Tissue-specific tagging of endogenous proteins in the fruit fly

Seeing is believing, and fluorescently tagged proteins have ushered in a major revolution in cell biology. Instead of observing the static components of dead cells fixed in plastic and reacted with dyes, tagged proteins fluorescing a variety of colors can be tracked in real time in live cells and organisms. We can peek at the previously only imaginable perpetual dynamism of life at the molecular level. In addition to turning us into spell-bound voyeurs, well-defined fluorescent tags also give us a hand-hold to isolate the binding partners of proteins of interest.

In a recent article by the Rodal lab reported in Biology Open, the authors report a new tagging methods designed to get rid of technological artifacts that can cause fluorescently tagged proteins to be expressed at the wrong time and place, and at the wrong levels. By using CRISPR mediated gene editing in fruit flies, they developed a novel approach to visualize any protein of choice in any tissue of choice at the level, localization and time that nature has intended. This method, dubbed T-STEP (for tissue-specific tagging of endogenous proteins), opens up novel experimental avenues to answer long-standing questions in several areas of neuroscience and cell biology, such as: how many different neurotransmitters are expressed in one neuronal circuit? Which tissue-type is a protein expressed in and when? What happens to a disease carrying mutant protein in a tissue of interest at endogenous levels?

As a proof of principle, two endosomal proteins, Vps35 (linked to Parkinson’s disease) and OCRL (linked to Lowe syndrome), which have never before been seen or localized in fruit flies, have now been visualized live at endogenous levels. Moreover, a Parkinson’s disease-specific mutation (D620N) in Vps35 has also been tagged with fluorescent proteins, opening up exciting new research avenues for interrogating binding partners and/or kinetics that may be altered during the diseased states.

In summary, T-STEP is an exciting novel tool that offers a simple and efficient method to tissue-specifically tag any protein at endogenous levels. Authors from the Rodal lab include Kate Koles (Research Scientist) and Anna Yeh ’16.

• Koles K, Yeh AR, Rodal AA. Tissue-specific tagging of endogenous loci in Drosophila melanogaster. Biol Open. 2015;5(1):83-9.

A facilitated diffusion confusion dissolution

To utilize the information contained within a cell’s genes, the enzyme RNA polymerase must find the beginning of each gene (the promoter).  Finding the beginning is a prodigious task:  RNAP must start at a particular base pair of DNA, but the cell contains millions of base pairs to choose from.  It has been proposed that gene-finding challenge is aided by a process termed ‘facilitated diffusion’ (FD).  In FD, RNA polymerase first binds to a random position on DNA and then slides along the DNA like a bead on a string until it encounters the target DNA sequence.

In a recently published study in PNAS (1), biophysicists Larry Friedman and Jeffrey Mumm worked with Prof. Jeff Gelles in the Brandeis Biochemistry department to test key predictions of the FD model.  They used a novel light microscope that Friedman and colleagues invented and built at Brandeis, a microscope that can directly observe the binding of an individual RNA polymerase to a single DNA.  The scientists studied the σ⁵⁴ RNA polymerase holoenzyme, an RNA polymerase found in most species of bacteria.  Surprisingly, none of the three predictions of the FD model that the experiments tested were found to be valid, demonstrating that target finding by the polymerase is not accelerated by sliding along DNA.  Friedman and colleagues instead propose that RNA polymerases are present in such large numbers that they can diffuse through the cell and efficiently bind to their target sites directly.  The absence of FD may explain how other proteins can bind to positions on the DNA that flank gene start sites and yet not interfere with RNA polymerase finding the gene.

Is this the end of the story? Not likely, given previous publications suggesting FD plays a role for some other DNA binding proteins. Using single-molecule techniques like those developed in the Gelles lab, scientists in next few years should give us a better idea if FD is very rare or very common. [editor: as a chemical engineer, I’m sad to see FD not have a role — it seemed like such a nice theory…]

Friedman LJ, Mumm JP, Gelles J. RNA polymerase approaches its promoter without long-range sliding along DNA.  Proc Natl Acad Sci U S A. 2013 May 29. [Epub ahead of print]

 

 

Rodal to Receive NIH New Innovator Award

The NIH recently announced that Assistant Professor of Biology Avital Rodal will be a recipient of the 2012 NIH Directors New Innovator Award. The award allows new, exceptionally creative and ambitious investigators to begin high impact research projects. Granted to early stage investigators, candidates are eligible for the award for up to ten years after the completion of their PhD or MD. The award emphasizes bold, new approaches, which have the potential to spur large scientific steps forward. This year’s award was made to fifty-one researchers, and provides each with 1.5 million dollars of direct research funding over five years.

The Rodal lab studies the mechanisms of membrane deformation and endosomal traffic in neurons as they relate to growth signaling and disease. Membrane deformation by a core set of conserved protein complexes leads to the creation of tubules and vesicles from the plasma membrane and internal compartments. Endocytic vesicles contain, among other cargoes, activated growth factors and receptors, which traffic to the neuronal cell body to drive transcriptional responses (see movie). These growth cues somehow coordinate with neuronal activity to dramatically alter the morphology of the neuron, and disruptions to both endocytic pathways and neuronal activity have been implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and Alzheimer’s disease.

Dr. Rodal hopes to determine how neuronal activity affects the in vivo function and biochemical composition of the membrane trafficking machinery, by examining the transport of fluorescently labeled growth factor receptors in chronically or acutely activated neurons at the Drosophila neuromuscular junction (NMJ). Her group will combine these live imaging studies with a proteomic analysis of endocytic machinery purified from hyper-activated and under-activated neurons. By investigating the interplay between neuronal activity, membrane deformation, and receptor localization in live animal NMJs, she hopes to gain a better understanding of the strategies that healthy neurons employ to regulate membrane trafficking events, and provide new insight into specific points of failure in neurodegenerative disease.

• see also story at Brandeis NOW

Using PhADE in single molecule fluorescence imaging

Anna Loveland, a postdoc in the Grigorieff Lab, has a new paper, “A general approach to break the concentration barrier in single-molecule imaging” that appeared today in Nature Methods online. The paper is based on her PhD work, which was done jointly in the labs of Antoine van Oijen and Johannes Walter at Harvard.

Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule’s signal. Loveland et al. employ a new imaging approach called PhADE (photoactivation, diffusion and excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. The authors labeled the eukaryotic DNA replication protein flap endonuclease 1 with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations.

Anna B Loveland, Satoshi Habuchi, Johannes C Walte & Antoine M van Oijen (2012) A general approach to break the concentration barrier in single-molecule imaging. Nature Methods