Letter

Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic

  • Nature volume 552, pages 410414 (21 December 2017)
  • doi:10.1038/nature25146
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Abstract

Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes1,2. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments2,3. It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe ‘coincidence-detecting’ sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.

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Acknowledgements

We thank D. Alessi, T. Balla, P. De Camilli, O. Gozani, L. Lavis, H. Stenmark, T. Takenawa and Y. Takuwa for reagents; J. R. Houser for maintaining the TIRF and spinning-disk microscopes; J. England for advice and support; members of our laboratory for help and encouragement; and in particular S. C. Harrison for discussions and editorial help. R.M. was supported by a National Defense Science and Engineering Graduate (NDSEG) Fellowship from the DoD Air Force Office of Scientific Research and E.S. by the National Natural Science Foundation of China (31770900, 31270884, 30900268), the Beijing Natural Science Foundation (5122026, 5092017) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2011087). S.U. is a Fellow at the Image and Data Analysis core at Harvard Medical School and thanks H. Elliott and D. Richmond for discussions, and acknowledges the MATLAB code repository received from the Computational Image Analysis Workshop supported by NIH grant GM103792. T.K. acknowledges support from the Janelia Visitor Program and thanks E. Betzig, E. Marino, T. Liu and W. Legant for help and advice in constructing and installing the lattice light-sheet microscope. Construction of the lattice light-sheet microscope was supported by grants from Biogen and Ionis Pharmaceuticals to T.K. The research was supported by NIH grant NIH R01 GM075252 to T.K.

Author information

Author notes

    • Robert Marsland III
    • , Eli Song
    •  & Raphael Gaudin

    Present addresses: Department of Physics, Boston University, 590 Commonwealth Ave, Boston, Massachusetts 02215, USA (R.M.); National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China (E.S.); Institute of Viral and Liver Disease—INSERM U1110, 3 rue Koeberlé, Strasbourg 67000, France (R.G.).

Affiliations

  1. Department of Cell Biology, Harvard Medical School, 200 Longwood Ave, Boston, Massachusetts 02115, USA

    • Kangmin He
    • , Srigokul Upadhyayula
    • , Benjamin R. Capraro
    • , Weiming Wang
    • , Raphael Gaudin
    •  & Tom Kirchhausen
  2. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, 200 Longwood Ave, Boston, Massachusetts 02115, USA

    • Kangmin He
    • , Srigokul Upadhyayula
    • , Eli Song
    • , Song Dang
    • , Benjamin R. Capraro
    • , Weiming Wang
    • , Wesley Skillern
    • , Raphael Gaudin
    • , Minghe Ma
    •  & Tom Kirchhausen
  3. Department of Pediatrics, Harvard Medical School, 200 Longwood Ave, Boston, Massachusetts 02115, USA

    • Kangmin He
    • , Srigokul Upadhyayula
    •  & Tom Kirchhausen
  4. Physics of Living Systems Group, Massachusetts Institute of Technology, 400 Technology Square, Cambridge, Massachusetts 02139, USA

    • Robert Marsland III

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Contributions

K.H. and T.K. designed experiments; K.H. generated the probe constructs, and performed and analysed the experiments using TIRF and spinning-disk confocal microscopy; S.U., K.H. and W.S. performed and analysed the experiments using the lattice light-sheet microscope; R.G. established gene-editing protocols; R.G., K.H., and W.W. designed constructs for gene-editing; K.H., E.S., S.D., R.G., W.W. and M.M. generated the gene-edited cell lines; B.R.C. purified the recombinant proteins and carried out the in vitro lipid-binding assay; K.H., R.M. and T.K. discussed the results and contributed to the final manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Kangmin He or Tom Kirchhausen.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

  2. 2.

    Supplementary Information

    This file contains a supplementary discussion and references.

  3. 3.

    Supplementary Data

    This file contains supplementary figure 1 – uncropped gels.

Videos

  1. 1.

    Recruitment dynamics of full-length Aux1 and Aux1 lacking its PTEN-like domain and the J-domain to endocytic clathrin-coated pits and vesicles.

