Interview part 2- Cellular connections: Where biology meets mechanosensing

Dr. Avinanda Banerjee’s interview with Bio Patrika hosting “Vigyaan Patrika”, a series of author interviews. Dr. Banerjee is presently working as research fellow in Skin Research Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore under Dr. Srikala Raghavan. Her present role involves working on testing novel biomaterials for skin grafts in burn patients. Before moving to Singapore, she obtained her first postdoctoral training in Dr. Srikala Raghavan’s lab at Institute for Stem Cell Science and Regenerative Medicine (inStem), Bangalore, India from 2016 to 2020. Where she worked on the role of vinculin in maintaining stem cell quiescence. She obtained her PhD degree from Calcutta University in the year 2016 under supervision of Dr. Kaushik Sengupta from Saha Institute of Nuclear Physics (SINP), Kolkata, India. Her doctoral works identified effects of Lamin A mutations in nuclear morphology, function in Dilated Cardiomyopathy. She obtained her master’s degree in Biotechnology from Bangalore University, India and her bachelor’s degree in Microbiology from Calcutta University. Her research interest revolves around different accepts of mechanobiology of cell and tissue. Here, Avinanda talks about her first co-author paper titled “Mechanical instability of adherens junctions overrides intrinsic quiescence of hair follicle stem cells” published in Dev Cell.

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How would you explain your paper’s key results to the non-scientific community?

Communication is not only an important aspect of human behavior, it is equally a crucial factor at the cellular level in unicellular and multicellular organisms. In multicellular organisms, if cells need to work together to form a tissue they need to communicate with each other. Cells in our body constantly communicate with each other as well as to their surroundings to modulate its internal functions. Tissues are maintained and restored by a specialized set of cells, which are capable of long-term self-renewal and differentiation, known as the resident stem cells (SCs). Functioning of SCs largely depends on their successful communication with the surrounding cells and environment (alternately termed as cellular niche). This communication involves detection of various signals from its niche. Responding to these signals by modulating its gene expression helps cells to determine its fate. These signals can be in multiple forms and flavors such as biochemical, electrical and more recently mechanical cues are being considered as the cellular signals to which cells respond. This ability of cells to sense mechanical signals from their niche is termed as mechanosensing. Translation of these mechanosignals into a cellular response is called mechanotransduction. Understanding the entire cascade of the process at the cellular level starting from sensing the mechanical force, then translating it into a meaningful response is the main basis of mechanobiology. The field of mechanobiology is gaining attention in life-science research for a couple of decades now. Studies have shown that both the mechanical properties and forces emerging from the niche can control cellular function and fate. Among many different ways through which cells can connect with each other are the cellular junctions. Recent studies have revealed that functions such as determining the shape of multicellular organisms and maintaining the homeostatic state of adult tissues requires a fine-tuning of the cell-cell junction responses and transmission of the applied mechanical load at these junctions. Cell adhesion systems are generally known to resist the forces that would otherwise tend to tear the tissue apart. Thus, it is presumably by definition, the biology of these junctions has evolved to achieve the mechanical nature of their function. One of the least explored cellular junctions in the field of mechanobiology is adherens junction (AJ). Though AJ has started to emerge as the main hub of force propagation within epithelial tissues, still how the function of SCs in these tissues regulated by mechanotransduction through AJ is poorly understood. In our recent study, we have studied the role of one of the key players in AJ called vinculin, a known mechanotransducer. Studies in mouse model have shown that complete loss of vinculin is lethal to the developing embryo and that the mouse embryo dies due to failure in the development of heart and nervous system.

To study the role of vinculin in AJ mediated mechanotransduction we have used skin, an epithelial tissue, as our model system. Skin is one of the tissues that bear maximum mechanical load and it is subjected to various mechanical insults on a regular basis. For the purpose of this study, we generated a mouse model, which lacks vinculin only in the skin epithelia. These conditional vinculin null mice (vin cKO) developed a sparse hair phenotype on their back skin. This phenotype led us to look more carefully in the hair follicle (HF), an appendage of the skin. Hair follicles in all mammals are maintained and regulated by a set of special cells called the hair follicle stem cells (HFSCs) which reside in a dedicated compartment of the HF called bulge region. HFSCs in vin cKO mice are not maintained in their dormant or quiescent states. HFs are known to cycle through dedicated stages called anagen (growth), catagen (regression) and telogen (rest or quiescent) to maintain the tissue lifelong. Absence of vinculin activates the HFSCs and brings them out of their quiescence. When we studied the ultrastructure of the bulge region of the HF in vin cKO mice using electron microscopy, we noticed cell to cell junctions are not formed properly in the stem cell compartment of the hair follicles. Due to this abnormal AJ junction formation, HFSCs are not able to efficiently communicate with its niche. A known phenomenon in the stem cell field is contact-mediated inhibition of cell proliferation that in simpler terms is the capacity of cells within a given space to regulate its growth responses in the context of cell-to-cell contact. Once the cell makes contact with its neighboring cell it sends a signal to stop growth or proliferation. In our system, since they are not forming normal AJ they are not only able to successfully make contact nor sense the neighboring cells, hence they are not receiving any signal to stop the proliferation and thus these SCs are pushed out of their quiescence. The next question we asked was what is the cause and relationship of these abnormal AJ in the vinculin null mice with the loss of quiescence of HFSCs. We decided to measure the force generated at the AJ formed in the vinculin null cells. Interestingly, they form very weak junctions compared to the control cells. Thus, the absence of vinculin from AJ leads to a formation of mechanically weak junctions, which impair the mechanotransduction ultimately leading to loss of quiescence in HFSCs. To further investigate the molecular mechanism involved in the loss of quiescence and junction strength we performed global gene expression profiling. Gene expression profiling in the vinculin null HFSCs revealed that a candidate protein YAP1 (a transcription factor and a potent mechanotransducer) and its targets are upregulated. Localization of YAP1 from cytoplasm to the nucleus is responsible for turning on genes specific for proliferation. YAP1 is known to be sequestered at the AJ in contact-inhibited stem cells. Thus, mechanically weak AJ in the absence of vinculin, being responsible for localization of YAP1 from the junctions to the nucleus, ultimately turn on the proliferative genes and results in the loss of HFSCs quiescence.

