Abstract
Cells migrating through complex three-dimensional environments experience considerable physical challenges, including tensile stress and compression. To move, cells need to resist these forces while also squeezing the large nucleus through confined spaces. This requires highly coordinated cortical contractility. Microtubules can both resist compressive forces and sequester key actomyosin regulators to ensure appropriate activation of contractile forces. Yet, how these two roles are integrated to achieve nuclear transmigration in three dimensions is largely unknown. Here, we demonstrate that compression triggers reinforcement of a dedicated microtubule structure at the rear of the nucleus by the mechanoresponsive recruitment of cytoplasmic linker-associated proteins, which dynamically strengthens and repairs the lattice. These reinforced microtubules form the mechanostat: an adaptive feedback mechanism that allows the cell to both withstand compressive force and spatiotemporally organize contractility signalling pathways. The microtubule mechanostat facilitates nuclear positioning and coordinates force production to enable the cell to pass through constrictions. Disruption of the mechanostat imbalances cortical contractility, stalling migration and ultimately resulting in catastrophic cell rupture. Our findings reveal a role for microtubules as cellular sensors that detect and respond to compressive forces, enabling movement and ensuring survival in mechanically demanding environments.
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Data availability
pET28a-Halotag-CNA35 is available from Addgene (#131128). All other data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.
Code availability
The numerical simulations were implemented in the programming language Julia. The source codes to reproduce the simulations visualized in Fig. 8 can be downloaded from the GitHub repository at https://github.com/alistairfalconer/NCB_Compression-Dependent-Microtubule-Reinforcement/.
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Acknowledgements
We thank A. Yap, D. Bryant, T. Wittmann, A.J. (Jeffrey) van Haren, S. Dumont, D. Barber, A. Molines and M. Samuels for discussion and comments on the manuscript. We apologize to colleagues whose work could not be cited due to space constraints. We also thank TRI Flow and Microscopy facility staff, D. Khalil, Y. Ding, S. Roy and A. Ju and IMB microscopy facility staff. We acknowledge L. Lavis for his contribution of Janelia Fluorophores. Research was conducted in a facility constructed with support from the Australian Cancer Research Foundation (ACRF)/Institute for Molecular Bioscience Cancer Biology Imaging Facility, which was established with the support of the ACRF. Part of the research was carried out at the TRI, which is supported by a grant from the Australian Government. This work was performed, in part, at the Queensland node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano- and micro-fabrication facilities for Australia’s researchers. This work was supported by Australian Research Council fellowship FT190100516 and Rebecca Cooper Medical Foundation grant PG2018168 (S.J.S.), Company of Biologists Travel Award JCSTF1903138 (R.J.J.). Australian Research Council grant DP180102956 (D.B.O.), Australian Research Council fellowship FT200100899 (M.D.W.), National Health and Medical Research Council APP1084893 and the Meehan Project Grant 021174 2017002565 (N.K.H.), National Health and Medical Research Council Senior Research Fellowship 1147364 (P.T.), Cancer Institute New South Wales Early Career fellowship (M.N.) and The National Institutes of Health, R35GM133522 (R.P.F.), RM1GM145399 (K.M.D. and G.D.) and Fellinger Krebsforschung grant SENSECARE 2023-07-0046 (A.J.L.).
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S.J.S. conceptualized and administered the project. R.J.J. designed, performed and analysed biological experiments with assistance from S.J.S. and M.D.W. C.J.S. contributed KASH reagent generation, experiments and data analysis. M.E.M. and R.J.J. contributed collagen hydrogel experiments. D.P.S. developed flow cytometry protocols for stable cell line establishment. A.D.F. and D.B.O. performed mathematical simulations, prepared mathematical figures and developed the mathematical model with input from S.J.S. R.J.J. and M.D.W. S.J.S., R.J.J., C.J.S., M.E.M. and M.D.W. prepared data and schematics for visualization. R.J.J. prepared video data for publication. P.T., M.N., K.M.D., G.D. and R.P.F. provided access to instrumentation, computing resources, analysis tools (light-sheet microscopes, FRET biosensors, data curation and software) and supervision. S.J.S., N.K.H. and D.B.O. were responsible for funding acquisition. S.J.S. and R.J.J. wrote the original draft. S.J.S., R.J.J., M.D.W., C.J.S., A.J.L., K.M.D., G.D., N.K.H. and D.B.O. reviewed the manuscript. S.J.S., R.J.J., M.D.W. and D.B.O. edited the manuscript.
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Extended data
Extended Data Fig. 1 Microtubule morphology in 2D and 3D collagen hydrogel models.
(a) 1205Lu melanoma cell CRISPR-edited to express meGFP-α-tubulin migrating in 2D. LUT inverted to enhance visibility of microtubule organization. (b) 1205Lu-meGFP-α-tubulin (orange heatmap) expressing cell migrating through a CNA35-Halo-JF549 labelled collagen I hydrogel (2.5 mg/mL; cyan heatmap) imaged by spinning disc confocal microscopy (SDCM). Visible distention of the cell body and nucleus can be observed where microtubules are seen to outline. (c) 1205Lu melanoma cell invading through a collagen hydrogel (DQ Collagen; white) labelled for F-actin (phalloidin, magenta) and nucleus (DAPI, cyan). Collagen aligns at cell-matrix attachment points (white arrowheads). The nucleus deforms as the cell invades (yellow arrowheads).
