The push and pull of mitochondrial gymnastics: mechanical force in mitochondrial dynamics.

Speaker: Benoît Kornmann
Department: Bionanoscience
Subject: The push and pull of mitochondrial gymnastics: mechanical force in mitochondrial dynamics.
Location: Room E – Building 22 (TU Delft)
Date: 17-03-2016

Author: Kristian Blom

At the 17th of March, I visited a Bionanoscience seminar given by Benoît Kornmann, group leader of the Kornmann Lab of ETH Zurich. The Kornmann lab studies the ultrastructural organization of the cell and the biology of organelles.

Prof. dr. Kornmann introduced the main topic of the seminar with a drawing of the internal structure of an eukaryotic cell. Such pictures are typically used in textbooks to get some more insight in the anatomy of the cell, but they are rather misleading. The missing factor in most textbook images is the entropy and dynamical behavior of the internal structure. During mitosis, this chaotic internal environment needs to be regulated to make sure that both daughter cells get approximately the same cellular content (e.g. cytoplasmic organelles). But the molecular mechanism that allow proper segregation of cytoplasmic organelles in human cells are poorly understood. The research group of Dr. Kornmann focused on the mitotic redistribution of mitochondria during mitosis.
First I shall summarize the conclusion of the research, after that I will tell something about the methods used to obtain the results of the research.

During mitosis, mitochondria move towards the equatorial plate before partitioning into the daughter cells. This movement is established by microtubule-based transport. The bond formed between the mitochondria and the microtubules is established by the proteins Miro and Cenp-F. Miro, a mitochondrial protein, is located at the outer membrane of the mitochondria. During cytokinesis, Miro recruits Cenp-F, which associates with the plus-end (growing tip) of growing microtubules. This protein-microtubule interaction ensures the mitochondrial transport towards the equatorial plate, and the redistribution of the mitochondria over the two daughter cells.

The Miro-Cenp-F interaction observed by the research group of Dr. Kornmann, came as a surprise. At first sight, the main focus was to identify Miro interactors at mitochondria. While analyzing the Miro complex, a lot of unrelated proteins were identified. So, to get rid of these unrelated proteins, they applied a strategy called SILAC: Stable isotope labeling by amino acids in culture. After this, only three proteins were observed: Miro1, Miro2 (together they form Miro) and Cenp-F. Together with statistical analyses, this observation strongly suggests that Miro forms a complex with Cenp-F.

Image 1: Localization of Cenp-F (Green) and mitochondria (Red) in the presence and absence of Miro1, Miro2 or both. Top: Cenp-F signal only. Middle: Overlay. Bottom: magnification of the boxed areas. Source: Mitotic redistribution of the mitochondrial network by Miro and Cenp-F, 2015, Benoît Kornmann.

 

To know whether Miro is the causing agent for the recruitment of Cenp-F to the mitochondria, the Kornmann Lab used complete loss-of-function of both Miro1 and Miro2. Using the CRISPR/Cas9 method, Miro1 was mutated. By knocking down Miro2 in the same cell line, Miro-less cells were created. This complete silencing of the Miro protein led to the disappearance of Cenp-F at the mitochondria, no matter which cell cycle the cells were in. At the same time, Cenp-F was expressed in other locations of the cell (nucleus, nuclear envelope, etc.), suggesting that Miro is necessary for Cenp-F recruitment. To show that Miro is not only necessary, but also sufficient for Cenp-F recruitment, they overexpressed Miro1 and Miro2. This overexpression led to increased Cenp-F recruitment at the mitochondria.

This blog is too short to discuss all the different methods used for this research, but still I would like to give a remark about the second method which I did explain. By overexpressing Miro1/2 and observing an increase of Cenp-F recruitment at the mitochondria, you can’t conclude that Miro is sufficient for Cenp-F recruitment from my point of view. Because, there could be some protein or other chemical agent which is needed above a certain threshold before Cenp-F recruitment becomes activated. The concentration of this hypothetical chemical agent doesn’t influence the increased Cenp-F recruitment when Miro1/2 is overexpressed, but it’s still necessary for Cenp-F recruitment.

 

 

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