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Macromolecular crowding acts as a physical regulator of intracellular transport

Nature Physics volume 16, pages 1144–1151 (2020)Cite this article

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Abstract

Eukaryotic cell processes depend on molecular motors to transport cargo along cytoskeletal filaments, and the response of these mechanoenzymes to external forces shapes their cellular function. These responses have been largely mapped out in dilute, in vitro media. The cytosol, however, is host to a high concentration of macromolecules, and this crowding can alter protein conformation, binding rates, reaction kinetics and therefore motor function. Here, we use live-cell and single-molecule imaging and optical tweezer force measurements to uncover the consequences of macromolecular crowding on cargo transport by kinesin-1 motors. Surprisingly, we find that crowding significantly slows transport by teams of motors, while having no effect on single-motor velocity. We find that this emergent property of kinesin teams results from the increased sensitivity of the individual motors to hindering load when in a crowded medium. We explain this increased sensitivity using a model where entropic forces due to crowding push the two kinesin heads together into a more compact configuration when the motor is poised to take a step. Our results highlight the importance of motor–motor interactions in cargo transport, explain the long-observed variability of cargo velocity and suggest the use of crowding as a control parameter to study kinesin’s mechanochemical cycle.

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Fig. 1: Crowding slows down transport of cargo hauled by multiple kinesins both in vivo and in vitro.
Fig. 2: Crowding has no effect on single-motor velocity or run length under zero force.
Fig. 3: Crowding changes the detachment sensitivity but not the velocity of kinesin under hindering load, while the motor’s detachment sensitivity under assisting load is unaltered.
Fig. 4: The velocity of cargo carrying multiple kinesins increased with assisting force, in contrast to the behaviour of single motors.
Fig. 5: A molecular model of how crowding could alter kinesin’s sensitivity to hindering force.

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Data availability

Source data are provided with this paper. All other data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported in part by the National Science Foundation grants PHY-1505020 and PHY-1915119 to G.T.S. We thank G. Esposito and D. Chaudhury for helpful discussions and access to instrumentation. The research was partially carried out using Core Technology Platform resources at New York University Abu Dhabi.

Author information

Author notes

  1. Guilherme Nettesheim

    Present address: Department of Physics, Cavendish Laboratory, University of Cambridge, Cambridge, UK

  2. Gabriel R. Jaffe

    Present address: Department of Physics, University of Wisconsin—Madison, Madison, WI, USA

  3. These authors contributed equally: Ibtissem Nabti, Chandrashekhar U. Murade, Gabriel R. Jaffe.

Authors and Affiliations

  1. Center for Nonlinear Dynamics and Department of Physics, The University of Texas at Austin, Austin, TX, USA

    Guilherme Nettesheim, Gabriel R. Jaffe & George T. Shubeita

  2. New York University Abu Dhabi, Abu Dhabi, United Arab Emirates

    Ibtissem Nabti, Chandrashekhar U. Murade & George T. Shubeita

  3. Burnett School of Biomedical Sciences, University of Central Florida, Orlando, FL, USA

    Stephen J. King

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Contributions

G.T.S. conceived the study, G.N., G.T.S., I.N. and C.U.M. designed and performed experiments and analysed data, G.N. created the analytic model for transport by teams of kinesin motors with contributions from G.T.S., G.R.J. developed data acquisition, feedback control and data analysis software, S.J.K. provided reagents and G.N. and G.T.S. wrote the paper. All authors discussed the results and implications and commented on the manuscript at various stages.

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Correspondence to George T. Shubeita.

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Peer review information Nature Physics thanks Erwin Peterman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The slowdown of lipid droplets in the shrunken Drosophila embryos is not due to increased viscous drag forces or lack of ATP.

a, The force–velocity curve for kinesin-1 (solid line) [Schnitzer et al. 39] and its derivative (dashed line) show that an unhindered motor (small force) is not very sensitive to force. Conversely, a motor that is insensitive to force, yet still moving relatively fast, must be unhindered. b, Lipid droplets moved by kinesin-1 were separated into two groups on the basis of their size as determined from the DIC microscopy images. The mean velocities of the large and small lipid droplets were indistinguishable both in the control (p-value = 0.38, Student’s t-test, Nsmall = 16, Nlarge = 41) and shrunken embryos (p-value = 0.44, Student’s t-test, Nsmall = 8, Nlarge = 23). This indicates that the slowdown in the shrunken embryos is not due to increased drag forces since drag forces would affect the large lipid droplets more severely. Error bars are SEM. c, Optical trapping stall force measurements on lipid droplets in the control embryos show that large and small lipid droplets are moved on average by the same number of motors since the average stall force is the same (p-value = 0.36, Student’s t-test Nsmall = 34, Nlarge = 56). Error bars are SEM. Lipid droplet diameter ranged between 350 nm and 1000 nm. To accentuate the difference in size for the analyses in (b) and (c), all droplets falling around the median size were not used in the comparison. This resulted in the mean diameter of the large group being approximately 1.5 times that of the small group. (d) Lipid droplets moved by kinesin-1 were tracked in permeabilized shrunken embryos in the presence of 2 mM ATP or 1 mM GTP in the incubation medium. Lipid droplets maintained their reduced speed suggesting that lack of ATP or GTP is not responsible for the slowdown.

