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Optogenetic manipulation of cellular communication using engineered myosin motors

Nature Cell Biology volume 23, pages 198–208 (2021)Cite this article

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An Author Correction to this article was published on 08 April 2021

An Author Correction to this article was published on 19 February 2021

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Abstract

Cells achieve highly efficient and accurate communication through cellular projections such as neurites and filopodia, yet there is a lack of genetically encoded tools that can selectively manipulate their composition and dynamics. Here, we present a versatile optogenetic toolbox of artificial multi-headed myosin motors that can move bidirectionally within long cellular extensions and allow for the selective transport of GFP-tagged cargo with light. Utilizing these engineered motors, we could transport bulky transmembrane receptors and organelles as well as actin remodellers to control the dynamics of both filopodia and neurites. Using an optimized in vivo imaging scheme, we further demonstrate that, upon limb amputation in axolotls, a complex array of filopodial extensions is formed. We selectively modulated these filopodial extensions and showed that they re-establish a Sonic Hedgehog signalling gradient during regeneration. Considering the ubiquitous existence of actin-based extensions, this toolbox shows the potential to manipulate cellular communication with unprecedented accuracy.

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Fig. 1: Design, characterization and applications of ATVs in live cells.
Fig. 2: Design and characterization of optoATVs in live cells.
Fig. 3: Applications of optoATVs in live cells.
Fig. 4: Characterization and applications of ATVs and optoATVs in mouse iN cells.
Fig. 5: Live confocal imaging reveals the presence of an extensive filopodial network in the axolotl blastema.
Fig. 6: Applications of optoATVs to manipulate filopodia dynamics in the axolotl blastema cause selective regeneration defects.
Fig. 7: Application of optoATVs in the axolotl blastema reveals the critical role of filopodia in re-establishing a Shh gradient during limb regeneration.

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Supporting data are available upon request to the corresponding author. Source data are provided with this paper.

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Acknowledgements

We thank Z. Bryant and P. Ruijgrok for discussion on the design and application of the motors and useful comments on the manuscript, the Cell Sciences Imaging Facility (CSIF) at Stanford University for technical assistance and the whole Barna Lab for helpful discussion and critical reading of the manuscript. The project was supported by awards R01HD088597 (M.B.) and U19MH104172 (M.W.) from the National Institutes of Health (NIH) and, in part, by award 1S10OD01227601 from the National Center for Research Resources (NCRR). The content of this Report is solely the responsibility of the authors and does not necessarily represent the official views of NIH or NCRR. Z.Z. is supported in part by an NIH P50 training grant. O.Z. is a Simons Fellow of the Helen Hay Whitney Foundation (HHWF) and in part supported by the Canadian Institutes for Health Research (CIHR) Fellowship. M.B. is a New York Stem Cell Foundation (NYSCF) Robertson Investigator and M.W. is a Howard Hughes Medical Institute (HHMI) Faculty Scholar.

Author information

Author notes

  1. Nicolas Denans

    Present address: Stowers Institute for Medical Research, Kansas City, MO, USA

  2. Yingfei Liu

    Present address: Institute of Neurobiology, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi, China

Authors and Affiliations

  1. Department of Chemical and Systems Biology, Stanford University, Stanford, CA, USA

    Zijian Zhang

  2. Department of Developmental Biology, Stanford University, Stanford, CA, USA

    Zijian Zhang, Nicolas Denans, Olena Zhulyn, Hannah D. Rosenblatt & Maria Barna

  3. Department of Genetics, Stanford University, Stanford, CA, USA

    Zijian Zhang, Nicolas Denans, Olena Zhulyn, Hannah D. Rosenblatt & Maria Barna

  4. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA

    Yingfei Liu & Marius Wernig

  5. Department of Pathology, Stanford University, Stanford, CA, USA

    Yingfei Liu & Marius Wernig

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Contributions

Z.Z., N.D. and M.B. conceived the project. N.D., Z.Z. and M.B. designed and constructed the expression vectors. Z.Z. and M.B. planned the experiments. Z.Z. and N.D. executed and analysed the experiments. Y.L., O.Z., H.D.R., M.W. and M.B. contributed to essential cell cultures, reagents and expertise. Z.Z. and M.B. wrote the manuscript with input from all authors. M.B. supervised all aspects of the work.

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Correspondence to Maria Barna.

