| Caged thiophosphate: Photoactivation |
Building blocks for synthesis of proteins and peptides that can be converted to stable phopsphorothioates |
Completed - Aemissegger et al., 2007 |
Imperiali |
| Photoactivation |
Fak/PTK2 - Caged phosphoTyr (Y*) - cPYFAK, probe sequence; VSETDDY*AEIIDEEDT-NH2 |
Completed - Humphrey et al 2005 |
Imperiali, Jacobson |
| Caged phospho-serine and cell cycle control |
Caged phosphoSerine (S*) to probe the role of phosphoSerine peptides in cell cycle control |
Completed - Nguyen et al.,2004 |
Imperiali |
| Photoactivation |
FlAsH-Cys4-cofilin - Demonstrate activity in vivo |
Completed - van Rheenen et al., 2007, CMC Update, Jan 2008 |
Condeelis |
| CALI |
Capping Protein -Simultaneous knock-down & rescue with EGFP labeled fusion protein |
Completed - Vitriol et al., 2007 |
Jacobson (Vitriol, Bear) |
| CALI |
EGFP-Cali of Mena |
Completed - Vitriol et al., 2007 |
Jacobson (Vitriol, Bear) |
| Photoactivation - caged phospho-amino acids |
Protein translation with a caged phosphoamino acid. General technology for unnatural amino acid mutagenesis with chemically acylated tRNAs developed |
Completed - Rothman et al., 2005 |
Imperiali (D. Dougherty) |
| Photoactivation |
Dually caged cofilin activity & fluorescent probe. Will permit tracing of uncaged cofilin in single cells |
In progress - Demonstrated activity of F1AsH loaded Cys4-cofilin in vitro; synthesized a black hole quencher that inhibits the activity of cofilin when derivatized to cys3 and quenches the fluorescence of F1AsH as determine spectrofluorimetrically |
Condeelis |
| Caged phospho-proteins |
Semi-synthetic myosin - Prepare MLC and mutants via semi-synthesis and evaluate in vitro |
In progress - MLC semi-synthesis completed, variants underway. Efforts to identify ideal caged variant still underway. |
Imperiali (Horwitz) |
| Photoactivation |
fluorescent caged cofilin to be used as an activatable biosensor |
In progress - Observe interactions between FlAsH-Cys4 cofilin and PIP2 or actin in vivo using FRET |
Condeelis |
| Caged phospho-proteins |
Semi-synthetic paxillin - Prepare Tyr31 mutants that mimic or inhibit phosphorylation and measure effects of uncaging in vitro |
In progress - paxillin probes prepared and currently being evaluated in cell-based systems at UVa |
Imperiali (Horwitz) |
| Photoactivation - Peptide probes |
Study of phosphorylation in AMPAR trafficking. C-terminal tail of GluR2 which interacts with PDZ domain of GRIP and PICK |
In progress - Peptide probes have been prepared and are being studied as agents for investigating receptor trafficking |
Imperiali (D. Choquet) |
| CALI |
Changed strategy to employ GST fused to fluorescent proteins to study mechanism of CALI with purified proteins. Employ a fluorescent protein-profilin fusion binding to VASP to study mechanism of CALI in cell lysates |
In progress - preliminary poster available. Progress in EGFP CALI. Publication in preparation |
Jacobson (McLean, Sligar) |
| Photoactivation |
fluor-cofilin caged in both its activity and fluorescence to be used as a caged biosensor |
Initiated |
Condeelis |
| Photoactivation |
VASP with caged phosphoserine at position 153 (via unnatural amino acid mutagenesis). Note: VASP knock out cell line available |
Terminated - Difficulty encountered with in vitro translation using chemically acylated tRNAs. Project not technically feasible at this time due to limited manpower |
Imperiali (F. Gertler) |
| CALI |
Use CALI methods to rapidly inactivate specific Rho family GTPases in cells subjected to knock-down/fluorescent rescue |
Under consideration |
Jacobson (Haugh) |
| CALI |
Itgb3 Beta3 integrin |
Under consideration |
Jacobson (B. Wherle-Haller) |
| CALI |
Talin |
Under consideration |
Jacobson (D. Critchley) |
| Photoactivation |
Cap G |
Under consideration; Feasibility studies underway |
