Hej!
Here I am again. It's been a while but I've been very busy. A lot has happened in the two weeks I've been here.
Maybe I should give a short and easy explanation of the project I'm working on:
In diabetics, the problem is often that the beta cells of the islets of langerhans in the pancreas do not excreet enough insulin. Researchers have noticed that in diabetic mice, there are less physical connections between mitochondria (aka the powerhouse of the cell) and the endoplasmic reticulum (aka the shipping facility of the cells). Imagine something like this: if the cells are healthy, the mitochondria and the endoplasmic reticulum like holding hands. But if they are ill (so diabetic) they don't. With less of these connections, they produce less insulin however, we don't know if this is the cause of less interaction between these 2 organelles, or if the drop in insulin production causes these to organelles to have less interaction. Kinda like: what was first, the chicken or the egg? However here we can simulate one of the conditions and see if that influences the other.
What we will be trying is the increase the physical interactions between these 2 organelles and see if this has an influence on the excretion of insulin or not. If it does, then that would be one step in the direction of: the physical interactions decreasing causes insulin drop. But of course to be sure of that, further research would still be required.
Next to the insulin production, we will also be monitoring the calcium signaling within the cells. Calcium regulates a lot of enzymes and proteins in the cell, and we want to see if this changes when these physical interactions change.
Anyways, a short summary of what I've been doing:
I created 2 plasmids (DNA in a ring shape) which should be translated into triple fusion proteins. Each of them contain a fluorescent marker, a protein that should only appear in certain organelles of the cell, and a protein that is used for optogenetics (using the Cry2-CIBN method).
I transformed them into bacteria, so I could produce a lot of the plasmids. I extracted the DNA out of the bacteria. I transfected Hela cells with the plasmids, and looked at these cells if they expressed these proteins through a confocal microscope. Here are some of the nice pictures I took:
Mitochondria with GFP:
Mitochondria with mApple:
Composite of the previous two pictures:
A short explanation of the figures above:
We wanted to check if our fusion protein was expressed in the right organelle, in this case mitochondria. So we had 1 plasmid of which was proven that it expressed at the right place (The GFP picture, picture 1). We also added our own plasmid with mApple (picture 2). To check if they express at the same place (which they obviously do), we overlapped the pictures in 2 colors (red and green) and where the picture is yellow/orange. The expression is at the same place. So we could conclude that in this case our own fusion protein expresses at the right place. (Not the complete explanation, other things should be taken into consideration, but as to make it not to difficult I'm not gonna bother you with it.
The same thought process as from the mitochondria was used with a protein in the endoplasmic reticulum, here are some pictures:
ER with GFP:
ER with mCherry:
Composite of the two previous pictures:
Here it is also very clearly visible that the cells at the side are only transfected with GFP plasmid, thus showing us green in the last picture.
Anyway, a lot of exciting stuff is coming my way in the near future, and I'm looking forward to it, so I'll end this blog post here!
See you soon!
~Cedric