Work done in the lab of Shubhasis Haldar, Department of Biological Sciences, Ashoka University
About author
Soham Chakraborty does research in single-molecule biophysics. His research work has an integrated approach from biology, physics and chemistry. As a young researcher, he has a strong influence on polymer physics and how protein molecules could behave as an intricate polymer under force. Soham is currently working on force-sensitive proteins and interested in how mechanical stability of these proteins are regulated during different cellular processes, in the lab of Dr. Shubhasis Haldar at Ashoka University. He will be further willing to connect these changes to cellular level to illustrate a comprehensive picture of cellular mechano transduction events.
Interview
How would you explain your research outcomes to the non-scientific community?
Cells in our body can migrate very efficiently in the tissue, which requires continuous cell interactions with their underneath substratum (called extracellular matrix). Cell migration is controlled by a force-driven brake system called focal adhesion (FA), which is a multiprotein assembly of almost 200 proteins. Proper functioning of FA as a well-defined molecular clutch depends on its constituent mechanical linkages. These linkages both transmit and transduce the mechanical force into the cells. These FA proteins mechanically interact with each other and control the cell migration through traction force. Furthermore, these mutual interactions modulate the force-sensitivity of these proteins, which becomes dysregulated in different pathological conditions. Therefore, it is of utmost importance to decipher what plausible factors can modulate this force-response of these proteins.
Since each single protein molecule has their own contribution to the overall FA dynamics and thereby the cell migration, hence force-response of a single protein is of prime importance to understand the underlying mechanism. To date, many attempts have been performed to understand this mechanism, however, were unable to disclose what happens at single molecule level. Interestingly, we from the structural mechanobiology lab at Ashoka University have successfully addressed this problem and provided a plausible single-molecule scenario regarding the force sensitivity of these focal adhesion proteins.
Proteins are the fundamental units of cells and therefore, functional and structural maintenance of proteins is a first line mechanism for cells to survive. Molecular chaperones are such protein molecules which assists the cellular proteins to maintain their structure-function relation. These chaperones are also present in focal adhesion and emerging evidence suggest their contribution in focal adhesion dynamics. While existing studies have reported that chaperones assist in the folding of cytoplasmic proteins and prevent their aggregations, the researchers from the current study find that chaperones have a mechanical role too.
A single protein molecule has a force sensitivity within piconewton (pN) range and these chaperones are able to modulate the force sensitivity of these FA proteins. What does force sensitivity actually mean for a protein? In simple terms, force sensitivity is defined as the capability of proteins to withstand a certain amount of force while performing their biological functions. Since proteins have mainly two states: folded and unfolded. The energy is restored in the folded protein molecule, which could unfold depending on the force magnitude. Within their capacity, the protein molecules remain folded and accordingly interact with other proteins. However, at higher mechanical load beyond their ability, these folded proteins become unfolded since they cannot restore the energy anymore. This mechanical response dictates proteins to act as mechanical switches, controlling their interaction with other proteins and thereby, reflects into cellular behaviour.
Talin is a central FA protein, which exhibits this force-dependent folding behaviour and concurrent interactions. Within its 13 rod domains (R1-R13), R3 has least mechanical stability and unfolds very low force of 5 pN, whereas R13 domain has higher mechanical stability, unfolding at 15 pN. For example, at <5 pN force, talin interacts with Rap1 interacting adaptor molecule (RIAM), whereas at >5 pN force, talin unfolds and interacts with vinculin. This mechanical switching is mirrored in cellular structure and accelerates cell migration. Interestingly, the research group of the current study has observed that molecular chaperone can change this mechanical stability of talin.
We have developed the covalent magnetic tweezers (CMT), the first of its kind in India, to understand this force dependent-interaction of talin with chaperones. They found unfoldase chaperones (stabilizes the protein at mainly unfolded state) destabilize the folded talin and unfolds it at lower force. By contrast, the foldase chaperones increase the mechanical stability of talin. Consequently, this chaperone-modulated stability prompts changes in whole FA dynamics and cell behaviour.
