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Elucidating the biomechanical aspects of mammalian Notch Signaling

  • Author(s): Ravindranath, Shreyas Raj
  • Advisor(s): Botvinick, Elliot
  • et al.
Abstract

Canonical Notch Signaling Pathway is a critical signaling pathway that is required for cell fate specification and as such for the development of cells of almost all type of tissues. This seemingly linear but increasingly complicated pathway has been implicated in several disorders and cancers. This is because both losses and gains in Notch signals are associated with improper downstream target gene activation leading to disorders. This peculiar signaling pathway comprises of Notch receptors (Notch) transactivated by its ligands on the apposing cells. In the basal state Notch exists in an auto-inhibited, unligated state and is activated only by ligand binding in trans. Upon binding, ligand exerts a physical pulling force to expose ADAM10 (A Disintegrin And Metalloproteinase) cleavage site (also known as S2 site) deeply buried inside three globular domains of the Notch mechano-sensitive unit known as Negative Regulatory Region (NRR). ADAM10 cleavage is immediately followed by intramembrane γ-secretase cleavage releasing the Notch intracellular domain (NICD). NICD translocates to nucleus and associates with transcription factors and activates several target genes in a global or a context dependent fashion. During maturation, Notch is cleaved into a heterodimer by furin in the trans-golgi network at a region known as S1 cleavage site within the NRR just few amino acids upstream of the S2 site. Abrogation of heterodimerization process by deletion of S1 cleavage leads to reduced signaling in mice causing cardiovascular abnormalities. Here using optical tweezers based dynamic force spectroscopy, we study the response of S1 deleted Notch compared to wild type heterodimerized Notch to observe differences in response to ligand generated forces. I have found that S1 deleted Notch requires more mechanical work reflective of transactivation energetics upon ligand pulling compared to wild type Notch highly correlating with the loss of signaling in the S1 deleted Notch mice. Another important aspect of mammalian Notch receptors is the existence of several receptors and ligand which exhibit combinatorial effects in a context dependent fashion. I have also explored the possibility of each receptor-ligand bond strength and pulling mechanics to explain signaling outcomes and effects of the pair in a particular context.

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