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Optical control of fast and processive engineered myosins in vitro and in living cells

Nature Chemical Biology volume 17, pages 540–548 (2021)Cite this article

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Abstract

Precision tools for spatiotemporal control of cytoskeletal motor function are needed to dissect fundamental biological processes ranging from intracellular transport to cell migration and division. Direct optical control of motor speed and direction is one promising approach, but it remains a challenge to engineer controllable motors with desirable properties such as the speed and processivity required for transport applications in living cells. Here, we develop engineered myosin motors that combine large optical modulation depths with high velocities, and create processive myosin motors with optically controllable directionality. We characterize the performance of the motors using in vitro motility assays, single-molecule tracking and live-cell imaging. Bidirectional processive motors move efficiently toward the tips of cellular protrusions in the presence of blue light, and can transport molecular cargo in cells. Robust gearshifting myosins will further enable programmable transport in contexts ranging from in vitro active matter reconstitutions to microfabricated systems that harness molecular propulsion.

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Fig. 1: Engineering monomeric myosin XI motors for high speed and strong optical response.
Fig. 2: Directional control of processive myosins in vitro.
Fig. 3: Optically controlled localization of myosin motors in live cells.
Fig. 4: Single-molecule imaging of engineered motor constructs in live cells.
Fig. 5: Optically controlled localization of motors and molecular cargos in live cells.

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Data availability

Data for all results reported in this study are freely available as follows. The cryo-EM map and atomic model for the myosin XI–F-actin complex have been deposited under accession numbers EMDB-22808 and PDB 7KCH, respectively. Source data are available on the journal website as spreadsheet files linked to Figs. 1–5 and Extended Data Figs. 3 and 4. Source data for Supplementary Figs. 3–11 are available from the Stanford Digital Repository (https://doi.org/10.25740/65j8-6114). All unique biological materials generated for this study, including plasmids for purification of engineered motors and for live-cell experiments, are available from the authors on request. Source data are provided with this paper.

Code availability

Custom code used in this study is freely available from the Stanford Digital Repository (https://doi.org/10.25740/65j8-6114).

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Acknowledgements

This work was supported by a Human Frontiers Science Program (HFSP) long-term fellowship to P.V.R., an NSF graduate research fellowship to S.Z., a W.M. Keck Foundation grant to Z.B. and M. Prakash, HFSP grant RPG0023/2014, NIH R01 GM114627 to Z.B., and Stanford Bio-X seed grants to Z.B. and M.Z.L. and to P.-S.H. and Z.B. Work performed at the Stanford Nano Shared Facilities (SNSF) was supported by the National Science Foundation under award no. ECCS-1542152. Cryo-EM work was conducted at the Simons Electron Microscopy Center (SEMC) and the National Resource for Automated Molecular Microscopy (NRAMM) located at the New York Structural Biology Center, supported by NIH grant no. P41 GM103310, NYSTAR, and Simons Foundation grant no. SF349247. We thank N. Denans, M. Barna and Z. Zhang for useful discussions and for conducting and sharing results on early tests of engineered motors in live cells, T. Aksel and C. Espenel for advice on gliding filament analysis, P. Mangeol for providing a custom version of the KymographClear software to enable processing of ROI sets, S. Sutton for providing purified F-actin and members of the Bryant laboratory for discussions and assistance.

Author information

Author notes

  1. Rajarshi P. Ghosh

    Present address: Howard Hughes Medical Institute, and Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

  2. These authors contributed equally: Paul V. Ruijgrok, Rajarshi P. Ghosh.

Authors and Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Paul V. Ruijgrok, Rajarshi P. Ghosh, Sasha Zemsky, Muneaki Nakamura, Robert Chen, Vipul T. Vachharajani, Alexander E. Chu, Namrata Anand, Raphael R. Eguchi, Po-Ssu Huang, Michael Z. Lin, Jan T. Liphardt & Zev Bryant