Model. Image credits Avinanda. WT bulge stem cells: Normal cell junctions, Quiescent, Cytoplasmic YAP. KO bulge stem cells: Abnormal cell junctions, Non-quiescent, Increased nuclear localization of YAP (green) providing mechanism for increased proliferation, Abbreviations: AJ-Adherens Junction, D-Desmosomes, IF-Intermediate filaments, HD-Hemidesmosomes, FA- Focal adhesion, N-Nucleus, ECM-Extra cellular matrix.

Insights from this study can be further applied for a better understanding of the development and homeostasis of tissues and organs that experience and generate mechanical forces, and the role of stem cells in regenerating these tissues.

What are the possible consequences of these findings for your research area?

Over the past couple of decades, there have been tremendous advances in stem cell research that has started to provide a broader understanding of how SCs function to maintain and repair tissue. There is also a lot of interest in elucidating how SCs respond to matrix stiffness, how the mechanical cues are converted into intracellular signaling cascades that impinge on gene expression changes, and how stem cell fates are determined. Thus, elucidating the molecular mechanisms by which stem cells use mechanical signals to communicate with their environment to decide their fate might pave the way for understanding how perturbations to these interactions can promote various disease states that arise from the dysregulation of stem cells. Moreover, understanding the role of various molecular players involved in this cell-to-cell junction mediated mechanotransduction may serve as targets for therapeutic interventions in many diseases. Insights from this study can be further applied for a better understanding of the development and homeostasis of tissues and organs that experience and generate mechanical forces, and the role of stem cells in regenerating these tissues. Finally, this knowledge can be extended in understanding the regulation of tissue mechanics to develop improved engineered tissue and novel biomaterials to form tissue repair and reconstructions.

What was the exciting moment (eureka moment) during your research?

Cells usually make contact with its niche through cell-to-cell junction called AJ and the cell to substratum junction called focal adhesion (FA). Vinculin is present at both of these junctions. Initially it was not clear to us that absence of vinculin from which of these junctions was causing the loss of quiescence phenotype. Once we performed the ultrastructure analysis, we identified gaps between the plasma membranes of two adjacent cells in the HFSC compartment. This was an indication that absence of vinculin from AJ is responsible for the loss of adherence between the plasma membranes of the adjacent cells. Finally, the force measurements of the junctions revealed the weak junction phenotype in the absence of vinculin. This helped us to confirm that it is indeed a phenotype stemming from the abnormal cell-to-cell junction.

What do you hope to do next?

I would like to further study the pathways involved in mechanical signal crosstalk that connects the cell periphery to the nucleus via the cytoskeletal system of the cell. Both the nuclear envelope and the cell junctions (AJ and FA) have been identified as potential mechanosensory hubs. This study could be of importance to understand the role of mechanotransduction in other tissues that undergo repeated stress (for example addressing its role in the satellite cells of muscles).

 Where do you seek scientific inspiration?

I draw my scientific inspiration from my supervisor Dr. Srikala Raghavan. She has inspired me to think differently and guided me to get a complete new perspective for every situation I stumbled upon during my journey of postdoctoral training. I am extremely fortunate to have her as my supervisor and mentor. It is a treat to work with Dr. Srikala Raghavan as she is very understands us well, is highly supportive, approachable and a brilliant scientist. Being in Srikala’s lab, I am constantly surrounded by extremely talented, intelligent and curious peers who inspire me in my day-to-day quest. At the end of the day, I have learnt that it is extremely important to be happy with the success of small experiments that keeps you motivated for the next day.

How do you intend to help Indian science improve?

Indian science and scientists are making their mark in the global science community. It is very important to nurture more number of young people to pursue their career in science be it in an academic or industrial background. I would like to focus my research more towards the translational aspect of mechanobiology. It is now well understood how communication at any scale is important, so it is imperative to say that communicating our scientific findings to the non-scientific community is essential for spreading the awareness and interest in doing science research. I would like to contribute to various science outreach programs specially one dedicated for school children. Any possible collaboration between the scientific and non-scientific community could result in a great outcome, which can prove extremely helpful for Indian Science.

Reference

Biswas R, Banerjee A, Lembo S, Zhao Z, Lakshmanan V, Lim R, Le S, Nakasaki M, Kutyavin V, Wright G, Palakodeti D, Ross RS, Jamora C, Vasioukhin V, Jie Y, Raghavan S. Mechanical instability of adherens junctions overrides intrinsic quiescence of hair follicle stem cells. Dev Cell. 2021;56(6):761-780.e7. doi: 10.1016/j.devcel.2021.02.020.

Dr. Srikala Raghavan lab: https://www.instem.res.in/faculty/srikala

Edited by: Ashwini Kumar

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