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Extended data

Extended Data Fig. 1 Characterization of ATV+ and ATV- in live cells.

a, Representative images of randomly chosen mouse fibroblasts expressing either (upper) ATV+ or (lower) ATV- with pmBFP chosen from 3 independent experiments. Scale, 20 µm. b, c, Mouse fibroblasts co-expressing sfGFP-ATV+, mKate2-ATV- and pmiRFP in the same cells. ATV+ and ATV- display significantly different localization patterns within the same cells. Representative images chosen from 3 independent experiments. Scale, 20 µm. Mouse fibroblasts expressing (d) sfGFP-ATV+ or (e) sfGFP-optoATV- were imaged under a total internal reflection (TIRF) microscope. d, Left: A kymograph of a cellular protrusion showing single molecule traces of sfGFP-ATV+ moving down the tip. Scale, 1 µm. Right: Quantification of the velocity of ATV+ moving within cellular protrusions. Single dots represent single molecule traces chosen from 3 independent experiments (N = 23 traces). e, Left: A kymograph of a cellular protrusion where single molecule traces of sfGFP-optoATV- retracting back to the cell body upon light stimulation. Scale, 100 nm. Right: Quantification of the velocity of activated optoATV- moving within cellular protrusions. Single dots represent single molecule traces chosen from 3 independent experiments (N = 16 traces). The graphs are shown as mean ± SEM.

Source data

Extended Data Fig. 2 Bidirectional transport of bulky transmembrane SHH receptors within filopodia using ATVs in live cells.

a, Expression of sPtch1-eGFP with or without co-expression of mKate2-ATV+ in C3H/10T1/2 cells. Representative images chosen from 3 independent experiments. Scale, 20 µm. b, Quantification of the relative concentration of sPtch1-eGFP at the tip compared to the cell body (N = 33 and 41 filopodia each), based on images from (a). c, Expression of Disp1-eGFP with or without co-expression of mKate2-ATV- in C3H/10T1/2 cells. Representative images chosen from 3 independent experiments. Scale, 20 µm. d, Quantification of the relative concentration of Disp1-eGFP at the tip compared to the cell body (N = 86 and 96 filopodia each), based on images from (c). Statistics determined by two-tailed Mann-Whitney test. Both bar graphs displayed as mean ± SEM.

Source data

Extended Data Fig. 3 Inducing filopodia growth by doxycycline in a stable Cos7 cell line.

a, A Cos7 cell line stably expressing mKate2-ATV+ and Espin1-sfGFP (linked with a T2A linker) under a TetOn-3G system was generated. b, The cells were imaged with or without 24 hours of 2 µg/ml doxycycline induction. Filopodia growth was only observed when doxycycline was applied. Representative images chosen from 3 independent experiments. Scale, 20 µm. pmBFP was used to visualize full cell morphology. c, Quantification of the filopodia density of each individual cell (N = 10 cells each). Statistics determined by two-tailed Mann-Whitney test. The bar graph displayed as mean ± SEM.

Source data

Extended Data Fig. 4 Preventing the outgrowth of mDia2M/A-mediated artificial filopodia using ATV- in live cells.

a, Expression of sfGFP-mDia2M/A with or without co-expression of mKate2-ATV- in Cos7 cells. The mutant actin nucleating protein mDia2M/A could generate artificial filopodia, while mDia2M/A trapped inside the cell body by mKate2-ATV- could not. Representative images chosen from 3 independent experiments. Scale, 20 µm. b, Quantification of the filopodia density of each individual cell (N = 10 cells each). Statistics determined by two-tailed Mann-Whitney test. The bar graph displayed as mean ± SEM.

Source data

Extended Data Fig. 5 GBP-GFP interaction is necessary for preserving native function of the transported cargo in live cells.

a, Expression of mKate2-ATV+ & sfGFP-mDia2M/A independently or the covalently linked cargo-motor in Cos7 cells. The prior is based on the binding interaction of GBP-GFP while the latter is based on covalent linkage. GBP-GFP interaction preserved the function of mDia2M/A in generating filopodia, while covalently linkage disrupted cargo function and cells formed fewer filopodia. The white dashed line delineates the smooth cell border. Representative images chosen from 3 independent experiments. Scale, 20 µm. b, Quantification of the filopodia density of each individual cell (N = 10 cells each). Statistics determined by two-tailed Mann-Whitney test. The bar graph displayed as mean ± SEM.

Source data

Extended Data Fig. 6 OptoATV+ could generate filopodia de novo by transporting MyoXtail to the cell border upon blue light stimulation in live cells.

a, Expression of mKate2-optoATV+ and eGFP-MyoXtail in Cos7 cells, before and after one pulse of 200 ms blue light every 10 s. de novo growth of filopodia could be readily observed after 2 minutes. The white dashed line delineates the cell border where the filopodium was grown. Representative images chosen from 3 independent experiments. Scale, 5 µm. b, Quantification of filopodia density of each cell before and after light activation (N = 10 cells each). Statistics determined by two-tailed Mann-Whitney test. The bar graph displayed as mean ± SEM.

