Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization

Journal name:
Nature Cell Biology
Volume:
14,
Pages:
1159–1168
Year published:
DOI:
doi:10.1038/ncb2597
Received
Accepted
Published online

Abstract

As the main microtubule-organizing centre in animal cells, the centrosome has a fundamental role in cell function. Surrounding the centrioles, the pericentriolar material (PCM) provides a dynamic platform for nucleating microtubules. Although the importance of the PCM is established, its amorphous electron-dense nature has made it refractory to structural investigation. By using SIM and STORM subdiffraction-resolution microscopies to visualize proteins critical for centrosome maturation, we demonstrate that the PCM is organized into two main structural domains: a layer juxtaposed to the centriole wall, and proteins extending farther away from the centriole organized in a matrix. Analysis of Pericentrin-like protein (PLP) reveals that its carboxy terminus is positioned at the centriole wall, it radiates outwards into the matrix and is organized in clusters having quasi-nine-fold symmetry. By RNA-mediated interference (RNAi), we show that PLP fibrils are required for interphase recruitment and proper mitotic assembly of the PCM matrix.

At a glance

Figures

  1. 3D SIM of proteins critical for centrosome maturation identifies two distinct structural domains within the PCM.
    Figure 1: 3D SIM of proteins critical for centrosome maturation identifies two distinct structural domains within the PCM.

    (a) Maximum intensity projections along the Z axis of centrioles from S2 cells labelled with antibodies against centriolar proteins obtained either with widefield or 3D SIM microscopy. Scale bar, 200 nm. (b) Summary schematic of the 3D subvolume iterative alignment strategy based on cross-correlation in real space used for alignment and analysis of experimental 3D volumes. See Methods for a detailed description. (c) 2D projections of the average aligned volumes. SAS-6 n = 10(antibodies anti-eGFP), SAS-4 n = 88 (antibodies against amino acids 2–150), ASL n = 21(amino acids 958–972 of ASL), PLP n = 82 (amino acids 1–381 of PLP-PB), SPD-2 n = 19(amino acids 375–695 of ASL), CNN n = 22 (amino acids 1–571 of CNN), γ-tubulin  n = 33. Scale bar, 200 nm. (d) Fluorescence intensity profiles from the centre of the centriole image outwards, measured from radially averaged 2D projections of average volumes of centrosomal proteins. (e) Radially averaged fluorescence intensity values obtained from individual centrosomal protein projected volumes were fitted to an offset Gaussian function to calculate the centre position and deviation of the distribution. (f) Top, centrosomes isolated from Drosophila syncytial blastoderm embryos were allowed to regrow microtubules with rhodamine–tubulin and stained with rabbit anti-PLP (amino acids 1–381 of PLP-PB) or mouse anti-γ-tubulin antibodies recognized with anti-mouse Alexa 488. Bottom, image gallery of centrosomes from Drosophila embryos stained with rabbit anti-PLP NTD and anti-rabbit Alexa 488. Scale bars, 500 nm. (g) High-pressure frozen Drosophila syncytial blastoderm embryos embedded in resin were sectioned (75 nm) and stained with rabbit antibody anti-PLP NTD (amino acids 1–381) and anti-rabbit immunogold labelled secondary antibodies (10 nm beads). The yellow circles highlight gold bead positions on the electron micrograph. The white lines highlight the putative position of the centriole wall. Scale bars, 100 nm.

