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Folding and Assembly of Cytoskeletal Proteins Under Force - from Single Molecule Studies of Dystrophin to Studies of Intact Cells University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Spring 2011 Folding and Assembly of Cytoskeletal Proteins Under Force - From Single Molecule Studies of Dystrophin to Studies of Intact Cells Christine Carag [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biophysics Commons Recommended Citation Carag, Christine, "Folding and Assembly of Cytoskeletal Proteins Under Force - From Single Molecule Studies of Dystrophin to Studies of Intact Cells" (2011). Publicly Accessible Penn Dissertations. 349. https://repository.upenn.edu/edissertations/349 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/349 For more information, please contact [email protected]. Folding and Assembly of Cytoskeletal Proteins Under Force - From Single Molecule Studies of Dystrophin to Studies of Intact Cells Abstract Changes in tertiary and quaternary structure of proteins within the actin cytoskeletal network are a likely way cells read mechanical signals from their environment. However, showing that these conformational changes occur as a result of mechanical stress and that such changes are important to the function of the cell is a major challenge. This thesis seeks to address these questions using a cohort of molecular biophysical and cell biological methods applied in increasingly complex contexts. First, the importance of force-driven unfolding to function and how changes in unfolding pathway correlate with diseased states was determined with single molecule Atomic Force Microscopy on nano-constructs of wild-type and mutant forms of dystrophin. Biophysical studies showed that the ability to fold into mechanically stable, spectrin-type helical bundle domains and the preservation of cooperative unfolding were common characteristics of functional truncated dystrophins. Second, a newly developed in-cell cysteine labeling technique demonstrated stress-enhanced repeat unfolding within spectrin in wild-type red blood cells under shear stress versus static conditions, thus demonstrating that forced unfolding is not just an in vitro phenomena. The importance of the cytoskeletal network to spectrin function was also demonstrated in mutant, 4.1R-null red blood cells, where the intrinsic properties of spectrin remain intact but the network integrity is compromised by absence of 4.1R. Loss of network integrity was evident in a decrease in spectrin unfolding under stress. Repeat unfolding was accompanied by changes in associations of spectrin with its binding partners in a time- and stress- dependent manner, indicating that the erythrocyte cytoskeleton exhibits a graded response to stress. Lastly, with cardiomyocytes derived from embryonic stem cells, the importance of stress to quaternary structure of actinin within the sarcomeric cytoskeleton and its effects on cell-wide function was tested in cells adhered to elastic substrates. Substrate stiffness sets the load on these spontaneously contracting cells, and differences in load lead to cytoskeletal reorganization with significant effects on cardiogenic development. Taken together, these findings present evidence of various cytoskeletal proteins – especially in the spectrin superfamily – as mediators of mechanical signaling within cell. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemical and Biomolecular Engineering First Advisor Dennis E. Discher Keywords Dystrophin, repeat domains, cysteine labeling, cardiomyocyte Subject Categories Biophysics This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/349 FOLDING AND ASSEMBLY OF CYTOSKELETAL PROTEINS UNDER FORCE - FROM SINGLE MOLECULE STUDIES OF DYSTROPHIN TO STUDIES OF INTACT CELLS Christine Carag Krieger A DISSERTATION in Chemical and Biomolecular Engineering Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2011 ___________________________________ Dennis E. Discher, Ph. D. Supervisor of Dissertation ___________________________________ Raymond J. Gorte, Ph. D. Graduate Group Chairman DISSERTATION COMMITTEE H. Lee Sweeney, Ph. D. John C. Crocker, Ph. D. Matthew J. Lazzara, Ph. D. Chairman of Physiology Associate Professor Assistant Professor To Taylor Without his patience and support this thesis would not have been accomplished. Acknowledgements First and foremost I would like to thank my advisor Dennis Discher. Through his indulgence I had the opportunity to work on many projects and learn several techniques. I will always be grateful for the exposure I had to different fields and their diverse ways of thinking. I would also like to thank Dan Safer, Nishant Bhasin, and Colin Johnson for giving me a good start at the