-07102007-181046
Date Deposited: 10 Nov 2011 19:50
Last Modified: 19 Dec 2016 14:36


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