  2. The molecular architecture of PLP.
    Figure 2: The molecular architecture of PLP.

    (a) Amino-acid map of Drosophila PLP isoforms predicted from the Flybase database. Sequence prediction of the coiled-coil conformation was performed with the software Coils using a window of 28 residues. Amino-acid stretches were considered coiled-coil if predicted with a probability of ≥70%. (b) Top, wild-type S2 cells co-stained with rabbit anti-PLP MD primary antibodies (amino acids 1,805–2,137, anti-rabbit Alexa 488) and with guinea-pig anti-PLP NTD primary antibodies (amino acids 1–381, anti-guinea-pig Alexa 555). Bottom, S2 cells expressing eGFP–PACT co-stained with mouse anti-eGFP monoclonal antibody (anti-mouse Alexa 488) and with rabbit anti-PLP NTD primary antibodies (amino acids 1–381, anti-rabbit Alexa 555). The centrosomes in the outlined regions in the large panel are shown at high magnification in the adjacent panels. DNA is labelled with DAPI stain. Scale bars, 1 μM (left), 500 nm (right). (c) 2D projections of the average aligned volumes. PLP NTD = 82; PLP MD n = 29; eGFP–PACT n = 9. (d) Fluorescence intensity profiles from the centre of the centriole image outwards measured from radially averaged 2D projections of average volumes of centrosomal proteins. Scale bar, 250 nm. (e) Radially averaged fluorescence intensity values obtained from individual centrosomal protein projected volumes were fitted to an offset Gaussian function to calculate the centre position and deviation of the distribution. (f) STORM image of centrioles from S2 cells stained with rabbit anti-PLP MD antibody (amino acids 1,805–2,137) and anti-rabbit secondary antibodies conjugated with Alexa 647/Alexa 405 dye pairs. Scale bar, 100 nm.

  3. PLP fibrils associated with mother centrioles form a gap where the daughter centriole assembles.
    Figure 3: PLP fibrils associated with mother centrioles form a gap where the daughter centriole assembles.

    (a) Rows 1 to 3, centrosomes from wild-type S2 cells co-stained with rabbit anti-PLP NTD antibodies (amino acids 1–381, anti-rabbit 555) together with mouse anti-SAS-4 antibodies (anti-mouse 488). Row 4, centrosomes from S2 cells expressing eGFP–SAS-6 co-stained with mouse anti-GFP antibodies (anti-mouse 488). together with rabbit anti-PLP NTD antibodies (anti-rabbit 555). Scale bar, 500 nm. (b) Volume rendering of G2 centrioles from a stained with mouse anti-SAS-4 and rabbit anti-PLP NTD shown from end-on and side views. (c) Gap distance measurements obtained from 3D SIM volumes of a subpopulation of G2 centrosomes stained with rabbit PLP NTD antibody. (d) Rendering of volume averages of G1 and G2 centrosomes stained with anti-PLP NTD antibody. G2 centrosomes were divided into two separate populations for subclass averaging of centrioles with an open gap or partially open gap. (e) STORM image of centrioles from S2 cells stained with rabbit anti-PLP NTD antibody (amino acids 1–381) and anti-rabbit secondary antibodies conjugated with Alexa 647/Alexa 405 dye pairs. Given the cluster variability, we used a blinded study to quantify how often a missing cluster could be recognized (58%) in STORM images of mother centrioles stained with anti-PLP antibody from cells blocked in G2.

  4. PLP is associated exclusively with mother centrioles until metaphase.
    Figure 4: PLP is associated exclusively with mother centrioles until metaphase.

    (a) S2 cells expressing eGFP–SAS-6 stained with mouse anti-GFP antibodies (anti-mouse 488) and with rabbit anti-PLP NTD (amino acids 1–381, anti-rabbit 555). DNA is labelled with DAPI stain. The white arrows point to the lack of or partial recruitment of PLP on daughter centrioles at metaphase. The blue arrow on the telophase panels shows complete recruitment of PLP on daughter centrioles. (b) Wild-type S2 cells stained with mouse anti-SAS-4 antibodies (anti-mouse 488) and rabbit anti-PLP NTD antibodies (amino acids 1–381, anti-rabbit 555). The centrosomes in the outlined regions in the large panel are shown at high magnification in the adjacent panels. DNA is labelled with DAPI stain. The white arrows point to the lack of or partial recruitment of PLP on daughter centrioles at metaphase. Note the green arrow on the metaphase panel showing the separation of SAS-4-stained centrioles compared with G2 cells. The blue arrow on the telophase panels shows complete recruitment of PLP on daughter centrioles. (c) Quantification of the number of SAS-4, SAS-6 clusters or PLP rings per centrosome per S2 cell. (G1 n = 26, G2 = 29,telophase = 10.) (d) S2 cells expressing eGFP–SAS-6 co-stained with guinea-pig anti-PLP NTD antibodies (amino acids 1–381, anti-guinea-pig 405), rabbit anti-GFP antibodies (anti-rabbit 488) and mouse anti γ tubulin (anti-mouse 555). Scale bars, 1 μM (a,b,d).

