Research ArticleCELL BIOLOGY

Dorsoventral polarity directs cell responses to migration track geometries

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Science Advances  31 Jul 2020:
Vol. 6, no. 31, eaba6505
DOI: 10.1126/sciadv.aba6505
  • Fig. 1 Cells migrate with different efficiencies through vertically and laterally confined microchannels.

    (A) Polarization of actin in HT-1080/LifeAct-GFP/H2B-mCherry fibrosarcoma cell invading the deep dermis along myofiber and fibrillar collagen-rich tissue structures. Images represent overview and detail obtained by multicolor multiphoton time-lapse microscopy 5 days after tumor implantation. Arrowheads, second harmonic generation (SHG)–positive collagen fibers. Asterisks, SHG-positive myofibers. Scale bar, 25 μm. (B) Normalized fluorescence intensities of LifeAct-GFP and SHG along the indicated line scan arrow from a representative cell. (C) Comparison of LifeAct-GFP peak intensities in individual cells relative to the position of myofiber (Myo) and collagen (Col) SHG signals (n = 10 cells; three mice). (D) Schematic representation of a cross-sectional view of vertical and lateral microchannels. (E) Dimensions of vertical and lateral channels, as measured by a profilometer (n = 40 channels). (F) Migration speeds of HT-1080 fibrosarcoma cells in lateral, vertical, and unconfined microchannels (n ≥ 241 cells; four independent experiments). (G) Phase-contrast image of contiguous microchannels. Cells first experience lateral confinement before transitioning to vertical confinement. Scale bar, 40 μm. (H) Migration speeds of HT-1080 cells inside contiguous channels experiencing first lateral and then vertical confinement (left) or vice versa (right) (n = 150 cells; three independent experiments). (I) Migration speeds of HT-1080 cells in lateral/vertical channels when the basal glass slide of the channel is coated with a thin layer of PDMS (n ≥ 101 cells; two independent experiments). Data represent the mean ± SD (E, F, H, and I) or median (C). **P < 0.01 relative to lateral/unconfined control; §§P < 0.05 relative to myofiber.

  • Fig. 2 Dorsoventral polarity determines the efficiency of cell migration in confinement.

    (A) Representative images of HT-1080 cell stained for actin on its ventral and dorsal surfaces on 2D. Scale bars, 5 μm. (B to D) Actin intensity on the ventral and dorsal surfaces of cells fixed and stained with actin phalloidin on 2D (n = 20 cells; two independent experiments) (B) or in lateral (n = 21 cells; two independent experiments) (C) and vertical (n = 53 cells; three or more independent experiments) (D) microchannels. Actin intensity was normalized to the ventral layer. (E) Schematic representation of a microfluidic device in which the 2D cell seeding area is orthogonal (YZ plane) to the typically used basal (XY plane) seeding region. (F) Migration speeds of HT-1080 cells in vertical and lateral microchannels, as assessed after seeding cells on a basal (XY) or orthogonal (YZ) seeding region (n ≥ 96 cells; four independent experiments). (G) Migration speeds of HT-1080 cells in lateral and vertical microchannels treated with poly-l-lysine (PLL) and methoxy poly(ethylene glycol) (mPEG) succinimidyl valerate (n ≥ 61 cells; three independent experiments). Data represent the mean ± SD; **P < 0.01 relative to ventral; ##P < 0.01 relative to vertical basal-XY; ¶¶P < 0.01 relative to lateral basal-XY; §§P < 0.01 relative to lateral orthogonal-YZ.

  • Fig. 3 Channel geometry mediates phenotypic switching of polarized cells by spatially regulating RhoA activity.

    (A) Representative XY/YZ images of a mesenchymal and blebbing cell fixed and stained with actin phalloidin (green) and Hoechst (blue) in lateral and vertical confinement, respectively. Scale bar, 5 μm. (B) Percentage of HT-1080 cells migrating with mesenchymal versus blebbing phenotypes in lateral and vertical confinement (n = 3 independent experiments; ≥20 cells per experiment). (C) Average phenotype score (0, mesenchymal; 1, blebbing) of live LifeAct-GFP–labeled HT-1080 cells during migration through contiguous channels (n = 50 cells; three independent experiments). (D) Donor fluorescence lifetime of RhoA activity biosensor inside vertical and lateral microchannels and on 2D, as measured by FLIM-FRET (n ≥ 27 cells; four independent experiments). (E) Spatial distribution of RhoA activity inside vertical microchannels as measured by FLIM-FRET (n ≥ 35 cells; five independent experiments). (F) Heat map of RhoA activity biosensor of representative cells inside vertical or lateral microchannels, as imaged by FLIM-FRET. ns, nanoseconds; DIC, differential interference contrast. Scale bars, 10 μm. (G) Percentage of control, Y27632-treated (10 μM), or constitutively active RhoA (Q63L)–expressing HT-1080 cells, migrating with mesenchymal versus blebbing phenotypes in lateral and vertical confinement (three or more independent experiments; ≥20 cells per condition). Values represent the mean ± SD (D and E) or the mean ± SEM (B, C, and G); *P < 0.05 and **P < 0.01 relative to lateral control; ##P < 0.01 relative to vertical control; §§P < 0.01 relative to 2D; $$P < 0.01 relative to vertical front; P < 0.05 and ¶¶P < 0.01 relative to vertical rear.