Extended Data Fig. 2 Validation of CLASP depletion phenotype.
(a) Immunoblot of lysates from 1205Lu melanoma cells expressing different shRNA constructs after seven days of puromycin selection. Tubulin was used as a loading control. Blots were probed with paralog specific antibodies to either CLASP1 or CLASP2. (b) Representative frames of timelapse images of 1205Lu melanoma cells expressing mCherry-H2B (nuclei, magenta) depleted of CLASP2, embedded in a collagen I hydrogel (2.5 mg/mL). (c) Cell migration tracks (spider plots) of control (non-targeting) and CLASP-depleted (CLASP1 shRNA) cells. n = 29 cells per condition. Data shown represents one experiment. Experiments were performed two times with similar results. Data displayed as spider plot. All data points displayed on graph. (d) Speed and Displacement measurements of cells migrating in collagen hydrogels. Track displacement from n = 165 control cells and n = 91 CLASP1 shRNA cells. Average track speed from n = 166 control cells and n = 91 CLASP1 shRNA cells. Data shown represents one experiment. Experiments were performed two times with similar results. Data displayed as violin plot indicating minimum, first quartile, median, third quartile, and maximum, with width indicating frequency of values. All data points displayed on graph. Statistical analysis by two-tailed Mann-Whitney Test. (e) Immunofluorescence of control (non-targeting) and CLASP1- and CLASP2-depleted 1205Lu melanoma cells labelled for annexin V (cyan) and nuclei (Hoechst; magenta). (f) Quantitation of the percentage of annexin v positive cells in control and CLASP-depleted cells. Parental n = 5, nt (non-targeting) n = 10, CLASP1 hairpins 32 n = 10, sh33 n = 10, and CLASP2 hairpins sh55 n = 10, sh58 n = 10, n = fields of view from low magnification images. Data shown represents one experiment. Experiments were performed two times with similar results. Data displayed as dot plot +/- s.d. All data points displayed on graph. (g) Control (parental), and alternative CLASP1 and 2 hairpins from Fig. 1d (shRNA #32, #33 and #55, 58, respectively), 1205Lu melanoma cells embedded in collagen I hydrogels (collagen, 2.5 mg/mL) labelled for acetylated α-tubulin (AcK40). AcK40 fluorescence intensity displayed as a LUT heatmap; purple indicating low with yellow-red indicating high. (h) Immunofluorescence of 2D control (parental, nt; non-targeting), CLASP1-(shRNA#32, #33) and CLASP2-depleted (#55, #58) 1205Lu melanoma cells labelled for acetylated α-tubulin (AcK40, contrast inverted). (i) Immunoblot of tubulin pelleting assay lysates of 1205Lu melanoma cells expressing non-targeting (control), CLASP1 or 2 shRNA constructs. CLASP depletion does not alter the levels of polymerized microtubules. (j) Representative images of control (non-targeting), CLASP1-depleted (CLASP1 shRNA #33) and cold treated 1205Lu cells stably coexpressing meGFP-α-tubulin and Snap-H2B labelled with JF646. (k) Quantitation of soluble to polymerized microtubules in conditions listed in j. n = 162 ROIs, pooled from 6 individual cells per condition. Data shown represents one experiment. Experiments were performed two times with similar results. Data displayed as a dot plot shows mean ± SD. Source numerical data and unprocessed original blots are available in Source data.
Extended Data Fig. 3 Characterization of CLASP and microtubule-dependent migration phenotypes in PDMS microchannels.