  5. Plp is required for the initial recruitment and proper 3D assembly of the PCM distal layer.
    Figure 5: Plp is required for the initial recruitment and proper 3D assembly of the PCM distal layer.

    (a) Left, Plp genomic region obtained from the Flybase database. The genes are labelled with the original nomenclature; in parenthesis are the isoform names from the present nomenclature in Fly base. The position of the sequences on Plp exons used for dsRNA production is annotated on the sequence. Right, western blot of extracts obtained from S2 cells treated for control or Plp dsRNA probed with rabbit anti-PLP NTD antibody. (b) S2 cells treated with control dsRNA or dsRNA specific for Plp exon 10 were stained with anti-CNN antibodies (anti-mouse 488), mouse anti-γ-tubulin antibodies or rabbit anti-SPD2 antibodies (anti-rabbit or mouse 555). Widefield images were collected with a ×60 objective and deconvolved with a nonlinear positivity-constrained iterative algorithm. The centrosomes in the outlined regions in the large panel are shown at high magnification in the adjacent panels. DNA was labelled with DAPI stain. (c) Mitotic S2 cells treated with dsRNA specific for Plp exon 10 were stained with guinea-pig anti-PLP NTD (anti-guinea-pig 555) and rabbit anti-CNN amino acids 1–571 (anti-rabbit 488). DNA was labelled with DAPI stain. SIM images were acquired in the camera linear range of response and are shown with identical intensity settings. (d) Volume rendering of control and Plp RNAi S2 cells shown in c. (e) Volumes obtained from stained S2 cells as in c were quantified for the volume occupied and their surface area with Chimera software (control n = 16, Plp RNAi n = 18; ***P<0.001). (f) Volumes were approximated to a spherical object to measure volume2/area3. Scale bars, 10 μM (b); 1 μM (c). Uncropped images of blots are shown in Supplementary Fig. S5.

  6. The molecular architecture of Kendrin/Pericentrin is similar to that of PLP.
    Figure 6: The molecular architecture of Kendrin/Pericentrin is similar to that of PLP.

    (a) Linear map of the amino-acid sequence of human Kendrin predicted from the UCSC database. The antibodies on the schematic show the location of the PLP protein sequence used for immunization. Sequence prediction of the coiled-coil conformation was performed with the software Coils using a window of 28 residues. Amino-acid stretches were considered coiled-coil if predicted with a probability of ≥70%. (b) Human RPE cells were labelled with antibodies against Kendrin NTD (amino acids 744–909, anti-rabbit 555) and Kendrin CTD (amino acids 3,197–3,336, anti-rabbit 555) with mouse acetylated-tubulin or mouse anti-γ-tubulin antibodies (anti-mouse 488). The centrosomes in the outlined regions in the large panel are shown at high magnification in the adjacent panels. Nuclei were labelled with DAPI stain. The white arrow points to the diffused population of Kendrin in interphase. (c) 2D projections of the average aligned volumes (Kendrin NTD n = 16, Kendrin CTD n = 15). Scale bar, 250 nm. (d) Fluorescence intensity profiles from the centre of the centriole image outwards measured from radially averaged 2D projections of average volumes of centrosomal proteins. (e) Radially averaged fluorescence intensity values obtained from individual centrosomal protein projected volumes were fitted to an offset mirrored Gaussian function to calculate the centre position of the distribution. (f) HeLa cells expressing eGFP–PACT were co-stained with antibodies against Kendrin NTD (amino acids 744–909, anti-rabbit 555) and anti-GFP (anti-mouse 488). Scale bars, 1 μm (b); 500 nm (f).

  7. Pericentrin-like protein forms elongated fibrils that extend radially from the centriole wall to support the 3D organization of the PCM.
    Figure 7: Pericentrin-like protein forms elongated fibrils that extend radially from the centriole wall to support the 3D organization of the PCM.

    During centrosome maturation, the PCM is organized in two distinct structural domains: a layer juxtaposed to the centriole wall, and proteins extending farther away from the centriole organized in a matrix. In this proximal PCM domain, we found elongated PLP fibrils that are anchored with the PACT domain at the centriole wall, with their amino terminus extending outwards. PLP is exclusively associated with mother centrioles until metaphase and forms a gap where the daughter centriole assembles. During centrosome maturation, PLP facilitates the proper 3D assembly of the PCM distal layer by organizing a shell of CNN molecules that is in place around the wall of mother centrioles from the interphase cell cycle stage.

