Experiment 2 part 3: Nuclei and cell division in wild-type and myo II- cells

You have seen that the myosin II mutant cells do not grow in suspension.  We will now further investigate the source of the phenotype.

Cell Lines: Mike will give you wild-type and myo- mutant cells that have been transfected with a NLS-tdTomato vector.  This probe when expressed in cells localizes to the nucleus of cells.

NLS=nuclear localization signal 

tdTomato = tandem-dimer Tomato. 

Tomato is a modified fluorescent protein that is red shifted and is  expressed as two copies of the fluorescent protein in the same polypeptide (a dimer of Tomato forms through intermolecular dimerization). 

Procedure: 

Monday or Tuesday: set up mutant and wild type in flasks on the shaking platform.  Remember, that they are going to be used on Thursday, so take note of how many generations of growth. Titrate the wild-type starting concentration so they are still growing happily on Thursday. Use more mutant cells because you now know they do not grow in suspension. 

Setting up the experiment

1)On THursday- capture time lapse movies of the mutant and wild-type cells as they settle from the shaking suspension onto the surface. You will use the green LED/G filter cube combination to see the nuclei. We have not done this experiment before, so the magnification, time factor etc is yours to determine.

2)Investigate why the cells did not grow, why they are larger than normal (can you measure how large?) and what happens when you give the cells a surface to attach to. 

Lab Writeup:

Can you now present a hypothesis that explains what happened with the growth curves on a surface and in suspension. How do these processes compare between mutant and wild-type, surface and suspension?

_________________________________________________________________________

Reagents/Media:

HL5 media

H50 Buffer

G418 Drug

Cell Strain(s):

NC4A2 and HK321

Other things needed for this experiment:

1. Cells expressing NLS-tdTomato

2.Ibidi dishes