High-Resolution Analysis of Synaptic Proteins

Summary

Principal Investigator: Daniel Chiu
Affiliation: University of Washington
Country: USA
Abstract: As the molecular components of organelles are understood in greater detail, there is a growing need for quantitative approaches to determine how changes in protein expression in cells affect organelle composition and function. The larger goal of this work is to develop sensitive single- molecule and nanofluidic techniques that allow quantification of organelle composition with increasing sensitivity and apply them to understanding synaptic vesicle recycling. In the last funding cycle, we developed a quantitative TIRF (total-internal-reflection fluorescence) microscopy technique to quantify the major membrane proteins of synaptic vesicles isolated from brain. A key feature of this approach is that it uses the entire distribution of calibration data, not just the average. A major finding of our work was that vesicle proteins fall into two classes, those that are generally monodispersed (vary little in protein number between vesicles) and those that are polydispersed (vary considerably between vesicles). This suggests that changes in the expression levels of monodispersed proteins are more likely to lead to pathological changes in vesicle functioning than changes in the expression of polydispersed proteins. The goal of the current proposal is to test this hypothesis. To do so we propose four aims: 1) Develop a high-throughput quantification system that utilizes a flow-through technique based on optical gradient flow focusing and single-molecule detection, 2) Develop an analytical scale vesicle purification process to enable analysis of synaptic vesicles from genetically manipulated neurons, 3) Test the effects of protein over expression on vesicle protein contents, and 4) Develop and utilize a quantitative fluorescence microscopy approach for quantification of protein number and variability at the level of the synapse. Completion of the above aims will result in both a new level of understanding of synaptic vesicle assembly and new powerful tools for manipulating and quantitating a wide range of sub-cellular organelles, protein complexes, and other biological nanoparticles. PUBLIC HEALTH RELEVANCE: Changes in synaptic vesicle protein expression are associated with both normal processes like learning and memory and with a number of pathologies ranging from schizophrenia to epilepsy. The goal of this work is to develop sensitive single-molecule and nanofluidic techniques that allow quantification of organelle composition with increasing sensitivity and apply them to understanding synaptic vesicle recycling. Gaining a better understanding of synaptic composition and function may provide the basis for the development of novel therapies for CNS dysfunction that act by modulating synaptic function;additionally, the tools we develop should find broad use in the analysis of other subcellular organelles.
Funding Period: ----------------2005 - ---------------2014-
more information: NIH RePORT

Top Publications

  1. pmc Deconvolving single-molecule intensity distributions for quantitative microscopy measurements
    Sarah A Mutch
    Department of Chemistry, University of Washington, Seattle, Washington 98195, USA
    Biophys J 92:2926-43. 2007
  2. pmc Lyophilization of semiconducting polymer dot bioconjugates
    Wei Sun
    Department of Chemistry, University of Washington, Seattle, Washington 98195, United States
    Anal Chem 85:4316-20. 2013
  3. doi Single-cell nanosurgery
    Maxwell B Zeigler
    Department of Chemistry, University of Washington, Seattle, WA, USA
    Methods Mol Biol 991:139-48. 2013
  4. pmc Single-axonal organelle analysis method reveals new protein-motor associations
    Allyson E Sgro
    Department of Chemistry, University of Washington, Seattle, WA 98195 1700, USA
    ACS Chem Neurosci 4:277-84. 2013
  5. pmc Single-molecule fluorescence quantification with a photobleached internal standard
    Jennifer C Gadd
    Department of Chemistry, University of Washington, Seattle, Washington 98195 1700, USA
    Anal Chem 84:10522-5. 2012
  6. pmc Analytical tools for characterizing heterogeneity in organelle content
    Robbyn K Anand
    Department of Chemistry, University of Washington, Seattle, WA 98195 1700, USA
    Curr Opin Chem Biol 16:391-9. 2012
  7. pmc High-throughput fluorescence-activated nanoscale subcellular sorter with single-molecule sensitivity
    Perry G Schiro
    Department of Chemistry, University of Washington, Seattle, Washington 98195, United States
    J Phys Chem B 116:10490-5. 2012
  8. pmc Determining the number of specific proteins in cellular compartments by quantitative microscopy
    Sarah A Mutch
    Department of Chemistry, University of Washington, Seattle, Washington, USA
    Nat Protoc 6:1953-68. 2011
  9. pmc Semiconducting polymer dots doped with europium complexes showing ultranarrow emission and long luminescence lifetime for time-gated cellular imaging
    Wei Sun
    Department of Chemistry, University of Washington, Seattle, WA 98195 USA
    Angew Chem Int Ed Engl 52:11294-7. 2013
  10. pmc Alzheimer's disease protein Abeta1-42 does not disrupt isolated synaptic vesicles
    Peter B Allen
    University of Washington, Department of Chemistry, Box 351700, Seattle, WA 98195, USA
    Biochim Biophys Acta 1782:326-34. 2008

