Unraveling hedgehog signaling by single-molecule tracking

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Since the first optical detection of single molecules in 1989, there has been a rapid growth in applications, especially in biophysics. Single-molecule techniques are well suited for investigating the heterogeneity of populations, which is of particular interest in living systems that are defined by their complexity. Single-molecule fluorescence microscopy incorporates three main advantages of fluorescence imaging: biocompatibility, target specificity, and extreme sensitivity, and then improves upon the spatial precision by an order of magnitude relative to traditional, diffraction-limited imaging (down to tens of nanometers). This dissertation describes the application of single-molecule tracking to the investigation of the Hedgehog signaling pathway in mammalian cells. Hedgehog signaling is an essential developmental signaling pathway. Aberrant signaling leads to birth defects and has been implicated in a wide variety of cancers. Despite its oncological importance, the precise molecular mechanisms of signal transduction remain unclear. Previous work showed that pathway transduction involves the translocation of signaling proteins into and out of the primary cilium, a small organelle that protrudes from the cell surface. By tracking individual pathway proteins Patched1 and Smoothened with high spatial and temporal resolution (30 nanometers, 100 Hz), we have discovered and characterized changes in their dynamic motions within the cilium when the pathway is active or inactive at the most basic, functional level. In particular, we have found that the propensity for these proteins to bind at the base and tip of the cilium is a function of the pathway activation state. Moreover, aspects of the behavior of Smoothened are anticorrelated with the behavior of Patched1. These results have helped elucidate pathway function, and are applicable to the development of specific anti-cancer drugs that aim to disrupt particular steps of Hedgehog signaling. Chapter 1 provides an introduction to single-molecule imaging and an overview of Hedgehog signaling. Chapter 2 details the construction and operation of a fluorescence microscope with single-molecule sensitivity and outlines the various methodology utilized in later chapters. Chapters 3 and 4 describe our investigation of the Hedgehog signaling transducers Smoothened and Patched1, respectively. Chapter 5 summarizes our results and highlights several promising directions for further investigation. Finally, Chapter 6 contains the source code and manuals for the tools developed in these projects.


Type of resource text
Form electronic; electronic resource; remote
Extent 1 online resource.
Publication date 2017
Issuance monographic
Language English


Associated with Weiss, Lucien Everett
Associated with Stanford University, Department of Chemistry.
Primary advisor Moerner, W. E. (William Esco), 1953-
Thesis advisor Moerner, W. E. (William Esco), 1953-
Thesis advisor Boxer, Steven G. (Steven George), 1947-
Thesis advisor Dai, Hongjie, 1966-
Advisor Boxer, Steven G. (Steven George), 1947-
Advisor Dai, Hongjie, 1966-


Genre Theses

Bibliographic information

Statement of responsibility Lucien Everett Weiss.
Note Submitted to the Department of Chemistry.
Thesis Thesis (Ph.D.)--Stanford University, 2017.
Location electronic resource

Access conditions

© 2017 by Lucien Everett Weiss
This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).

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