Work done in the lab of Dr. Sivaram V S Mylavarapu at the Regional Centre for Biotechnology
About author
Dr. Amrita Kumari pursued her Bachelor’s (honours) in Biotechnology from Amity University, Noida and Masters in Biotechnology from IIT Bombay. For her PhD, she joined Dr. Sivaram Mylavarapu’s laboratory at the Regional Centre for Biotechnology, Faridabad. For her doctoral work, she studied the role of the motor dynein during cell division, which was published in the prestigious Journal of Cell Biology (JCB) and was featured on the cover of the December 2021 JCB issue. She loves communicating science to various audiences through posters, talks, blogs, outreach programs etc. She has won several national and international awards for presenting her research work at various platforms during her PhD tenure. Amrita considers herself a perennially hungry bookworm and finds solace between pages of any classic science non-fiction book available on the planet. When not in the lab, she can be found in coffee shops either reading books or listening to people’s life experiences.
Interview
How would you explain your research outcomes to the non-scientific community?
Our lab focuses on how tiny vehicles called molecular motors function inside our cells during mitosis. Mitosis, the vital process of cell division that produces two identical cells from the mother cell, is required for embryonic development, stem cell regeneration, tissue repair and to maintain general homeostasis in our body. The molecular motor “dynein” is known to play a crucial role during mitosis by ferrying mitosis-specific molecular cargoes towards the negative ends of molecular tracks in the cell called microtubules. Interestingly, during the non-dividing phase of interphase, dynein mainly carries membrane-enclosed cargoes on microtubule tracks, but switches cargoes dramatically while entering mitosis (fig.1). That makes dynein a virtual “super motor” of the cellular system.
Dynein is known to undergo a specific chemical modification named phosphorylation at the beginning of mitosis. Out of four dynein protein components, the Light Intermediate Chain 1 (LIC1) subunit undergoes the maximum amount of phosphorylation. The new phosphorylation tag is essential to erase dynein’s earlier identity of an “interphase motor” by making it shed off its interphase cargoes and bind to its new mitotic cargoes. We were very curious to understand how putting a chemical tag on a subunit confers the dynein motor the new acquired ability to bind to new cargoes.
The three-dimensional structure of the LIC1 protein consists of a compactly folded N-terminal domain, but a largely unstructured and “floppy” C-terminal region. The flexible C-terminal domain contains many phosphorylation sites; imagine a fairy light wire (LIC1 amino acid stretch) rolled into a ball at one end, while the other is loose and hangs free. The loose end of the wire has many “small switching bulbs (phosphorylation sites)” clustered in a small stretch. The location of these phospho-switches in the flexible region makes them easily accessible to molecular signals in the cell. We discovered that switching off three specific phosphorylation sites on this C-terminus of LIC1 during mitosis results in various cell division defects that are signatures of cancerous cells.
We also wanted to know whether dynein is able to bind to its mitotic cargoes in the absence of phosphorylation. A major way dynein engages with diverse cargoes is by binding to different “adaptor proteins”, each with their signature “protein binding sockets” where matching cargoes can fit for dynein to carry. We wondered how phosphorylation helps dynein choose among different adaptors in dividing cells. We discovered that one local population of phosphorylated dynein interacts with a particular adaptor named spindly present on the chromosomes, enabling this fraction of dynein to ferry a specific set of cargoes from the center of the cell to the two opposite poles of the dividing cell. However, another population of phosphorylated LIC1 attracts the interesting isomerase enzyme Pin1, which recognizes these phosphorylated regions and subsequently “twists” the shape of the protein at adjacent regions, resulting in various downstream functional consequences. In the case of dynein, this interaction changes the conformation of this motor protein and increases its affinity for a different mitotic adaptor, Hook2. Now this population of dynein is directed towards a specific cellular location (spindles or poles of dividing cells) and participates in ferrying a separate set of cargoes. In essence, the pattern and intensity of the turned-on bulbs (phospho sites) on the ferry light (LIC1 protein) directs dynein to choose the right cargo at the right time (fig.1).
