Genetically encoded imaging tools for investigating cell dynamics at a glance.

The biology of a cell is the sum of many highly dynamic processes, each orchestrated by a plethora of proteins and other molecules. Microscopy is an invaluable approach to spatially and temporally dissect the molecular details of these processes. Hundreds of genetically encoded imaging tools have been developed that allow cell scientists to determine the function of a protein of interest in the context of these dynamic processes. Broadly, these tools fall into three strategies: observation, inhibition and activation. Using examples for each strategy, in this Cell Science at a Glance and the accompanying poster, we provide a guide to using these tools to dissect protein function in a given cellular process. Our focus here is on tools that allow rapid modification of proteins of interest and how observing the resulting changes in cell states is key to unlocking dynamic cell processes. The aim is to inspire the reader's next set of imaging experiments.

[1]  Pere Roca-Cusachs,et al.  Mechanical strain stimulates COPII‐dependent secretory trafficking via Rac1 , 2022, The EMBO journal.

[2]  M. Leptin,et al.  Optogenetic activators of apoptosis, necroptosis, and pyroptosis , 2022, The Journal of cell biology.

[3]  Miguel Hernández-González,et al.  Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation , 2022, bioRxiv.

[4]  A. Akhmanova,et al.  Opto-katanin, an optogenetic tool for localized, microtubule disassembly , 2021, Current Biology.

[5]  Oriol Vinyals,et al.  Highly accurate protein structure prediction with AlphaFold , 2021, Nature.

[6]  S. Royle,et al.  Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes , 2021, bioRxiv.

[7]  L. Benedetti.  Optogenetic Tools for Manipulating Protein Subcellular Localization and Intracellular Signaling at Organelle Contact Sites , 2021, Current protocols.

[8]  Mihaela Jagrić,et al.  Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces , 2021, eLife.

[9]  K. Roberts,et al.  Selective targeting of non-centrosomal AURKA functions through use of a targeted protein degradation tool , 2020, Communications Biology.

[10]  Viktor Stein,et al.  Linker Engineering in the Context of Synthetic Protein Switches and Sensors. , 2020, Trends in biotechnology.

[11]  J. Marvin,et al.  Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells , 2020, eLife.

[12]  K. Scheffler,et al.  The prophase oocyte nucleus is a homeostatic G-actin buffer , 2020, bioRxiv.

[13]  Gabrielle Larocque,et al.  Intracellular nanovesicles mediate α5β1 integrin trafficking during cell migration , 2020, bioRxiv.

[14]  T. Umehara,et al.  Rational design and implementation of a chemically inducible heterotrimerization system , 2020, Nature Methods.

[15]  R. Deshaies,et al.  Harnessing the Power of Proteolysis for Targeted Protein Inactivation. , 2020, Molecular cell.

[16]  R. Bayliss,et al.  Defining endogenous TACC3–chTOG–clathrin–GTSE1 interactions at the mitotic spindle using induced relocalization , 2020, Journal of Cell Science.

[17]  T. Schwarz,et al.  The light-sensitive dimerizer zapalog reveals distinct modes of immobilization for axonal mitochondria. , 2019, Nature Cell Biology.

[18]  Talley J. Lambert,et al.  FPbase: a community-editable fluorescent protein database , 2019, Nature Methods.

[19]  Gabrielle Larocque,et al.  Unintended perturbation of protein function using GFP nanobodies in human cells , 2019, Journal of Cell Science.

[20]  Gabrielle Larocque,et al.  Tumor protein D54 defines a new class of intracellular transport vesicles , 2019, The Journal of cell biology.

[21]  A. Bowman,et al.  Evidence for the nuclear import of histones H3.1 and H4 as monomers , 2018, The EMBO journal.

[22]  S. Royle,et al.  FerriTag is a new genetically-encoded inducible tag for correlative light-electron microscopy , 2018, Nature Communications.

[23]  Wilfried Weber,et al.  OptoBase: A Web Platform for Molecular Optogenetics. , 2018, ACS synthetic biology.

[24]  D. Stephens,et al.  ER-to-Golgi trafficking of procollagen in the absence of large carriers , 2018, bioRxiv.

[25]  James E. Bradner,et al.  The dTAG system for immediate and target-specific protein degradation , 2018, Nature Chemical Biology.

[26]  Alexander Deiters,et al.  Optochemical Control of Biological Processes in Cells and Animals. , 2018, Angewandte Chemie.

[27]  Melina Schuh,et al.  A Method for the Acute and Rapid Degradation of Endogenous Proteins , 2017, Cell.

[28]  Gabrielle Larocque,et al.  New tools for “hot-wiring” clathrin-mediated endocytosis with temporal and spatial precision , 2017, The Journal of cell biology.