References

  1. Bettencourt-Dias, M. & Glover, D. M. Centrosome biogenesis and function: centrosomics brings new understanding. Nat. Rev. Mol. Cell Biol. 8, 451463 (2007).
  2. Nigg, E. A. & Stearns, T. The centrosome cycle: centriole biogenesis, duplication and inherent asymmetries. Nat. Cell Biol. 13, 11541160 (2011).
  3. Azimzadeh, J. & Marshall, W. F. Building the centriole. Curr. Biol. 20, R816R825 (2010).
  4. Bettencourt-Dias, M. et al. Centrosomes and cilia in human disease. Trends Genet. 27, 307315 (2011).
  5. Vorobjev, I. A. & Chentsov Yu, S. Centrioles in the cell cycle. I. Epithelial cells. J. Cell. Biol. 93, 938949 (1982).
  6. Rieder, C. L. The centrosome cycle in PtK2 cells: asymmetric distribution and structural changes in the pericentriolar material. Biol. Cell. 44, 117132 (1982).
  7. Bornens, M. et al. Structural and chemical characterization of isolated centrosomes. Cell Motil. Cytoskeleton. 8, 238249 (1987).
  8. Mogensen, M. M. et al. Microtubule minus-end anchorage at centrosomal and non-centrosomal sites: the role of ninein. J. Cell Sci. 113, 30133023 (2000).
  9. Bornens, M. Centrosome composition and microtubule anchoring mechanisms. Curr. Opin. Cell Biol. 14, 2534 (2002).
  10. Moritz, M. et al. Microtubule nucleation by γ-tubulin-containing rings in the centrosome. Nature 378, 638640 (1995).
  11. Kollman, J. M. et al. Microtubule nucleation by γ-tubulin complexes. Nat. Rev. Mol. Cell Biol. 12, 709721 (2011).
  12. Palazzo, R. E. et al. Centrosome maturation. Curr. Top Dev. Biol. 49, 449470 (2000).
  13. Decker, M. et al. Limiting amounts of centrosome material set centrosome size in C. elegans embryos. Curr. Biol. 21, 12591267 (2011).
  14. Andersen, J. S. et al. Proteomic characterization of the human centrosome by protein correlation profiling. Nature 426, 570574 (2003).
  15. Muller, H. et al. Proteomic and functional analysis of the mitotic Drosophila centrosome. EMBO J. 29, 33443357 (2010).
  16. Li, J. B. et al. Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene. Cell 117, 541552 (2004).
  17. Kilburn, C. L. et al. New tetrahymena basal body protein components identify basal body domain structure. J. Cell. Biol. 178, 905912 (2007).
  18. Keller, L. C. et al. Proteomic analysis of isolated Chlamydomonas centrioles reveals orthologs of ciliary-disease genes. Curr. Biol. 15, 10901098 (2005).
  19. Pelletier, L. et al. Centriole assembly in Caenorhabditis elegans. Nature 444, 619623 (2006).
  20. Guichard, P. et al. Procentriole assembly revealed by cryo-electron tomography. EMBO J. 29, 15651572 (2010).
  21. Paintrand, M. et al. Centrosome organization and centriole architecture: their sensitivity to divalent cations. J. Struct. Biol. 108, 107128 (1992).
  22. Li, S. et al. Three-dimensional structure of basal body triplet revealed by electron cryo-tomography. EMBO J. 31, 552562 (2012).
  23. Anderson, R. G. The three-dimensional structure of the basal body from the rhesus monkey oviduct. J. Cell. Biol. 54, 246265 (1972).
  24. O’Toole, E. T. et al. Three-dimensional organization of basal bodies from wild-type and delta-tubulin deletion strains of Chlamydomonas reinhardtii. Mol. Biol. Cell 14, 29993012 (2003).
  25. Wiese, C. & Zheng, Y. A new function for the γ-tubulin ring complex as a microtubule minus-end cap. Nat. Cell Biol. 2, 358364 (2000).
  26. Keating, T. J. & Borisy, G. G. Immunostructural evidence for the template mechanism of microtubule nucleation. Nat. Cell Biol. 2, 352357 (2000).
  27. Moritz, M. et al. Three-dimensional structural characterization of centrosomes from early Drosophila embryos. J. Cell. Biol. 130, 11491159 (1995).
  28. Ibrahim, R. et al. Electron tomography study of isolated human centrioles. Microsc. Res. Tech. 72, 4248 (2009).
