Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells

View ORCID ProfileShalin B. Mehta, Molly McQuilken, Patrick La Riviere, Patricia Occhipinti, Amitabh Verma, Rudolf Oldenbourg, Amy S. Gladfelter, Tomomi Tani
doi: https://doi.org/10.1101/068767
Shalin B. Mehta
Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA02543
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Shalin B. Mehta
Molly McQuilken
Department of Biological Sciences, Dartmouth College, Hanover, NH03755Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Patrick La Riviere
Department of Radiology, University of Chicago, Chicago, IL60637
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Patricia Occhipinti
Department of Biological Sciences, Dartmouth College, Hanover, NH03755Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Amitabh Verma
Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA02543
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Rudolf Oldenbourg
Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA02543Physics Department, Brown University, Providence, RI02912
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Amy S. Gladfelter
Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA02543Department of Biological Sciences, Dartmouth College, Hanover, NH03755Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Tomomi Tani
Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA02543
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: ttani@mbl.edu
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Preview PDF
Loading

Abstract

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells, yet remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within, ∼350nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide new insights into how micron-scale ordered assemblies emerge from nanoscale molecules in living cells.

Significance Statement In living cells, the 3D architecture of molecular assemblies such as chromosomes, lipid bilayers, and the cytoskeleton is regulated through the interaction among their component molecules. Monitoring the position and orientation of constituent molecules is important for understanding the mechanisms that govern the structure and function of these assemblies. We have developed an instantaneous fluorescence polarization microscope to track the position and orientation of fluorescently labeled particles, including single molecules, which form micron-scale macromolecular assemblies in living cells. Our imaging approach is broadly applicable to the study of dynamic molecular interactions that underpin the function of micron-scale assemblies in living cells.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
Back to top
PreviousNext
Posted August 12, 2016.
Download PDF

Supplementary Material

Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
Share
Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells
Shalin B. Mehta, Molly McQuilken, Patrick La Riviere, Patricia Occhipinti, Amitabh Verma, Rudolf Oldenbourg, Amy S. Gladfelter, Tomomi Tani
bioRxiv 068767; doi: https://doi.org/10.1101/068767
Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells
Shalin B. Mehta, Molly McQuilken, Patrick La Riviere, Patricia Occhipinti, Amitabh Verma, Rudolf Oldenbourg, Amy S. Gladfelter, Tomomi Tani
bioRxiv 068767; doi: https://doi.org/10.1101/068767

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Biophysics
Subject Areas
All Articles
  • Animal Behavior and Cognition (1540)
  • Biochemistry (2499)
  • Bioengineering (1756)
  • Bioinformatics (9720)
  • Biophysics (3927)
  • Cancer Biology (2990)
  • Cell Biology (4230)
  • Clinical Trials (135)
  • Developmental Biology (2651)
  • Ecology (4124)
  • Epidemiology (2033)
  • Evolutionary Biology (6930)
  • Genetics (5239)
  • Genomics (6531)
  • Immunology (2205)
  • Microbiology (7004)
  • Molecular Biology (2780)
  • Neuroscience (17399)
  • Paleontology (127)
  • Pathology (432)
  • Pharmacology and Toxicology (712)
  • Physiology (1067)
  • Plant Biology (2514)
  • Scientific Communication and Education (646)
  • Synthetic Biology (835)
  • Systems Biology (2698)
  • Zoology (438)