Detail Information

Publications19

  1. pmc Deconvolving single-molecule intensity distributions for quantitative microscopy measurements
    Sarah A Mutch
    Department of Chemistry, University of Washington, Seattle, Washington 98195, USA
    Biophys J 92:2926-43. 2007
    ..This methodology has been tested with the avidin-biocytin binding system for which the best-fit distribution of biocytins in the sample puncta was in good agreement with a bulk determination of the avidin-biocytin binding ratio...
  2. pmc Lyophilization of semiconducting polymer dot bioconjugates
    Wei Sun
    Department of Chemistry, University of Washington, Seattle, Washington 98195, United States
    Anal Chem 85:4316-20. 2013
    ..These results indicate lyophilization may be a preferred approach for storing and shipping Pdot bioconjugates, which is an important practical consideration for ensuring Pdots are widely adopted in biomedical research...
  3. doi Single-cell nanosurgery
    Maxwell B Zeigler
    Department of Chemistry, University of Washington, Seattle, WA, USA
    Methods Mol Biol 991:139-48. 2013
    ....
  4. pmc Single-axonal organelle analysis method reveals new protein-motor associations
    Allyson E Sgro
    Department of Chemistry, University of Washington, Seattle, WA 98195 1700, USA
    ACS Chem Neurosci 4:277-84. 2013
    ..This work confirms a previously discovered association between KIF1A and p38 and shows that KIF1A also transports SV2A- and VAMP2-containing carrier vesicles...
  5. pmc Single-molecule fluorescence quantification with a photobleached internal standard
    Jennifer C Gadd
    Department of Chemistry, University of Washington, Seattle, Washington 98195 1700, USA
    Anal Chem 84:10522-5. 2012
    ..6 single fluorophores. SynaptopHluorin vesicles, which contain the environmentally sensitive green fluorescent protein, exhibited an average of 4.4 single green fluorescent proteins per vesicle...
  6. pmc Analytical tools for characterizing heterogeneity in organelle content
    Robbyn K Anand
    Department of Chemistry, University of Washington, Seattle, WA 98195 1700, USA
    Curr Opin Chem Biol 16:391-9. 2012
    ..We then highlight the advantages that droplet microfluidics offers for probing subcellular heterogeneity...
  7. pmc High-throughput fluorescence-activated nanoscale subcellular sorter with single-molecule sensitivity
    Perry G Schiro
    Department of Chemistry, University of Washington, Seattle, Washington 98195, United States
    J Phys Chem B 116:10490-5. 2012
    ..Utilizing a near-nanoscale channel system, we were able to achieve upward of 91% recovery of desired objects with a 99.7% purity...
  8. pmc Determining the number of specific proteins in cellular compartments by quantitative microscopy
    Sarah A Mutch
    Department of Chemistry, University of Washington, Seattle, Washington, USA
    Nat Protoc 6:1953-68. 2011
    ..As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes...
  9. pmc Semiconducting polymer dots doped with europium complexes showing ultranarrow emission and long luminescence lifetime for time-gated cellular imaging
    Wei Sun
    Department of Chemistry, University of Washington, Seattle, WA 98195 USA
    Angew Chem Int Ed Engl 52:11294-7. 2013
    ..The long fluorescence lifetime of the Pdots was used to distinguish them from other red fluorescence emitting nanoparticles, and improve the signal-to-noise ratio for time-gated cellular imaging. PVK=poly(9-vinylcarbazole). ..
  10. pmc Alzheimer's disease protein Abeta1-42 does not disrupt isolated synaptic vesicles
    Peter B Allen
    University of Washington, Department of Chemistry, Box 351700, Seattle, WA 98195, USA
    Biochim Biophys Acta 1782:326-34. 2008
