Methods
I.) Distinct Modes of motility have different requirements for Rho and ROCK activity
a. Methods
BE (colon carcinoma), LS174T (colon carcinoma), SW962 (squamous cell carcinoma), WM266.4 (melanoma), A375P, and A375m2(melanoma) cells were analyzed to test for cell invasion relative to the controls used.
The two controls used were TAT-C3 (which inactivates the RhoA, RhoB, and RhoC ) and Y27632 (which inhibits ROCKI and ROCKII).
The five cell types/lines were placed in matrigel (a matrix consisting of collagen IV, Laminin and some growth factors) in the presence of either of the two controls to see how their movement or ability to invade the matrigel was affected.
To analyze the morphologies of the different types of tumor cells invading the matrigel, analysis by confocal microscopy was done. The microscope allowed the imaging of the cells and allowed determination of their morphologies to be determined.
The results from the matrigel studies were also analyzed in an actual organism. In this case, nude mice were used as the test subject. Tumor cells (BE andA375m2) were injected into the flanks of male nude mice. After the tumors were roughly 1cm in diameter, they were removed, prepared, and analyzed using indirect immunofluorescence microscopy. Confocal microscopy analysis was also done, in which the samples were placed under a coverslip with an oil immersion.
II.) RhoA and ROCK signalling promote rounded cell motility
a. Methods for determination
Out of the five cells lines that were used to determine cell motility in the 2-D/linear environment, the A375m2 and BE cells (used because they have pronounced rounded and elongated morphologies, respectively) were used to analyze morphology in a 3-d environment. For in vitro analysis, a thick layer of matrigel (simulating a 3-D tumor environment) was used.
These two cell types were placed in the simulating 3-D matrigel environment in the presence of TAT-C3 and Y27632 and their reactions to these two controls were analyzed.
A375P cells were used to investigate how activating Rho and ROCK signalling effects tumor cells.
The following movies are cells moving in a 2-D linear environment: Clip 1, Clip 2
To view the clips, use either Quicktime, RealPlayer or Windows Media Player
III.) Analysis of Rounded Cell Movement
a. Methods:
To see the movement of cells in a 3-D matrix, confocal microscopy was used on A375m2 cells.
Based on previous studies showing that the Golgi apparatus and MTOC are located towards the front of the cell, a study was done to analyze this theory. BE and A375m2 cells were used. After plating these two cell types, the A375m2 cells were analyzed after 8 hours and the BE cells after 16hours to locate the discussed components and their relative locations within the cell.
The following time lapse movies represent the cells moving in a 3-D matrigel: Clip 1, Clip 2
To view the clips, use either Quicktime, RealPlayer or Windows Media Player
IV.) Rounded motility does not require pericellular proteolysis
a. Methods
Protease inhibitors were used to determine if extracellular proteases are required for rounded cell motility. The inhibitors GM6001, leupeptin, aprotinin, and calpeptin were the inhibitor controls used for this type of analysis.
These inhibitors were put in the presence of the five cell lines being analyzed throughout the study
In the presence of the inhibitors, the cell types were placed in the matrigel and their progress observed.
Introduction