Cell-permeable organic fluorescent probes for live-cell long-term super-resolution imaging reveal lysosome-mitochondrion interactions

Characterizing the long-term nanometer-scale interactions between lysosomes and mitochondria in live cells is essential for understanding their functions but remains challenging due to limitations of the existing fluorescent probes. Here, we develop cell-permeable organic fluorescent probes for lysosomes with excellent specificity and high photostability. We also use an existing Atto 647N dye with high brightness and excellent photostability to achieve specific labeling of mitochondria in live cells. Using these probes, we obtain dual-color structured illumination microscopy (SIM) images of dynamic physical lysosome-mitochondrion interactions in live cells at an ~90-nm resolution over a long time course of ~13 min. We successfully record the consecutive dynamic processes of lysosomal fusion and fission, as well as four types of physical lysosome-mitochondrion interactions by super-resolution imaging. Our probes provide an avenue for understanding the functions and the dynamic interplay of lysosomes and mitochondria in live cells.Studying interactions between lysosomes and mitochondria in living cells is difficult due to the limitations of existing probes. Here, the authors develop new cell-permeable fluorescent probes to image the dynamics of lysosomes and their physical interactions with mitochondria using super-resolution microscopy.

[1]  Bor Luen Tang,et al.  SIRT7 and hepatic lipid metabolism , 2015, Front. Cell Dev. Bio..

[2]  M. Davidson,et al.  Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics , 2015, Science.

[3]  F. Cordelières,et al.  A guided tour into subcellular colocalization analysis in light microscopy , 2006, Journal of microscopy.

[4]  Julie M Huynh,et al.  Mucolipidosis Type IV Protein TRPML1‐Dependent Lysosome Formation , 2015, Traffic.

[5]  Kangqiang Qiu,et al.  Real-time tracking mitochondrial dynamic remodeling with two-photon phosphorescent iridium (III) complexes. , 2016, Biomaterials.

[6]  M. Schrader,et al.  The different facets of organelle interplay—an overview of organelle interactions , 2015, Front. Cell Dev. Biol..

[7]  Kees Jalink,et al.  Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy , 2016, PloS one.

[8]  C. Tacchetti,et al.  Mitochondria and Melanosomes Establish Physical Contacts Modulated by Mfn2 and Involved in Organelle Biogenesis , 2014, Current Biology.

[9]  J. Audet,et al.  Flow cytometric screening of cell-penetrating peptides for their uptake into embryonic and adult stem cells. , 2007, Analytical biochemistry.

[10]  H. McBride,et al.  A Vesicular Transport Pathway Shuttles Cargo from Mitochondria to Lysosomes , 2012, Current Biology.

[11]  M. Liao,et al.  Quantitative proteomics using stable isotope labeling with amino acids in cell culture reveals protein and pathway regulation in porcine circovirus type 2 infected PK-15 cells. , 2012, Journal of proteome research.

[12]  E. Arriaga,et al.  Mitochondrial turnover and aging of long-lived postmitotic cells: the mitochondrial-lysosomal axis theory of aging. , 2010, Antioxidants & redox signaling.

[13]  Christopher J. Obara,et al.  Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER , 2016, Science.

[14]  U. Brunk,et al.  The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis. , 2002, European journal of biochemistry.

[15]  R. Rodenburg,et al.  Trolox-sensitive reactive oxygen species regulate mitochondrial morphology, oxidative phosphorylation and cytosolic calcium handling in healthy cells. , 2012, Antioxidants & redox signaling.

[16]  B. Tang,et al.  Real-time monitoring of the mitophagy process by a photostable fluorescent mitochondrion-specific bioprobe with AIE characteristics. , 2015, Chemical communications.

[17]  J. Nunnari,et al.  The Emerging Network of Mitochondria-Organelle Contacts. , 2016, Molecular cell.

[18]  Yu-Hui Zhang,et al.  Labeling lysosomes and tracking lysosome-dependent apoptosis with a cell-permeable activity-based probe. , 2012, Bioconjugate chemistry.

[19]  N. Raimundo,et al.  Mechanisms of communication between mitochondria and lysosomes. , 2016, The international journal of biochemistry & cell biology.

[20]  M L Walsh,et al.  Localization of mitochondria in living cells with rhodamine 123. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R. Youle,et al.  Mechanisms of mitophagy , 2010, Nature Reviews Molecular Cell Biology.

[22]  S. McKinney,et al.  Nonblinking and long-lasting single-molecule fluorescence imaging , 2006, Nature Methods.

[23]  M. Poot,et al.  Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. , 1996, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[24]  R. Gottlieb,et al.  Autophagy in health and disease. 5. Mitophagy as a way of life. , 2010, American journal of physiology. Cell physiology.

[25]  D. Chan,et al.  Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases , 2009, Human molecular genetics.

[26]  Daniel J. Klionsky,et al.  Autophagy fights disease through cellular self-digestion , 2008, Nature.

[27]  M. Gustafsson.  Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

[28]  S. Ryter,et al.  Autophagy in human health and disease. , 2013, The New England journal of medicine.

[29]  Fei Yang,et al.  Direct cytosolic delivery of cargoes in vivo by a chimera consisting of D- and L-arginine residues. , 2012, Journal of controlled release : official journal of the Controlled Release Society.

[30]  Jan Vogelsang,et al.  On the mechanism of Trolox as antiblinking and antibleaching reagent. , 2009, Journal of the American Chemical Society.

[31]  J. Luzio,et al.  Endocytic Delivery to Lysosomes Mediated by Concurrent Fusion and Kissing Events in Living Cells , 2005, Current Biology.

[32]  M. van der Laan,et al.  Cellular metabolism regulates contact sites between vacuoles and mitochondria. , 2014, Developmental cell.

[33]  M. Schuldiner,et al.  A dynamic interface between vacuoles and mitochondria in yeast. , 2014, Developmental cell.

[34]  N. Dolman,et al.  Tools and techniques to measure mitophagy using fluorescence microscopy , 2013, Autophagy.

[35]  Ian M. Dobbie,et al.  SIMcheck: a Toolbox for Successful Super-resolution Structured Illumination Microscopy , 2015, Scientific Reports.

[36]  Christian Eggeling,et al.  Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy. , 2010, Chemistry.

[37]  M. Colombo,et al.  A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. , 2001, Journal of cell science.

[38]  Lingyan Ping,et al.  Protein profiling of active cysteine cathepsins in living cells using an activity-based probe containing a cell-penetrating peptide. , 2012, Journal of proteome research.

[39]  R. Youle,et al.  Parkin is recruited selectively to impaired mitochondria and promotes their autophagy , 2008, The Journal of cell biology.

[40]  K. Turksen.  Autophagy in Health and Disease , 2018, Stem Cell Biology and Regenerative Medicine.

[41]  S. Rodríguez-Enríquez,et al.  Selective degradation of mitochondria by mitophagy. , 2007, Archives of biochemistry and biophysics.

[42]  M. Davidson,et al.  Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination , 2012, Proceedings of the National Academy of Sciences.

[43]  M. Gustafsson,et al.  Super-resolution 3D microscopy of live whole cells using structured illumination , 2011, Nature Methods.

[44]  Jiahuai Han,et al.  Imaging of intracellular acidic compartments with a sensitive rhodamine based fluorogenic pH sensor. , 2011, The Analyst.