Elucidating the Biophysical Mechanisms of Notch Activation Open Access

Abstract

The Notch signaling pathway is an evolutionarily conserved mechanism for cell-cell communication that regulates many aspects of development and adult tissue homeostasis. For signal transduction to occur, membrane-anchored ligand and receptor molecules make direct contact at the interface of two apposing cell surfaces and set into motion a series of proteolysis events. Aberrant Notch signaling is linked to a number of developmental diseases and cancers, but there is still much unknown regarding the molecular mechanisms of receptor activation. A proposed mechanotransduction model hypothesizes that a mechanical force is required to unfold a portion of the receptor and reveal a key cleavage site. However, methods to directly measure cell exerted tension have only recently been established and relating force to downstream biochemical signaling remains a significant challenge.

In this dissertation, surface-based activation of Notch was used to address long-standing questions regarding the role of direct mechanical intervention as well as spatial and temporal inputs in activating the signaling pathway. The second chapter of this thesis focused on the development of a new technique for disrupting receptor spatial organization. Using dip-pen nanolithography, an optically transparent polymer (PDAC) was patterned onto a glass surface. The nanoscale polymer features impeded lipid diffusion and allowed for laterally mobile ligand molecules to be located adjacent to immobilized ECM proteins. The manipulation of receptor spatial organization altered the clustering of Notch and EGFR.

The third chapter centered on understanding how Delta ligand properties influenced Notch activation. While factors such as ligand density, orientation and surface anchoring chemistry were all explored, the most important parameter influencing receptor activation was lateral ligand mobility. Intriguingly, less diffusive ligand molecules (diffusion coefficient less than 0.1 μm²/s) resulted in significantly higher levels of downstream Notch activation.

The final chapter concentrated on modifying the tethered Delta ligand to create a DLL4 tension sensor with high sensitivity in order to detect Notch generated force and relate this to activation. The current data strongly suggest that the tension exerted by the cell is below the limit of detection as ligand-receptor dissociation is observed before extension of the linker region. There is still much to learn regarding the role of physical inputs in Notch signaling, but the development of new tools and methods continues to advance our understanding of this ubiquitous and versatile pathway.

Table of Contents

Chapter 1: The Notch Signaling Pathway...1

1.1 Introduction...2 1.1.1 Historical background...2 1.1.2 Biomedical relevance...4 1.2 What is currently known about the mechanism of Notch signaling?...4 1.2.1 Core components of Notch signaling...4 1.2.2 Models of activation...7 1.2.3 Role of oligomerization...9 1.3 Current methods used to study Notch and other juxtacrine systems...10 1.4 Aim and scope of the dissertation...12 1.5 References...13 Chapter 2: Nanoscale Patterning for Manipulating the Spatial Organization of Proteolipid Membranes...20 2.1 Introduction...21 2.2 Results and discussion...25 2.2.1 Selection of polymer "ink" and procedure for patterning...25 2.2.2 PDAC impedes lipid diffusion...26 2.2.3 Characterization of patterned polymer...28 2.2.4 Cell response to ligand functionalized nanopatterns...31 2.2.5 E-beam nanopatterns to restrict Delta-Notch movement...34 2.3 Conclusions...36 2.4 Materials and methods...37 2.4.1 Materials...37 2.4.2 DPN and AFM experiments...37 2.4.3 Supported bilayer formation...38 2.4.4 Fluorescence imaging...38 2.4.5 Cell experiments...39 2.4.6 Determination of pY levels...39 2.5 References...40 Chapter 3: Membrane Tethered Delta Activates Notch and Reveals a Role for Spatio-Mechanical Regulation of the Signaling Pathway...48 3.1 Introduction...49 3.2 Results and discussion...51 3.2.1 Delta-Notch binding on fluid membranes...51 3.2.2 Formation and dynamics of Delta-Notch clusters...55 3.2.3 Determination of DLL4-Notch1 binding ratio...61 3.2.4 Biological activity of surface tethered DLL4...68 3.2.5 Role of lateral ligand mobility of Notch activation...72 3.3 Conclusions...77 3.4 Materials and methods...78 3.4.1 Preparation of small unilamellar vesicles...78 3.4.2 Assembly of supported lipid membranes...79 3.4.3 DLL4 ligand labeling with Alexa Fluor dye...79 3.4.4 Design and expression of DLL4-mCherry...80 3.4.5 Biotin ligase modification of DLL4-mCherry...80 3.4.6 Live cell imaging...81 3.4.7 Binding specificity of DLL4 functionalized membranes...81 3.4.8 Calibration curves and determination of F factor...82 3.4.9 Data analysis for stoichiometry measurements...82 3.4.10 Activation of Notch reporter cell line...83 3.4.11 Immunostaining and analysis of NICD localization...83 3.4.12 Biotin-functionalized glass for non-fluid DLL4 surfaces...84 3.4.13 Physisorbed DLL4 on glass...85 3.4.14 Measurement of ligand diffusion coefficient...85 3.5 References...85 Chapter 4: Tension Sensing in the Notch Signaling Pathwa y...93 4.1 Mechanotransduction: Translating force into an intracellular message...94 4.1.1 Introduction...94 4.1.2 Cytoskeleton-associated mechanotransducers: Integrins and cadherins...95 4.1.3 Lipid-bilayer mediated mechanosensing: Mechanosensitive ion channels...98 4.2 Current methods to measure and study cellular forces...99 4.2.1 Atomic force microscopy (AFM)...100 4.2.2 Optical tweezers...102 4.2.3 Magnetic tweezers...103 4.2.4 Genetically encoded FRET force sensors...103 4.2.5 Molecular tension-based fluorescence microscopy (MTFM)...106 4.2.6 Comparing different methods for measuring force...108 4.3 Results and discussion...110 4.3.1 Designing a Notch tension sensor...110 4.3.2 Incorporation of genetically encoded tension sensor into DLL4...111 4.3.3 Semi-synthetic tension probes...113 4.3.3.1 Expressed protein ligation: DLL4 α-thioester and Cys-PEG24-biotin...113 4.3.3.2 Sortase mediated ligation:DLL4-mCherry-LPXTG and GGG-PEG24-QSY9-biotin...116 4.3.3.3 Sortase mediated ligation:DLL4-LPXTG and DNA hairpin121 4.4 Conclusions...128 4.5 Materials and methods...129 4.5.1 Expression of DLL4 ligands...129 4.5.2 Genetically encoded tension sensor...130 4.5.3 Semi-synthetic tension probe...131 4.5.3.1 Expression and purification of mCherry α-thioester...131 4.5.3.2 Expressed protein ligation: mCherry α-thioester and Cys-PEG24-biotin...131 4.5.3.3 Expression and purification of Sortase A (SrtA)...132 4.5.3.4 Click chemistry reaction to produce oligoglycine modified DNA...133 4.5.3.5 Sortase mediated ligation: DLL4-mCherry-LPXTG and DNA hairpin...134 4.6 References...134 Chapter 5: Conclusions and Perspectives...142 5.1 Summary...143 5.2 Disrupting receptor spatial organization...143 5.3 What is the role of force?...144 5.4 Future outlook...146 5.5 Other contributions and curriculum vitae...147 5.6 References...148

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, General
Keyword	Supported membrane Fluorescence Juxtacrine
Committee Chair / Thesis Advisor	Salaita, Khalid, Emory University
Committee Members	Lutz, Stefan, Emory University Dyer, Brian, Emory University

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