| CALI |
Capping Protein - (simultaneous knock-down and rescue with EGFP labeled fusion protein) |
Completed - Vitriol et al., 2007 (CMC Update Article) |
Jacobson (Vitriol, Bear) |
| CALI |
Changed strategy to employ GST fused to fluorescent proteins to study mechanism of CALI with purified proteins. |
Completed - McLean et al., 2009 (CMC Update Article) |
Jacobson (McLean & Sligar) |
| CALI |
Mena - (EGFP-Cali) |
Completed - Vitriol et al., 2007 (CMC Update Article) |
Jacobson (Vitriol, Bear) |
| Photoactivation |
Fak/PTK2 - (caged phosphoTyr (Y*) - cPYFAK, probe sequence; VSETDDY*AEIIDEEDT-NH2) |
Completed - Humphrey et al., 2005 |
Imperiali, Jacobson |
| Photoactivation |
FlAsH-Cys4-cofilin - (demonstrate activity in vivo) |
Completed - van Rheenen et al., 2007 (CMC Update Article) |
Condeelis |
| 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 (CMC Update Article) |
Imperiali (D. Dougherty) |
| Photoactivation - (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 - (caged thiophosphate) |
Building blocks for synthesis of proteins and peptides that can be converted to stable phosphorothioates |
Completed - Aemissegger et al., 2007 |
Imperiali |
| CALI |
Modeling the EGFP-capping protein CALI-induced protrusive phenotype and achieving understanding of why intensities used for FRAP of capping protein do not produce the same effect. |
Completed, publication in progress |
Jacobson (Kapustina & Loew) |
| Photoactivation |
VASP - (with caged phosphoserine at position 153, via unnatural amino acid mutagenesis. Note: VASP knock out cell line available) |
On hold - 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 |
In vitro CALI of TagRFP-T and mOrange2 (from Tsien lab)-GST fusions to test whether more photostable fluorescent proteins make better CALI reagents |
In progress |
Jacobson (McLean & Sligar) |
| CALI |
Test of whether CALI of YFP-Hem1 in neutrophil-like cells alters their direction of migration by disrupting Hem-1 “waves”. |
In progress |
Jacobson (Orion Weiner) |
| Photoactivation |
Dually caged cofilin activity and 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 |
| Photoactivation |
fluor-cofilin - (caged in both its activity and fluorescence to be used as a caged biosensor) |
In progress |
Condeelis |
| 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 |
| Photoactivation - (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 - (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) |
| Photoactivation |
Cap G |
Under consideration - Feasibility studies underway |
Jacobson (G. Marriott) |
| 2-color cross correlation analysis with RICCS and TIRF |
Develop software for the analysis and implement with paxillin-FAK |
Completed - RICS Digman et al., 2009, TIRF in progress (CMC Update Article) |
Gratton, Horwitz, Wiseman |
| Cross correlation number and brightness (ccN&B) |
Detection of molecular interaction between two species |
Completed - RICS Digman et al., 2009 (CMC Update Article) |
Gratton, Horwitz, Wiseman |
| High resolution velocity mapping |
Develop software for spatial temporal derivative analysis - (implement analysis on adhesions in migrating cells) |
Completed - Digman et al., 2008 (CMC Update Article) |
Gratton, Horwitz |
| In Vivo Imaging |
Development of biological systems for in vivo imaging approaches |
Completed - Murase and Horwitz, 2002; Murase and Horwitz, 2004; Carbonell et al., 2005; Sakakibara and Horwitz, 2006 (CMC Update Article) |
Horwitz |
| Photoactivation |
Caged phosphoTyr (Y*) - cPYFAK, probe sequence - (VSETDDY*AEIIDEEDT-NH2) |
Completed - Humphrey et al., 2005 |
Imperiali, Jacobson |
| Raster image correlation spectroscopy (RICS) |
Development and use of raster image correlation spectroscopy |
Completed - Digman et al., 2005 (CMC Update Article); Digman et al., 2005 (CMC Update Article); Brown et al., 2008 (CMC Update Article); Digman and Gratton, 2009 |
Gratton, Horwitz, Wiseman |
| Scanning Confocal & TIRF |
Determine molecular number, clustering & aggregate size |
Completed - Dalal et al., 2008 (CMC Update Article); Digman et al., 2008, Scanning completed, TIRF in progress (CMC Update Article) |
Gratton, Horwitz, Wiseman |
| Scanning-FCS |
Measure concentration and dynamics of molecules within and adjacent to adhesions |
Completed - Digman et al., 2005 (CMC Update Article) |
Gratton (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) |
| 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 (CMC Update Article); Brown et al., 2006 (CMC Update Article) |
Horwitz, Wiseman |
| TIRF - (time correlation analysis for binding dynamics) |
Eliminate bleaching & blinking contributions (k-space) |
Completed - Kolin et al., 2006; Durisic et al., 2007 |
Wiseman |
| 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., 2008 |
Gratton, Horwitz |
| TIRF and two color imaging |
Quantitative data generation for modeling |
Completed - Choi et al., 2008 (CMC Update Article) |
Horwitz (Mogilner) |
| Traction force mapping |
Substrate characterization and pattern printing technology for polyacrylamide substrates |
Completed - Damljanović et al., 2005, tutorial available |
Jacobson |
| Two photon ICCM |
Alpha5/alpha-actinin interactions in static, diffusive and directionally moving protein complexes |
Completed - Wiseman et al., 2004 |
Horwitz (P. Wiseman) |
| Cross correlation analysis of adhesion components - (apply N&B and ccRICS to nascent adhesions using: integrin-talin, integrin-a-actinin, and FAK-Paxillin) |
|
In progress |
Horwitz |
| Field effect transistor (FET) local adhesion strength sensing |
Modeling EGF signaling network using CMAP. Developing models binding and enzymatic reactions within CMAP approach. Use them as building blocks of a signaling network. |
In progress |
Jacobson (V. Misra) |
| 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) |
| 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) |
| Two color high resolution velocity mapping |
Implement 2-color STICCS to measure flux of aggregated proteins |
In progress |
Horwitz, Wiseman |
| Photoactivation & ICS |
Combining Flash activation probe technologies with correlation spectroscopy techniques |
Under consideration - 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 |
Under consideration - Explore feasibility of applying one and two color RICS to collagen gels and cells in vivo |
Gratton, Horwitz |
| Visualizing adhesions in 3 dimensions - (Develop methods for visualizing GFP derivatives in migrating cells in collagen gels.) |
|
Under consideration - Paxillin and vinculin have been seen in 3-D collagen gels as nascent and elongated adhesions. |
Horwitz |