  2. ChEM-H, Stanford University, Stanford, CA, USA

    Rajarshi P. Ghosh, Raphael R. Eguchi, Po-Ssu Huang & Jan T. Liphardt

  3. Bio-X Institute, Stanford University, Stanford, CA, USA

    Rajarshi P. Ghosh, Po-Ssu Huang, Michael Z. Lin, Jan T. Liphardt & Zev Bryant

  4. Cell Biology Division, Stanford Cancer Institute, Stanford, CA, USA

    Rajarshi P. Ghosh & Jan T. Liphardt

  5. Program in Biophysics, Stanford University, Stanford, CA, USA

    Sasha Zemsky, Vipul T. Vachharajani & Alexander E. Chu

  6. Laboratory of Structural Biophysics and Mechanobiology, The Rockefeller University, New York, NY, USA

    Rui Gong & Gregory M. Alushin

  7. Department of Neurobiology, Stanford University, Stanford, CA, USA

    Lin Ning & Michael Z. Lin

  8. Department of Biochemistry, Stanford University, Stanford, CA, USA

    Raphael R. Eguchi

  9. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA

    Zev Bryant

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  1. Paul V. Ruijgrok
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Contributions

P.V.R., M.N., R.C. and V.T.V. designed and produced engineered myosin constructs. P.V.R., S.Z., M.N. and V.T.V. designed and performed in vitro measurements. P.V.R. and S.Z. developed analysis code and analyzed in vitro data. R.P.G. designed and performed research in fibroblasts and epithelial cells, including the design and production of constructs and cell lines and consulting on analysis approaches. P.V.R. analyzed live-cell data and consulted on live-cell imaging. R.G. designed and performed cryo-EM research and analyzed structural data. L.N. designed and performed research in neurons. R.R.E., N.A. and A.E.C. designed and purified the FRET construct and contributed structural modeling expertise. Z.B., J.T.L., G.M.A., M.Z.L. and P.-S.H. supervised research. All authors discussed the results and contributed to writing the manuscript.

Corresponding author

Correspondence to Zev Bryant.

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Extended data

Extended Data Fig. 1 Cryo-EM analysis of F-actin decorated with rigor state myosin XI.

a, Cryo-EM map of the rigor state of the Chara myosin XI catalytic domain CM11L2+4 CD746 bound to F-actin, reconstructed at 4.3 Å. b, The gold-standard Fourier shell correlation (FSC) curve for the 3D reconstruction. The average resolution is estimated at the FSC value of 0.143. c, Local resolution estimation of the 3D reconstruction. d, Cryo-EM map of two actin and one myosin subunits with the ribbon model of myosin fitted. The terminal helix of the myosin converter domain (residues 738-746) is highlighted in cornflower blue; this helix can be extended by a fused lever arm as indicated by the dashed line.

Extended Data Fig. 2 Structural models and predicted stroke vectors.

a,b, Structural models of MyLOVChar and MyLOVChar4, displaying representative pre-stoke (transparent) and post-stroke (solid) models from a set of 50 top-scoring structures modeled using RosettaRemodel (Methods and Supplementary Table 1), with annotated actin polarity. Post-stroke models are based on an actin-bound rigor stroke structure of Chara corallina myosin XI structure (Fig. 1b and Extended Data Fig. 1). Pre-stroke structure models built from myosin V pre-stroke crystal structure were aligned to the actin-bound myosin XI rigor. Idealized power stroke sizes and directions in the dark state (arrows below structures) and in the lit state (arrows above structures) were calculated by projecting the motion of the effective tip of the lever arm along the actin filament for the 50 top-scoring structures, and recording the mean. The effective tip of the lever arm was taken as the position of the C-terminal residues in the dark state, and as the position of the last rigid element on the LOV2 domain in the lite state (G516) c, Tabulation of calculated stroke sizes and optical stroke modulation Mstroke for structural models of MyLOVChar-MyLOVChar5. Negative values indicate a stroke directed to the actin minus end. The stroke modulation Mstroke was calculated as 1- sdark/slit. A modulation larger than 1 indicates a directional reversal of the stroke.

