Gerard Marriott

Professor
Ph.D., 1987, University of Illinois

Contact Information
Email: gm@physiology.wisc.edu
(608) 262-6309 Phone
(608) 265-5512 Fax

Lab members: as of March. 2008
 
Postdocs: Shu Mao, Richard Perrins, Chutima Petchprayoon, Hui Wang
 
Technician: Jing Ran

Research Interests
 

Research Objective: To understand the molecular basis of complex biomolecular processes through the development and application of new approaches in biophotonics and chemical biology.

Optical probes to study the molecular basis of cell motility (PI: NIH R01 12/06~11/10): This recently funded project aims to understand motility in terms of the regulation of protein interactions at the leading edge of a motile cell. The multi-disciplinary approach integrates novel probes including optical switches and multiplexed imaging technologies, including photochromic FRET, to rapidly map, measure and manipulate distributions and activities of specific cytoskeleton proteins and their complexes with high spatial resolution and apply mathematical modeling to correlate these measurements to cell function. In particular, we focus on understanding how localized and dynamic changes in the concentration of specific ligands affect the regulation of local interactions between actin and actin binding proteins and how these events are coupled to the global response of motility.

Mechanism of action and regulation of force generating molecular machines: New quantitative microscope imaging techniques, including time resolved delayed fluorescence (Marriott et al, 1991;1994), FRET (Yan & Marriott, 2003a,b), fluorescence polarization (Yan & Marriott, 2003b) and optical probes (Sakata et al, 2005a,b) are being used to quantify structural dynamics of tropomyosin and troponin I and to generate 3-D maps of the calcium and myosin triggered conformational transitions that underlie the regulation of thin filaments. These studies are being performed on normal and mutated forms of cTm implicated in hypertrophic cardiomyopathies. These studies also include the development and application of optical methods to directly and/or indirectly rapidly control the activity of molecular motor protein in nanoscale devices, within cardiac muscle fibers and within artificial cell systems.

In vivo labeling technologies: (Co-PI. HFSPO. 12/05~11/08)
Research activities focus on:

• Application of a genetic encoded labeling technology (AGT transferase) to link diverse chemical and optical probes to specific fusion proteins in cells. This technology is being used to image changes of cytoskeleton protein interactions during cell protrusion.
• Developing new probes for in vivo labeling of genetically encodable proteins and MS analysis as part of a functional proteomic analysis of cell motility.
• New genetically encoded proteins for functional proteomics, FRET and intravital imaging

Control of protein conformation at the level of single molecules: (Co-PI; DARPA. 12/06~03/08) (Continued annual renewals based on meeting milestones) The objective of this project is to control single protein molecule activity through real-time, reversible manipulation and measurement of protein conformations. Biophotonics and protein engineering techniques are used for single molecule based fluorescence sensing of anthrax antigen-mediated binding of a SCA gainst anthrax lethal toxin. Real-time control of these conformational transitions will be achieved using optical switches on the SCA. These studies pave the way for nanoscale devices that will exploit the remarkable specificity and diversity of protein interactions, activity and function.
 
A second funded project aims to control ion channel activity in the TRPV1 channel protein. Site directed mutagenesis is being used to introduce a pair of cysteine residues that will be crosslinked with a bi-maleimido optical switch. Optical manipulation of the switch between the SP and MC states will be used to control the flow of calcium ions through the channel poor. The engineered channel will also include a genetically-encoded optical readout for calcium ions on its C-terminus.

Molecular Heliographs: Encryption and decoding of protein identity: PI. (Luke Lee, co-PI). R01 (4.1.08~3.31.13) Award pending.

Abstract: The objective of the proposed research is to advance single molecule-based, multiplexed imaging and analysis of the distributions and interactions of proteins through the development of new genetically-encoded fluorescent protein complexes and nanoparticle-based optical probes. The past decade has witnessed staggering advances in the development and application of optical microscopy for functional proteomics; in particular it is now possible to image the distributions and interactions of single genetically encoded fluorescent proteins with nanometer resolution within a living cell. On the other hand, multiplexed imaging and analysis of protein function is limited to a handful of labeled proteins, rather than the tens to hundreds that regulate specific signaling pathways and cellular processes. This limitation will be overcome in this proposal through the design and development of new classes of synthetic, genetically encoded and nanoparticle probes that can be used to encode protein identity. Parallel developments in an associated optical decoding imaging technology should lead to an unparalleled improvement in multiplexed imaging of proteins within living cells. The project is guided by two specific aims:

Specific Aim 1: To develop hybrid genetically-encoded fluorescent proteins harboring synthetic- and protein-based optical switches for multiplexed imaging and identification of proteins involved in motility.

