Public Abstract

DE-SC0019313: In Planta Single-Molecule Imaging and Holographic Force Spectroscopy to Study Real-Time, Multimodal Turnover Dynamics of Polysaccharides and Associated Carbohydrate Metabolites

Award Status: Inactive

Institution: Rutgers, The State University of New Jersey, Piscataway, NJ
UEI: M1LVPE5GLSD9
DUNS: 001912864

Most Recent Award Date: 07/27/2021
Number of Support Periods: 3
PM: Kulkarni, Resham

Current Budget Period: 09/01/2020 - 08/31/2022
Current Project Period: 09/01/2018 - 08/31/2022
PI: Lee, Sang-Hyuk

Supplement Budget Period: N/A

Public Abstract

Cellular production of complex polysaccharides from simple carbohydrate-based precursor molecules serves critical roles in a variety of metabolic processes essential to the survival of every living organism. In the context of photosynthetic organisms like plants and algae, cell wall polysaccharides synthesis is particularly of interest for emerging biomass and bioenergy related applications. Despite a long history of research in plant cell biology, our understanding of in planta cell wall synthesis and its regulation is far from complete due to the lack of high-resolution multimodal microscopy-based tools suitable for in vivo plant cell imaging. In this project, a multidisciplinary team of scientists from Rutgers University, Vanderbilt University, and Oak Ridge National Laboratory aims to develop an innovative multimodal single-molecule manipulation/imaging optical instrument through integration of holographic optical tweezers, super-resolution fluorescence microscopy, single-particle tracking, and surface-enhanced Raman spectroscopy (SERS) to investigate in vivo cell wall regeneration of protoplasts from Arabidopsis and Poplar. The goal of this project is to concurrently study cellular metabolic processes relevant to polysaccharides synthesis, using multiple in vivo single-molecule assays, as follows: 1) force spectroscopy on the nascent polysaccharides chain extruding out of polysaccharide synthase complexes (PSC) through cell membranes using optical tweezers, 2) single-molecule-counting super-resolution localization microscopy on fluorescently labeled PSC, 3) single-particle tracking of PSC, 4) and SERS-sensing of sugar metabolites using plasmonic gold nanostars attached to PSC. The multimodal imaging assays will be first developed and validated in vitro, and then adapted for in vivo assays. Comparative study of both in vitro and in vivo data will not only allow systematic validation of the proposed approach, but also provide new insights into previously unknown in vivo regulatory mechanisms of cell wall biosynthesis. This novel approach will reveal in vivo plant cell wall polysaccharides synthesis processes with unprecedented molecular-level details through the simultaneous characterization of the structure, dynamics, and function of single enzyme complexes as well as intracellular sugar metabolites flux. The results from this project will greatly advance the mechanistic and holistic understanding of in vivo cell wall synthesis, which will accelerate the development of better transgenic crops for bioenergy related applications. Moreover, the new toolbox, combining powerful advanced microscopy assays with cell/protein engineering, will have broader impacts on molecular and cellular biology fields by paving the way for multimodal single-molecule studies in native cellular environments.

Cellular production of complex polysaccharides from simple carbohydrate-based precursor molecules serves critical roles in a variety of metabolic processes essential to the survival of every living organism. In the context of photosynthetic organisms like plants and algae, cell wall polysaccharides synthesis is particularly of interest for emerging biomass and bioenergy related applications. Despite a long history of research in plant cell biology, our understanding of in planta cell wall synthesis and its regulation is far from complete due to the lack of high-resolution multimodal microscopy-based tools suitable for in vivo plant cell imaging. In this project, a multidisciplinary team of scientists from Rutgers University, Vanderbilt University, and Oak Ridge National Laboratory aims to develop an innovative multimodal single-molecule manipulation/imaging optical instrument through integration of holographic optical tweezers, super-resolution fluorescence microscopy, single-particle tracking, and surface-enhanced Raman spectroscopy (SERS) to investigate in vivo cell wall regeneration of protoplasts from Arabidopsis and Poplar.

The goal of this project is to concurrently study cellular metabolic processes relevant to polysaccharides synthesis, using multiple in vivo single-molecule assays, as follows: 1) force spectroscopy on the nascent polysaccharides chain extruding out of polysaccharide synthase complexes (PSC) through cell membranes using optical tweezers, 2) single-molecule-counting super-resolution localization microscopy on fluorescently labeled PSC, 3) single-particle tracking of PSC, 4) and SERS-sensing of sugar metabolites using plasmonic gold nanostars attached to PSC. The multimodal imaging assays will be first developed and validated in vitro, and then adapted for in vivo assays. Comparative study of both in vitro and in vivo data will not only allow systematic validation of the proposed approach, but also provide new insights into previously unknown in vivo regulatory mechanisms of cell wall biosynthesis.

This novel approach will reveal in vivo plant cell wall polysaccharides synthesis processes with unprecedented molecular-level details through the simultaneous characterization of the structure, dynamics, and function of single enzyme complexes as well as intracellular sugar metabolites flux. The results from this project will greatly advance the mechanistic and holistic understanding of in vivo cell wall synthesis, which will accelerate the development of better transgenic crops for bioenergy related applications. Moreover, the new toolbox, combining powerful advanced microscopy assays with cell/protein engineering, will have broader impacts on molecular and cellular biology fields by paving the way for multimodal single-molecule studies in native cellular environments.