Creating fluorescent probes targeting the genome and key bio-pathways

There are four research areas in the Department of Pharmaceutical Chemistry. Creating fluorescent probes targeting the genome and key bio-pathways is a research challenge within protein and cellular engineering.

The challenge

Since the late 20th century, scientists have been able to track the locations, movements, and activities of specific types of protein molecules inside cells by labeling them with fluorophores (molecules that absorb and then emit specific wavelengths of light). This is often achieved by immunofluorescence—linking fluorophores with antibodies designed to bind to the proteins of interest. Thus proteins can be tracked by combining microscopes with appropriate wavelength laser light sources. In addition, the engineering of different-colored fluorophores allows the combined tracking of multiple types of proteins and their interactions.

Fully tracking the precise locales, timing, and extent of important biological events in the cell via fluorescent labels, including in the genome, would be vital to guiding effective therapeutic interventions.

Example of our methods and research include

New probes for fluorescence microscopy of epigenetics and intracellular signalling

Department scientists are improving upon fluorescent reporting of cellular processes, using protein, nucleic acid, and cellular engineering approaches to develop new probes. Specifically, they are adapting new techniques to probe specific DNA sequences for the visualization of their dynamics and are synthesizing smaller, more precise immuno-labeling for tracking key intracellular signal transmissions—in both cases, without disturbing the targeted processes. Thus they are converting key biological questions into imaging processes.

“Imagenomics”: genome dynamics via CRISPR/dCas9 visualization

While the genetic sequences in our cells’ nuclei are fixed, gene expression (the encoding of proteins that carry out the activities of life) is not. The genome’s functional output is regulated by its dynamic interactions with proteins and RNAs, including enzymes’ chemical modifications to histones—spool-like proteins that DNA is wound around.

Such interactions can alter the spatial arrangement and packaging of the chromatin (the complex of DNA and proteins), which, in turn, can regulate which genes are accessible for activation. Such conformational change by the genome, altering gene activity, underlies both the cell’s healthy ability to respond to endogenous signals and environmental stimuli and, when it goes awry, the inappropriate genetic expression underlying many diseases.

While some epigenetic modifications may be detected, identified, and located via methods such as mass spectrometry, scientists have not been able to directly and visually monitor these dynamic processes of chromatin remodeling in living cells and map them to overall DNA sequences and dynamics in real time. This would fundamentally advance our understanding of genetic regulation toward potential therapeutic interventions.

schematic of CRISPR system
Cell, Vol. 155, Issue 7, 19 Dec 2013

Schematic diagram of CRISPR/Cas system genome engineering technique, repurposed for dynamic imaging: an endonuclease-deficient dCas9 enzyme tagged with EGFP (enhanced green fluorescent protein) combines with a structurally optimized small guide RNA (sgRNA) to label genomic loci (a gene or specific DNA sequence) for imaging

In collaboration with UCSF colleagues, department scientists are developing methods to view, monitor, and map genomic modifications and dynamics (epigenomics) in living cells and even living animals via fluorescence microscopy. This involves adapting a recently developed genome engineering technique, based on the CRISPR system, for visualization.

CRISPR stands for Clustered Regularly Interspaced Palindromic Repeats. It is a mechanism used by bacteria to immunize themselves against viruses (bacteriophages) by taking part of an invader’s DNA sequence and inserting it into its own for future recognition and resistance. When the guide RNA transcribed from these repeats combines with the Cas9 nuclease enzyme, the CRISPR system can cut DNA sequences complimentary to the guide RNA.

While this method has been used to edit and regulate specific genes, department research is applying it to visualization of specific genomic loci by deactivating the DNA-cutting (endonuclease) aspect of the Cas9, making dCas9, and fusing it with fluorescent proteins.

Department scientists and their collaborators have demonstrated broad applications for an imaging-optimized CRISPR system as a platform for viewing specific genomic elements and their dynamics in living mammalian cells. Their work and findings include:

  • Establishing comparable target sequence flexibility and detection accuracy/efficiency to the fluorescence situ hybridization (FISH) technique, but with the advantage of being able to label features in living cells.
  • Generation of cloned human cell lines expressing fused proteins combining nuclease-deactivated Cas9 (dCas9) and enhanced green fluorescent protein (EGFP).
  • Visualization of human telomeres in different cell types: These stabilizing caps at the ends of eukaryotic chromosomes are composed to 5,000 to 15,000 base pairs with a repeated nucleotide sequence of TTAGGG (human), thus allowing the recruitment of numerous dCas9-EGFP proteins with a single sgRNA sequence.
  • Imaging of protein-coding genes in different cell types: Researchers targeted the MUC4 and MUC1 genes, which encode mucin glycoproteins that combine with others to form protective mucus gels and have been associated with cancers and other diseases. They demonstrated they could detect gene copy numbers in living cells by targeting a tandem repetitive region as well as a specific five kilo-base non-repetitive region of MUC4.

CRISPR imaging of telomeres and genes
Cell, Vol. 155, Issue 7, 19 Dec 2013

(Above) Demonstration of the CRISPR imaging of both telomeres and protein-encoding genes, MUC4 and MUC1, in human cell lines: (Left group) HeLa derived from cervical cancer cells. (Right group) RPE, retinal pigment epithelium cells. The sgGAL4 (small guide RNA at far left) has no cognate target in the human genome and was used a negative control. The E1, E2, and E3 labels reference specific gene regions being targeted. (Below) Schematic of the human MUC4 gene showing target sequences.