Extended Data Fig. 3 Velocity traces of gliding filament assays on MyLOVChar4 at variable blue light intensity.

The traces are selected from the dataset used to compose Fig. 1h in the main text. Each trace displays the frame-to-frame velocity at the annotated blue light irradiance (box in the top right corner), averaged over all tracked polarity-labeled filaments in 2-3 consecutive repeats of the stimulation sequence. The initial dark velocity vdark,i was taken as the mean velocity of the time before the start of the blue light stimulus, indicated by the left gray dashed lines, and annotated for the bottom plot. The subsequent parts of the trace were globally fit to an exponential rise and decay, extracting the characteristic rates kon and koff, the steady-state lit velocity vlit (black dash-dotted lines), and the final velocity vdark,f (right gray dashed lines, annotated for bottom plot). In Fig. 1e and Fig. 1h the dark velocity vdark (black dashed lines) was reported, taken as the mean of vdark,i and vdark,f.

Source data

Extended Data Fig. 4 Dose-dependent conformational changes in a minimal LOV2-containing lever arm construct.

a, Block diagram and idealized molecular cartoon of a minimal lever arm construct with N-terminal SNAP-tag and C-terminal HaloTag fusions (approximate positions shown). HaloTag was labeled with TMR (donor) and SNAP-tag with SNAP-Cell 647-SiR (acceptor) for fluorescence measurements. b, Schematic of light-dose-dependent population of conformational states of the construct. Transparent elements in the lit state indicate increased flexibility. Positions of the fluorescent dyes are annotated (D: donor, A: acceptor). c, Fluorescence emission spectra normalized to the peak donor emission. Inset: extracted acceptor emission, obtained by subtracting the normalized emission from a donor-only sample from the normalized emission of the FRET sample. d, Schematic of the light stimulus experiment in bulk in solution. e-f,Time traces of fluorescence intensity immediately after a removal of a blue light stimulus (irradiance: 10 mW/cm2), with continued excitation of the donor dye. e, Fluorescence intensity collected at the emission maximum of the acceptor (665 nm). f, Fluorescence intensity collected at the emission maximum of the donor (570 nm), recorded on an independent run of the stimulus sequence, on the same sample as d using the same blue light irradiance. g, Amplitude of the normalized intensity difference of acceptor channel after recovery from a light stimulus as function of irradiance of the blue light, showing two replicate datasets (circular and square data points) taken on different days. Insets: time-dependent traces for measurements at the annotated irradiances. Black lines in e-f and insets in g are single exponential decays with amplitude A and characteristic time t. Listed in pairs (A, t), parameter values are e: (−7 %, 18 s), f: (+3 %, 20 s), g (left): (−2 %, 20 s), g (center): (−5 %, 20 s), g (right): (−7%, 18 s) s). Black line in g is a saturation curve with A=7 % at saturation, with half-saturation occurring at a dose of 1.6 mW/cm2. The average decay time <t> over all n = 20 experiments was 19 ± 1 s (mean ± s.e.m.).

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–11, Note 1 and Tables 1 and 2.

Reporting Summary

Supplementary Video 1

Rotating view of a post-stroke model of MyLOVChar bound to actin. The model was constructed by fusion of a cryo-EM reconstruction of rigor Chara corallina myosin XI (Extended Data Fig. 1) with crystal structures for domains in the engineered lever arm, via flexible backbone modeling with RosettaRemodel.

Supplementary Video 2

Gliding filament assays of MyLOVChar at 2 mM ATP. The blue background in frames 100–300 (at times 13.5–40.5 s) indicates the period when blue stimulation is turned on, with irradiance at the sample of 11 mW cm−2. The false-color video displays the fluorescence of actin filaments (labeled with TMR-phalloidin) in green. The red channel displays the Cy5 fluorescence from short gelsolin-capped filament seeds that mark the (+)-ends of the actin filaments. The overlay in white displays the result of the automated tracking analysis, marking the objects that were detected as polarity-labeled filaments with a circle around the actin (+)-end and a line along the detected filament.

