Biology; Biophysics; Biotechnology; Chemistry; Nanotechnology; Optics and Photonics
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 (
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.
- Zoncu R, Perera RM, Balkin DM, Pirruccello M, Toomre D, De Camilli P. A phosphoinositide switch controls the maturation and signaling properties of APPL endosomes. Cell. 2009 Mar 20;136(6):1110-21. PubMed PMID: 19303853; PubMed Central PMCID: PMC2705806.
- Perera RM, Zoncu R, Lucast L, De Camilli P, Toomre D. Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages. Proc Natl Acad Sci U S A. 2006 Dec 19;103(51):19332-7. Epub 2006 Dec 8.
- Sebastian R, Diaz ME, Ayala G, Letinic K, Moncho-Bogani J, Toomre D. Spatio-temporal analysis of constitutive exocytosis in epithelial cells. IEEE/ACM Trans Comput Biol Bioinform. 2006 Jan-Mar;3(1):17-32.
- Goss JW, Toomre DK. Both daughter cells traffic and exocytose membrane at the cleavage furrow during mammalian cytokinesis. J Cell Biol. 2008 Jun 30;181(7):1047-54. Epub 2008 Jun 23. PubMed PMID: 18573914; PubMed Central PMCID: PMC2442215.
- Toomre D, Steyer JA, Keller P, Almers W, Simons K. Fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy. J Cell Biol. 2000 Apr 3;149(1):33-40. PubMed PMID: 10747085; PubMed Central PMCID: PMC2175107.
- Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol. 2000 Oct;1(1):31-9. Review. Erratum in: Nat Rev Mol Cell Biol 2001 Mar;2(3):216. PubMed PMID: 11413487.