Derek K. Toomre, Ph.D

Assistant Professor of Cell Biology Assistant Member, Ludwig Institute for Cancer Research
Phone: (203) 785-4319 Lab: (203) 785-5057 CINEMA Lab: (203) 785-7371 Fax: (203) 785-3559 e-mail: derek.toomre@yale.edu		Department of Cell Biology Yale University School of Medicine 333 Cedar Street PO Box 208002 New Haven, CT 06520-8002 <Courier Address> 333 Cedar Street, SHM C-227 (Lab: SHM C-229) New Haven, CT 06510-3206

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Cellular Imaging and Analysis of Polarized Membrane Traffic

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 cell morphogenesis.

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.

Figure 1: New Views and Insights with Fluorescent Imaging of Cells and Tissue.
Top left: Complex structural organization of tissue as seen in from section of human colon using A33, an antibody developed by the LICR and further characterized here.
Top Right: Multispectral imaging of a cell with markers for the nucleus (blue), clustered lipid raft marker (green) and actin (red).
Bottom left: Red tracks of vesicles as they move along microtubules (green) in living cells (see Toomre et al, 1999 [PDF] ).
Bottom right: High resolution view of a single vesicle (only ~100 nanometers across) fusing with the cell surface, using a specialized TIRFM imaging. The time-lapse series is shown in 1 second intervals (left-to-right) and when the vesicle fuses an enormous flash is seen (see Toomre et eal., 2000 [PDF]).

Link: The CINEMA Lab "Cinema Imaging Using New Microscopy Approaches"

Selected Publications

Zoncu R, Perera RM, Sebastian R, Nakatsu F, Chen H, Balla T, Ayala G, ToomreD, De Camilli PV.  Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate.
Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3793-8. Epub 2007 Feb 27.

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.

Hadjidemetriou S, Toomre D, Duncan JS. Segmentation and 3D reconstruction of microtubules in total internal reflection fluorescence microscopy (TIRFM).
Med Image Comput Comput Assist Interv Int Conf Med Image Comput Comput Assist
Interv. 2005;8(Pt 1):761-9.

Hua W, Sheff D, Toomre D, Mellman I. Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging.
J Cell Biol. 2006 Mar 27;172(7):1035-44.

Gong LW, Di Paolo G, Diaz E, Cestra G, Diaz ME, Lindau M, De Camilli P, Toomre D.
Phosphatidylinositol phosphate kinase type I gamma regulates dynamics of large dense-core vesicle fusion.
Proc Natl Acad Sci U S A. 2005 Apr 5;102(14):5204-9. Epub 2005 Mar 25.

Krylyshkina O, Anderson KI, Kaverina I, Upmann I, Manstein DJ, Small JV, Toomre DK.
Nanometer targeting of microtubules to focal adhesions.
J Cell Biol. 2003 Jun 9;161(5):853-9. Epub 2003 Jun 2.

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.

Keller P, Toomre D, Diaz E, White J, Simons K. Multicolour imaging of post-Golgi sorting and trafficking in live cells.
Nat Cell Biol. 2001 Feb;3(2):140-9.