[29]  D. Toomre,et al.  Novel ecto-tagged integrins reveal their trafficking in live cells , 2017, Nature Communications.

[30]  Zeno Lavagnino,et al.  Quantitative Assessment of Fluorescent Proteins , 2016, Nature Methods.

[31]  Frank Jülicher,et al.  Polarized endosome dynamics by spindle asymmetry during asymmetric cell division , 2015, Nature.

[32]  Laura Klewer,et al.  Chemically induced dimerization: reversible and spatiotemporal control of protein function in cells. , 2015, Current opinion in chemical biology.

[33]  D. Sahlender,et al.  Contributions of epsinR and gadkin to clathrin-mediated intracellular trafficking , 2015, Molecular biology of the cell.

[34]  I. E. Smith,et al.  Catalytic in vivo protein knockdown by small-molecule PROTACs. , 2015, Nature chemical biology.

[35]  Michael A. Lampson,et al.  Optogenetic control of organelle transport using a photocaged chemical inducer of dimerization , 2015, Current Biology.

[36]  Lukas C. Kapitein,et al.  Optogenetic control of organelle transport and positioning , 2015, Nature.

[37]  Brian Kuhlman,et al.  Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins , 2014, Proceedings of the National Academy of Sciences.

[38]  S. Munro,et al.  The specificity of vesicle traffic to the Golgi is encoded in the golgin coiled-coil proteins , 2014, Science.

[39]  M. Lampson,et al.  Recruitment of Mad1 to metaphase kinetochores is sufficient to reactivate the mitotic checkpoint , 2014, The Journal of cell biology.

[40]  Stephen J. Royle,et al.  Protein adaptation: mitotic functions for membrane trafficking proteins , 2013, Nature Reviews Molecular Cell Biology.

[41]  Stefan R. Pulver,et al.  Ultra-sensitive fluorescent proteins for imaging neuronal activity , 2013, Nature.

[42]  Edward F. Harry,et al.  Specific removal of TACC3–ch-TOG–clathrin at metaphase deregulates kinetochore fiber tension , 2013, Journal of Cell Science.

[43]  R. Vale,et al.  Biophysical Mechanism of T Cell Receptor Triggering in a Reconstituted System , 2012, Nature.

[44]  Franck Perez,et al.  Synchronization of secretory protein traffic in populations of cells , 2012, Nature Methods.

[45]  Josiah P. Zayner,et al.  TULIPs: Tunable, light-controlled interacting protein tags for cell biology , 2012, Nature Methods.

[46]  M. Affolter,et al.  Fluorescent fusion protein knockout mediated by anti-GFP nanobody , 2011, Nature Structural &Molecular Biology.

[47]  T. Corson,et al.  Small-Molecule Hydrophobic Tagging Induced Degradation of HaloTag Fusion Proteins , 2011, Nature Chemical Biology.

[48]  M. Ehlers,et al.  Rapid blue light induction of protein interactions in living cells , 2010, Nature Methods.

[49]  S. Lukyanov,et al.  Fluorescent proteins and their applications in imaging living cells and tissues. , 2010, Physiological reviews.

[50]  Taekjip Ha,et al.  Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics , 2010, Nature.

[51]  D. Sahlender,et al.  Rapid Inactivation of Proteins by Rapamycin-Induced Rerouting to Mitochondria , 2010, Developmental cell.

[52]  Toru Komatsu,et al.  Organelle-Specific, Rapid Induction of Molecular Activities and Membrane Tethering , 2010, Nature Methods.

[53]  Tatsuo Fukagawa,et al.  An auxin-based degron system for the rapid depletion of proteins in nonplant cells , 2009, Nature Methods.

[54]  Atsushi Miyawaki,et al.  Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression , 2008, Cell.

[55]  M. Cristina Cardoso,et al.  A Versatile Nanotrap for Biochemical and Functional Studies with Fluorescent Fusion Proteins*S , 2008, Molecular & Cellular Proteomics.

[56]  Guillermo Ayala,et al.  Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate , 2007, Proceedings of the National Academy of Sciences.

[57]  L. Banaszynski,et al.  A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules , 2006, Cell.

[58]  Tobias Meyer,et al.  An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways , 2005, Nature Methods.

[59]  David Zenisek,et al.  Coupling between Clathrin-Coated-Pit Invagination, Cortactin Recruitment, and Membrane Scission Observed in Live Cells , 2005, Cell.

[60]  Chang‐Deng Hu,et al.  Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis , 2003, Nature Biotechnology.

[61]  Gero Miesenböck,et al.  Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins , 1998, Nature.

[62]  R. Tsien,et al.  Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin , 1997, Nature.

[63]  S. Schreiber,et al.  Controlling signal transduction with synthetic ligands. , 1993, Science.