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eScholarship
Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2014)

The Notch signaling pathway is required in sensing the local cellular environment during multicellular development. Because Notch utilizes juxtacrine contact between two cells and relies upon a set of protease cleavage events, understanding the distribution and dynamics of the receptor during activation are of particular importance in order to understand its regulation. Notch must not just be expressed and present at the cell surface, but must be at the right place at the right time for proper activation to occur. Here we invent and then use monovalent Quantum Dots (mQDs) to target and monitor human Notch1 on the surface of live mammalian cells. We track and observe the dynamics of the receptor and compare the diffusion and distribution of Notch with other structural features at the surface of the cell. We find Notch to be slow and confined in its diffusion at the cell surface. In comparing temporal dynamics with static high-resolution static microscopy of the receptor we find Notch to be excluded from particular regions on the surface. These regions of exclusion include focal adhesions as determined by co-imaging Paxillin and Notch. In an attempt to determine the mechanism of exclusion we systematically pared domains from the receptor and identify the extracellular Notch regulatory region (NRR) as playing a significant role in distributing the receptor at the cell surface.

Additional work tries to quantitate the emergent behaviors observed in intercellular interactions taking place in heterogeneous collections of tissues. We repurpose single molecule tracking techniques used to track single molecules in order to track and identify the distribution and dynamics of cells to arrive at quantitative descriptions of multicellular interactions. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.

    Cover page: Dynamics of Single Human Notch1 Receptors at the Surface of Live Mammalian Cells
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2012)
    Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.
      Cover page: Programmed Cell-to-Cell Variability in Ras Activity Triggers Emergent Behaviors during Mammary Epithelial Morphogenesis
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2016)
      Tools capable of imaging and perturbing mechanical signaling pathways with fine spatiotemporal resolution have been elusive, despite their importance in diverse cellular processes. The challenge in developing a mechanogenetic toolkit (i.e., selective and quantitative activation of genetically encoded mechanoreceptors) stems from the fact that many mechanically activated processes are localized in space and time yet additionally require mechanical loading to become activated. To address this challenge, we synthesized magnetoplasmonic nanoparticles that can image, localize, and mechanically load targeted proteins with high spatiotemporal resolution. We demonstrate their utility by investigating the cell-surface activation of two mechanoreceptors: Notch and E-cadherin. By measuring cellular responses to a spectrum of spatial, chemical, temporal, and mechanical inputs at the single-molecule and single-cell levels, we reveal how spatial segregation and mechanical force cooperate to direct receptor activation dynamics. This generalizable technique can be used to control and understand diverse mechanosensitive processes in cell signaling. VIDEO ABSTRACT.
        Cover page: A Mechanogenetic Toolkit for Interrogating Cell Signaling in Space and Time
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2022)
        Adherens junctions (AJs) create spatially, chemically and mechanically discrete microdomains at cellular interfaces. Here, using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering and mechanical loading, we find that AJs also organize proteolytic hotspots for γ-secretase with a spatially regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand-receptor engagement (LRE microdomains) to initiate receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP microdomains). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of lipid-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP microdomains, AJs prevent inappropriate enzyme-substrate interactions and suppress spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiation of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of other large cell-surface receptors such as amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis and disease.
          Cover page: Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling
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