Specific Aim 2: To develop nanoparticle probes harboring synthetic optical switches and specific capture groups for surface enhanced Raman scattering-based, multiplexed imaging of cell surface receptors implicated in motility.
 
The proposed research will lead to new and powerful approaches for multiplexed imaging and analysis of membrane receptors and cytoskeleton proteins within living cells that should improve our understanding of the mechanisms underlying complex cell processes such as receptor-mediated signaling and cell motility.

New optical probes to study cell motility: (NIH R01 12/06~11/10)

Optical Lock-in Detection (OLID) Imaging microscopy using synthetic and genetically-encoded optical switches:
We have developed a new approach to rapidly and reversibly modulate protein fluorescence and protein interactions using a family of optical switches based on nitroBIPS, spironaphthoxazines (Fig 1A; Sakata et al, 2005) and the genetically-encoded protein, Dronpa. NitroBIPS undergoes high-fidelity, rapid and reversible, optically driven transitions between a non-fluorescent spiro (SP)-state and a fluorescent merocyanine (MC) state (Fig. 1A; Sakata et al, PNAS, 2005). MC-fluorescence provides a readout of the state of the switch. Controlling transitions between each state of the switch with a defined perturbation waveform of UV- and visible light allows us to modulate MC-fluorescence with the same waveform. This “AC” component of the signal is isolated from “DC” background sources by lock-in detection and fitting the fluorescence intensity within each pixel to the reference perturbation waveform over many cycles of optical switching (Fig. 2A,B). Images of the “AC” component of the signal or correlation coefficient, have a much higher signal to background ratio compared to intensity images, as can be seen in images of an optical switch stained xenopus spinal cord explant (Fig. 2C,D).

A.
B. C.

Fig. 1: Top. Optically-driven, reversible transitions in nitroBIPS-like optical switches. (A), Left, Montage of MC-fluorescence in cells of a xenopus spinal cord explants and (Right), profile of the modulated MC-fluorescence intensity within a ROI for the cell shown in the montage.(B), MC-intensity image of C12-nitroNIPS (Top), a membrane specific optical switch in a xenopus spinal cord explants and (C), corresponding image of the “AC” component or correlation coefficient of the optical switch probe.

Optical switches for metal ions and cell calcium:
Part of our approach to studying the role of Ca2+ in the regulation of cell protrusion involves the design, synthesis and in vivo characterization of a class of Ca2+ optical switches that bind tightly to Ca2+ in the MC but not the SP state (Fig. 2 Left). Rapid and reversible manipulation of Ca2+ in cells is being realized by 1- and 2-photon mediated transitions between the SP and MC states.

Combinatorial optical switches for applications in biology and biomaterials: Large numbers of functionalized optical switches are being synthesized by using combinatorial chemistry. High efficiency coupling of members of a family of indolines with members of a family of salicylaldehydes affords diverse BIPS-like optical switches. For example, a selection of the 40 possible divalent metal ion optical switches in our library is shown in figure 3. By varying the geometry and distance between the chelating groups it has been possible to control divalent metal ion affinity between the SP and MC states (Sakata et al, 2008) in vitro and in vivo. We are also working on bifunctional switches to crosslink polymers in order to control microfluidic devices as well as related functionalized probes that may be used to optically and reversibly control pH (swelling) and dielecltric properties of polymers and surfaces. These probes are also being attached to nanoparticles for applications in sensing and optical encryption of nanoparticle identity with Luke Lee at UC-Berkeley.

Fig. 3: Generation of diverse optical switches using combinatorial chemistry: For example, switches that differ in their affinity for metal ions in the SP and MC states were generated by reacting indolines (top row) with salicylaldehydes (left column). Other optical switches with different functionalities eg groups that differ in their pKa, dielectric properties are being made using a similar approach

Our current library of indolines and salicyaldehydes/nitronaphthols will yield a library of ~900 distinct optical switches. We are also coordinating our efforts to expand our library of indolines, nitrosonapthols or salicylaldehydes to generate 10,000 unique switches.