Extended Data Fig. 2 Kinesin teams slow down with increasing crowder concentration.

a, Trajectories of beads carried by kinesin-1 teams in control and crowded media. b, Histogram of bead velocity measured from a linear fit to the whole trajectory rather than fitting segments of constant velocity as done in Fig. 1d in the main text. The two histograms show the same pronounced slowdown in the crowded medium. c,The velocity of beads carrying teams of kinesins-1 drops progressively with increasing crowder concentration (left) but no slowdown is observed when a single kinesin-1 motor moved the bead in media crowded with BSA or Ficoll 70. Error bars are SEM. d, The drag force on a 500 nm cargo bead moved within a bead diameter from the surface at various speeds in crowded media is measured using the optical trap. At the typical motor speeds smaller than 1 µm/s, the drag force is smaller than 0.1 pN, too small to slow down kinesin. Assuming Stokes’ drag, the viscosity in BSA and Ficoll 70 is smaller than 25 mPa.s and 7 mPa.s, respectively. Given the proximity to the surface where drag forces are increased compared to bulk, these values of viscosity are upper limits.

Extended Data Fig. 3 The slowdown of kinesin teams is not due to increased ionic strength or osmolality, reduced pH, spurious surface effects, or reduced kinesin length and is not rescued by increased ATP or reduced salt concentrations.

a, The pH of the crowded media was measured using a ratiometric pH indicator (SNARF-4F 5-(and-6)-Carboxylic Acid). For both 30% BSA and 15% Ficoll, the indicator signal places the pH between 6.7 and 6.9. This is consistent with the pH meter measurement of 6.88 for these media. The line passing through the indicator calibration data (black squares) serves as a guide to the eye. b, The velocity of beads hauled by multiple kinesin motors was measured under various buffer conditions to rule out the possibility that changes in the effective salt or ATP concentrations, pH, or osmolality in the crowded medium are causing the slowdown. Each condition was assayed side-by-side on a dual-channel chamber with the standard control or crowded medium to which it is compared in the figure. Doubling the salt concentration, matching the osmolality to that of the crowded buffer using sorbitol, or reducing the pH to 6.0 do not result in the slowdown observed in the presence of crowders. Similarly, the slowdown in crowded buffer is not rescued by increased ATP or reduced salt concentrations. c, It was also conceivable that crowding promotes non-specific interactions of the motors with the chamber surface. Such interactions would add a hindering external force to the cargo, thus slowing it down. Repeating the experiment with microtubules elevated from the surface as described in the methods in the Supplementary Information showed the same reduction in cargo velocity in the crowded solution, leading us to rule out surface effects. d, We tested whether crowding promotes kinesin bound to the beads to fold around its hinge which could affect the activity and the mechanical properties of the motors. To that end, a full-length enzymatically inactive kinesin mutant that binds to microtubules but is unable to move along was diluted to the single molecule level on beads, and the jiggling of the tethered bead once the motor bound the microtubule was recorded. A shorter kinesin should result in a smaller extent of bead jiggling which we quantify using the standard deviation of the position perpendicular to the microtubule measured over a period of 8 minutes for 6–8 different beads. While crowding results in a slightly smaller extent of jiggling, the difference is not statistically significant (p = 0.35, Student t-test). Error bars are S.E.M.

Supplementary information

Supplementary Information

Materials and methods; method to measure motor force-dependent run length using a stationary trap; analytical model for multiple-motor transport subject to assisting force; model of kinesin’s force-dependent stepping explaining the motor’s unchanged zero-force run length and its increased sensitivity to force in the presence of crowders.

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Nettesheim, G., Nabti, I., Murade, C.U. et al. Macromolecular crowding acts as a physical regulator of intracellular transport. Nat. Phys. 16, 1144–1151 (2020). https://doi.org/10.1038/s41567-020-0957-y

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