  • Fig. 4 Optimal levels of contractility promote efficient cell migration in confinement.

    (A) Migration phenotypes of HT-1080 vehicle control and blebbistatin-treated cells (n = 3 independent experiments; ≥20 cells per condition). (B) Migration speeds of vehicle control and blebbistatin-treated (2 or 50 μM) HT-1080 cells (n ≥ 61; three independent experiments). (C) Migration phenotypes of scramble control (SC) and MIIA- and/or MIIB-knockdown HT-1080 cells (n ≥ 3 independent experiments; ≥20 cells per condition). (D) Images and (E) quantification of perinuclear myosin (XY plane, dorsal surface) from representative HT-1080 cells expressing MIIA-GFP and stained with Hoechst on 2D or in vertical (±2 μm of blebbistatin) or lateral confinement. White arrowheads indicate representative myosin fibers (n = 3 independent experiments). Scale bars, 2 μm. (F and G) Average number (F) and area (G) of paxillin-GFP–labeled FAs on 2D and inside lateral and vertical microchannels (n ≥ 24 cells; four independent experiments). (H) Migration phenotypes of vehicle control and low-dose (0.25 μM) FAK-treated HT-1080 cells in vertical and lateral confinement (n = 4 independent experiments; ≥20 cells per experiment). (I) Migration speeds of vehicle control and low-dose FAK-treated HT-1080 cells (n ≥ 170 cells; three independent experiments) in vertical and lateral confinement. Values represent the mean ± SD (B, F, G, and I) or the mean ± SEM (A, C, E, and H). *P < 0.05 and **P < 0.01 relative to lateral control; #P < 0.05 and ##P < 0.01 relative to vertical control.

  • Fig. 5 The nucleus becomes stiffer and acts as a mechanical barrier in vertical confinement.

    (A) Nuclear entry time of H2B-mCherry–labeled LMNA-KD (knockdown) or scramble control HT-1080 cells (n ≥ 143; three independent experiments) in lateral and vertical channels. (B) Representative heat map of Brillouin shift for 2D, vertically, and laterally confined cells. Scale bars, 10 μm. (C) Nuclear Brillouin shift for scramble control, LMNA-KD–treated, blebbistatin (2 μM)–treated, and TSA (100 ng/μl)–treated HT-1080 cells in 2D, lateral, and vertical confinement (n ≥ 13 cells; two or more independent experiments). (D) Nuclear Brillouin shift of cells in the vertical and lateral segments of contiguous microchannels, where cells experienced vertical and subsequently lateral confinement (n ≥ 9 cells; two independent experiments). (E) Heat map of Brillouin shift for an individual cell migrating, first, through the vertical and, subsequently, the lateral segment of a contiguous microchannel. Scale bars, 10 μm. Values represent the mean ± SD; **P < 0.01 relative to lateral control; ##P < 0.01 relative to vertical control; §§P < 0.01 relative to 2D control.

  • Fig. 6 Nuclear stiffness regulates RhoA activity and cell migration phenotype in confinement.

    (A) Migration speeds of HT-1080 scramble control and LMNA-KD cells (n ≥ 149; three independent experiments) using two different shRNA sequences. (B) RhoA activity of scramble control and LMNA-KD HT-1080 cells, as measured by FLIM-FRET (n ≥ 14 cells; two or more independent experiments). (C) Migration phenotypes of scramble control and LMNA-KD HT-1080 cells, as assessed after fixing and staining cells with actin phalloidin (n ≥ 3 independent experiments; ≥20 cells per condition). (D) Migration phenotypes of vehicle control and TSA-treated (100 ng/μl) HT-1080 cells, as assessed after fixing and staining cells with actin phalloidin (n ≥ 3 independent experiments; ≥10 cells per condition). (E) Quantification of phosphorylated LMNA (pLMNA) per cell for vehicle control versus blebbistatin-treated (2 μm) cells, as measured from the average intensity projection of cells fixed and stained for pLMNA (n ≥ 29 cells; two independent experiments). Values represent the mean ± SD (A, B, and E) or means ± SEM (C and D). *P < 0.05 and **P < 0.01 relative to lateral control; #P < 0.05 and ##P < 0.01 relative to vertical control; §P < 0.05 relative to 2D control.

Supplementary Materials

  • Supplementary Materials

    Dorsoventral polarity directs cell responses to migration track geometries

    Emily O. Wisniewski, Panagiotis Mistriotis, Kaustav Bera, Robert A. Law, Jitao Zhang, Milos Nikolic, Michael Weiger, Maria Parlani, Soontorn Tuntithavornwat, Alexandros Afthinos, Runchen Zhao, Denis Wirtz, Petr Kalab, Giuliano Scarcelli, Peter Friedl, Konstantinos Konstantopoulos

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