Speaker: Benoit Kornmann
Department: School of and biochemistry, Georgia Tech Department
Subject: The Push and Pull of Mitochondrial Gymnastics
Location: Department of Bionanoscience, TU-Delft
Author: Mirte Golverdingen
Benoit Kornmann works in a Cell Biology lab which is interested in dynamics on organelles. Their research mostly focusses on the mitochondria.
He started his talk with a simple picture of the cell. However, this picture is not how a real life cell is like. There are a lot of organelles in one cytoplasm and therefore the cytoplasm is very crowded. The organelles are completely intertwined in a compact environment. Furthermore, the organelles in the cell move, this means that there is a dynamic environment in the cell.
Kornmann focusses mainly on the mitochondrial dynamics. Mitochondria can be transported through the cell along cytoskeletal tracks. They have also the ability to fuse together and to fission apart. All these actions need mechanical forces to do their work. Kornmann focusses on the origin and the effects of these mechanical forces.
Mitochondrial transport is based on microtubules transport. It is promoted by Miro, kinesin and dynein. Miro interacts with the mitochondria and is in contact with the kinesin and the dynein. Kinesin is a motor protein that moves to the + end of the microtubules. While the motor protein dynein moves to the – end of the microtubules.
Kornmann’s research group showed that miro interacts with the protein Cenp-F. This protein is expressed in the nucleus during the S, G2 and the mitosis phase of the cell cycle. During mitosis Cenp-F first moves to the nuclear envelope following by the movement to the kinetochore. During the telophase and cytokinesis Cenp-F is requited to the mitochondria. We can also see this by using the CRISPR-Cas technique. If you silence miro by using this technique, the Cenp-F is not distributed to the mitochondria.
Cenp-F is a very large protein and it is made of a lot of coiled-coils. It has two microtubules binding domains at the N and C terminus of the protein. However, there is no binding site for motor proteins, so what then is the mechanical force that results in the movement of mitochondria? Recent research showed that the growing of microtubules can also do mechanical work. Therefore, the research group of Kornmann asked the question: does Cenp-F harness this force?
If Cenp-f does use the mitochondria to create mechanical force it needs to be located at the tip of the microtubules. Kornmann showed that the protein indeed is located on the tip of the microtubules. He did this by using super-resolution microscopy and immune-fluorescence. Eb1 was one of the labeled proteins and it is located on the microtubule. Cenp-F also labeled with another color. The images from the microscope showed that Cenp-F indeed was found at the top of the microtubules. Kornmann also used TIRF microscopy to confirm his claim.
Kornmann concluded from these result that that miro is interacting with the Cenp-F protein. Cenp-F is then interacting with the tip of the microtubules. It is a very simple idea that justifies the results. They also reconstructed an in vitro system by using a stable microtubule seed on a plate. On this they put growing dynamic microtubules. They also added a glass bead that was coated with Cenp-F. They used microscopy to follow the bead and they saw the growth and shrinkage of the bead. This also confirmed the hypothesis that Cenp-F binds to the tip of the microtubules.
The network of mitochondria in the cell is in constant movement. How do the organelles avoid entangling with each other? Kornmann did research to this question by using the bacteria Shingella flexerni Once this bacterium is in a cell it high jacks the actin system, so they can infect the neighboring cell. When a bacteria bump into a mitochondrion, the mitochondrion will undergo fission. It is a biochemical process where Drp1 plays also a great role. The Drp1 protein makes a ring that activates the fission and splits the cell. They controlled if Drp1 is needed for this fission in the cell and it indeed was. When Drp1 in the mitochondria was inactivated by using the CRISPR-CAS method the mitochondria could not undergo fission. However, they can undergo fusion
How is the bacterium sensed? How does Drp1 know that it assembled right where the bacterium hits the mitochondria? Kornmann thought this could be sensed by a change in the mechanical forces on the Drp1 molecule. So the mitochondria feel that they are squeezes. By using Atomic Force Microscopy with a round tip they could put a force on a very flat cell. By applying a force across the cell membrane they were able to cause mitochondrial fission. Could this also be done on a more natural way than ATM?
For this question they let cells grow on a patterned surface. In your body your cells also do not grow on a flat plane. They used the edge of a groove on a vinyl gramophone record to grow cells on. They looked how the separation of mitochondria on the edge was organized for the wild type and for Drp1 CRISPR-CAS and Drp1 siRNA silenced cells. When the cell did not have the Drp1 protein the mitochondrion did not undergo the fission process. However, the wild type did undergo fission on the edge of the groove. Furthermore, every mitochondrion that went over the edge did undergo the fission process. Kornmann also showed that the mechanical force on the mitochondria fission site recruits Drp1 to this site. So fission can take place.
During this talk it became again clear that a cell is much more complex than you can imagine. The cytoplasm is very crowded and, therefore, the mitochondria have developed a mechanism to be able to move through the cell without being damaged. I did not know before that mitochondria cells can undergo fission. Moreover, I never realized that they need the microtubules to travel through the cell. It is exciting that the Kornmann group could show us in a simple way by using interesting new techniques as CRISPR-CAS how microtubules are able to travel through the crowded cytoplasm.