Here we present an interview with Dr Rajarshi Chakrabarti, a post-doctoral researcher in the field of actin/mitochondria dynamics, working in professor Henry Higgs’ Laboratory based at Department of Biochemistry and Cell Biology, in the Geisel School of Medicine at Dartmouth. Dr Rajarshi has made valuable contributions that have improved understanding of the mechanism of mitochondria division. One of such contributions was published in 2018 in the Journal of Cell Biology and was also reviewed in an application note.
The Higgs Lab is a pioneer in the field of actin dynamics. A key research goal of the laboratory is to understand the role of actin filaments in organellar dynamics. When Dr Rajarshi Chakrabarti joined the professor Henry Higgs lab 2015 he started to work on the mitochondria/actin field for his postdoctoral studies. According to Rajarshi “Mitochondria are interesting organelles since they cannot be synthesized de novo. Thus, to maintain a healthy number of mitochondria in cells, the former have to undergo cycles of fusion and division. This dynamicity is highly controlled and regulated: any deviation from the norm results in several diseases. Thus, I was interested to understand the basic mechanism of mitochondrial dynamics.
During his research work in the Higgs lab, Rajarshi investigated the mitochondria division mechanism, following up on professor Higgs lab earlier work. It had been previously shown that an ER-bound actin polymerization factor was required for mitochondrial division. Dr Rajarshi Chakrabarti further helped elucidate the mechanism of mitochondrial division (Figure 2). For complete mitochondrial division, a succession of extremely complex and fast events must happen which will culminate into Inner Mitochondrial Membrane (IMM) division, followed by Outer Mitochondrial Membrane (OMM) division, and will originate two mitochondria from an existing mitochondrion.
Nevertheless, his research is not easy: imaging mitochondria can be challenging. “Mitochondria are highly dynamic organelles and measuring calcium dynamics in these fast-moving organelles requires considerable speed in the equipment to be used. Additionally, mitochondria are sites of ATP as well as of ROS production. We also need to remember that too much laser irradiation during imaging can be fatal for the cells.”
Considering the challenges described above for mitochondrial imaging, it is obvious that both photobleaching and phototoxicity are a serious concern while imaging live mitochondria. The researchers need to use very low laser powers and short acquisition times in order to keep the cells and mitochondria healthy during the studies. In fact, for the majority of his live-cell studies,
Dr Rajarshi Chakrabarti prefers to use a confocal spinning disk microscope, because he is analyzing very fast events (on the order of a few seconds). “Confocal spinning disk gives me the liberty of temporal resolution with minimum photobleaching to complete these kinds of studies. Using the Zyla camera on the dragonfly additionally improves the quality of the image as well.” Parameters such as speed of acquisition, low light acquisition, resolution, simultaneous double color imaging are all extremely important for analyzing mitochondria dynamics. The Dragonfly confocal system’s ability to acquire channels simultaneously allowed the researcher to acquire four-color live imaging data, and he even realized that” there wasn´t any bleed through among the channels”.
We further wanted to know: what the essential parameters required for an imaging system in mitochondria research? Dr Rajarshi replied that: “essential parameters for an imaging system in mitochondria research, or even in research overall are: 1- Speed of acquisition, 2- Limited photobleaching, 3- Limited bleed-through between channels and 4- Ability to perform multi-position imaging with accuracy.” In conclusion, Dragonfly helps in the study of mitochondria division because the system integrates speed of acquisition coupled to little or no photobleaching. These characteristics helped Rajarshi immensely during mitochondrial imaging.
Finally, we ask Dr Rajarshi: are there any other comments you would like to add about the Dragonfly confocal system? “Dragonfly is a wonderful system to perform both live and fixed cell imaging. Additionally, the software interface is crisp and has all the important tabs that you need to work with at your fingertips. This makes the interface less cluttered and easy to use.
Figure 2 - Model for mitochondrial division. Image courtesy of Dr Chakrabarti. The following steps will promote mitochondria division: 1) ER-bound INF2 mediated actin filaments is required for ER to mitochondria calcium transfer. 2) INF2- mediated actin filaments enhance ER-Mitochondria contact to facilitate this process. 3) Elevation of mitochondrial calcium is required for mitochondrial division. 4) Elevated mitochondrial division gives rise to Inner Mitochondrial Membrane (IMM) constriction that specify sites of mitochondrial division. 5) During a complete mitochondrial division, IMM division precedes before OMM division.