Improved superresolution microscopic technique allows researchers to view vesicle maturation into endosomes. Image provided by Wendell Lim.
Understanding the tightly controlled processes in cells at the single-cell level has been a challenge. One approach has been to use superresolution microscopy techniques. In a paper just out in the Proceedings of the National Academy of Science, researchers have improved a superresolution microscopy to be able to track the movement and change in the molecular composition of vesicles as they mature into endosomes.
The introduction of superresolution microscopy in 2006 did away with the diffraction-limit problem of conventional optical microscopy. The different forms of superresolution microscopy, STORM, PALM and FPALM, all use photoactivatable fluorophores that are switched on and off. “Many people have assumed that it would be straightforward to use superresolution microscopy to count the absolute number of molecules in a given structure,” explains Bo Huang at the University of California, San Francisco. “The reality is much more complicated.”
Huang explains that these fluorescent proteins blink, so researchers inadvertently count the blinking fluorescent proteins more than once. There is also the problem of undercounting when the fluorescent proteins don’t fully mature.
So Huang, in collaboration with Wendell Lim, also at UCSF, developed an intracellular calibration approach: They first measured photophysical parameters in cells under imaging conditions that corrected for blinking. Then, they counted the number of molecules in protein complexes with known stoichiometry. Next, they calculated how many fluorescent proteins didn’t mature. Once they had this calibration in place, the investigators were ready to count absolute numbers of molecules.
Lim explains that vesicle trafficking is a great example of a dynamic process in which it’s hard to pin down the numbers of molecules. “While we know most of the key markers associated with distinct states along the pathway, relatively little quantitative information is known about the density of molecules in each individual vesicle and particular stages,” he says. “This is in large part due to the fact that it is hard to resolve individual vesicles/endosomes by conventional microscopy and hard to count the numbers of molecules associated with each vesicle.” A perfect test case to see how well the new technique works.
The investigators established the number of phosphatidylinositol 3-phosphate binding sites in individual vesicles of single cells. PI3P is a key player in endocytosis and a marker for vesicle maturation. The investigators found that, while vesicle composition and movement were tightly regulated within a cell, the process was more stochastic between different cells. More importantly, Huang and Lim’s team worked out some mechanistic details of endocytosis, finding that PI3P production happens before the vesicles fuse into larger endosomes.
Huang says the method is amenable to more than just vesicle trafficking: “This method could have wide applications in measuring the stoichiometry of protein-protein interactions and the composition of protein complexes, for example, oligomerization state of membrane receptors.”
