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Open Forum on Cell Signaling

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Highlights from the Session: New Strategies for Imaging Protein Localization and Dynamics

1 July 2008

Nancy R. Gough

This session took place Monday, 7 April, and the speakers described various techniques for detecting the location of molecules within cells, the movement of molecules within cells or at the membrane, the activity of proteins, or the posttranslational modification of proteins. There were six speakers: Jin Zhang (Johns Hopkins University), Brent Martin of the Cravatt lab (Scripps Research Institute), Jay Groves (University of California, Berkeley), May C. Morris (CNRS-CRBM, Research Center of Macromolecular Biochemistry); Sophia Breusegem of the Doctor lab (University of Colorado Health Sciences Center), and Ronald Raines (University of Wisconsin, Madison)

Dynamic Visualization of Signaling Activities in Living Cells- Jin Zhang

Dr. Zhang is interested in how specificity is achieved in a signaling network. Spatiotemporal regulation is a key to creating signaling specificity. Protein kinase A (PKA) is as an example of a spatially-restricted signaling molecule whose location is controlled by the anchoring proteins known as AKAPs. Traditional biochemical methods or immunocytochemistry do not allow spatiotemporal information to be gathered. However, biosensors (see Gaits and Hahn) were developed to allow monitoring of signals in live cells. For example, a reporter comprised of a phosphobinding domain + substrate peptide that separate two fluorophores will in the presence of a kinase and ATP lead to changes in FRET (fluorescence resonance energy transfer). AKAR1, 2, and 3 are biosensors that report on PKA activity.

Zhang and colleagues use two reporters (one for PKA called AKAR and one for cAMP based on the cAMP-regulated GTPase Epac that is called Epac-ICUE) to monitor both Epac and PKA signaling in pancreatic beta cells. One caveat to using these sensors is that they may alter the cellular signaling properties if they compete for cAMP or PKA.

Beta cells exhibit an oscillatory cAMP signal (see Borodinsky and Spitzer). Oscillatory changes were recorded with Epac-ICUE and AKAR reported that PKA activity also oscillated. If H89 was applied to inhibit PKA, then calcium oscillations ceased. ICUEPID is a three-fluorophore reporter that reports on PKA and has also an Epac domain. Experiments with ICUEPID indicated that these two regions of the reporter were not activated with the same oscillatory pattern, but that one precedes the other.

The AKAR sensors have been used in several other systems. When the AKAR sensor was used to monitor PKA activity in fibroblasts that were migrating into a wounded region of a monolayer, membrane-localized AKAR revealed that PKA was activated at the leading edge of the cell. The AKAR system has been adapted for high-throughput screening assays for drugs that alter PKA activity. There is work underway to engineer reporters for phosphatase activity and other types of posttranslational modifications, as well as new types of "tunable" reporters that use three fluorophores with transfer from the first to the second to the third.

Proteomic Profiling of Dynamic Palmitoylation- B. R. Martin

Palmitoylation occurs on cysteines, and is a dynamic and reversible modification that anchors proteins to membranes. One of the first approaches to do a global analysis of palmitoylation used acyl-biotinyl exchange chemistry [Wan et al., Nature Protocols 2, 1573 (2007)] and identified ~50 proteins in yeast. Palmitoyl acyl transferases (PATs) use C:16 or C:18 fatty acids, whereas myristoylation enzymes use shorter-chain fatty acids (see Resh). 17-ODYA, which is an alkyne that is sold by Sigma as a cytochrome p450 inhibitor, can be used to specifically label palmitoylated proteins because of its size and the presence of the reactive alkyne. When this chemical is added to cells, it becomes incorporated into acyl CoA, and then becomes covalently attached to proteins. If "click chemistry" is then used to attach rhodamine or biotin (which would be used for streptavidin purification) to the 17-ODYA-tagged proteins, then these tagged proteins can be visualized on a gel (rhodamine) or analyzed by mass spectrometry (biotin). This approach, called MudPIT, was used to identify the palmitoylated profile in Jurkat T cells and with this method, the palmitoylated proteins that are known to be part of the T cell receptor signaling complex were identified.

Imaging the Mechanics of Signal Transduction in Membranes- J. T. Groves

Dr. Groves is interested in how lipids and proteins on the surfaces of cells, especially at points of cell-cell contact or cell-substrate contact, contribute to the organization of molecular signaling structures as well as the cell's response to the contact (see Groves). The T cell immune synapse, which is the organized complex of proteins and lipids at the site of contact between a T cell and an antigen-presenting cell, is spatially organized in such a way that the center of the synapse is closely apposed membranes (15 nm), and then the membranes around it are farther apart (42 nm). The overall size is large (close to 5 micrometers).