  29. Moritz, M. et al. Recruitment of the γ-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds. J. Cell. Biol. 142, 775786 (1998).
  30. Schnackenberg, B. J. et al. Reconstitution of microtubule nucleation potential in centrosomes isolated from Spisula solidissima oocytes. J. Cell Sci. 113, 943953 (2000).
  31. Ou, Y. Y. et al. Higher order structure of the PCM adjacent to the centriole. Cell Motil. Cytoskeleton 55, 125133 (2003).
  32. Ou, Y. & Rattner, J. B. A subset of centrosomal proteins are arranged in a tubular conformation that is reproduced during centrosome duplication. Cell Motil. Cytoskeleton. 47, 1324 (2000).
  33. Gustafsson, M. G. et al. Three-dimensional resolution doubling in wide-fieldfluorescence microscopy by structured illumination. Biophys. J. 94, 49574970 (2008).
  34. Huang, B. et al. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810813 (2008).
  35. Huang, B., Bates, M. & Zhuang, X. Super-resolution fluorescence microscopy. Annu. Rev. Biochem. 78, 9931016 (2009).
  36. Gogendeau, D. & Basto, R. Centrioles in flies: the exception to the rule? Semin. Cell Dev. Biol. 21, 163173 (2010).
  37. Schermelleh, L. et al. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320, 13321336 (2008).
  38. Nakazawa, Y. et al. SAS-6 is a cartwheel protein that establishes the 9-fold symmetry of the centriole. Curr. Biol. 17, 21692174 (2007).
  39. Rodrigues-Martins, A. et al. DSAS-6 organizes a tube-like centriole precursor, and its absence suggests modularity in centriole assembly. Curr. Biol. 17, 14651472 (2007).
  40. Gopalakrishnan, J. et al. SAS-4 provides a scaffold for cytoplasmic complexes and tethers them in a centrosome. Nat. Commun. 2, 359 (2011).
  41. Kirkham, M. et al. SAS-4 is a C. elegans centriolar protein that controls centrosome size. Cell 112, 575587 (2003).
  42. Van Breugel, M. et al. Structures of SAS-6 suggest its organization in centrioles. Science 331, 11961199 (2011).
  43. Kitagawa, D. et al. Structural basis of the 9-fold symmetry of centrioles. Cell 144, 364375 (2011).
  44. Gillingham, A. K. & Munro, S. The PACT domain, a conserved centrosomal targeting motif in the coiled-coil proteins AKAP450 and pericentrin. EMBO Rep. 1, 524529 (2000).
  45. Kawaguchi, S. & Zheng, Y. Characterization of a Drosophila centrosome protein CP309 that shares homology with Kendrin and CG-NAP. Mol. Biol. Cell 15, 3745 (2004).
  46. Martinez-Campos, M. et al. The Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis. J. Cell. Biol. 165, 673683 (2004).
  47. Conduit, P. T. et al. Centrioles regulate centrosome size by controlling the rate of CNN incorporation into the PCM. Curr. Biol. 20, 21782186 (2010).
  48. Erickson, H. P. Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy. Biol. Proced. Online 11, 3251 (2009).
  49. Xu, K., Babcock, H. P. & Zhuang, X. Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat. Methods 9, 185188 (2012).
  50. Lau, L. et al. STED microscopy with optimized labeling density reveals 9-fold arrangement of a centriole protein. Biophys. J. 102, 29262935 (2012).
  51. Dzhindzhev, N. S. et al. Asterless is a scaffold for the onset of centriole assembly. Nature 467, 714718 (2010).
  52. Rogers, G. C. et al. The SCF Slimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication. J. Cell. Biol. 184, 225239 (2009).
  53. Loncarek, J. et al. Control of daughter centriole formation by the pericentriolar material. Nat. Cell Biol. 10, 322328 (2008).
  54. Wang, W. J. et al. The conversion of centrioles to centrosomes: essential coupling of duplication with segregation. J. Cell. Biol. 193, 727739 (2011).
  55. Dobbelaere, J. et al. A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila. PLoS Biol. 16, e224 (2008).
  56. Rogers, G. C. et al. A multicomponent assembly pathway contributes to the formation of acentrosomal microtubule arrays in interphase Drosophila cells. Mol. Biol. Cell 19, 31633178 (2008).
