An international team led by a UofT lab has revealed a novel regulatory system behind cell migration and shown how the different parts work together.
This publication in the journal Current Biology adds to our understanding of how healthy bodies are maintained, as cell migration is crucial to the developing body and to immune cells traveling to the site of injury.
How do cells push out into their environment?
Ernest Iu, a graduate student in Cell & Systems Biology, decided to probe how calcium controls the structure and dynamics of the actin filaments at the leading edge of migrating cells.
Actin filaments are rods that grow within the cell to push out against the cell membrane, leading to flaps called ‘lamellipodia’ that promote movement. Scientists for years have noticed the importance of calcium ions in the regulation of lamellipodia dynamics, but how exactly this regulation occurs has remained unclear.
In a technical tour-de-force, Iu and his colleagues in the Plotnikov lab exposed novel details of what’s happening under the hood of calcium-dependent cell migration. This work is published as “A TRPV4-dependent calcium signaling axis regulates lamellipodial actin architecture to promote cell migration”.
Iu’s supervisor, Professor Sergey Plotnikov, is enthusiastic about this work: “Previous investigations into the biological roles of calcium signaling pathways have been largely limited to observing cellular responses. We have revealed the machinery behind a long-sought signaling mechanism that regulates the actin cytoskeleton in cell migration.”
Technical innovations demonstrate cellular machinery in unprecedented detail
Cell migration is usually observed on cells growing on glass, but the ragged edges of the cell make quantitative analysis difficult. Iu’s technique circumvented this shortcoming by observing round cells as they settled onto the bottom of the dish and spread out in a circle.
He showed slower spreading in cells without the TPRV4 channel compared to control cells, demonstrating that this calcium channel was important for lamellipodia. Iu then proceeded to look inside the cell to reveal an intricate mechanism of interacting proteins dependent on TRPV4.
Initial results showed that inhibiting TPRV4 inhibited the RhoA protein. “I thought this was a dead end,” states Plotnikov. “How do you study the functional connections of two seemingly unrelated proteins? But Ernest was too stubborn to stop.”
Iu’s perseverance first identified a molecular switch that controls other proteins, known as a kinase CaMKII. CaMKII that becomes active when the TRPV4 channel is activated.
Next, Iu innovated a technically challenging procedure to isolate proteins from the migrating region of the cell. Iu and colleague Alexander Bogatch placed the cells on a fine mesh and provoked the cells to grow lamellipodia through the mesh.
They then collected these microscopic protrusions to isolate the proteins at the tip for mass spectrometry analysis. The success of this advanced cell fractionation and mass spectrometry experiment was the result of a global collaborative effort with the Humphries lab in Manchester, made possible by generous support from the University of Manchester-University of Toronto Joint Research Fund (now the MMT Research Fund).
Defining all the components of a spatially confined signaling circuit for cell movement
The team showed that the RhoA protein in the protrusions reacted to calcium influx and that RhoA activity was dependent on activation by the CaMKII protein.
Iu brought the study full circle by showing the RhoA/CaMKII calcium sensor was linked through the TEM4 protein and demonstrating that this signalling hub controlled the actin filaments pushing out the lamellipodia to the TRPV4-containing cell edges.
Further collaborations with the Tanentzapf lab at University of British Columbia revealed that Iu’s results in cultured cells in vitro were also supported ex vivo in migrating mouse melanoblasts.
Iu therefore fully defined a spatially confined calcium signaling circuit that orchestrates actin cytoskeletal organization in the lamellipodia cellular domain as part of his PhD in Cell & Systems Biology.
The next steps for the Plotnikov lab are to determine how TRPV4 is activated and the clinical significance of TRPV4-dependent cell migration, particularly given the critical role of cell migration in pathological conditions such as atherosclerosis and cancer metastasis.
Dr Ernest Iu has earned the Yen fellowship at the University of Chicago to conduct his postdoctoral research under the mentorship of Professor Margaret Gardel, a world-leading expert in cell and tissue biophysics. Congratulations on this impressive work!