  • Using CRISPR imaging to monitor telomere length and dynamics in living cells. This work included detection of experimentally induced elongation. (Telomere shortening has been associated with cellular aging and with cancer-related genomic instability.) In addition, time-lapse microscopy tracked the movement of telomeres in living cells and confirmed that the labeling did not disrupt such dynamics.

tracking telomere movements in living cells
Cell, Vol. 155, Issue 7, 19 Dec 2013

Tracking telomere movements in live RPE (retinal pigment epithelium) cells via CRISPR imaging. (Scale bar = five micrometers, i.e., millionths of meters). Three panels (right) show trajectories of three telomeres’ movements over hundreds of frames. (Scale bars = 200 nanometers, i.e., billionths of meters).

  • Using time-lapse CRISPR imaging of the labels on specific genomic loci to track chromosome dynamics and reorganization during cell division.

Discovering new locations and timing for signalling by G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) are biologically and pharmacologically vital subjects of study. This family of transmembrane proteins bind with extracellular signaling molecules such as hormones or neurotransmitters, then transmit information into cells (signal transduction), via changes in their shape (conformation) that release the G-protein from a complex attached to a part of the GPCR inside the cell. The G-protein (specifically its Gs alpha subunit) then activates specific enzymes that, in turn, trigger biochemical sequences (pathways) that can greatly alter cell activities such as metabolism, division, or gene expression.

Such prime biological roles make GPCRs relevant to the treatment of many disorders, including cancers, diabetes, heart disease, asthma, and allergies. Indeed, one published analysis notes that 30 percent of all marketed drugs act on them; another that they are targeted by 40 percent of all prescription drugs.

GPCRs have traditionally been seen as generating their G-protein-coupled signaling strictly from their locations in cells’ outermost walls (plasma membranes) —with the signaling thought to end after intracellular proteins called arrestins bind to the GPCRs, inhibit them, and target them for internalization. In the latter process, known as endocytosis, the receptors are drawn into pockets formed in the cell membrane (clathrin-coated pits) that are pinched off into containers (vesicles) called endosomes for transport, breakdown, and/or recycling to the cell membrane.

However, in recent years a number of studies proposed that the GPCRs for certain ligands (e.g., thyroid-stimulating hormone, dopamine) continue their signaling activity from within endosomes. This could suggest additional or different effects and locations at which to target GPCRs with drugs.

That hypothesis had not been directly tested until department scientists in collaboration with UCSF, national, and international colleagues applied recently developed fluorescence microscopy probes to obtain photographic and video evidence that GPCRs are, indeed, signaling from endosomes after their internalization.

Specifically, the researchers examined beta-adrenergic receptors (β2-AR)—prototypical GPCRs that bind extracellular ligands such as adrenaline (epinephrine). The β2-AR-associated G-proteins released from complex inside cells activate specific enzymes to stimulate the cAMP-dependent pathway, which ultimately dilates blood vessels and respiratory bronchi, increases heart rate, increases the breakdown of energy stored as fat or glycogen—which can all be part of the body’s stress response.

Scientists here used nanobodies—the binding domains of antibodies from camels (which are single rather than double chain, thus especially small and flexible), genetically expressed by bacteria. The nanobodies were engineered to selectively bind to either the active conformations of the beta-adrenergic receptors or to an intermediate of the activated β2-AR-associated G-proteins. The nanobodies were, in turn, fused to enhanced green fluorescent protein.

schemes for detecting conformational activation
Nature, 28 Mar 2013

Engineered nanobodies fused to green fluorescent protein (Nb80-GFP and Nb37GFP) selectively bind to: A] active conformations of the receptor (β2-AR); or B] active conformations of the G-protein’s Gs subunit that is released to trigger signaling pathway.

Researchers used the adrenergic receptor activator (agonist) isoprenaline in combination with the fluorescent activity reporters to probe and track the activation of the β2 adrenergic receptors and their associated G-proteins in living mammalian cells.

The scientists found the receptors were, indeed, not only activated in the cells’ plasma membranes after the initial application of the agonist but also, in a second phase several minutes later—after arrestin inhibition and internalization. The GPCR is activated again in vesicles known as early endosomes, as detected via β2-AR-associated G-proteins active on those endosomes’ surfaces. Thus, those internalized receptors’ activity contribute to a temporally distinct and significant further activation of the cells’ cAMP pathway.

The findings not only provided direct support for GPCR signaling from endosomes, thereby revising a long-held tenet of molecular pharmacology, but also demonstrated a strategy for probing the location and timing of dynamic protein conformational changes in living cells.

model for two-phase GCPR activation
Nature, 28 Mar 2013

Internalized β2-ARs contribute to the acute cAMP response

A] Model for two phases of nb80-GFP recruitment by the temporally and spatially distinct GCPR (β2-AR) activations, first at the plasma membranes (yellow ball is receptor agonist) and then at endosomes

B] Model for two-phase β2-AR Gs activation, at plasma membrane and then at endosomes, separated by endocytic event