Why make libraries of optical switches? First, members in our library will be used to identify interesting chelates for calcium, zinc and other metal ions; Second, we propose to screen our switches to identify compounds exhibiting a diverse spectrum of spectroscopic, photochemical and functional activities that will find applications for example as probes for PALM and frequency encoded photochromic FRET and optically switchable drugs. Third, the ability to incorporate diverse functional and reactive groups into our photochromes is expected to yield compounds with applications in biomaterials and bioengineering e.g. optically controlled microfluidic valves, and optically responsive surfaces for biorecognition.

In vivo labeling using genetically encoded cell chemistry (HFSPO: 11/04~10/08)
Current research in this area is focused on meeting the following objectives:

• Application of a new genetically encoded labeling technology (AGT transferase) to link diverse chemical and optical probes to specific fusion proteins in cells. This technology is being used to image changes of cytoskeleton protein interactions during cell protrusion.
• Developing new probes for in vivo labeling of genetically encodable proteins and MS analysis as part of a functional proteomic analysis of cell motility.
• New genetically encoded proteins for functional proteomics, FRET and intravital imaging.

Selenocysteine, the 21st amino acid: Cell chemistry and functional proteomics
Selenocysteine, a natural amino acid is found in a small subset of mitochondrial proteins. Selenocysteine is introduced into proteins via an unusual mechanism that involves a selenocysteine (Sel) insert in the mRNA that recognizes specific proteins and Sel-tRNA. We are developing a genetically encodable approach to introduce Sel at any site within cytoskeleton proteins. This will be realized by expressing Sel-encoded genes and exploiting the highly reactive nature of Sel with chloro and fluoro-containing probes. We expect to introduce a diverse family of optical probes and biosensors into encoded proteins using in vivo Sel chemistry that include fluorophores, crosslinking and positron-emitting molecules. This approach should represent a significant advance over other in vivo labeling technologies not the least because the Sel tag is a single amino acid.

Light directed activation of "caged" proteins
We introduced the technique of light directed activation of caged proteins including molecular motors (myosin) and components of the force-producing actin cytoskeleton to perturb the activity and interactions of biomolecules in cells – this approach is routinely to study the function of specific proteins during motility with mm and msec resolution (Marriott, 1994; Marriott & Heidecker, 1996).

Single molecule thermo-optical control of protein conformation and activity: DARPA (12/06~03/08: Annual renewal based on meeting milestones)
We are developing an approach for real-time, reversible control and measurement of the interaction between single molecules of the anthrax lethal antigen and an engineered single chain antibody. New optical probes and protein engineering is used to sense ligand-mediated changes in antibody conformation via FRET and environmental sensing. Real-time control of these conformational transitions is designed to modulate the interaction by a factor of >10 between the SP and MC states..

Nanoscale molecular sorting devices (R21: resubmission 03.08)
Principles from synthetic biology are being used to fabricate a free-running, nanoscale, molecular device to identify and sort toxins, viruses and bacteria from a mixture. The device is constructed on a microfabricated surface harboring nanoscale tracks of actomyosin that pass through a sample reservoir containing a mixture of antigens including viruses and toxin proteins. Actin filaments directly or indirectly labeled with multiple capture groups are used to transport the antigens to specific unloading sites on the track that contain immobilized antibody against the antigen. Unloading (sorting) of the antigen from the actin filament at unloading sites is achieved by using an antibody having a higher affinity for the antigen and long complex lifetime. Upto 10 unloading sites will be fabricated onto the closed circle actomyosin track. The unloading event will be monitored by using fluorescence detection or SPR imaging. The sorting device is considered free running, since it is driven by ATP hydrolysis. We envision using optical switches conjugates of myosin at specific sites on the track to control the movement of the actin filament (e.g. for unloading).

Mechanism of force production in motor proteins: (NIH R01 12/03~11~07):
New quantitative microscope imaging techniques, including time resolved delayed fluorescence (Marriott et al, 1991;1994), FRET (Yan & Marriott, 2003a,b), fluorescence polarization (Fig. 4; Yan & Marriott, 2003b) and optical probes (Sakata et al, 2005a,b) are being used to quantify structural dynamics of tropomyosin and troponin I and to generate 3-D maps of the calcium and myosin triggered conformational transitions that underlie the regulation of thin filaments. These studies are being performed on normal and mutated forms of cTm implicated in hypertrophic cardiomyopathies. These studies are also proving useful in developing new approaches for the optical control of molecular motors in nanoscale devices (above) and within artificial cells (above)

Fig. 4: Mapping functional protein motions within single actin filaments: A), Model of the actomyosin motility system depicting labeled actin molecules in a filament; B), FRET analysis of proximity between equivalent sites on actin sliding on myosin; C), FPIM analysis of protein motions in single actin filaments: Calculated polarization image showing highly ordered TMR-Phalloidin on a single filament.