    ..From these experiments, we conclude that isolated synaptic vesicles are not disrupted by Abeta1-42...
  11. pmc Sizing subcellular organelles and nanoparticles confined within aqueous droplets
    Jennifer C Gadd
    Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195 1700, USA
    Anal Chem 80:3450-7. 2008
    ....
  12. doi Calcium-assisted glass-to-glass bonding for fabrication of glass microfluidic devices
    Peter B Allen
    Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, USA
    Anal Chem 80:7153-7. 2008
    ..This technique is compatible with inexpensive unpolished glass and is limited in resolution by the PDMS etch guide used and the intrinsic properties of isotropic etching...
  13. doi Controlling mass transport in microfluidic devices
    Jason S Kuo
    Department of Chemistry, University of Washington, Seattle, Washington 98195 1700, USA
    Annu Rev Anal Chem (Palo Alto Calif) 4:275-96. 2011
    ..We also highlight several in vitro biological studies in the areas of organ engineering, cancer, and blood coagulation that have benefited from accurate control of mass transfer...
  14. pmc Large structural change in isolated synaptic vesicles upon loading with neurotransmitter
    Kristi L Budzinski
    Department of Chemistry, University of Washington, Seattle, Washington, USA
    Biophys J 97:2577-84. 2009
    ..Vesicles lacking SV protein 2A (SV2A) did not manifest a change in size after loading with glutamate, indicating that SV2A is required for this phenomenon...
  15. pmc Pressure-driven laminar flow switching for rapid exchange of solution environment around surface adhered biological particles
    Peter B Allen
    Dept of Chemistry, University of Washington, Seattle, WA 98195, USA
    Lab Chip 10:727-33. 2010
    ..This technique adds to the microfluidic tool box that is currently available for manipulating the solution environment around biological particles and molecules...
  16. pmc Ultrasensitive and high-throughput fluorescence analysis of droplet contents with orthogonal line confocal excitation
    Gavin D M Jeffries
    Department of Chemistry, University of Washington, Seattle, Washington 98195 1700, USA
    Anal Chem 82:9948-54. 2010
    ..Using this method, we were able to analyze picomolar concentrations of analytes contained within picoliter-volume droplets at a rate of greater than 350 droplets per second...
  17. pmc A compact and highly fluorescent orange-emitting polymer dot for specific subcellular imaging
    Fangmao Ye
    Department of Chemistry, University of Washington, Seattle, WA, USA
    Chem Commun (Camb) 48:1778-80. 2012
    ..The small particle size, high brightness, and the ability to highly specifically target subcellular structures make the CN-PPV dots promising probes for biological imaging and bioanalytical applications...
  18. pmc Protein quantification at the single vesicle level reveals that a subset of synaptic vesicle proteins are trafficked with high precision
    Sarah A Mutch
    Department of Chemistry and Pharmacology, University of Washington, Seattle, WA 98195, USA
    J Neurosci 31:1461-70. 2011
    ..These findings place constraints on models of protein function at the synapse and raise the possibility that changes in vesicle protein expression affect vesicle composition and functioning...
  19. pmc A high-throughput method for generating uniform microislands for autaptic neuronal cultures
    Allyson E Sgro
    Department of Chemistry, University of Washington, Seattle, WA 98195 1700, USA
    J Neurosci Methods 198:230-5. 2011
    ..The stamping method we report can be used to increase the number of single-neuron islands per culture and aid in the rapid visualization of microislands...