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Dynamic polyhedral actomyosin lattices remodel micron-scale curved membranes during exocytosis in live mice

Nature Cell Biology volume 21, pages 933–939 (2019)Cite this article

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Abstract

Actomyosin networks, the cell’s major force production machineries, remodel cellular membranes during myriad dynamic processes1,2 by assembling into various architectures with distinct force generation properties3,4. While linear and branched actomyosin architectures are well characterized in cell-culture and cell-free systems3, it is not known how actin and myosin networks form and function to remodel membranes in complex three-dimensional mammalian tissues. Here, we use four-dimensional spinning-disc confocal microscopy with image deconvolution to acquire macromolecular-scale detail of dynamic actomyosin networks in exocrine glands of live mice. We address how actin and myosin organize around large membrane-bound secretory vesicles and generate the forces required to complete exocytosis5,6,7. We find that actin and non-muscle myosin II (NMII) assemble into previously undescribed polyhedral-like lattices around the vesicle membrane. The NMII lattice comprises bipolar minifilaments8,9,10 as well as non-canonical three-legged configurations. Using photobleaching and pharmacological perturbations in vivo, we show that actomyosin contractility and actin polymerization together push on the underlying vesicle membrane to overcome the energy barrier and complete exocytosis7. Our imaging approach thus unveils a force-generating actomyosin lattice that regulates secretion in the exocrine organs of live animals.

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Fig. 1: Contractile polyhedral GFP-NMIIA lattice on fused SGs during regulated exocytosis in vivo.
Fig. 2: Dynamic F-actin polyhedral lattice on fused SGs during regulated exocytosis in vivo.
Fig. 3: The actomyosin lattice scaffolds centripetally growing F-actin as the SG shrinks.
Fig. 4: NMIIA lattice comprises bipolar filaments and does not require F-actin for recruitment.

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Data availability

The authors declare that the main data supporting the findings of this study are available within the article and its Supplementary Information files. Source data for Figs. 1d,e, 2d,f,g, 3c,d and 4b,c have been provided as Supplementary Table 2. Extra data are available from the corresponding authors on request.

Code availability

All custom codes are available from the corresponding authors on request.

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Acknowledgements

This research was supported by the NIH, NCI Center for Cancer Research Intramural Research Program (ZIA BC 011682) and the NIDCD Intramural Research Program (Z01 DC 000002). We thank E. Balzer at Nikon for help with the Nikon spinning-disc microscope, C. Combs at the NHLBI Light Microscopy Core for help with the STED microscope, I. Belyansteva at the NIDCD for help with the Zeiss Airyscan microscope and R. Cui for general laboratory help.

Author information

Authors and Affiliations

  1. Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA

    Seham Ebrahim, Desu Chen, Max Weiss, Lenka Malec, Yeap Ng & Roberto Weigert

  2. Laboratory of Cell Structure and Dynamics, National Institute on Deafness and other Communication Disorders, NIH, Bethesda, MD, USA

    Ivan Rebustini, Evan Krystofiak & Bechara Kachar

  3. Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA

    Longhua Hu & Jian Liu

  4. School of Medical Sciences, UNSW, Sydney, New South Wales, Australia

    Andrius Masedunskas, Edna Hardeman & Peter Gunning

  5. Intracellular Membrane Trafficking Section, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD, USA

    Roberto Weigert

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  1. Seham Ebrahim
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Contributions

R.W., S.E. and B.K. designed the experiments. S.E., M.W., L.M., Y.N. and I.R. performed the experiments. E.H., P.G. and A.M. provided intellectual insights throughout the project. E.K. and B.K. carried out the TEM. S.E., D.C. and B.K. analysed the experiments. L.H. and J.L. performed the quantitative 3D image analysis. S.E., R.W. and B.K. wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Bechara Kachar or Roberto Weigert.

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Integrated supplementary information

Supplementary Figure 1 Actin and NMIIA coat fused SGs in salivary and pancreatic acinar cells during regulated exocytosis.

a, Maximum intensity Z-stack projections from the salivary gland of a mouse expressing an mTomato-fused membrane marker showing the membranes from the canalicular APM (arrow) diffused into the SG membrane after fusion (magenta), and labeled with phalloidin to delineate F-actin (green), which coats the SG membrane. b, F-actin (magenta) and GFP-NMIIA (green) decorate SGs fused to the canaliculi (arrows) during regulated exocytosis in the salivary gland. c, F-actin (magenta) and GFP-NMIIA (green) decorate SGs fused to the canaliculi (arrows) during regulated exocytosis in the pancreas. d, Change in NMIIA cage diameter, fluorescence intensity, and density over time during integration for the representative SG in Fig. 1d. e, Intravital time-lapse microscopy of fused SG from pancreas of GFP-NMIIA mouse depicted using “Red Fire” lookup table to highlight intensity differences. Yellow arrowheads point to GFP-NMIIA head puncta. Experiments were repeated in 7 (a, b) and 3 (c, e) mice independently Scale bars: 1 µm.