Supplementary Video 3

Gliding filament assays of MyLOVChar2 at 2 mM ATP. The blue background in frames 100–300 (at times 13.5–40.5 s) indicates the period when blue stimulation is turned on, with irradiance at the sample of 11 mW cm[−2. The false-color video displays the fluorescence of actin filaments (labeled with TMR-phalloidin) in green. The red channel displays the Cy5 fluorescence from short gelsolin-capped filament seeds that mark the (+)-ends of the actin filaments. The overlay in white displays the result of the automated tracking analysis, marking the objects that were detected as polarity-labeled filaments with a circle around the actin (+)-end and a line along the detected filament.

Supplementary Video 4

Gliding filament assays of MyLOVChar3 at 2 mM ATP. The blue background in frames 100–300 (at times 13.5–40.5 s) indicates the period when blue stimulation is turned on, with irradiance at the sample of 11 mW cm−2. The false-color video displays the fluorescence of actin filaments (labeled with TMR-phalloidin) in green. The red channel displays the Cy5 fluorescence from short gelsolin-capped filament seeds that mark the (+)-ends of the actin filaments. The overlay in white displays the result of the automated tracking analysis, marking the objects that were detected as polarity-labeled filaments with a circle around the actin (+)-end and a line along the detected filament.

Supplementary Video 5

Gliding filament assays of MyLOVChar4 at 2 mM ATP. The blue background in frames 100–300 (at times 13.5–40.5 s) indicates the period when blue stimulation is turned on, with irradiance at the sample of 11 mW cm−2. The false-color video displays the fluorescence of actin filaments (labeled with TMR-phalloidin) in green. The red channel displays the Cy5 fluorescence from short gelsolin-capped filament seeds that mark the (+)-ends of the actin filaments. The overlay in white displays the result of the automated tracking analysis, marking the objects that were detected as polarity-labeled filaments with a circle around the actin (+)-end and a line along the detected filament.

Supplementary Video 6

Gliding filament assays of MyLOVChar5 at 2 mM ATP. The blue background in frames 100–300 (at times 13.5–40.5 s) indicates the period when blue stimulation is turned on, with irradiance at the sample of 11 mW cm−2. The false-color video displays the fluorescence of actin filaments (labeled with TMR-phalloidin) in green. The red channel displays the Cy5 fluorescence from short gelsolin-capped filament seeds that mark the (+)-ends of the actin filaments. The overlay in white displays the result of the automated tracking analysis, marking the objects that were detected as polarity-labeled filaments with a circle around the actin (+)-end and a line along the detected filament.

Supplementary Video 7

Detail from Supplementary Video 5, with annotated (−)-end directed motility and directional reversal of an individual actin filament.

Supplementary Video 8

Single-motor tracking of processive motility in MyLOVChar4~1R~TET-HaloTag motors, at 10 μM ATP. The false-color video displays fluorescence from the motors in red, superimposed on a static background of the immobilized actin filaments in green and the actin (+)-ends in yellow. The blue background indicates the period when the blue light was turned on. The motors were labeled with Alexa 660 and excited at 633 nm. The actin (+)-ends were labeled with Cy5 and also imaged with 633 nm excitation, before the start of the motor video. Actin filaments were labeled with TMR-phalloidin and imaged with 532 nm excitation after the acquisition of the motor video.

Supplementary Video 9

Gold nanoparticle tracking of MyLOVChar4L2(+4)~1R~TET. Three successively acquired videos are shown for the same region of interest. The false-color videos display fluorescent motors (green) and gold nanoparticle scattering (yellow) superimposed on a static background of immobilized actin filaments (red). Actin (+)-ends are annotated with white circles. Actin labeled with Alexa Fluor 633 was imaged before the start of the videos. Unidirectional (+)-end directed engineered Nicotiana tabacum myosin XI tetramers (NM11CD738 2R~1R~TET) labeled with TMR (top) were imaged before the gold nanoparticles, and were used to score the directionality of the actin filaments. Gold nanoparticles were then imaged with blue light on (middle) for one 100-second video and blue light off (bottom) for one 100-second video. Data were collected at 7 μM ATP.