Dr. Groves is interested in how the position of the T cell receptor (TCR) on the surface influences its signaling. He created a hybrid live-cell and supported-membrane synapse, using artificial membranes on spatially-restricted supports, and then added a cell to interact with this (see Mossman et al.). At early time points following cell contact, the TCRs start to cluster and their movement is unimpeded, but at later times the barriers on the membrane supports influence the organization of the immune synapse.

Various types of imaging can be used in conjunction with these hybrid systems: total internal reflection fluorescence (TIRF) microscopy, epifluorescence microscopy, and fluorescence cross-correlation spectroscopy (FCCS). In real time, TIRF reveals that the immune synapse forms from multiple small TCR signaling complexes and they move around the imposed boundaries readily. TIRF allows single-cluster tracking and then movement of individual clusters can be combined into an ensemble tracking record. The movement of TCR clusters toward the immune synapse is oscillatory, and once they form an immune synapse, the TCRs all show collective oscillations with a period matches that of calcium oscillations.

The actin cytoskeleton appears to flow past the TCR clusters that encounter the barriers, which suggests a frictional coupling mechanism. The adhesion molecules (ICAM and LFA) that ultimately make up the ring around the TCRs at the immune synapse are also driven toward the immune synapse. The migrating TCR clusters and adhesion molecule clusters are mutually exclusive (no TCRs migrate with ICAM and LFA) even when they are moving toward the synapse. Clustering of LFA appears to control where it sorts (Fab-labeled LFA does not form a tight donut around the TCRs at the immune synapse, mAb-labeled LFA forms a tight ring around TCRs at the immune synapse, and crosslinked mAb-labeled LFA goes to the center of the immune synapse with the TCRs.) Actin flow is the only necessary driving force and clustered molecules are "dragged" along more effectively.

Sensors of Mitosis- May C. Morris

Her group is developing sensors of mitotic kinases and phosphatases. CDK1-cyclinB1 is regulated by phosphorylation (phosphorylated forms are inactive, dephosphorylated forms are active). The players in this system also undergo dynamic subcellular localization. Some of the nongenetically-encoded (those not based on proteins) fluorescent sensors include peptide-based or nucleotide-based sensors, as well as sensors that are targeted to a particular protein interaction domain. The structural and sequence information of cyclin B, Cdc25, and CDK1 were used to develop sensors for the CDK1-cyclin B complex, activated CDK2, activated CD25 phosphatases. These sensors were introduced into cells using cell-penetrating peptides.

Microvillar Protein Trafficking and Dynamics Imaged by TIRF Microscopy in Living Cells- Sophia Y. Breusegem

The Doctor lab is interested in phosphate homeostasis in the kidney and in the trafficking of phosphate tansporters. Transporter abundance at the cell surface is regulated by luminal phosphate and hormones, and is controlled by endocytosis and exocytosis. They have developed a way to use TIRF to detect apical proteins and image the microvilli and the clefts between the microvilli. With this system, they determined that if the actin cytoskeleton is poisoned with jasplakinolide, then the two transporters are differentially affected: one has its internalization inhibited and the other has its internalization promoted in response to PTH (parathyroid hormone). They have also monitored the movement of PDZ-containing proteins, some of which interact with the transporters. Shank2E (a PDZ-containing protein) appears to move up and down the microvilli.

Latent Fluorophores for Biomolecular Imaging- R. T. Raines

Ribonuclease A (RNase A) is a secreted protein and is now being developed as a drug for blocking the flow of biochemical information from DNA to protein. For example, Onconase is an RNase found in the Northern leopard frog that is in phase III clinical trials. Onconase is selectively toxic to tumor cells, whereas mammalian RNase A is not toxic. One reason for the lack of toxicity is that mammalian cells have a horseshoe-shaped ribonuclease inhibitor protein. The interaction between RNase A and ribonuclease inhibitor occurs with femtomolar affinity (which is orders of magnitude higher than antigen-antibody interactions). Onconase does not interact with ribonuclease inhibitor at all. So, if one could engineer an RNase A that can evade ribonuclease inhibitor, this protein may kill tumor cells. Onconase treatment of nude mice with an implanted tumor resulted in tumor regression, but also caused massive weight loss, which was related to renal toxicity.

With that introduction to RNase A. Dr. Raines switched gears to discuss how he is developing probes to try to understand the mechanism of toxicity. Onconase and engineered RNase A are selectively toxic to cancer cells and are not toxic to normal cells. An enormous amount of RNase A binds to the surface of cells in culture, thus Raines' group developed probes that are not fluorescent until the protein is taken into the cell by endocytosis. They took a page from the "trimethyl-lock" mechanism of drug delivery to engineer these latent fluorophores that are unlocked by exposure to the intracellular esterases and thus are only visible after they get inside cells. These were then used to show that charge-neutralized RNase A gets into the cells poorly, whereas the normal +6 charged RNase A is readily endocytosed. This may even be a mechanism by which tumor-cell selectivity is achieved, because tumor cells are more anionic than are nontumor cells.

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