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Updated: Feb 15, 2026
Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy
Published on: August 20, 2018
Distribution of TMV movement protein in single living protoplasts immobilized in agarose
1The Scripps Research Institute, Division of Plant Biology, Department of Cell Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Abstract:
Recent studies of the tobacco mosaic virus (TMV) P30 movement protein (MP) fused with green fluorescent protein (GFP) during TMV infection described the involvement of elements of the cytoskeleton and components of the endoplasmic reticulum (ER) in the intracellular trafficking of MP:GFP from the sites of synthesis in the cytoplasm to plasmodesmata. To examine in real-time the pattern of synthesis, accumulation and degradation of MP:GFP, we developed a method to immobilize protoplasts in agarose such that they are maintained alive for extended periods of time. The pattern of MP:GFP accumulation in single living protoplasts visualized by confocal laser scanning microscopy (CLSM) was parallel to that previously described in a population of protoplasts harvested at different times post-infection. Additionally, a network of weakly fluorescent filaments, which are apparently different from microtubules, was observed to surround the nucleus and these filaments were associated with fluorescent bodies (previously identified as ER-derived structures). Later in infection, the fluorescent bodies increased in size and coalesced to form larger structures that accumulated near the periphery of the cells while highly fluorescent non-cortical filaments were observed distributed in the cytoplasm. The putative involvement of these filaments in targeting the fluorescent bodies to the periphery of the cell is discussed. Studies of single, embedded protoplasts make it possible to observe changes in amount and subcellular localization of viral and other proteins.
Insights
Researchers visualized tobacco mosaic virus movement protein (MP:GFP) dynamics in living plant cells. They observed novel filament networks involved in transporting viral components to cell peripheries, offering insights into viral spread.
Area of Science:
- Plant Virology
- Cell Biology
- Molecular Plant Pathology
Background:
- Tobacco mosaic virus (TMV) movement protein (MP) transport is crucial for viral spread.
- Previous studies implicated cytoskeleton and endoplasmic reticulum (ER) in MP trafficking.
- Real-time observation of MP dynamics in living cells was lacking.
Purpose of the Study:
- To visualize the real-time synthesis, accumulation, and degradation of MP fused with GFP (MP:GFP).
- To investigate the intracellular trafficking pathways of MP:GFP in living plant cells.
- To identify novel cellular structures involved in TMV MP transport.
Main Methods:
- Developed a method to immobilize and maintain living protoplasts in agarose for extended observation.
- Utilized confocal laser scanning microscopy (CLSM) to visualize MP:GFP dynamics in single, embedded protoplasts.
- Compared observations in single protoplasts with previous population-based studies.
Main Results:
- Observed MP:GFP accumulation patterns mirroring previous population studies in real-time.
- Identified a novel network of non-microtubule filaments surrounding the nucleus, associated with ER-derived bodies.
- Documented the enlargement and peripheral accumulation of fluorescent bodies, with cytoplasmic filaments potentially aiding transport.
Conclusions:
- The developed method allows for real-time observation of viral protein dynamics in living plant cells.
- Novel filament networks and their association with ER-derived structures play a role in TMV MP intracellular trafficking.
- Findings provide new insights into the mechanisms of viral cell-to-cell movement.
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