Phase separation in SAP97, a synaptic protein and its recruitment of the Ca2+ sensor, Calmodulin, into condensates.

Work done in the lab of Prof. Deepak Nair at Centre for Neuroscience, IISc, Bengaluru

About author

Premchand Rajeev did his Bachelor of Forestry from Forest College and Research Institute, Mettupalayam, Tamil Nadu in 2014. Then, in 2016, he joined the Centre for Neuroscience at IISc to pursue his PhD degree in Neuroscience. He joined the Nano-Org lab of Deepak Nair with a curious mind to study and understand the nanoscale organisation of synaptic proteins using super resolution microscopy techniques such as dSTORM, STED, and PALM. In his doctoral work, he studied the organization of the synaptic protein SAP97, which is implicated in multiple psychiatric disorders. Outside his scientific pursuits, he is an accomplished classical dancer.

Interview

How would you explain your research outcomes to the non-scientific community?

An organism’s learning and memory are progressive properties from the neural signaling mechanisms of the nervous system, where the intracellular transmission of signals is regulated by junctions called synapses. In chemical synapses, the chemical messengers (Neurotransmitters) in presynapse, receptors (Proteins) in post synapse,and their associated molecules work in coordination to establish, regulate, and modify the signal transmission.

SAP97 is one of the many proteins that function as scaffolds at the postsynapse to fixate the receptor proteins adjacent to the presynaptic region. The loss of function of this protein has been linked to various psychiatric disorders, including schizophrenia, autism spectrum disorders and bipolar disorder. In this study, we investigated the nanoscale organization of SAP97 in vivo and in vitro conditions and provided a model for the organization mechanism to extract the biophysical characteristics of the protein aggregates using dSTORM, a super-resolution microscopy technique. We found that SAP97 follows a first-order phase transition in heterologous cells and hippocampal pyramidal neurons. Additionally, the C-terminus spliced variants of SAP97 showed differential kinetics in response to intracellular Ca2+ fluctuations in an isoform-specific manner.

Further, we purified the C-terminal region of these isoforms that steers the interaction with the Ca2+ sensor, Calmodulin (CaM) and performed in vitro liquid-liquid phase separation. We observed spontaneous aggregation of molecules of both isoforms of SAP97, and these aggregates were capable of recruiting Calmodulin in the presence of Ca2+. This study provides insights into an intrinsic capability of a synaptic protein, localized at the periphery of postsynaptic density, to co-condense a Ca2+ sensor in the presence of Ca2+.

How do these findings contribute to your research area?

Liquid-liquid phase separation is an emerging field where the synaptic compartments are perceived as membrane-less organelles. The study brings in the affinity of SAP97 molecules to homotopically condense at nanomolar concentrations and the recruitment of Ca2+ bound CaM into clusters, which changes our perspective of CaM interaction with scaffolds. This work provides insights into the molecular interactions and biophysical properties of SAP97 enabling us to close in on its isoform specific differential dynamics, nanoscale distribution and the molecular signatures in cell systems and solutions. This opens opportunities to explore more into its impacts on the receptor dynamics that controls the frequency and amplitude of synaptic signals, which ultimately determine the efficacy of signaling.

“This study provides insights into an intrinsic capability of a synaptic protein, localized at the periphery of postsynaptic density, to co-condense a Ca2+ sensor in the presence of Ca2+.”

What was the exciting moment during your research?

The whole journey was a roller coaster ride with highs and lows, but the moment that took the prize was when I did the protein purification for 4 months and finally saw the molecules getting phase separated. It was an “Eureka” moment, to be remembered forever.

What do you hope to do next?

So far, I have been focusing on the nanoscopic organizations of synaptic proteins and their dynamics. Furthermore, I hope to expand my skills in the fields of electrophysiology and behaviour to get a panoramic view of the nuances in neurobiology.

Where do you seek scientific inspiration from?

I seek inspiration from the past. It fascinates me to visualize our journey from the wilderness to a person reading this digital document, and yet the wheels are turning. “The fire, once discovered, the rains cannot put it out, still burning in us to fuel up the persistent efforts to an endless journey”.

How do you intend to help Indian science improve?

I believe that our education system needs restructuring to generate more scientific and critical minds. We are the products of an education system, once implemented to produce efficient clerks in the colonial era. On comparing the number of graduates and postgraduates with the number enrolling in research, the system seems to be killing the enthusiasm and curiosity in students. I would urge students to come out of their comfort zones and to achieve their full potential. Furthermore, the future of neuroscience demands multidisciplinary approaches where I would try to the best of my ability to collaborate and incorporate multiple viewpoints of solutions to a problem.

Reference

Rajeev, P., Singh, N., Kechkar, A. et al. Nanoscale regulation of Ca2+ dependent phase transitions and real-time dynamics of SAP97/hDLG. Nat Commun 13, 4236 (2022). https://doi.org/10.1038/s41467-022-31912-1

Copy Editor

Sukanya Madhwal

Ph.D. student at inStem Bangalore

Sukanya hails from a small town, kotdwara in Uttarakhand. She completed her Masters’ degree in Biotechnology from Banasthali Vidyapith, Rajasthan. After this, she served as a graduate teacher for one year at P.G. College, Kotdwara, Uttarakhand. In 2014, she qualified for the JGEEBILS exam conducted by NCBS/TIFR and joined as a Research Scholar (Ph.D. student) in Dr. Tina Mukherjee’s lab at inStem. Currently, she continued working as a bridging post-doc in Dr. Tina Mukherjee’s lab. Besides work, she loves reading non-fiction books, enjoys gardening, and cooking delicious food.

For interview related queries, write to us at interview.biopatrika@gmail.com