Like a blind man with a cane, a cell on the move pushes out thin, stiff spikes called filopodia to probe the environment around it. “Filopodia are involved in wound healing, axon guidance, embryo development and cancer cell invasion, making them relevant for medicine,” explained Thankiah Sudhaharan, an Advanced Microscopy Expert at the A*STAR Microscopy Platform of the Skin Research Institute Singapore (SRIS).
However, filopodia have proven challenging to observe under the microscope, since they are very thin and can extend, contract and move quickly. Now, Sudhaharan and his colleagues have found a way to use superresolution techniques to map out how proteins are arranged in filopodia, giving new insight into the structure and function of these cellular protrusions.
Sudhaharan was interested in a protein called IRSp53, which recruits structural protein actin modulators to assemble the internal scaffolding of filopodia. A part of the IRSp53 protein—known as the I-BAR domain—is known to have a role in shaping the cell membrane into a thin spike.
To study how IRSp53 mediates all these functions, Sudhaharan’s team engineered cells that expressed fluorescent IRSp53 protein and cells producing just the fluorescent I-BAR domain. They then used two superresolution techniques to visualize the proteins inside the cells’ filopodia, obtaining images with resolutions two to five times better than conventional microscopy.
The first, called direct STochastic Optical Reconstruction Microscopy (dSTORM), harnesses the ability to switch fluorescent molecules on and off, so that instead of all the proteins fluorescing at once and having their signals merged, the researchers could observe individual molecules blinking randomly at precise spots.
In 3D Structured Illumination Microscopy (3D-SIM), the second technique, the cell is illuminated with a finely striped pattern of light which interacts with the fluorescent structures inherent in the cell to produce interference. Repeating this process from different angles produces different patterns of interference, which can be computationally assembled into a superresolution 3D image.
“The combination of dSTORM and 3D-SIM let us build up a fine-grained map of protein locations, allowing us to identify the differences between dynamic and non-dynamic filopodia for the first time,” said Sudhaharan. Using this method, the team demonstrated that IRSp53 was located only at the edges of the filopodia, making them flexible and retractable, while the I-BAR domain in isolation was scattered more evenly, resulting in far more rigid filopodia.
“This finding opens up significant new pathways in the study of filopodia since many other proteins involved in their structure, function and dynamics still need studying at single-molecule resolution,” Sudhaharan added.