Cells can pick up molecules from their environment through the process of endocytosis, where a region of the outer cell membrane bends inward and pinches off, forming a small sphere within the cell called a vesicle. Molecular machineries can then unload the cargo the vesicle holds for use within the cell. Many molecules the cell needs cannot pass through the membrane without this process, making it essential for their survival. In many types of cells, the process of endocytosis is slow, taking tens of seconds to minutes. In neurons, it happens much more rapidly -- as fast as 50 milliseconds. Interestingly, despite this difference in speed, the same set of proteins is required for the endocytotic process in neurons and non-neuronal cells. In order to explain this, scientists in the field assume that the role of endocytotic proteins do not change with the speed of endocytosis. However, in a recent investigation published in Molecular Biology of the Cell, researchers led by postdoctoral fellow Dr. Kumud Poudel in the Bai Laboratory (Basic Sciences Division) show that the endocytosis protein Endophilin likely plays distinct roles in rapid and slow endocytosis due to time limits.
The protein Endophilin plays an important role in endocytosis in many cell types including neurons. It belongs to a large group of related proteins that alter membrane curvature and overall shape. Research has shown that Endophilin has the ability to sense membrane curvature, to induce local membrane deformation, and to sculpt membrane into long hollow tubes, called tubulation (see Figure). However, it was unknown whether Endophilin can perform all of these functions at any time or sequentially throughout the process, and how these different tasks fit into the physiological time frame of the process. Dr. Poudel and his colleagues found that Endophilin performs different tasks sequentially to induce large-scale membrane tubulation. They found that the protein induces change in curvature after it arrives and initially binds the membrane.
To monitor the binding of Endophilin to membrane, the researchers made use of fluorescence-resonance energy transfer (FRET), a process that leads to a distinct fluorescent signal when two different fluorescent proteins, a donor and acceptor, are close together, within 10nm or less. They incorporated one fluorescent tag onto Endophilin and the other into the membrane lipid and detected Endophilin binding to membrane vesicles in test tubes by measuring fluorescent signal. Using a stopped-flow mixing apparatus coupled with fluorescence detection, they were able to take rapid snapshots and gather information about Endophilin binding to curved membrane vesicles over time, yielding kinetic information. They also monitored Endophilin binding to vesicles with various diameters, made by passing membrane through different diameter needles, and found that Endophilin prefers to bind vesicles with higher curvature (smaller diameter).
Next they wanted to investigate how quickly Endophilin can induce local membrane deformation. To do this, they used a different fluorescent tag on the lipid in the vesicle membranes. This fluorescent molecule, called laurdan, dims when it is exposed to water. Upon membrane curvature, the lipids in the membrane are pulled further apart from each other and exposed to the water in the solvent. Thus, by measuring the loss of lipid-bound laurdan fluorescence, the researchers can track the bending of vesicle membranes. Interestingly, Dr. Poudel and his colleagues found that membrane deformation occurs approximately 30 milliseconds after the binding of Endophilin to the vesicles, as measured previously using FRET. Thus, the bending activity of Endophilin appears to occur in a second phase following the binding phase.
The researchers asked whether the curving activity depended on the concentration of Endophilin or on a mechanism other than collision with membrane. They found that while Endophilin-membrane binding accelerated with increasing concentration, the ability of Endophilin to bend membrane, on the other hand, did not depend on the concentration of Endophilin in the solution. These data suggest that Endophilin undergoes a sensing-to-bending transition, a previously unknown feature of membrane bending proteins.
Finally, the scientists wanted to measure how long it takes Endophilin to make large-scale membrane changes. To capture this, they performed electron microscopy of samples of Endophilin and membrane vesicles that had been mixed and incubated together for 3 seconds, 2 minutes and 30 minutes. They found that tubules appeared in the 2 minute samples and that many small diameter vesicles appeared in the 30 minute sample. Therefore, while the binding and local membrane deformation reactions occur within a fraction of a second, the large tubules and highly curved vesicles are made over a period of several seconds to minutes.
These findings reveal that Endophilin transforms from a curvature sensor to a membrane bender, and then subsequently induces local deformation. The large-scale changes such as tubulation occur over multiple seconds to minutes. Thus, Endophilin’s action is likely to depend on the duration of the individual endocytosis process. This improves our understanding of the role of Endophilin in neuron communication and may help guide therapeutic efforts for neurological disorders.
Poudel KR, Dong Y, Yu H, Su A, Ho T, Liu Y, Schulten K, Bai J. 2016. "A time-course of orchestrated endophilin action in sensing, bending, and stabilizing curved membranes." Molecular Biology of the Cell. Epub ahead of print.
This research was funded by the National Institutes of Health, FHCRC New Development Fund, and an American Heart Association Fellowship. Computer time was provided by the Texas Advanced Computing Center funded by the National Science Foundation.