Jacobson (G. Marriott) |
| Scanning Confocal & TIRF |
Determine molecular number, clustering & aggregate size |
Completed - Dalal et al., 2008, Digman et al., 2008, Scanning completed, TIRF in progress |
Gratton,Horwitz,Wiseman |
| Traction force mapping |
Substrate characterization and pattern printing technology for polyacrylamide substrates |
Completed - Damljanovic et al., 2005 - tutorial available |
Jacobson |
| Spatial-temporal directional ICM (STICS) |
Developed theory for spatial-temporal ICM. Simulations to verify theory. Maps of directed motion of alpha5 integrin and alpha-actinin, speed and direction. |
Completed - Hebert, et al, 2005; Brown, et al, 2006 |
Wiseman, Horwitz |
| Raster image correlation spectroscopy (RICS) |
Development and use of raster image correlation spectroscopy |
Completed - Digman et al., 2005, Digman, et al, 2005(b); Digman, et al, 2007 |
Gratton,Horwitz,Wiseman |
| Scanning-FCS |
Measure concentration and dynamics of molecules withing and adjacent to adhesions |
Completed - Digman et al., 2005 |
Gratton,(Horwitz) |
| High resolution velocity mapping |
Develop software for spatial temporal derivative analysis;and implement analysis on adhesions in migrating cells |
Completed - Digman et al., 2007 |
Gratton,Horwitz |
| TIRF time correlation analysis for binding dynamics |
Optimize parameters for data analysis ; Develop fitting models to differentiate between binding and slow diffusion; Prototype with paxillin to measure dynamics; |
Completed - Digman et al., 2007 |
Horwitz;Gratton |
| TIRF time correlation analysis for binding dynamics |
Eliminate bleaching & blinking contributions (k-space) |
Completed - Durisic et al., 2007, and Kolin et al., 2006 |
Wiseman |
| Photoactivation |
Caged phosphoTyr (Y*) - cPYFAK, probe sequence; VSETDDY*AEIIDEEDT-NH2 |
Completed - Humphrey et al., 2005 |
Imperiali, Jacobson |
| In Vivo Imaging |
Development of biological systems for in vivo imaging approaches |
Completed - Murase & Horwitz 2002, Murase & Horwitz 2004, Carbonell et al. 2005, Sakakibara & Horwitz 2006 |
Horwitz |
| Spatial ICM |
Extend ICM to live cell imaging. Measure cluster size and aggregation of integrins, and aggregation of alpha-actinin. Develop versitile software for calculations. |
Completed - Wiseman et al., 2004 |
Gratton,(Wiseman) |
| 2-color cross correlation analysis with RICCS and TIRF |
Develop software for the analysis and implement with paxillin-FAK |
Completed- RICS and submitted, TIRF in progress |
Gratton; Wiseman; Horwitz |
| Two photon ICCM |
Alpha5/alpha-actinin interactions in static, diffusive and directionally moving protein complexes. |
Completed - Wiseman et al., 2004 |
Horwitz;(P. Wiseman) |
| Two color high resolution velocity mapping |
Implement 2-color STICCS to measure flux of aggregated proteins |
In progress |
Wiseman, Horwitz |
| Traction force mapping |
Testing of small molecular compounds that inhibit Focal Adhesion Kinase (FAK) on Traction Force development |
In progress - preliminary poster available Progress in Traction Mapping |
Jacobson (Rajfur, Tilghman, Parsons) |
| Traction force mapping |
Data input to modeling initiative |
In progress - Trials of traction force mapping of keratocytes on polyacrylamide substrates combined with uncaging Thymosin Beta 4 underway. Preliminary poster available Progress in Traction Mapping |
Jacobson (Mogilner) |
| Photoactivation & ICS |
Combining Flash activation probe technologies with correlation spectroscopy techniques |
Proposed - Explore feasibility of applying cofilin probes, caged phosphor-paxillin probes and caged phosphor-MLC probes from aboce with ICS imamginng approaches |
Horwitz;(Gratton) |
| RICS in 3D |
Apply raster image correlation spectroscopy |
Proposed - Explore feasibility of applying one and two color RICS to collagen gels and cells in vivo |
Gratton; Horwitz |
