The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy

Protein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy Förster resonance energy transfer (FLIM-FRET) and stimulated emission depletion (STED) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.

[1]  Joerg Bewersdorf,et al.  Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy. , 2010, Biophysical journal.

[2]  W. Almers,et al.  Tracking SNARE Complex Formation in Live Endocrine Cells , 2004, Science.

[3]  Laurent Cognet,et al.  Super-resolution microscopy approaches for live cell imaging. , 2014, Biophysical journal.

[4]  Grant J. Jensen,et al.  Magnetosomes Are Cell Membrane Invaginations Organized by the Actin-Like Protein MamK , 2006, Science.

[5]  D. Schüler,et al.  A simple light scattering method to assay magnetism in Magnetospirillum gryphiswaldense , 1995 .

[6]  D. Ingber,et al.  Mechanotransduction across the cell surface and through the cytoskeleton , 1993 .

[7]  Michael Unser,et al.  DeconvolutionLab2: An open-source software for deconvolution microscopy. , 2017, Methods.

[8]  Christian Tardif,et al.  Fluorescence lifetime imaging nanoscopy for measuring Förster resonance energy transfer in cellular nanodomains , 2019, Neurophotonics.

[9]  D. Holdstock.  Past, present--and future? , 2005, Medicine, conflict, and survival.

[10]  S. Hell,et al.  Fluorescence nanoscopy in cell biology , 2017, Nature Reviews Molecular Cell Biology.

[11]  S. Hell,et al.  Sharper low-power STED nanoscopy by time gating , 2011, Nature Methods.

[12]  U. Heyen,et al.  Growth and magnetosome formation by microaerophilic Magnetospirillum strains in an oxygen-controlled fermentor , 2003, Applied Microbiology and Biotechnology.

[13]  D. Schüler,et al.  Segregation of prokaryotic magnetosomes organelles is driven by treadmilling of a dynamic actin-like MamK filament , 2016, BMC Biology.

[14]  Rafael Yuste,et al.  Fluorescence microscopy today , 2005, Nature Methods.

[15]  D. Schüler,et al.  Probing the Mechanical Properties of Magnetosome Chains in Living Magnetotactic Bacteria , 2014, Nano letters.

[16]  S. Lukyanov,et al.  GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. , 2004, Molecular biology and evolution.

[17]  Binary phase masks for easy system alignment and basic aberration sensing with spatial light modulators in STED microscopy , 2017, Scientific Reports.

[18]  A. Hyman,et al.  Cell Polarity: The importance of being polar , 1995, Current Biology.

[19]  Jerker Widengren,et al.  Stimulated Emission Depletion Microscopy. , 2017, Chemical reviews.

[20]  Roger D Kamm,et al.  Measuring molecular rupture forces between single actin filaments and actin-binding proteins , 2008, Proceedings of the National Academy of Sciences.

[21]  D. Schüler,et al.  Loss of the actin‐like protein MamK has pleiotropic effects on magnetosome formation and chain assembly in Magnetospirillum gryphiswaldense , 2010, Molecular microbiology.

[22]  Damien Faivre,et al.  An acidic protein aligns magnetosomes along a filamentous structure in magnetotactic bacteria , 2006, Nature.

[23]  P. Graumann.  Dynamics of bacterial cytoskeletal elements. , 2009, Cell motility and the cytoskeleton.

[24]  X. Zhuang,et al.  Breaking the Diffraction Barrier: Super-Resolution Imaging of Cells , 2010, Cell.

[25]  D. Faivre,et al.  From bacteria to mollusks: the principles underlying the biomineralization of iron oxide materials. , 2015, Angewandte Chemie.

[26]  Jonghee Yoon,et al.  A Bacteria‐Based Remotely Tunable Photonic Device , 2017 .

[27]  Ulrike Endesfelder,et al.  From single molecules to life: microscopy at the nanoscale , 2016, Analytical and Bioanalytical Chemistry.

[28]  R. Day,et al.  Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy , 2011, Nature Protocols.

[29]  Alberto Diaspro,et al.  STED super-resolved microscopy , 2018, Nature Methods.

[30]  D. Faivre,et al.  Interaction of Proteins Associated with the Magnetosome Assembly in Magnetotactic Bacteria As Revealed by Two-Hybrid Two-Photon Excitation Fluorescence Lifetime Imaging Microscopy Förster Resonance Energy Transfer , 2013, The journal of physical chemistry. B.

[31]  D. Schüler,et al.  The Acidic Repetitive Domain of the Magnetospirillum gryphiswaldense MamJ Protein Displays Hypervariability but Is Not Required for Magnetosome Chain Assembly , 2007, Journal of bacteriology.

[32]  José-Angel Conchello,et al.  Fluorescence microscopy , 2005, Nature Methods.

[33]  R. Dobarzić,et al.  [Fluorescence microscopy]. , 1975, Plucne bolesti i tuberkuloza.

[34]  Niko Hildebrandt,et al.  How to Apply FRET: From Experimental Design to Data Analysis , 2013 .

[35]  Joseph A Loo,et al.  Investigation of stable and transient protein–protein interactions: Past, present, and future , 2013, Proteomics.

[36]  D. Faivre.  Formation of magnetic nanoparticle chains in bacterial systems , 2015, MRS Bulletin.

[37]  J. Willemse,et al.  Positive control of cell division: FtsZ is recruited by SsgB during sporulation of Streptomyces. , 2011, Genes & development.

[38]  C. Fan,et al.  Effects of donor and acceptor's fluorescence lifetimes on the method of applying Förster resonance energy transfer in STED microscopy , 2018, Journal of microscopy.

[39]  Alberto Diaspro,et al.  Evaluating image resolution in stimulated emission depletion microscopy , 2018 .

[40]  Paul R. Selvin,et al.  The renaissance of fluorescence resonance energy transfer , 2000, Nature Structural Biology.

[41]  D. Schüler,et al.  Magnetosome biogenesis in magnetotactic bacteria , 2016, Nature Reviews Microbiology.

[42]  Leigh G. Monahan,et al.  Super-resolution imaging of the bacterial cytokinetic protein FtsZ. , 2011, Micron.

[43]  Markus Meister,et al.  Physical limits to magnetogenetics , 2016, eLife.

[44]  T. Bonhoeffer,et al.  Live-cell imaging of dendritic spines by STED microscopy , 2008, Proceedings of the National Academy of Sciences.

[45]  D. Schüler,et al.  Biologically controlled synthesis and assembly of magnetite nanoparticles† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4fd00240g Click here for additional data file. , 2015, Faraday discussions.