Since chaperone overexpression is common in any pathological condition including cancer metastasis, where talin-centered FA dynamics play a central role; this force-dependent interaction signify a plausible mechanical effect of chaperone through inducing a domain-locked talin response in cell migration and consequently in its dysregulation.
How do these findings contribute to your research area?
Chaperone contribution to adhesion at both cell-extracellular matrix and cell-cell interfaces has long been suspected. Since most of these proteins are mechanosensitive, chaperone interactions with them might occur in a force-dependent manner; however, the underlying mechanism of these protein-chaperone interactions remains elusive. We showed for the very first time that chaperones interact with these force-sensitive focal adhesion proteins and tune their mechanical stability: unfoldase chaperones decrease mechanical stability, while foldase chaperones increase the stability.
Demonstrating this mechanical aspect of chaperone-adhesion protein would be very important to understand the overall chaperone contribution in focal adhesion dynamics (at cell-matrix interfaces).
“We have developed the covalent magnetic tweezers (CMT), the first of its kind in India, to understand this force dependent-interaction of talin with chaperones.”
What was the exciting moment during your research?
Our journey through these previous years was no less than a rollercoaster ride, with an initial disappointment. At first, we were not able to standardize the experiments and instrumentations. We overcame these frustrations by working day and night in the lab for 3-4 months and at last, we were very happy. But soon we reached a deadline when our current papers with the previous one got rejected from several journals and given a huge amount of reviewer’s comments. Thankfully, these comments with some new experiments made the avenue of our work more interesting and gave a novel mechanical aspect of chaperones involvement in focal adhesion.
What do you hope to do next?
The impact of any project depends on how many small interesting facts it explores since one starts to find his/her question. Our “Structural Mechanobiology Lab” has opened many different ways to uncover the mechanical aspect of cellular proteins and how their mechanical properties could be changed through different biochemical modulators such as post translational modification, chaperone and drug interactions. Our lab has already started to explore these force responses of mechanosensitive proteins while tuning their structure-function dynamics.
After finishing my Ph. D (if hopefully!), I will try to pursue postdoctoral research to perform the single-molecule force study in animal models and in vitro cell line conditions.
Where do you seek scientific inspiration from?
Research was not my first dream. Unfortunately, due to some issues, I could not join a medical institute even after securing an excellent rank in medical entrance and then only my mind started to ponder for research in biomedical science. Thereafter, I did both my bachelors and Master’s in microbiology from University of Calcutta. Since biology and physics had always been my favorite subjects since school, I always wanted to implement these two subjects in my research and it came true during my PhD. This made me confident in the field of single-molecule biophysics. This interdisciplinary field has groomed me as an insatiable reader and a digital artist. My cornerstones are my supervisor Dr. Shubhasis Haldar, who keeps pushing my limits beyond my expectations, and my inspiring colleagues (Especially Deep and Souradeep) who support me with their suggestions during the “chai-pani” break.
How do you intend to help Indian science improve?
Though Indian Science is making a strong comeback internationally, few things need to be fixed quickly to strengthen their mark. The collaborative and risk-taking approach are exciting, unravelling some challenging issues in biomedical sciences. However, there is still a prominent communication gap between academia and industry, which is hampering the translative and applicative science and its globalization. The society should know the importance of scientists for the society (not only in biological science but also in other fields). I have heard a few times: “You are a microbiologist. Okay, so you can only perform some pathological test. Don’t do masters and try to get a job in the government sector”. We need to get out of this crude thing and let society know the significance of scientific enterprise. I strongly intend to bridge this gap in Indian society in my upcoming academic career.
Reference
Chakraborty, S., Chaudhuri, D., Banerjee, S. et al. Direct observation of chaperone-modulated talin mechanics with single-molecule resolution. Commun Biol 5, 307 (2022) .https://www.nature.com/articles/s42003-022-03258-3
Edited by: Nivedita Kamath