Introduction
In eukaryotic cells, actin filaments form cytoskeleton, a network of two types of fibrous structures, filaments and tubules. Cytoskeleton plays various key roles in cells such as the arrangement of cellular organelles, keeping the shape of cell, and power generation for cellular movement accompanying cell morphological changes in occasions such as cell division, muscle contraction and ciliary movement. In cells, many kinds of proteins associated with cytoskeleton precisely regulate the distribution and dynamic behavior of filaments; therefore, some kind of drug stimulation causes remarkable changes in the number, length, distribution, movement and stability of cytoskeleton filaments. The following describes the analysis of morphological changes in cytoskeleton caused by drug stimulation, using the “Morphology Analysis” protocol.
Fig1. Recognition of the cytoskeleton of MRC5 cell (Experiment 1)
A-1 and B-1 are original images, and A-2 and B-2 are recognized images.
Fig2. Morphological change due to A10 cell apoptosis(Experiment 2)
(1) Original images and analysis images
Original image (a) and analysis image (c) of control cells
Original image (b) and analysis image (d) of Staurosporine (10μM) treated cells
Left : (2) Number of cytoskeletons
Right : (3) Average cytoskeleton length and variation
(4) Cytoskeleton length distribution
(5) Cytoskeleton direction
(Horizontal direction as 0 degrees)
Experiment procedure
Experiment1
- MRC5 cells were seeded on 96-well plates at 20,000 cells/well, cultured for 24 hours, fixed with formaldehyde and stained actin with phalloidin.
Experiment2
- A10 cells were seeded on 96-well plates at 10,000 cells/well, cultured for 24 hours, and induced apoptosis by adding staurosporine (0-10μM, 2 hours).
- Fixed the cells with formaldehyde and stained actin with phalloidin.
The following were common procedure in both experiments:
- Images were captured using the CellVoyager CV6000 under the following conditions:
- Magnification: 40x objective lens
- Images captured per well: 1
- Exposure time (Alexa 488): 800msec
- The captured images were analyzed using the “Morphology Analysis” protocol.
- Cytoskeleton regions were identified from the original images.
- Feature quantities of identified cytoskeleton were measured.
Results and Conclusion
Changes in MRC5 cytoskeletal formation when apoptosis was induced were analyzed, using the “Morphology Analysis” protocol. As a result, significant differences from the control were identified in the number of actin filaments and the distribution of filament lengths (Fig. 2). Since the structural changes in cytoskeleton occur in cell migration, the “Morphology Analysis” protocol will be a useful tool for the study of the inflammatory response and also cancer metastasis.
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