Design of optical probes to study structure and regulation of the actin cytoskeleton (NIH R01 12/06~11/10)
We have elucidated the mechanistic basis for actin targeted macrolide drugs that include kabiramide C (KabC). These studies include high resolution structures of the G-actin-KabC complex and detailed biochemical and chemical biological analyses (Fig. 5A,B; Klenchin et al, 2003; Tanaka et al, 2003; Perrins et al, 2008) of KabC that serve as sensitive and specific probes of events at the (+)-end of the actin filament (Fig. 5C,D; Petchprayoon et al, 2004). These probes are used in an integrated approach to understand how local changes in molecular interactions are coupled to the global response of motility.

Fig. 5: Left, X-ray structure of KabC-Gactin at 1.45 A.; middle, structures of fluorescent derivatives of KabC; Right, Confocal imaging in live HeLa cells show the probe is labeled at sites of actin polymerization.

Selected Publications:

• Hui Wang, Shu Mao, Joseph M Chalovich, and Gerard Marriott(2008) Tropomyosin dynamics in cardiac thin filaments: A multi-site Foerster resonance energy transfer and anisotropy study. Biophys. J. BioFAST: February 29, 2008. doi:10.1529/biophysj.107.121129
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• Richard D. Perrins, Giuseppe Cecere, Ian Paterson and Gerard Marriott (2008) Synthetic Mimetics of Actin-binding Macrolides: Rational Design of Actin-targeted Drugs. Chemistry and Biology. 15, 287–294
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• Shu Mao, Richard KP Benninger, Yuling Yan, Chutima Petchprayoon, David K Jackson, Christopher J Easley, David W Piston, and Gerard Marriott(2008) Optical lock-in detection of fluorescence resonance energy transfer using synthetic and genetically-encoded optical switches. Biophys. J. BioFAST: February 15, 2008. doi:10.1529/biophysj.107.124859
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• Tomoyo Sakata, David K. Jackson, Shu Mao, and Gerard Marriott (2008) Optically Switchable Chelates: Optical Control and Sensing of Metal Ions. J. Org. Chem. 73 (1), 227 -233, 2008
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• Petchprayoon C., Asato Y., Higa T., Garcia-Fernandez L.F., Pedpradab S., Marriott G., Suwanborirux K. &Tanaka J. (2006) Four new kabiramides from the Thai sponge. Pachastrissa nux. Heterocycles 69, 447.
   
• Welham, NV, Marriott*, G, Bless, DM (2006). Proteomic profiling of rat thyroarytenoid muscle. JSLHR 49, 671-685. (*corresponding author).
   