Supplementary Video 10

Light-dependent localization of motors in live cells. The same optical stimulation assay is shown as in Fig. 3c, on a culture of fibroblast cells expressing MyLOVChar4R~1R~TET-SNAP-tag motors, fluorescently labeled with a far-red dye (SNAP-Cell 647-SiR). At each time point, the video displays the maximum intensity projection of a set of confocal z-stacks, acquired in a laser-scanning confocal microscope. The blue background indicates the period when the blue laser, co-scanning with the 639 nm excitation laser, was turned on (at 1% of nominal power).

Supplementary Video 11

Co-imaging of motor localization and cell morphology. The video displays simultaneous confocal fluorescence imaging and differential interference contrast on a mouse fibroblast cell expressing MyLOVChar4R~1R~TET-SNAP-tag, imaging the fluorescence of the far-red dye (SNAP-Cell 647-SiR). Differential interference contrast was acquired in the transmission path of the laser-scanning confocal microscope, with the laser light used to excite fluorescence (wavelength 639 nm).

Supplementary Video 12

Reduction of the population of cytoplasmic motors during optically controlled recruitment of motors to cellular protrusions. The video displays the fluorescence of MyLOVChar4R~1R~TET motors in a culture of mouse fibroblasts, during a period with blue light stimulation. Shown data are from a single plane from an acquired confocal z-stack, excited in the far red (639 nm wavelength). The blue background indicates the period when the blue light was turned on (a blue laser line in the laser-scanning confocal microscope, at 1% of nominal power).

Supplementary Video 13

Light-dependent localization of motors in hippocampal neurons. The video displays wide-field fluorescence images of MyLOVChar4~1R~TET~mRuby3 in cultured rat hippocampal neurons. The same optical stimulation assay is shown as in the top row of Fig. 3h. Fluorescence was excited with a xenon arc lamp filtered at 568/20 nm, and blue light stimulation (indicated by the blue background) was provided using an LED light source with an irradiance of approximately 10 mW cm−2.

Supplementary Video 14

Imaging of single motor complexes in live cells. The video corresponding to data in Fig. 4c. The motors are complexes formed by two tagged MyLOVChar4~1R~TET constructs: a dominant construct that is not imaged and a minor population of MyLOVChar4~1R~TET~ArrayG16X, which can bind up to 16 copies of the cytoplasmic population of wild-type green fluorescent protein (mwtGFP). The video was acquired at a frame rate of 10 Hz, with HILO illumination using a 488 nm laser.

Supplementary Video 15

Imaging of single motor complexes in live cells, showing detail on a different cell than in Supplementary Video 14, displaying motor trajectories in a single cellular protrusion. The motors are complexes formed by two tagged MyLOVChar4~1R~TET constructs: a dominant construct that is not imaged, and a minor population of MyLOVChar4~1R~TET~ArrayG16X, which can bind up to 16 copies of the cytoplasmic population of wild-type green fluorescent protein (mwtGFP). The video was acquired at a frame rate of 10 Hz, with HILO illumination using a 488 nm laser.

Supplementary Video 16

Light-dependent localization of motors and protein cargos in human breast epithelial cells. The videos display an optical stimulation assay from Fig. 5b on a MCF10a stable cell line expressing MyLOVChar4R1~1R~TET~SNAP~DFHR motors and a protein cargo. The motor construct is fluorescently labeled with a far-red dye (SNAP-Cell 647-SiR). Each frame is a maximum intensity projection of a confocal z-stack acquired in a laser-scanning confocal microscope. Blue light stimulation is provided by the 488 nm excitation used to visualize the eGFP cargo (that is sections of videos with visible fluorescence in green indicate periods of blue light stimulation), and motors are imaged with 639 nm excitation. These dual stable cell lines have been selected on antibiotics but not flow sorted, and show heterogeneous expression levels of both constructs. Enrichment of motors and cargos in cellular protrusions is visible in cells with strong expression of both components; cells with visible cargo expression but low motor expression appear green and do not show strong enrichment of cargo in protrusions, while cells with visible motor expression but low cargo expression appear red and show motor enrichment.