Our research reveals some of the intricacies underlying the design of energy-efficient molecular super motors inside a cell. Cells do not spend extra energy in making more proteins to meet the new workload during mitosis. Rather, they temporarily add specific chemical tags (such as phosphorylation) to the existing proteins to modulate their shapes and local surface chemistries, eventually providing layers of functional regulation. Imagine each phosphorylation site as a small bulb in the long fairy light wire (amino acid stretch of protein), with thousands of such wires present in the cell. They can light up during mitosis so as to produce different lighting patterns with different intensities (waves, flickers, pulses) in space and time to produce the desired output in the cell. Isn’t that super smart! This journey certainly gave us a sense of the efficient utilization of resources, which is indispensable to every aspect of life on this planet.
How do these findings contribute to your research area?
The hyper phosphorylation of the Light Intermediate Chain 1 (LIC1) subunit had been long suspected as a potential cause for making dynein lose most of its interphase cargoes while entering into mitosis. Despite that, the molecular mechanism behind this event was yet to be discovered. We showed for the first time how phosphorylation of LIC1 enables dynein to interact with different adaptor molecules and assemble into biochemically distinct complexes. These distinct complexes are required for a plethora of functions that dynein needs to perform during the short span of mitosis.
Studying the secrets of the superpowers of the dynein motor would be very interesting as well as useful. This would help us appreciate the precision and efficiency employed by nature to design one of the most efficient motors on this planet. This would also give us insights into rationally designing approaches to curb aberrant cell divisions, which are hallmarks of many cancers.
“Our research reveals some of the intricacies underlying the design of energy-efficient molecular super motors inside a cell.”
What was the exciting moment during your research?
Our journey through this project was no less than a rollercoaster ride filled with both disappointments and surprises. Among many successive triumphs, one of the most memorable ones was when we found a direct phosphorylation-based interaction between LIC1 and the prolyl isomerase Pin1. The next step was to find the molecular pathway this complex was involved in. But soon we reached a seeming dead end when we could not see the expected proteins pulling down with Pin1 from mitotic lysates. After wallowing for a while, we dug deep into the literature to find out about a lesser-known dynein complex that specifically participates in counteracting opposing forces from other motors. We probed for these protein members in the pulldown assays, which ultimately led us to discover a new dynein functional pathway that is not processive, rather a high load-bearing state of dynein performing an exclusive set of functions. This finding changed the course of our work and paved the way towards discovering a really interesting and novel aspect of dynein function during mitosis.
What do you hope to do next?
The success of any project depends on how many interesting leads it generates while searching for the answers we set out to find in the beginning. This work has left more questions to be answered for than the number of answers it has found. Our work has opened many different avenues to uncover the role of various evolutionarily conserved post translational modifications (PTMs) on the LIC molecule. The different permutations and combinations of these PTMs are expected to exponentially increase the number of distinct dynein motors and in turn, increase the different dynein-adaptor-cargo complexes to cover the broad spectrum of dynein functions across the cell cycle. The lab has already started following some of these leads towards unraveling the mystery of dynein’s incredible multi-functionality.
I will be joining my postdoctoral lab soon to work on yet another interesting aspect of mitotic dynamics.
Where do you seek scientific inspiration from?
Biology had always been my favorite subject, but I discovered my deep love for science literature during my PhD and that turned me into a voracious reader lately. I used to be completely blown away by the way all the fine details were weaved into interesting narratives in those books. That sparked the curiosity in me to keenly observe my surroundings with great awe and appreciation. My insatiable curiosity got nurtured and groomed by my very encouraging and inspiring supervisor and colleagues in the lab. As a result I started seeing the magic in every creation around me. Now it keeps me motivated to understand the what, why and how of everything that supports our life on this planet and to get a glimpse of the incredible intelligence working through every atom across an unfathomable scale.
How do you intend to help Indian science improve?
Indian science is making a mark on the world map like never before. This is an extremely exciting and motivating time to participate in and contribute to Indian science. The new crop of scientists is bringing a more ambitious, collaborative and risk-taking approach to work on some really interesting and challenging questions that need our immediate attention. Through my postdoctoral training, I would like to deepen my scientific expertise and imbibe leadership qualities, which will help me in contributing my share towards the community.
Reference
Kumari A, Kumar C, Pergu R, Kumar M, Mahale SP, Wasnik N, Mylavarapu SVS. Phosphorylation and Pin1 binding to the LIC1 subunit selectively regulate mitotic dynein functions. J Cell Biol. 2021 Dec 6;220(12):e202005184.
Edited by: Anjali Mahilkar