• Chutima Petchprayoon, Khanit Suwanborirux, Junichi Tanaka, Yuling Yan, Tomoyo Sakata, and Gerard Marriott (2005) Fluorescent Kabiramides: New Probes to Quantify Actin in Vitro and in Vivo. Bioconjugate Chem; ASAP Web Release Date: 13-Sep-2005; (Article) DOI: 10.1021/bc050006j
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• Klenchin, VA, King, R., Tanaka, J. Marriott, G and Rayment, I. (2005) Structural Basis of Swinholide A Binding to Actin. Chemistry and Biology 12, 287-291
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• Sakata, T., Yan, Y. & Marriott, G. Optical switching of dipolar interactions on proteins. (2005). PNAS 102, 4759-2764
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• Sakata, T. Yan Y. & Marriott, G. A family of site selective optical switches. (2005). J. Organic Chem 70., 2009 - 2013
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• Lee, H.J., Yan, Y., Marriott*, G & Corn, R. (2005). Quantitative functional analysis of protein interactions on surfaces. (*corresponding author). J. Physiology (Lond.). 2005 563: 61-71.
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• Petchprayoon, C., Khanit, S., Sakata, T. and Marriott, G. (2005). Synthesis and biochemical and cellular characterization of a kabiramide C harboring a pendent amino group. J. Natural Product Chemistry. 68. 157 - 161
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• Tanaka, J., Yan, Y., Choi, J, Bai, J., Klenchin, VA., Rayment, I and Marriott, G. Biomolecular mimicry in the actin cytoskeleton: Mechanisms underlying the cytotoxity of kabiramide C and related macrolides. PNAS (USA). 100, 13851-13856. (2003)
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• Klenchin, VA., Allingham, J., King, R. Tanaka, J., Marriott, G. and Rayment, I. High resolution crystal structure of the kabiramide-actin complex. Nature Structural Biology. In press. (2003).
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• Yan, Y and Marriott, G. Analysis of Protein Interactions using Fluorescence Technologies. Curr. Opin. Chem. Biol. 7, 1-6. (2003).
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• Schneider, N., Weber, I, Faix, J, Prassler, J., Muller-Taubenberger, A., Kohler, J., Gerisch G. and Marriott, G. A Lim Protein Involved in the Progression of Cytokinesis and Regulation of the Mitotic Spindle. Cell Motility and the Cytoskeleton 56. 130-139. (2003).
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• J. Wegner, H.J. Lee, G. Marriott, and R.M.Corn. Fabrication of Histidine-Tagged Fusion Protein Arrays for Surface Plasmon Resonance Imaging Studies of Protein-Protein and Protein-DNA Interactions. Anal. Chem. 75(18); 4740-4746 (2003).
   
• Yan, Y. & Marriott, G. FRET Image Microscopy and Fluorescence Polarization Image Microscopy Based Measurements of Molecular Proximity and Molecular Orientation for Fluorescent Probes on Single Actin filaments sliding on Myosin. Methods in Enzymology (in press) (2002).
   
• Faix, H., Lottspeich, F., Mintert, U. and Marriott, G. Cortexillin I is a key target for Rac1A in signaling pathways that lead to cytokinesis and cell motility EMBO J. 20, 3705-3715 (2001).
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• J. Tanaka, Marriott, G, Higa, T and Higa, T. Cacofurans A and B. New Furanoditerpenes from a marine sponge. J. Natural Products 64, 1468-1470 (2001).
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• Roy, P., Rajfur, Z., Jones, D. Marriott, G. & Jacobson, K. Local Photorelease of caged thymosin b4 in locomoting keratocytes causes cell turning. J. Cell Biology 153, 1035-1048 (2001).
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• Stocker, S., Hiery, M. and Marriott, G. Phototactic migration of Dictyostelium cells is linked to a new type of gelsolin-related protein. Mol. Biol. Cell (1999).
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• Choidas, A., Jungbluth, A., Sechi, A., Ullrich, A. and Marriott, G. The suitability and application of a GFP-actin fusion protein for long-term imaging of the organization and dynamics of the cytoskeleton in Mammalian cells. Eur. J. Cell Biol. 77, 81-90. (1998).
   
• Prassler, J., Murr, A. Stocker, S., Faix, J. and Marriott, G. Molecular and cellular characterization of a LIM-domain protein from the cytoskeleton of Dictyostelium. Mol. Biol. Cell. 9, 554-559. (1998).
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• Ottl, J., Gabriel, D. and Marriott, G. Synthesis, Characterization and Application of a New Class of Photocleavable Heterobifunctional Crosslinking Reagent. Bioconjugate Chemistry (1998). 9, 143-151
   
• Miyata, H., Kinosita, K. and Marriott, G. Cooperative Association of Actin Monomers and Crosslinked Actin Oligomers in Filaments. J. Biochemistry. 121, 527-533. (1997).
   
• Marriott, G. and Heidecker, M. Light-directed Activation of the Actin-Activated ATPase of a Caged Heavy Meromyosin. Biochemistry 35, 3170-3174. (1996).
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• Heidecker, M., Yan-Marriott, Y and Marriott, G. Proximity Relationships and Structural Dynamics of the Phalloidin Binding Site of Actin Filaments in Solution and on Single Actin Filaments on Heavy Meromyosin. Biochemistry 34, 11017. (1995).
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• Review of Biophotonics: A 2-Volume issue in Methods in Enzymology. by B.R. Masters. Journal of Biomedical Optics Volume10. Jan/Feb 2005
   
• The Biophotonics Cluster Program
   
• Graduate Course for Fall 2004: Principles of Biophotonics
   

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