Derek K. Toomre PhD
Associate Professor of Cell Biology; Director of the YALE 'CINEMA' Laboratory (Cellular Imaging using New Microscopy Approaches)
Cell Biology; Exocytosis; Endocytosis; Membrane Traffic; Tethering; Microscopy; Live Cell Imaging; TIRFM; Single molecule
A long-term goal of my laboratory is to understand how the exo-endocytic cycle is spatially regulated during cellular morphogenesis. Two major focuses are the spatial-temporal regulation of membrane traffic in adipocytes in response to insulin signaling and in fibroblasts during cell migration. To elucidated mechanisms that regulate membrane trafficking, my laboratory uses advanced imaging and analysis coupled with RNAi knockdown of key regulator machinery (e.g. exocyst complex). Innovative live cell TIRFM and spinning-disk confocal imaging at the 'CINEMA' imaging center are used to visualize directly, at the single-vesicle level, where and how membrane traffic is delivered and regulated. As impaired vesicle exocytosis is associated with type 2 diabetes and abnormal cell motility is associated with metastasis and cancer, the knowledge gained with this research will be highly significant in understanding the pathologies of these diseases.
Extensive Research Description
Cellular Imaging and Analysis of Polarized Membrane Traffic. A major goal of my laboratory is to develop and apply new and state-of-the-art multidimensional optical methods to better understand the basic mechanisms of polarized membrane trafficking and spatial-temporal control of endo-exocytosis.
One important challenge facing modern biology is to understand how individual biochemical reactions are integrated in space and time. Increasingly, new vital probes and optical methods has begun to provide unique mechanistic insight into how molecules, vesicles, organelles and whole cells are (re)organized in response to internal and external cues. This is especially relevant for the dynamic process of membrane traffic and the cytoskeleton in cell polarity - key areas of our interest. Insight into how cells both establish and lose polarity are also essential for understanding disease processes such as metastasis. In particular we are applying the optical methods of Total Internal Reflections Fluorescence Microscopy (TIRFM) and 4D (3D + time) multicolor spinning-disk confocal imaging to directly address, at the single-vesicle level, where and how polarized membrane traffic is delivered. TIRFM imaging (also called evanescent wave microscopy) can selectively illuminate an extremely thin optical section (< 50 nm) of the lower surface of the cell (reviewed in Toomre and Manstein, 2001[PDF]; for Java Tutorials see http://www.olympusmicro.com/primer/techniques/fluorescence/tirf/tirfhome.html.) It offers unsurpassed signal-to-noise and permits single-vesicle visualization and quantification of exocytic docking and fusion.
Specifically, using advance optical methods our lab is exploring the following related topics:
1) organization and coordination of exocytosis and cytoskeleton in polarized cells and
2) coupling of exo- and endocytosis and molecular mechanisms that regulate this process. For instance, TIRFM imaging has lead to a number of novel observations including imaging of constitutive exocytosis (and the surprising presence of exocytic ‘hot-spots’ for fusion on the cell surface) and nanometer targeting of microtubule plus ends to the cell surface and focal adhesions. To facilitate these and other studies multicolor TIRFM instruments, a 4D spinning disk confocal and electrophysiology instrumentation has been recently implemented here as part of “The CINEMA Lab” ("Cinema Imaging Using New Microscopy Approaches"), with support from Ludwig Institute for Cancer Research (LICR), various grants, Yale and the private sector. We are also collaborating with other groups at Yale (Dr. Jim Duncan’s group, Dept. of Biomedical Engineering) and overseas (Elena Diaz, Spain) to develop novel software to detect, analyze and make computational cellular models of these processes.