scholarly journals Photobleaching Regions of Living Cells to Monitor Membrane Traffic

2011 ◽  
Vol 2011 (11) ◽  
pp. pdb.prot066563-pdb.prot066563 ◽  
Author(s):  
E. L. Snapp ◽  
P. Lajoie
Keyword(s):  
Membrane Traffic ◽  
Living Cells

Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules in living cells

1999 ◽  
Vol 112 (1) ◽  
pp. 21-33 ◽  
Author(s):  
D. Toomre ◽  
P. Keller ◽  
J. White ◽  
J.C. Olivo ◽  
K. Simons
Keyword(s):  
Plasma Membrane ◽  
Protein Trafficking ◽  
Membrane Traffic ◽  
Living Cells ◽  
Tubular Structures ◽  
Trans Golgi Network ◽  
Vesicular Stomatitis ◽  
Golgi Network ◽  
Color Visualization

The mechanisms and carriers responsible for exocytic protein trafficking between the trans-Golgi network (TGN) and the plasma membrane remain unclear. To investigate the dynamics of TGN-to-plasma membrane traffic and role of the cytoskeleton in these processes we transfected cells with a GFP-fusion protein, vesicular stomatitis virus G protein tagged with GFP (VSVG3-GFP). After using temperature shifts to block VSVG3-GFP in the endoplasmic reticulum and subsequently accumulate it in the TGN, dynamics of TGN-to-plasma membrane transport were visualized in real time by confocal and video microscopy. Both small vesicles (<250 nm) and larger vesicular-tubular structures (>1.5 microm long) are used as transport containers (TCs). These TCs rapidly moved out of the Golgi along curvilinear paths with average speeds of approximately 0.7 micrometer/second. Automatic computer tracking objectively determined the dynamics of different carriers. Fission and fusion of TCs were observed, suggesting that these late exocytic processes are highly interactive. To directly determine the role of microtubules in post-Golgi traffic, rhodamine-tubulin was microinjected and both labeled cargo and microtubules were simultaneously visualized in living cells. These studies demonstrated that exocytic cargo moves along microtubule tracks and reveals that carriers are capable of switching between tracks.

Imaging of Organelle Membrane Systems and Membrane Traffic in Living Cells

2006 ◽  
Vol 2006 (6) ◽  
pp. pdb.prot4603-pdb.prot4603
Author(s):  
J. Lippincott-Schwartz ◽  
E. L. Snapp
Keyword(s):  
Membrane Traffic ◽  
Living Cells ◽  
Membrane Systems ◽  
Organelle Membrane

scholarly journals Imaging of Membrane Systems and Membrane Traffic in Living Cells

2011 ◽  
Vol 2011 (11) ◽  
pp. pdb.top066548-pdb.top066548
Author(s):  
E. L. Snapp ◽  
P. Lajoie
Keyword(s):  
Membrane Traffic ◽  
Living Cells ◽  
Membrane Systems

scholarly journals Targeted Chemical Disruption of Clathrin Function in Living Cells

2003 ◽  
Vol 14 (11) ◽  
pp. 4437-4447 ◽  
Author(s):  
Howard S. Moskowitz ◽  
John Heuser ◽  
Timothy E. McGraw ◽  
Timothy A. Ryan
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Time Scales ◽  
Membrane Traffic ◽  
Living Cells ◽  
Normal Function ◽  
Fk506 Binding Protein ◽  
Important Protein ◽  
Cross Linker ◽  
Specific Inhibitors

The accurate assignment of molecular roles in membrane traffic is frequently complicated by the lack of specific inhibitors that can work on rapid time scales. Such inhibition schemes would potentially avoid the complications arising from either compensatory gene expression or the complex downstream consequences of inhibition of an important protein over long periods (>12 h). Here, we developed a novel chemical tool to disrupt clathrin function in living cells. We engineered a cross-linkable form of clathrin by using an FK506-binding protein 12 (FKBP)-clathrin fusion protein that is specifically oligomerized upon addition of the cell-permeant cross-linker FK1012-A. This approach interrupts the normal assembly-disassembly cycle of clathrin lattices and results in a specific, rapid, and reversible ∼70% inhibition of clathrin function. This approach should be applicable to a number of proteins that must go through an assembly-disassembly cycle for normal function.

