Research ArticleMICROROBOTS

Control of molecular shuttles by designing electrical and mechanical properties of microtubules

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Science Robotics  27 Sep 2017:
Vol. 2, Issue 10, eaan4882
DOI: 10.1126/scirobotics.aan4882
  • Fig. 1 Schematic representation of MT sorting under a given electric field.

    Designing MT properties controls the trajectories of kinesin-propelled MTs. Various types of MTs were polymerized under different conditions in microtubes. When an electric field, E, is applied perpendicular to gliding MTs, their gliding directions are gradually oriented toward the anode. MTs are transported toward different destinations corresponding to their stiffness and surface charge density.

  • Fig. 2 Control of MT persistence length by polymerizing conditions.

    (A) Time course of MT length in the presence of 30 μM (red diamonds) and 10 μM tubulin concentrations (black triangles). Means ± SD are shown. Solid lines show the best fit to an exponential function using the least-squares method. Dashed lines show least-squares fitting of lines for the first several minutes (R2 > 0.92). (B) The schematic representation of the measurement system. MTs were partially immobilized onto a biotin-coated substrate by streptavidin and biotinylated MT seeds. (C) Sequential images of a fluctuating MT (red). The left segment (light green) was immobilized. Scale bar, 5 μm. Images were (D) binarized and (E) skeletonized with the FIESTA software. (F) Superposition of the whole shape of a fluctuating MT for each frame. (G) Box plots of Lp for MT-1 (n = 114 measurements), MT-2 (n = 128), MT-3 (n = 100), MT-4 (n = 86), and MT-5 (n = 126). Red plus signs are outliers that are above the third quartiles by 1.5 interquartile ranges. There are no significant differences between identical lowercase letters by the Steel-Dwass test at a critical value of P < 0.01.

  • Fig. 3 Control of MT gliding directions via the bottom-up designing of MT electrical and mechanical properties.

    (A) Sequential images of a gliding MT-2 (highlighted). An electric field of 5.3 kV m−1 was applied from the right- to the left-hand side of images. White arrows indicate the leading tip of the MT. Scale bars, 10 μm. (B) Trajectory and fitted result for the MT shown in (A). The leading tip was tracked (blue dots) and fitted with Eq. 1 as a red line (R2 = 0.99) to obtain RMT. (C) Box plots of normalized RMT for MT-1 (n = 32), MT-2 (n = 141), MT-5 (n = 62), and MT-5′ (n = 62). RMT were normalized to mean RMT for MT-2. Red plus signs are outliers that are above the third quartiles by 1.5 interquartile ranges. No significant differences were observed between MT-1 and MT-2 (a). MT-5 (b) and MT-5′ (c) showed significant differences with (a) and with each other, as determined by the Steel-Dwass test at a critical value of P < 0.01. MT trajectories followed Eq. 1 with R2 > 0.98.

  • Fig. 4 Demonstration of MT sorting by integrating the top-down design of PDMS device with the bottom-up design of MT properties.

    (A) Schematic representation of the PDMS device. The device consisted of three areas: MT landing area (between reservoirs C and D), MT sorting area (between reservoirs A and B), and MT alignment area (between MT landing and sorting areas). Channels in the alignment area were 40 μm in length and 5 μm in width. The channel height was 10 μm. (B) Image of the fabricated PDMS device. Red ink indicates the channels. Scale bar, 3 mm. (C) Bright-field image of the cross section of MT landing, MT alignment, and MT sorting areas. Channels are filled with ink without leakage. Scale bar, 50 μm. (D) Sequential images of MT immobilization in the MT landing area. Flow generated by pressure differences among reservoirs prevented MTs from escaping to the MT alignment area. White solid lines represent the wall. Scale bar, 20 μm. Fluorescent images of MTs in (E) the MT landing area and (F) MT alignment area. Scale bar, 20 μm. Histograms of MT orientation angles in (G) MT landing (n = 70) and (H) MT alignment areas (n = 35). Probabilities are represented by blue bars. MT gliding directions were aligned in the MT alignment area with significant decreases in the SDs of the orientation angles. Bin width = π/19 rad. (I) MT-2 (purple dashed lines) and MT-5 (red solid lines) were sorted with 57.7% efficiency (n = 129). (J) MT-2 (purple dashed lines) and MT-5′ (orange solid lines) were sorted with 80.4% efficiency (n = 159). Blue triangles represent the PDMS separation wall. An electric field of 3 kV m−1 was applied in the negative x direction. The upper right corners of the MT alignment area are set to origin.

  • Table 1 The five MTs investigated.
    NameMT-1MT-2MT-3MT-4MT-5
    Growth rateSlowFast
    NucleotideGMPCPPGTP
    Tau bindingYesNoYesNo

Supplementary Materials

  • robotics.sciencemag.org/cgi/content/full/2/10/eaan4882/DC1

    Supplementary Text

    Fig. S1. Histogram of MT length at each sampling time.

    Fig. S2. Details for measuring Lp of MTs.

    Fig. S3. Histograms of Lp for MT-1 to MT-5.

    Fig. S4. Scatterplots of Lp versus length for MT-1 to MT-5.

    Fig. S5. Histograms of RMT for MT-1, MT-2, MT-5, and MT-5′.

    Fig. S6. Scatterplots of RMT versus length for MT-1, MT-2, MT-5, and MT-5′.

    Fig. S7. Design of the separation wall.

    Table S1. Parameter nomenclature.

    Movie S1. An example of fluctuating MT-5.

    Movie S2. Sorting of MT-2 and MT-5.

    Movie S3. Sorting of MT-2 and MT-5′.

  • Supplementary Materials

    Supplementary Material for:

    Control of molecular shuttles by designing electrical and mechanical properties of microtubules

    Naoto Isozaki, Hirofumi Shintaku, Hidetoshi Kotera, Taviare L. Hawkins, Jennifer L. Ross, Ryuji Yokokawa*

    *Corresponding author. Email: ryuji{at}me.kyoto-u.ac.jp

    Published 27 September 2017, Sci. Robot. 2, eaan4882 (2017)
    DOI: 10.1126/scirobotics.aan4882

    This PDF file includes:

    • Supplementary Text
    • Fig. S1. Histogram of MT length at each sampling time.
    • Fig. S2. Details for measuring Lp of MTs.
    • Fig. S3. Histograms of Lp for MT-1 to MT-5.
    • Fig. S4. Scatterplots of Lp versus length for MT-1 to MT-5.
    • Fig. S5. Histograms of RMT for MT-1, MT-2, MT-5, and MT-5′.
    • Fig. S6. Scatterplots of RMT versus length for MT-1, MT-2, MT-5, and MT-5′.
    • Fig. S7. Design of the separation wall.
    • Table S1. Parameter nomenclature.
    • Legends for movies S1 to S3

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). An example of fluctuating MT-5.
    • Movie S2 (.avi format). Sorting of MT-2 and MT-5.
    • Movie S3 (.avi format). Sorting of MT-2 and MT-5′.

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