Peeping into the oxidative state of germline stem cells

 
In simplest terms, respiration is a process by which organisms breathe in oxygen and exhale carbon-dioxide. At the molecular level respiration aids in generation of energy in the form of Adenosine tri-phosphate (ATP) from chemical reactions involving electron transfer through molecular oxygen. These reactions primarily take place in the mitochondria of the cells and during the process derivatives of oxygen or reactive oxygen species (ROS) are generated. ROS are highly reactive byproducts which include superoxide anion (O₂•^–), peroxide (O₂•^-2) and hydroxyl ion (OH^–) or radical (OH•). They rapidly react and in most cases damage vital macromolecules like DNA, RNA, lipids and proteins. This mode of cellular damage is one of the primary causes for loss of homeostasis and can lead to rapid organismal aging. There are several cellular mechanisms that detoxify ROS, for instance, enzymes like superoxide dismutases, catalases, glutaredoxins and peroxiredoxins that actively neutralize ROS into less or completely harmless end products. Glutathione, a tripeptide present in cells, brandishes a sulfhydryl group that is a reactive target for ROS attack. Hydrogen peroxide is mitigated via non-enzymatic Fenton reactions that require Cu2+ or Fe2+.

Stem cells are undifferentiated cells that divide asymmetrically and differentiate into specific cell types, which then collectively form and function as a tissue. Cellular aging causes loss of cellular function leading to disruption of tissue homeostasis, thus contributing to organismal aging. Stem cells act as a pool of nascent cells that can replace damaged cells in a tissue, however, stem cells are no exception to cellular aging. For example, excess ROS can accelerate stem cell aging. The basic cell biological processes like transcription and translation (gene expression profile) and cell cycle of stem cells differ drastically from other cell types; this gives reason to investigate whether the way they manage ROS is any different. Germline stem cells are a type of stem cells that give rise to cells that form the egg or sperms. Unlike the soma, these stem cells are unique as they are responsible for the maintenance of the germline, which is perpetual across generations. ROS and mitochondria interplay is crucial in a normal case itself but it is consequential in case of female germline stem cells. Mitochondria are maternally inherited and the quality of mitochondria that will be transferred to the next generation determines the success of fertilization and early development. An interesting question being addressed in the lab is: “If autophagy contributes to germline stem cell aging and in what way”?  ROS is another factor that contributes to cellular aging and autophagy is crucial for alleviating ROS, hence it is important to study ROS in the context of germline stem cell aging.

The most common method to study ROS in biological systems is the use of fluorescent dyes whose properties are subject to their oxidative state. For example, JC-1 dye localizes to mitochondria and emits red fluorescence when mitochondria are in reduced state (polarized), and emit green fluorescence when mitochondria are oxidized (depolarized). Approach of using these dyes is powerful but limited by specificity and invasiveness to the system, many of which are prone to oxidative artefacts that misrepresent the actual redox state. These limitations were overcome by the most popular fluorescent molecule that revolutionized cell biology; the green-fluorescent protein (GFP). GFP was modified into redox sensitive GFP (roGFP) such that its fluorescent properties are a function of the redox state within the cell. Moreover, linking proteins involved in the redox sensing or response like Grx1 (glutaredoxin) and Orp1 (oxidant receptor peroxidase 1) not only results in faster relay of the redox state, but also imparts specificity at subcellular level. One of the main advantages is that these are genetically encoded and can be modified to be targeted to a particular cellular compartment; this is a tremendous improvement over traditional methods specially when study of in vivo cellular states are involved. Simone Albrecht and colleagues have generated redox probes in Drosophila by linking Grx1 and Orp1 to roGFP2. These probes can be used to study the in vivo redox state across all major somatic tissues. We modified these probes such that they constitutively express specifically in the germline including the germline stem cells, hence enabling us to study the redox homeostasis of germline stem cells.

To validate these probes, the germline tissue was exposed to chemicals that oxidize or reduce it at saturating levels. Measuring the fluorescence intensity of roGFP2 (fused to Grx1 and Orp1) at 405nm and 488nm provides a ratio that is indicative of the dynamic range. This dynamic range is a measure of the extent to which the probe can reflect how much the tissue can be either oxidized or reduced. In most physiological conditions, the redox potential for germline tissue  is expected to be within this dynamic range. Our efforts are now focused on measuring the redox potential within the germline stem cells in absence of autophagy and moderate upregulation of autophagy.

Group Leader Dr Bhupendra V. Shravage
Dr. B. V. Shravage completed his doctoral studies under the guidance of Prof. Dr. Siegfried Roth at the Institute for Developmental Biology, University of Cologne, Germany.  He worked on TGF-beta and EGF networks involved in patterning of dorsal chorion structures in Drosophila. He moved to UMass Medical School for his post-doctoral studies on Autophagy in cancer and cell death with Prof. Eric Baehrecke. During his tenure at UMass, they discovered the multi-functional role of Atg6 (Beclin1 in mammals) in various membrane-trafficking pathways and hematopoiesis in Drosophila. Since 2014 Dr. B. V. Shravage is working as a Scientist at the Agharkar Research Institute, Pune, India and affiliated Savitribai Phule Pune University, Pune, India. His research work is currently focused on understanding molecular regulation and function of autophagy in the germline.

Kiran S. Nilangekar
Kiran Nilangekar is a PhD student working under the guidance of Dr. Bhupendra V. Shravage at the Developmental Biology group in MACS-Agharkar Research Institute, Pune, India. He is working on autophagy and its involvement in germline stem cell maintenance. Before starting his PhD, he worked at the Cytoskeleton and Cell Shape lab at IISER-Pune after finishing his Master’s degree in Virology.

Research Objectives
Dr B.V. Shravage’s lab focuses on understanding the molecular mechanisms of autophagy mediated maintenance of cellular homeostasis in germline stem cells.

Funding
Grant No. ECR/2015/000239, Science and Engineering Board, Department of Science and Technology ,  BT_PR12718_MED31_298_2015, Department of Biotechnology, Government of India and (BT/HRD/35/02/2006 RLS 2013-2014) Ramalingaswami Fellowship, Department of Biotechnology, Government of India.