Real-time microviscosity measurement tools for the cell

Summary

Principal Investigator: Mark A Haidekker
Abstract: DESCRIPTION (provided by applicant): Microviscosity in the cell membrane and the cell cytoplasm are important parameters in cell signaling and disease. Presently, viscosity on the microscopic scale is determined by fluorescence anisotropy, fluorescence recovery after photobleaching (FRAP), or magnetic nanoparticles. A group of viscosity-sensitive fluorescent molecules, generally termed molecular rotors, allows a new, fast, and convenient approach with minimum requirements of instrumentation and very high temporal and spatial resolution. However, molecular rotors pose one disadvantage - they are intensity based, and local concentration gradients may reduce measurement accuracy. The proposed research program builds on a recently developed ratiometric dye (J.Am.Chem.Soc. 2006;128: 398-399) in which a molecular rotor and a fluorescent reference unit form a covalently linked dye pair for resonance energy transfer (RET). This ratiometric dye has the potential to overcome limitations posed by concentration gradients and optical properties. The overall goal of the proposed research is to develop specific fluorescent ratiometric viscosity sensors to be used in phospholipid bilayers, cell membranes, and the cell cytoplasm. We propose the synthesis, characterization, and optimization of membrane-targeted as well as cytoplasm-targeted ratiometric molecular rotors. The experimental approach includes the testing and application of the new viscosity sensitive dyes in model phospholipid bilayers, red cell ghosts, and living cells. This testing approach allows us to characterize the new probes in environments of increasing complexity. The outcome of the proposed research will be the availability of a series of real-time, microscale viscosity probes for cellular environments with a wide range of applications. Some examples where the new viscosity probes will be useful are: - Analysis of the involvement of the cell membrane in cell signaling under fluid shear stress (vascular endothelial cells) - Analysis of changes in cell membrane biomechanics in atherosclerosis - Studies involving membrane lipid rafts - Analysis of the role of cytoplasmic viscosity in the cryopreservation of cells While it is not the goal of this application to actually apply molecular rotors in the above examples, we will provide the necessary probes as well as their methods of use for investigators involved in any fields listed above or related. Public Health Relevance Statement: Cell membrane and cytoplasmic viscosity are of high relevance to cell signaling (e.g. blood pressure regulation) and to various disease states (e.g. altered membrane viscosity related to atherosclerosis, cell malignancy, hypercholesterolemia, and diabetes). We propose to develop new, ultrafast and ultra-high resolution methods to determine changes in membrane and cytoplasm viscosity using fluorescent molecular rotors. With these new tools, studies involving viscosity in the cell will be accelerated or made possible in the first place, thus enabling faster study and better understanding of cell signaling processes and the cellular foundations of various disease states. Cell membrane and cytoplasmic viscosity are of high relevance to cell signaling (e.g. blood pressure regulation) and to various disease states (e.g. altered membrane viscosity related to atherosclerosis, cell malignancy, hypercholesterolemia, and diabetes). We propose to develop new, ultrafast and ultra-high resolution methods to determine changes in membrane and cytoplasm viscosity using fluorescent molecular rotors. With these new tools, studies involving viscosity in the cell will be accelerated or made possible in the first place, thus enabling faster study and better understanding of cell signaling processes and the cellular foundations of various disease states.
Funding Period: ----------------2009 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. pmc Environment-sensitive behavior of fluorescent molecular rotors
    Mark A Haidekker
    Faculty of Engineering, 597 D, W, Brooks Drive, University of Georgia, Athens, GA 30602, USA
    J Biol Eng 4:11. 2010
  2. pmc Self-calibrating viscosity probes: design and subcellular localization
    Marianna Dakanali
    Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, MC 0358, La Jolla, CA 92093 0358, USA
    Bioorg Med Chem 20:4443-50. 2012
  3. pmc Imaging of flow patterns with fluorescent molecular rotors
    Adnan Mustafic
    Faculty of Engineering, Driftmier Engineering Center, University of Georgia, Athens, GA 30602, USA
    J Fluoresc 20:1087-98. 2010
  4. pmc Detection of liposome membrane viscosity perturbations with ratiometric molecular rotors
    Matthew E Nipper
    University of Georgia, Faculty of Engineering, Athens, GA 30602 4435, USA
    Biochimie 93:988-94. 2011
  5. pmc Synthesis and evaluation of self-calibrating ratiometric viscosity sensors
    Hyung Jo Yoon
    Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive MC 0358, La Jolla, CA 92093 0358, USA
    Org Biomol Chem 9:3530-40. 2011
  6. pmc Intrinsic and extrinsic temperature-dependency of viscosity-sensitive fluorescent molecular rotors
    Sarah Howell
    Faculty of Engineering, University of Georgia, 597 D W Brooks Drive, Athens, GA 30602, USA
    J Fluoresc 22:457-65. 2012

Detail Information

Publications8

  1. pmc Environment-sensitive behavior of fluorescent molecular rotors
    Mark A Haidekker
    Faculty of Engineering, 597 D, W, Brooks Drive, University of Georgia, Athens, GA 30602, USA
    J Biol Eng 4:11. 2010
    ..Furthermore, current applications of molecular rotors as microviscosity sensors are reviewed, and engineering aspects are presented on how measurement accuracy and precision can be improved...
  2. pmc Self-calibrating viscosity probes: design and subcellular localization
    Marianna Dakanali
    Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, MC 0358, La Jolla, CA 92093 0358, USA
    Bioorg Med Chem 20:4443-50. 2012
    ..For instance, hydrophilic dye 12 shows a homogeneous distribution inside the cell and represents a suitable probe for viscosity measurements in the cytoplasm...
  3. pmc Imaging of flow patterns with fluorescent molecular rotors
    Adnan Mustafic
    Faculty of Engineering, Driftmier Engineering Center, University of Georgia, Athens, GA 30602, USA
    J Fluoresc 20:1087-98. 2010
    ..With its high sensitivity and high spatial and temporal resolution, imaging of flow patterns with molecular rotors may become a useful tool in microfluidics, flow measurement, and control...
  4. pmc Detection of liposome membrane viscosity perturbations with ratiometric molecular rotors
    Matthew E Nipper
    University of Georgia, Faculty of Engineering, Athens, GA 30602 4435, USA
    Biochimie 93:988-94. 2011
    ..We suggest that the new self-calibrating dyes can be used for real-time viscosity sensing in liposome systems with the advantages of lifetime measurements, but with low-cost steady-state instrumentation...
  5. pmc Synthesis and evaluation of self-calibrating ratiometric viscosity sensors
    Hyung Jo Yoon
    Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive MC 0358, La Jolla, CA 92093 0358, USA
    Org Biomol Chem 9:3530-40. 2011
    ..We also present an application of such ratiometric dyes for the detection of membrane viscosity changes in a liposome model...
  6. pmc Intrinsic and extrinsic temperature-dependency of viscosity-sensitive fluorescent molecular rotors
    Sarah Howell
    Faculty of Engineering, University of Georgia, 597 D W Brooks Drive, Athens, GA 30602, USA
    J Fluoresc 22:457-65. 2012
    ..To use molecular rotors for the quantitative determination of viscosity or microviscosity, the exponent x needs to be determined for each dye-solvent combination...