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Imaging & Photomanipulation - Activities

Objectives

  • to develop photoactivation methodologies for migration molecules
  • to develop novel strategies for probing phosphorylation events including caged phosphopeptides and phosphoproteins
  • to develop and characterize new CALI reagents
  • to extend development of traction force assays to be applicable to a wider range of cell types and combine these traction force maps with photomanipulative techniques to develop quantitative data for the modeling initiative
  • to develop correlation and related methodologies to produce spatial maps of molecular concentration, interactions, and dynamics
  • to extend photomanipulation and imaging technologies to in vivo systems
  • to provide imaging expertise and facilities to the Consortium and the migration community and assist in the development of new probes or technologies for researchers who are not in the Consortium
  • to develop patterned substrates for cell imaging

Imaging Probe Developments | Imaging Technology Development

Imaging Probe Developments

Technology Description Status / Publication Contact (Collaborators)
CALI CALI improvements Completed - McLean et al., 2009 (CMC Update Article) Jacobson (McLean & Sligar)
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)
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 - Kapustina et al., 2010 (CMC Update Article) Jacobson (Kapustina & Loew)
CALI Test of whether CALI of YFP-Hem1 in neutrophil-like cells alters their direction of migration by disrupting Hem-1 “waves”. Completed Jacobson (Orion Weiner)
Photoactivation Dually caged cofilin activity and fluorescent probe - (will permit tracing of uncaged cofilin in single cells) Completed - 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 Fak/PTK2 - (caged phosphoTyr (Y*) - cPYFAK, probe sequence; VSETDDY*AEIIDEEDT-NH2) Completed - Humphrey et al., 2005 (CMC Update Articles: 1, 2) Imperiali, Jacobson
Photoactivation FlAsH-Cys4-cofilin - (demonstrate activity in vivo) Completed - van Rheenen et al., 2007 (CMC Update Article) Condeelis
Photoactivation fluor-cofilin - (caged in both its activity and fluorescence to be used as a caged biosensor) Completed - van Rheenen et al., 2009 (CMC Update Article) Condeelis
Photoactivation fluorescent caged cofilin - (to be used as an activatable biosensor) Completed - Observe interactions between FlAsH-Cys4 cofilin and PIP2 or actin in vivo using FRET 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-proteins) Semi-synthetic myosin - (prepare MLC and mutants via semi-synthesis and evaluate) Completed - Goguen et al., 2011 (CMC Update Article) Imperiali
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
Photoactivation - (peptide probes) Study of phosphorylation in AMPAR trafficking - (C-terminal tail of GluR2 which interacts with PDZ domain of GRIP and PICK) Completed - Peptide probes have been prepared and are being studied as agents for investigating receptor trafficking Sainlos et al., 2013 (CMC Update Article) Imperiali (D. Choquet)
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Imaging Technology Developments

Technology Description Status / Publication Contact (Collaborators)
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 analysis of adhesion components Completed - Choi et al., 2011 (CMC Update Article) Horwitz
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
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. Completed Jacobson (V. Misra)
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 (CMC Update Articles: 1, 2) 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 (CMC Update Articles: 1, 2) 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 Articles: 1, 2); 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 Data input to modeling initiative Completed - 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 Completed - Poster available: Progress in Traction Mapping Jacobson (Rajfur, Tilghman, Parsons)
Traction force mapping Substrate characterization and pattern printing technology for polyacrylamide substrates Completed - Damljanović et al., 2005, tutorial available Jacobson
Two color high resolution velocity mapping Implement 2-color STICCS to measure flux of aggregated proteins Completed - Toplak et al., 2012 (CMC Update Article) Horwitz, Wiseman
Two photon ICCM Alpha5/alpha-actinin interactions in static, diffusive and directionally moving protein complexes Completed - Wiseman et al., 2004 (CMC Update Articles: 1, 2) Horwitz (P. Wiseman)
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