Supplementary Video 17

Light-dependent localization of motors and FERM~NB113~eGFP cargo in human breast epithelial cells The videos display an optical stimulation assay on an MCF10a stable cell line expressing MyLOVChar4R1~1R~TET~SNAP~DFHR motors and FERM~NB113~eGFP cargo. The motor construct is fluorescently labeled with a far-red dye (SNAP-Cell 647-SiR). Each frame is a maximum intensity projection of a confocal z-stack acquired in a laser-scanning confocal microscope. Blue light stimulation is provided by the 488 nm excitation used to visualize the eGFP cargo (that is, sections of videos with visible fluorescence in green indicate periods of blue light stimulation), and motors are imaged with 639 nm excitation. These dual stable cell lines have been selected on antibiotics but not flow sorted, and show heterogeneous expression levels of both constructs. Enrichment of motors and cargos in cellular protrusions is visible in cells with strong expression of both components; cells with visible cargo expression but low motor expression appear green and do not show strong enrichment of cargo in protrusions, while cells with visible motor expression but low cargo expression appear red and show motor enrichment.

Supplementary Video 18

Light-dependent localization of motors and integrin β3~NB113~eGFP cargo in human breast epithelial cells. The videos display an optical stimulation assay from Fig. 5d on an MCF10a stable cell line expressing MyLOVChar4R1~1R~TET~SNAP~DFHR motors and FERM~NB113~eGFP cargo. The motor construct is fluorescently labeled with a far-red dye (SNAP-Cell 647-SiR). Each frame is a maximum intensity projection of a confocal z-stack acquired in a laser-scanning confocal microscope. Blue light stimulation is provided by the 488 nm excitation used to visualize the eGFP cargo (that is, sections of videos with visible fluorescence in green indicate periods of blue light stimulation), and motors are imaged with 639 nm excitation. These dual stable cell lines have been selected on antibiotics but not flow sorted, and show heterogeneous expression levels of both constructs. Enrichment of motors and cargos in cellular protrusions is visible in cells with strong expression of both components; cells with visible cargo expression but low motor expression appear green and do not show strong enrichment of cargo in protrusions, while cells with visible motor expression but low cargo expression appear red and show motor enrichment.

Supplementary Video 19

Light-dependent localization of motors and integrin β3~NB113~eGFP cargo in human breast epithelial cells. The videos display an optical stimulation assay on an MCF10a stable cell line expressing MyLOVChar4R1~1R~TET~SNAP~DFHR motors and integrin β3~NB113~eGFP cargo. The motor construct is fluorescently labeled with a far-red dye (SNAP-Cell 647-SiR). Each frame is a maximum intensity projection of a confocal z-stack acquired in a laser-scanning confocal microscope. Blue light stimulation is provided by the 488 nm excitation used to visualize the eGFP cargo (that is, sections of videos with visible fluorescence in green indicate periods of blue light stimulation), and motors are imaged with 639 nm excitation. These dual stable cell lines have been selected on antibiotics but not flow sorted, and show heterogeneous expression levels of both constructs. Enrichment of motors and cargos in cellular protrusions is visible in cells with strong expression of both components; cells with visible cargo expression but low motor expression appear green and do not show strong enrichment of cargo in protrusions, while cells with visible motor expression but low cargo expression appear red and show motor enrichment.

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Ruijgrok, P.V., Ghosh, R.P., Zemsky, S. et al. Optical control of fast and processive engineered myosins in vitro and in living cells. Nat Chem Biol 17, 540–548 (2021). https://doi.org/10.1038/s41589-021-00740-7

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