Imaging of
keratin dynamics during
the cell cycle and in response to phosphatase inhibition
Contents:
I.
Introduction
II.
Materials and Instrumentation
A. Special Reagents
B. Cell Lines
C. Microscopes
D. Culture Chamber
III.
Procedures
A.
Generation of Cell Lines Producing Fluorescent Keratin Filaments
1. Preparation of cDNA
Constructs
2. Generation of Stable
Cell Lines
3. Characterization of
Cell Lines
B.
Imaging of Keratin Filament Dynamics in Interphase
1. Preparation of Cells
2. Image Acquisition
C.
Special Aspects of Recording in Mitotic Cells
D.
Imaging in the Presence of Phosphatase Inhibitors
1. Determination of
Suitable Drug Concentration
2. Examination of
Reversibility
3. Light Dependency
E.
Fluorescence Recovery after Photobleaching
F.
Data Analysis
1. Preparation of Movies
2. 3D-Reconstructions
3. Quantification of
Soluble Pool
4. Quantification of
Fluorescence Recovery
5. Diagrammatic Representations
of Time-Dependent Fluorescence
a. Mobility
Diagrams
b. Time-Space
Diagrams
c. Intensity
Diagrams
IV. Pearls
and Pitfalls
A. Transient versus Stable
Transfection
B. Epifluorescence Microscopy versus
Confocal Laser Scan Microscopy
C. Effects of Light
V.
Discussion and Concluding Remarks
VI.
References