04/12/2021
By Susan Pryputniewicz

The Biomedical Engineering and Biotechnology program invites you to attend a Doctoral Dissertation defense by Christopher Tsiros on “Ca2+-Enhanced Interactions Between Skeletal Titin’s N2A Region and Actin Filaments.”

Ph.D. Candidate: Christopher Tsiros
Defense Date: Thursday, April 22, 2021
Time: 12:30 - 3:30 p.m.
Location: This will be a virtual defense via Zoom. Those interested in attending should contact the student christopher_tsiros@student.uml.edu and committee advisor matthew_gage@uml.edu at least 24 hours prior to the defense to request access to the meeting.

Committee Chair (Advisor): Matthew Gage, Ph.D., Associate Professor, Chemistry, University of Massachusetts Lowell

Committee Members:

  • Jeffrey Moore, Ph.D., Professor, Biological Science, University of Massachusetts Lowell
  • Michael Graves, Ph.D., Associate Professor, Biological Science, University of Massachusetts Lowell
  • Nicolai Konow, Ph.D., Assistant Professor, Biological Science, University of Massachusetts Lowell

Brief Abstract:

The sliding filament model has been the foundation of our understanding of muscle contraction, explaining contraction as the Ca2+-dependent formation of cross-bridges between actin and myosin filaments. While this has been the prevailing model for muscle contraction for over 50 years, a weakness of model is its inability to account for all the measurable forces observed in muscle contraction. Recently, it has been suggested that interactions between the titin filament and actin could be the missing component in existing models. The N2A region of titin consists of four Ig domains (I80-I83) and an insertion sequence located in between I80 and I81. This region is uniquely located between titin’s Ig-domain region that is elongated under low forces and it’s PEVK region, which is extended under higher forces. A deletion at the junction between the N2A and PEVK regions results in muscular dystrophy with mystosis (mdm) in mice, diminishing contractile properties in skeletal muscle. Based on these observations, it has been hypothesized that the N2A region might be a site of interaction between titin and actin. We demonstrate that recombinantly-expressed N2A constructs co-sediment with filamentous actin, supporting the potential association between N2A and actin. In addition, in vitro motility data demonstrates reduced actin filament velocity in the presence of N2A and the degree of velocity reduction is enhanced by addition of Ca2+, further supporting a binding interaction between N2A and actin in active muscle contraction. N2A-actin binding was also observed with dynamic force spectroscopy (DFS) experiments. Higher rupture forces observed in the presence vs. absence of Ca2+ suggest that the N2A-actin interaction is stabilized by Ca2+. Further co-sedimentation experiments were performed on different subsections of the N2A region in order to assess which domains within the N2A region were critical for actin binding. Loss of either I82 or I80 resulted in complete loss of binding to actin. Based on these results, we conclude that the primary binding site on N2A is in I82 and a secondary site may exist in either I80, the insertion sequence or a combination of both. High performance liquid chromatography (HPLC) analysis of actin-N2A samples in the presence of Ca2+ identified a second N2A-actin peak that is not observed either in the absence of Ca2+ or in constructs with I83 deleted. We hypothesize that Ca2+ improves the binding between N2A and actin by stabilizing the I83 domain, based on the known role of Ca2+ in stabilizing this domain. In summary, we conclude that the I82 domain contributes to N2A-actin binding and this binding is enhanced by the stabilization of the I83 domain by Ca2+. This previously unobserved mechanism supports the hypothesis that titin-actin interactions are the missing element in current accepted models of muscle contraction.

All interested students and faculty members are invited to attend the online defense via remote access.