beginning of my graduate career, former lab members Florian Rehfeldt and Rich Tsai for making lab a fun place to work, Allison Zajac and Beca Tenney for teaching me what it means to be a cell biologist, and finally Alexis Wallen for her continuous support. ii ABSTRACT FOLDING AND ASSEMBLY OF CYTOSKELETAL PROTEINS UNDER FORCE - FROM SINGLE MOLECULE STUDIES OF DYSTROPHIN TO STUDIES OF IN INTACT CELLS Christine Carag Krieger Dennis E. Discher Changes in tertiary and quaternary structure of proteins within the actin cytoskeletal network are a likely way cells read mechanical signals from their environment. However, showing that these conformational changes occur as a result of mechanical stress and that such changes are important to the function of the cell is a major challenge. This thesis seeks to address these questions using a cohort of molecular biophysical and cell biological methods applied in increasingly complex contexts. First, the importance of force-driven unfolding to function and how changes in unfolding pathway correlate with diseased states was determined with single molecule Atomic Force Microscopy on nano- constructs of wild-type and mutant forms of dystrophin. Biophysical studies showed that the ability to fold into mechanically stable, spectrin-type helical bundle domains and the preservation of cooperative unfolding were common characteristics of functional truncated dystrophins. Second, a newly developed in-cell cysteine labeling technique demonstrated stress-enhanced repeat unfolding within spectrin in wild-type red blood cells under shear stress versus static conditions, thus demonstrating that forced unfolding is not just an in vitro phenomena. The importance of the cytoskeletal network iii to spectrin function was also demonstrated in mutant, 4.1R-null red blood cells, where the intrinsic properties of spectrin remain intact but the network integrity is compromised by absence of 4.1R. Loss of network integrity was evident in a decrease in spectrin unfolding under stress. Repeat unfolding was accompanied by changes in associations of spectrin with its binding partners in a time- and stress- dependent manner, indicating that the erythrocyte cytoskeleton exhibits a graded response to stress. Lastly, with cardiomyocytes derived from embryonic stem cells, the importance of stress to quaternary structure of actinin within the sarcomeric cytoskeleton and its effects on cell-wide function was tested in cells adhered to elastic substrates. Substrate stiffness sets the load on these spontaneously contracting cells, and differences in load lead to cytoskeletal reorganization with significant effects on cardiogenic development. Taken together, these findings present evidence of various cytoskeletal proteins – especially in the spectrin superfamily – as mediators of mechanical signaling within cell. iv TABLE OF CONTENTS Title Page Chapter 1 1 Spectrin proteins, the mechanical mediators of the cell Chapter 2 22 Exon-skipped dystrophins for treatment of Duchenne muscular dystrophy: mass spectrometry mapping of most exons and cooperative domain designs based on single molecule mechanics Chapter 3 60 Cysteine Shotgun Mass Spectrometry (CS-MS) of Cells reveals molecular pathways of stress-enhanced unfolding and dissociation of the cytoskeleton Chapter 4 100 Early cardiomyocytes need work for continued differentiation Chapter 5: Conclusions 133 v LIST OF TABLES Title Page Table 2.1: Potential dystrophin deletants created by exon skipping 47 Table 2.2: Predicted unfolding lengths of domains in dystrophin nano-constructs 48 Table 2.3: Thermal denaturation of dystrophin nano-constructs 52 Table 2.4: Tryptic Peptides of Dystrophin detected by Mass Spectrometry 59 Table 3.1: Rate constants from fLL fits of wild-type and 4.1R-null ghosts 94 Table 3.2: Cys labeling ratios for spectrin peptides quantified by Mass 95 Spectrometry. Table 3.3: Ion flux data of mass-spectrometry detected peptides, α, β-Spectrin 96 Table 3.4: Predicted accessibility of cysteines within repeat domains 98 Table 3.5: Cys labeling ratios for ankyrin peptides quantified by Mass 99 Spectrometry. vi LIST OF FIGURES Title Page Figure 1.1: Structure of spectrin superfamily proteins 16 Figure 1.2: Schematic of the spectrin repeat domain 17 Figure 1.3: Force-dependent Linderstrom-Lang Equation 18 Figure 1.4: Cooperative unfolding in hinged and hinge-free rod domains 19 Figure 1.5: Cardiomyocyte and matrix contraction 20 Figure 1.6: Schematic of strained states for soft gels, E* gels, and stiff gels 21 Figure 2.1: Full-length and representative truncated, Becker MD dystrophin in the 44 dystrophin-glycoprotein complex. Figure 2.2: Phasing of DMD exons and protein domains with
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