    Gene-edited CLTA-TagRFP+/+ SUM159 cells transiently expressing either full-length (EGFP-Aux1) or Aux1 lacking its PTEN-like and J-domains (EGFP-Aux1(420-814)) imaged at their bottom surfaces by spinning-disk confocal microscopy every 2 s during 5 min. To facilitate visualization, the EGFP channels in the right panels were shifted laterally by 6 pixels.

  2. 2.

    Recruitment dynamics of the Aux1-based PtdIns(4,5)P2 sensor to endocytic clathrin-coated pits and vesicles.

    Gene-edited CLTA-TagRFP+/+ SUM159 cells transiently expressing the Aux1-based PtdIns(4,5)P2 sensor EGFP-PH(PLC1)-Aux1 or the general PtdIns(4,5)P2 sensor EGFP-PH(PLC1) imaged at their bottom surfaces by spinning-disk confocal microscopy every 2 s during 5 min. To facilitate visualization, the EGFP channels in the right panels were shifted laterally by 6 pixels.

  3. 3.

    Recruitment dynamics of the Aux1-based PtdIns3P sensor to clathrin-coated pits and vesicles.

    Gene-edited CLTA-TagRFP+/+ SUM159 cell transiently expressing the Aux1-based PtdIns3P sensor EGFP-2xFYVE(Hrs)-Aux1 imaged at its bottom surface by TIRF microscopy every 1 s during 5 min. To facilitate visualization, the EGFP channel in the right panel was shifted laterally by 6 pixels.

  4. 4.

    Recruitment dynamics of the Aux1-based PtdIns4P sensor to clathrin-coated pits and vesicles.

    Gene-edited CLTA-TagRFP+/+ SUM159 cell transiently expressing the Aux1-based PtdIns4P sensor EGFP-P4M(DrrA)-Aux1 imaged at its bottom surface by TIRF microscopy every 1 s during 5 min. To facilitate visualization, the EGFP channel in the right panel was shifted laterally by 6 pixels.

  5. 5.

    Recruitment dynamics of the Aux1-based PtdIns(3,4)P2 sensor to clathrin-coated vesicles and to uncoated vesicles.

    Gene-edited CLTA-TagRFP+/+ SUM159 cell transiently expressing the Aux1-based PtdIns(3,4)P2 sensor EGFP-2xPH(TAPP1)-Aux1 imaged at its bottom surface by TIRF microscopy every 1 s during 5 min. To facilitate visualization, the EGFP channel in the right panel was shifted laterally by 6 pixels.

  6. 6.

    Absence of Rab5 molecules in coated pits and coated vesicles.

    Double gene-edited CLTA-TagRFP+/+ and EGFP-Rab5c+/+ SUM159 cell imaged at its bottom surface near the leading edge by TIRF microscopy every 1 s during 5 min. To facilitate visualization, the EGFP channel in the right panel was shifted laterally by 6 pixels.

  7. 7.

    Recruitment of Rab5 to uncoated clathrin-derived endocytic carriers.

    Gene-edited EGFP-Rab5c+/+ SUM159 cells transiently expressing the Aux1-based PtdIns(3,4)P2 sensor mCherry-2xPH(TAPP1)-Aux1 were imaged by lattice light-sheet microscopy in 3D (time series of 300 s in duration, where each time point was a stack of 41 planes spaced ~261 nm apart imaged at ~2.5 s intervals between stacks). The temporal changes in the three dimensional position and content of the fluorescent objects containing Rab5c and the PtdIns(3,4)P2 sensor were determined by automated 3D detection and tracking. The 3D time series plot shows the three dimensional position as a function of time of 1265 traces detected in 16 cells, color-coded from red to green as the ratio of the fluorescence signals of recruited PtdIns(3,4)P2 sensor with respect to Rab5c. a-c, The first frame of each of the aligned traces start at position (x,y,z) normalized to (0,0,0). Appearance of directed movement, coincident with a significant increase in the step-size of the displacement, followed capture of increased amounts of Rab5c; they are shown as single track (panel a), all tracks with a 5 frame rolling window (panel b), and cumulative trajectories (panel c).

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