Biocurious is a weblog about biology, quantified.

Hot off the Press: Fluorescent timers

by PhilipJ on 21 January 2009

While we’re waiting for the most exciting future biophysics tool to get built, there are all kinds of practical improvements to current-generation microscopy that would still be exciting and useful. We’ve talked a lot about increasing the spatial resolution of optical microscopy, but it would also be useful to have tools for temporal measurements of dynamics in a living cell. Many cellular processes occur on temporal scales of many hours, and being able to track such processes with current-generation optical microscopes would still give exciting information about intracellular trafficking and dynamics. To do this, a tagging system that would give temporal information as well as spatial information is necessary.

A paper in the February issue of Nature Chemical Biology discusses just such a technique. Subach et al’s Monomeric fluorescent timers that change color from blue to red report on cellular trafficking introduces a new fluorescent timer system based on directed evolution of mCherry, a protein that fluoresces in the red. By carrying out mutagenesis on selected amino acids that interact with the chromophore of the GFP-like protein, it was possible to generate variants of the protein that had differing maturation rates for the chromophore (leading to different fluorescence intensities over time), as well as a temporal shift in the fluorescence spectrum from blue to red. Different mutants resulted in fast-, medium-, and slow-fluorescent timer molecules where the maturation rate of the fluorescence change varied as their namesake.

An example of the maturation of the slow-FT reporter fused to a protein in mammalian cells is shown in the figure below. This shows the ratio of blue to red fluorescence as a function of time in vivo, where even in the complex environment of the cell, the temporal properties of the fluorescent timers remain intact.

fluorescent timer colours

This system of fluorescent reporters was then used to study the dynamics of lysosome-associated membrane protein 2A, a protein that must reach lysosomes either via the Golgi complex directly, or via endocytosis from the plasma membrane. The medium-FT molecules had an ideal timescale to study the LAMP-2A intracellular dynamics, and the figure below shows the result of expressing a LAMP-2A-medium-FT fusion protein in mammalian cells.

FTs in the cell

Careful analysis of the localization and colours of fluorescence made it possible to conclude that a primarily indirect pathway for LAMP-2A trafficking is taken, based on the blue-to-red ratios both near the plasma membranes and in the Golgi. While the details of this specific experiment are complex, the above figure shows that this is a very powerful new technique to study both localization and temporal dynamics in vivo, and a range of fluorescent timer mutants with fast to slow dynamics should give the ability to study a wide range of intracellular processes. Indeed, the authors point out a number of interesting things to study in their conclusion:

FT proteins will also allow for identification of recycling events among compartments, temporal tracking of molecules before and after a particular cellular event (without the need for additional labeling or artificial photoswitching) and timing of particular intracellular post-translational modifications traceable by fluorescent procedures such as ubiquitination and farnesylation.

The optical microscope has a lot of science left to do.



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