title: Bridging length scales to measure polymer assembly.
abstract: Time-resolvable quantitative measurements of polymer concentration are very useful to elucidate protein polymerization pathways. There are numerous techniques to measure polymer concentrations in purified protein solutions, but few are applicable in vivo. Here we develop a methodology combining microscopy and spectroscopy to overcome the limitations of both approaches for measuring polymer concentration in cells and cell extracts. This technique is based on quantifying the relationship between microscopy and spectroscopy measurements at many locations. We apply this methodology to measure microtubule assembly in tissue culture cells andXenopusegg extracts using two-photon microscopy with FLIM measurements of FRET. We find that the relationship between FRET and two-photon intensity quantitatively agrees with predictions. Furthermore, FRET and intensity measurements change as expected with changes in acquisition time, labeling ratios, and polymer concentration. Taken together, these results demonstrate that this approach can quantitatively measure microtubule assembly in complex environments. This methodology should be broadly useful for studying microtubule nucleation and assembly pathways of other polymers.
authors.affiliation: John A. Paulson School of Engineering and Applied Science, Harvard University, Cambridge, MA 02138 bryankaye1@gmail.com.|FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Bryan
authors.initials: B
authors.lastname: Kaye
authors.affiliation: John A. Paulson School of Engineering and Applied Science, Harvard University, Cambridge, MA 02138.|FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Tae Yeon
authors.initials: TY
authors.lastname: Yoo
authors.affiliation: John A. Paulson School of Engineering and Applied Science, Harvard University, Cambridge, MA 02138.|FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Foster
authors.affiliation: John A. Paulson School of Engineering and Applied Science, Harvard University, Cambridge, MA 02138.|FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Che-Hang
authors.initials: CH
authors.lastname: Yu
authors.affiliation: John A. Paulson School of Engineering and Applied Science, Harvard University, Cambridge, MA 02138.|FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138.|Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Needleman
mesh_terms: D000818:Animals; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D008870:Microtubules / Q000378:metabolism; D017785:Photons; D058105:Polymerization; D011108:Polymers / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014404:Tubulin / Q000378:metabolism; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D011108:Polymers; D014404:Tubulin