16 January 2008. It’s not all that surprising to see flashing blue lights in the red light district—unless you are talking cell biology. In the January 11 Nature Chemical Biology, researchers led by Vladislav Verkhusha at the Albert Einstein College of Medicine, New York, introduced new fluorescent timers—proteins that gradually shift fluorescence emission, in this case from the blue to the red end of the spectrum. Far from using them to monitor seamy behavior, however, the researchers dispatched the blue-red lights to trace trafficking of LAMP-2A, a chaperone that is involved in autophagy. Their findings help resolve an ongoing debate about whether LAMP-2A travels directly to endosomes from the Golgi, or detours through the cell membrane. “Our studies confirm that this indirect membrane route is a major trafficking pathway,” said Verkhusha in an interview with ARF. Not only does the finding help researchers come to better grips with autophagy, which has become a major area of interest to Alzheimer and Parkinson disease researchers, but the new fluorescent tools might also prove useful more generally for tracing the movements of presenilin, BACE, and other molecules involved in the pathology of AD or other neurodegenerative diseases.
Verkhusha’s fluorescent timers are derivatives of mCherry, a red fluorescent protein that is itself derived from DsRed, a protein made by the sea anemone Discosoma striata. Like the widely used green fluorescent protein (GFP), the chromophore in DsRed is formed by an autocatalytic rearrangement of the protein backbone. Unlike GFP, however, DsRed is a tetramer. This makes it a less practical tool and also renders it mildly cytotoxic because of the stress it puts on cells that overexpress it. DsRed—in particular DsRed-E5, a fluorescent timer (FT) that shifts its emission peak over time—has found some use as a biological reporter. However, a monomeric version of DsRed-E5 would prove even more useful.
Verkhusha and colleagues have now developed a suite of such monomers, and they differ in the speed of their blue-red conversion. Joint first authors Fedor and Oksana Subach—another husband-and-wife team in the field—used targeted saturation mutagenesis to substitute amino acids thought to influence chromophore maturation. They obtained a series of mutant proteins with blue-to-red fluorescence conversion half-lives in the 0.25- to 10-hour range. The authors used these fast, medium, and slow fluorescent timers to trace LAMP-2A trafficking using a FT-LAMP-2A chimera. Using a pulse-chase approach, the Subachs and colleagues found that the nascent blue-fluorescing chimeras appeared in the Golgi, the plasma membrane, and, to a much lesser extent, in endosomal compartments. The red-shifted fluorescent probe never showed up at the plasma membrane, predominating instead in endosomal compartments and lysozomes. The findings indicate that LAMP-2A chimeras transit first through the Golgi and plasma membrane as young blue fluorophores, before maturing and ending up in the red zone of the lysozomes and endosomal compartments.
“This work nicely validates, in a very elegant way, the findings of Paul Matthews,” said Ralph Nixon, New York University School of Medicine. Nixon was not involved in the study, but his lab studies the role of autophagy in AD. Mathews, now at NYU as well, showed, while at Johns Hopkins University in Baltimore, that the membrane glycoprotein LEP 100 traffics to lysosomes via the plasma membrane (see Mathews et al., 1992). That had been suggested for LAMP-2A also because small amounts of the protein have been found at the cell surface in steady-state snapshots.
The work raises the question of whether LAMP-2A has some functional role at the cell membrane, despite its fleeting presence there. “Nature is usually so economical that you would not expect to see a protein on the cell surface if it is not needed there,” suggested Verkhusha. “I would not be surprised if someone finds out that LAMP-2A has a functional role at the cell membrane.”
“In terms of the broader implication, these tools will be very valuable in looking at trafficking patterns of other proteins,” Nixon said. To fully deliver on that promise, however, these fluorescent timers will need some technical refinement first. While the quantum yield (a measure of the proportion of energy released as fluorescence) of the blue forms is respectable (0.3 to 0.4), it is low for the red forms (0.05-0.09). This makes the probes perfectly adequate for cellular studies, said Verkhusha, but means they would be less impressive for deep tissue imaging in mammals.
In addition, the blue-red conversion is on the order of hours, not minutes, meaning the probes might prove less useful for tracing proteins with very short half-lives. For example, the half-life of the amyloid precursor protein APP in cells is less than an hour, said Nixon. “I wouldn’t discount APP entirely, but the half-lives are not in ideal range,” he added. “I think these probes would be more valuable for studying other proteins, including presenilin and BACE, that have half-lives in the order of hours. Certainly this has relevance to trafficking of a number of proteins of interest to the AD field.”
Another possibility is that the probes could prove useful in studying proteins that get stuck in certain compartments as a result of some underlying pathology. “That might be an attractive thing for us to do since we have been looking at the slowed degradation of proteins through autophagy, and any number of proteins might be studied in that particular way,” said Nixon (see ARF related news story).—Tom Fagan.
Subach FV, Subach OM, Gundorov I, Morozova KS, Piatkevich KD, Cuervo AM, Verkhusha VV. Monomeric fluorescent timers that change color from blue to red report on cellular trafficking. Nat Chem Biol. 2009 January 11. Abstract