  57. Choi, Y. K. et al. CDK5RAP2 stimulates microtubule nucleation by the γ-tubulin ring complex. J. Cell. Biol. 191, 10891095 (2010).
  58. Miyoshi, K. et al. Pericentrin, a centrosomal protein related to microcephalic primordial dwarfism, is required for olfactory cilia assembly in mice. FASEB J. 23, 32893297 (2009).
  59. Takahashi, M. et al. Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring γ-tubulin ring complex. Mol. Biol. Cell 13, 32353245 (2002).
  60. Delaval, B. & Doxsey, S. J. Pericentrin in cellular function and disease. J. Cell. Biol. 188, 181190 (2010).
  61. Rauch, A. et al. Mutations in the pericentrin (PCNT) gene cause primordial dwarfism. Science 319, 816819 (2008).
  62. Dictenberg, J. B. et al. Pericentrin and γ-tubulin form a protein complex and are organized into a novel lattice at the centrosome. J. Cell. Biol. 141, 163174 (1998).
  63. Ou, Y., Zhang, M. & Rattner, J. B. The centrosome: the centriole-PCM coalition. Cell Motil. Cytoskeleton. 57, 17 (2004).
  64. Mahen, R. & Venkitaraman, A. R. Pattern formation in centrosome assembly. Curr. Opin. Cell Biol. 24, 1423 (2012).
  65. Flory, M. R. & Davis, T. N. The centrosomal proteins pericentrin and kendrin are encoded by alternatively spliced products of one gene. Genomics 82, 401405 (2003).
  66. Lawo, S., Hasegan, M., Gupta, G. D. & Pelletier, L. Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material. Nat. Cell Biol.http://dx.doi.org/10.1038/ncb2591 (2012).
  67. Sonnen, K. F., Schermelleh, L., Leonhardt, H. & Nigg, E. A. 3D-structured illumination microscopy provides novel insight into architecture of human centrosomes. Biol. Open 000, 112 (2012).
  68. Mennella, V. et al. Motor domain phosphorylation and regulation of the Drosophila kinesin 13, KLP10A. J. Cell. Biol. 186, 481490 (2009).
  69. Rogers, S. L. et al. Molecular requirements for actin-based lamella formation in Drosophila S2 cells. J. Cell. Biol. 162, 10791088 (2003).
  70. Goshima, G. et al. Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316, 417421 (2007).
  71. McDonald, K. L. Electron microscopy and EM immunocytochemistry. Method. Cell Biol. 44, 411444 (1994).
  72. Beaudoin, G. M.3rd et al. Afadin, a ras/rap effector that controls cadherin function, promotes spine and excitatory synapse density in the hippocampus. J. Neurosci. 32, 99110 (2012).

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Affiliations

  1. Department of Biochemistry and Biophysics and the Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California 94158, USA

    • V. Mennella,
    • B. Keszthelyi,
    • F. Kan &
    • D. A. Agard
  2. Electron Microscope Laboratory, University of California, Berkeley, California 94720, USA

    • K. L. McDonald
  3. Department of Pharmaceutical Chemistry and Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94158, USA

    • B. Chhun &
    • B. Huang
  4. Department of Cellular and Molecular Medicine, Arizona Cancer Center, University of Arizona, Tucson, Arizona 85724, USA

    • G. C. Rogers

Contributions

V.M. conceived the strategy, designed and performed experiments, analysed data and wrote the paper; B.K. and V.M., with input from others at UCSF, developed the 3D volume alignment and analysis procedure; K.L.M. performed the immuno-electron microscopy experiments; B.C. helped with STORM data acquisition; F.K. helped with immunostaining experiments; G.C.R. provided reagents and constructs; B.H. advised on data analysis and STORM experiments and discussed results; D.A.A. advised on data analysis, discussed results and wrote the paper.

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The authors declare no competing financial interests.

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