Supplementary Figure 2 Characterization of x-y-z resolution using our spinning disc microscopy setup.

a, Single image using our microscopy setup, with no deconvolution (details in Methods) of DNA origami-based GATTA-Confocal Nanorulers, which each carry two fluorescent marks comprising AlexaFluor®-488, with a mark-to-mark distance of 270 nm39–41 . b, The image in (a) was analyzed with GATTAnalysis software, which uses an automated spot finder and fits two 2D-Gaussians to each structure to determine the center-to-center distance and the FWHM of the spots39–41 (example of boxed nanoruler in a displayed in plot). All data in the field are represented in the histogram, with mean, standard deviation (StDev) and standard error (SEM), calculated automatically by the software, displayed in plot. N= 78 Nanorulers. c, Slice view of a cropped region from Fig. 4g, of GFP-NMIIA signal on secretory granule surface (no deconvolution), showing xy, xz and yz planes. Red rectangles delineate the image pixel size in xy (0.05 μm) and z (0.150 μm), derived by NIKON Elements® software from the optics and camera pixel size. d, A 1.6 x 1.55 x 1.2 μm 3D volume-view of the same region. Note: interpolation between pixels by Elements results in smaller pixel size in final display of the 3D-volume snapshot. The “3D-measurement” module in NIKON Elements® Analysis Software was used to first detect centroids of each fluorescent punctum and then measure the distance between adjacent puncta in 3D space. Centroid-to-centroid distances are represented in the volume view image by straight lines and corresponding values listed (right).

Supplementary Figure 3 Actomyosin lattices observed in unprocessed spinning disc images, as well as by other imaging modalities.

a-d, Corresponding raw images for Fig. 1f-i, showing the GFP-NMIIA lattice is resolvable even before image deconvolution. These experiments were repeated respectively in 7 (f,g), 3 (h), and 3 (i) mice independently. e and f, Corresponding raw images for Fig. 2c and 3a, respectively, showing the F-actin (e) and actomyosin (f, F-actin- magenta and GFP-NMIIA- green) lattices are resolvable even before image deconvolution. These experiments were repeated respectively in 3 (e) and 7 (f) mice independently. g, Raw spinning disc image of fused SGs in pancreatic acinar cells labeled with GFP-NMIIA (green) and phalloidin to delineate F-actin (magenta). This experiment was repeated in 3 mice independently. h, 3D view of an Airyscan acquisition of the GFP-NMIIA lattice around a fused SG. i, 3D view of a structured illumination microscopy (SIM) acquisition of the actomyosin lattice coating a fused SG (F-actin, magenta and GFP-NMIIA, green). These experiments were repeated respectively in 3 (h) and 2 (i)mice independently. Scale bars: 1 µm.

Supplementary Figure 4 Characterizing the F-actin lattice around SGs during regulated exocytosis in vivo.

a, Left- A quantitative 3D model of the F-actin lattice around fused SGs derived from analysis and quantification of fixed, phalloidin-labeled images of fused SGs from salivary gland. Right- A schematic of the F-actin lattice to highlight lattice “arms” and “vertices”. b, c, Distribution of F-actin lattice arm-length (b) and inter-arm angle (c). d – f, Live time-lapse acquisitions of GFP-LifeAct dynamics around fused SGs from transgenic mice (d) were deconvolved (e) to improve image resolution. FI profiles along a line drawn through the SG center (f) depict smoother Gaussians and more accurate measurements obtained from deconvolved images. This experiment was repeated in 4 mice independently. Scale bars: 1 µm.

Supplementary information

Supplementary Information

Supplementary Figures 1–4, Supplementary Table and Supplementary video titles/legends and Supplementary references.

Reporting Summary

Supplementary Table 1

Microscope setups used to acquire images and movies from fixed 128 samples and live animals.

Supplementary Table 2

Statistics source data.

Supplementary Video 1

Time-lapse of regulated exocytosis in salivary gland of live mice expressing GFP-NMIIA.

Supplementary Video 2

Zoom in on GFP-NMIIA lattice on single SG (maximum intensity projection) during regulated exocytosis in salivary gland of live mouse.

Supplementary Video 3

SG from Supplementary Video 2 before and after image deconvolution.

Supplementary Video 4

Zoom in on GFP-NMIIA lattice on single SG (3D-rendering) during regulated exocytosis in salivary gland of live mouse.

Supplementary Video 5

Time-lapse of regulated exocytosis in pancreas of live mice expressing GFP-NMIIA.

Supplementary Video 6

Zoom in on GFP-NMIIA lattice on single SG (3D-rendering) during regulated exocytosis in pancreas of live mouse.

Supplementary Video 7

FRAP of GFP-NMIIA lattice around SG during assembly/addition (SG diameter = 1.4 um) phase in live mouse.

Supplementary Video 8

FRAP of GFP-NMIIA lattice around SG during Contractility/Crosslinking (SG diameter = 0.9 um) phase in live mouse.

Supplementary Video 9

FRAP of GFP-NMIIA lattice around SG during shrinking (SG diameter = 0.7 um) phase in live mouse.

Supplementary Video 10

3D-rendering of a Z-stack acquisition of GFP-NMIIA lattice around fused SG.

Supplementary Video 11

3D-rendering of a Z-stack acquisition of F-actin lattice around fused SG.

Supplementary Video 12

Time-lapse of regulated exocytosis in salivary gland of live mice expressing GFP-LifeAct.

Supplementary Video 13

Zoom in on GFP-LifeAct lattice on single SG during regulated exocytosis in live mouse.

Supplementary Video 14

3D-rendering of a Z-stack acquisition of F-actin (red) and GFP-NMIIA (green) lattices around fused SG.

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Ebrahim, S., Chen, D., Weiss, M. et al. Dynamic polyhedral actomyosin lattices remodel micron-scale curved membranes during exocytosis in live mice. Nat Cell Biol 21, 933–939 (2019). https://doi.org/10.1038/s41556-019-0365-7

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