Source data

Extended Data Fig. 7 Mouse induced neuronal (iN) cells mature within 14 days of induction.

Immunostaining of maturing iN cells at day 5 (upper lane) or mature iN cells at day 14 (lower lane). Representative images chosen from 3 independent experiments. iN cells readily express the pan-neuronal marker MAP2 at day 5, but only express SYN1 until fully mature at day 14. Scale, 100 µm (upper) or 50 µm (lower).

Extended Data Fig. 8 Electroporation is efficient in the axolotl blastema.

Electroporated axolotl blastema tissue was harvested and stained in Hoechst solution prior to mounting and confocal imaging. a, The cell nucleus is clearly stained by Hoechst and used to indicate total cell number inside the field of view. Representative image chosen from 3 independent experiments. Scale, 20 µm. b, Quantification of cell density based on either Hoechst-stained nucleus number or pmKate2-labelled cell number (N = 10 fields of view each). Approximately 65% of blastema cells were efficiently electroporated. The bar graph displayed as mean ± SEM.

Source data

Extended Data Fig. 9 The actin-based filopodial extensions in the axolotl blastema are gradually developed during regeneration.

Electroporation of the high-affinity F-actin probe utrophin calponin homology domain fused to eGFP (UCHD-eGFP) and LifeAct-iRFP into axolotl blastema. a, Live imaging of UCHD-eGFP inside axolotl blastema at 3 dpa. Representative images chosen from 3 independent experiments. The z-stack of the images are pseudocolor-coded to show the morphology at different depths. Scale, 20 µm. b, Histogram plotting the distribution of filopodia lengths at 3 dpa and 7 dpa based on the UCHD-eGFP fluorescence images (N = 63 filopodia each).

Source data

Extended Data Fig. 10 The selective defects in the regenerated posterior digits are not caused by differences in the axolotl background or the light/dark cycle.

(a) Quantification of the non-amputated right forelimb digit length of the axolotls at 35 dpa (N = 6 animals each). (b) Quantification of the regenerated left forelimb digit length ratio of the axolotls that were electroporated with pmKate2 only, with either 0 hour or 24 hours of light provided for 35 days (N = 4 animals each). Statistics determined by two-tailed Welch’s ANOVA test or two-tailed t-test. Both bar graphs displayed as mean ± SEM.

Source data

Supplementary information

Reporting Summary

Supplementary Video 1

Single molecule imaging of ATV+ in cellular extensions. Total internal reflection (TIRF) microscopy was used to record single molecule traces of sfGFP-ATV+ in a C3H/10T1/2 cell. Single particles could be observed moving towards filopodia tips, where sfGFP-ATV+ accumulated and formed bright puncta. Images were acquired every 200 ms, for 20 s. Scale, 10 µm.

Supplementary Video 2

Activation of optoATV+ in live cells. mKate2-optoATV+ was expressed in C3H/10T1/2 cells and remained inactive and evenly distributed at dark. Pulses of 200 ms blue light were applied when indicated in the video to activate optoATV+. Bright puncta of optoATV+ represented the accumulation of optoATV+ at filopodia tips post activation. The bright puncta gradually dissolved in the dark as oligomerization became reversed. Images were acquired every 1 min, for 60 min. Scale, 20 µm.

Supplementary Video 3

Activation of optoATV+ could transport sPtch1 to filopodia tips upon blue light stimulation in live cells. mKate2-optoATV+ and sPtch1-eGFP were expressed in C3H/10T1/2 cells. OptoATV+ remained inactive and sPtch1-eGFP was dominantly distributed in the cell body at dark. Pulses of 200 ms blue light were applied after every image acquisition to activate the transportation of sPtch1-eGFP by optoATV+. Bright puncta of green fluorescence represented the accumulation of transported sPtch1-eGFP at filopodia tips post activation. Images were acquired every 2 min, for 10 min. Scale, 5 µm. pmiRFP was used to visualize full cell morphology.

Supplementary Video 4

Activation of optoATV+ could generate filopodial extensions in Cos7 cells by transporting MyoXtail. mKate2-optoATV+ and MyoXtail-eGFP were expressed in Cos7 cells. Acquisition of images and activation of optoATV+ by pulses of 200 ms of blue light were both set at once every 30 s. The outgrowth of filopodial extensions could be observed after 1 min. Scale, 5 µm. pmiRFP was used to visualize full cell morphology.

Supplementary Data 1

Full nucleotide sequences for the plasmids generated in this study.

Source data

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Zhang, Z., Denans, N., Liu, Y. et al. Optogenetic manipulation of cellular communication using engineered myosin motors. Nat Cell Biol 23, 198–208 (2021). https://doi.org/10.1038/s41556-020-00625-2

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