scholarly journals The dynamic cell: Livecell imaging and membrane traffic pathways

2010 ◽  
Vol 32 (3) ◽  
pp. 4-7
Author(s):  
Mark Jepson ◽  
Paul Verkade ◽  
Paul Martin ◽  
George Banting
Keyword(s):  
Mammalian Cell ◽  
Nuclear Architecture ◽  
Membrane Traffic ◽  
Living Cells ◽  
Cellular Organization ◽  
Subcellular Organelles ◽  
Dynamic Cell ◽  
Static Images

Text books published 15 or more years ago presented lovely cartoons to illustrate the organization of a mammalian cell, highlighting the different subcellular organelles, the cytoskeletal components and the nuclear architecture. Many still do, but the better ones complement these static images with animations and/or movies from imaging of living cells (see Figure 1). The cartoons provide a reason able overview of cellular organization, but they cannot capture the fact that cells are highly dynamic entities with ongoing constitutive and regulated movement between compartments.

scholarly journals Time-Lapse Imaging of Membrane Traffic in Living Cells

2011 ◽  
Vol 2011 (11) ◽  
pp. pdb.prot066555-pdb.prot066555 ◽  
Author(s):  
E. L. Snapp ◽  
P. Lajoie
Keyword(s):  
Membrane Traffic ◽  
Time Lapse ◽  
Living Cells ◽  
Time Lapse Imaging

Fluorescence ratio imaging of dynamic intracellular ionic signals

1988 ◽  
Vol 46 ◽  
pp. 42-43
Author(s):  
R. Y. Tsien ◽  
A. Minta ◽  
M. Poenie ◽  
J.P.Y. Kao ◽  
A. Harootunian
Keyword(s):  
Binding Sites ◽  
Living Cells ◽  
Molecular Engineering ◽  
Ph Indicators ◽  
Highly Sensitive ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Technical Advances ◽  
Indicator Dyes ◽  
Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics

1988 ◽  
Vol 46 ◽  
pp. 118-119
Author(s):  
K. Jacobson ◽  
A. Ishihara ◽  
B. Holifield ◽  
F. Zhang
Keyword(s):  
Fluorescence Microscopy ◽  
Cell Biology ◽  
Extended Period ◽  
Dynamic Structure ◽  
Living Cells ◽  
Membrane Dynamics ◽  
Molecular Cell Biology ◽  
Image Averaging ◽  
Single Living Cells ◽  
Single Living

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.

Multimode light microscopy and fluorescence-based reagents as tools for the study of chemical and molecular dynamics of living cells and tissues

1992 ◽  
Vol 50 (1) ◽  
pp. 418-419
Author(s):  
D. L. Taylor
Keyword(s):  
Living Cells ◽  
Cellular Level ◽  
Molecular Techniques ◽  
Cellular Functions ◽  
Macromolecular Assemblies ◽  
Cell Functions ◽  
Molecular Events ◽  
Yield Information ◽  
Full Knowledge ◽  
Structural Methods

Cells function through the complex temporal and spatial interplay of ions, metabolites, macromolecules and macromolecular assemblies. Biochemical approaches allow the investigator to define the components and the solution chemical reactions that might be involved in cellular functions. Static structural methods can yield information concerning the 2- and 3-D organization of known and unknown cellular constituents. Genetic and molecular techniques are powerful approaches that can alter specific functions through the manipulation of gene products and thus identify necessary components and sequences of molecular events. However, full knowledge of the mechanism of particular cell functions will require direct measurement of the interplay of cellular constituents. Therefore, there has been a need to develop methods that can yield chemical and molecular information in time and space in living cells, while allowing the integration of information from biochemical, molecular and genetic approaches at the cellular level.

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