(a) Simplified graphical schematic of constriction microchannel design. (b) LUT inverted timelapse sequences (spinning disc confocal microscopy) of control (non-targeting), CLASP1 and CLASP2-depleted 1205Lu cells endogenously expressing tagged microtubules (meGFP-α-tubulin). (c) Cell types that prefer A1 migration modes do not rupture in constriction microchannels in the absence of CLASP1. Non-targeting; teal shades and CLASP1 shRNA; purple shades), U251 glioma, TIF telomerase-immortalized normal fibroblasts, RPE1 retinal pigment epithelial cells. U251 control n = 45 cells, CLASP shRNA n = 41 cells. TIF control n = 45 cells, CLASP shRNA n = 57 cells. RPE1 Control n = 45 cells, CLASP shRNA n = 49 cells. Data shown represents one experiment. Experiments were performed two times with similar results. The box-and-whisker plot shows median, first and third quartile (box) and 95% confidence intervals (notches) with whiskers extending to the furthest observations within 1.5 times the interquartile range. Dots are all individual data points. Purple dots indicate dead cells, light grey dots indicate live cells. Statistical analysis by two-tailed Mann-Whitney Test. (d) Control and CLASP-depleted 1205Lu cells migrating in straight channels (5 ×10-microns). (e) Quantitation of the number of control (non-targeting) and CLASP1-depleted (CLASP1 shRNA) cells per field of view (FOV) present in straight channels after 20 hours. Control n = 11, CLASP1 shRNA n = 18 field of views. Data shown represents one experiment. Experiments were performed two times with similar results. Data displayed as violin plot indicating minimum, first quartile, median, third quartile, and maximum, with width indicating frequency of values. All data points displayed on graph. Statistical analysis by two-tailed Mann-Whitney Test. (f) Quantitation of the percentage of cell survival in straight channels after 20 hours., Control n = 11, and CLASP1 shRNA n = 18 fields of views. Data shown represents one experiment. Experiments were performed two times with similar results. Data displayed as a bar chart, mean ∓SD. Statistical analysis by two-tailed Mann-Whitney Test. (g) Representative image of a CLASP1-depleted 1205Lu cell endogenously co-expressing meGFP-α-tubulin (grey; contrast inverted) and Snapf-H2b-JF549 (nuclei; magenta) migrating in confinement. Annexin positivity (annexin-V-647; cyan) occurs at 11 h. (h) Representative images of 1205Lu cells migrating in constriction circular array microchannels treated with microtubule targeting agents, nocodazole and taxol. Concentrations displayed on panel. Control treatment is DMSO. (i) Quantitation of h. Control n = 11, 3.3 µM Nocodazole n = 8, 100 nM Nocodazole n = 10, 1 µM Taxol n = 10 individual fields of view. Data shown represents one experiment. Experiments were performed two times with similar results. The box-and-whisker plot shows median, first and third quartile (box) and 95% confidence intervals (notches) with whiskers extending to the furthest observations within 1.5 times the interquartile range. All data points displayed on graph. Statistical analysis by Kruskal-Wallis test with two-stage step-up Benjamini, Kriefer and Yekutieli correction method to control False Discovery Rate. q-values shown on graph. (j) Example of a nuclear translocation stages- early, mid and post constriction phases. Nuclear bleb depicts nuclear herniation event as a result of constriction. Source numerical data are available in Source data.
Extended Data Fig. 4 EB1 spatio-temporal dynamics in confinement microchannels.
(a) Representative images of EB1 temporal dynamics in a cell undergoing nuclear transmigration. EB1 tracks are displayed as a Temporal projection of 0–360 seconds. Nucleus outlined in teal. (b) Analysis of EB1 comet mean track speed in 2D and in the front and rear compartment of cells undergoing nuclear transmigration in microchannels. Mean track speeds from n = 60 tracks per condition. Data shown represents one experiment. Experiments were performed two times with similar results. Statistical analysis by two-tailed Mann-Whitney Test. (c) Correlation of the mean EB1 comet track speed and EB1 track distance for pre, mid and post nuclear constriction phases in front and rear cell compartments from data in b. Statistics test by Pearson correlation coefficient. Source numerical data are available in Source data.
Extended Data Fig. 5 CLASP depletion results in increased membrane rupture and propidium iodide influx in nuclear constricting conditions.
Representative images of (a) control (non-targeting) and (b) CLASP1-depleted (CLASP1 shRNA) 1205Lu cells subjected to axial confinement (3-micron height). Images were acquired at indicated time intervals. An increase in Propidium iodide (P.I. magenta) positive nuclei in cells counterstained with cell permeable Hoechst (nuclei, grey) was used to detect a loss of membrane integrity. (c) Quantitation of membrane integrity loss as measured by propidium iodide positive nuclei over an extended 20-hour time course. Measurements of P.I. positive nuclei for control (n = 6, 5, 3, 3, 4 independent fields of view for 0, 5, 60, 420, and 1440 min, respectively). CLASP1 KD (n = 5, 5, 1, 1, 2 independent fields of view for 0, 5, 60, 420, and 1440 min, respectively, low magnification images). Graph shows mean ± SD. Data shown represents one experiment. Experiments were performed two times with similar results. Representative images of 1205Lu cells endogenously expressing meGFP-α-tubulin and the nuclear marker Snapf-H2B-(Snap-JF646) subjected to (d) 3.7-micron and (e) 11-micron axial confinement. (f) Time-course images comparing P.I. influx (positive nuclei counterstained for Hoechst 33342) unconfined conditions vs., 3.7-micron and 11-micron axial confinement. (g) Quantitation of PI positive nuclei (percentage) over 20-hour time course. Measurements of PI positive nuclei for control (no confinement) are from n = 10 independent fields of views per each time point. 3.7 µm confinement measurements were from n = 10, 10, 7, 10, 10, 10, 10 and 10 independent fields of view for 0, 5, 30 min, 1, 2, 4, and 24 hours, respectively. 11 µm confinement measurements were from n = 10, 9, 10, 10, 10 and 10 independent fields of view for 0, 5, 30 min, 1, 2, 4, and 24 hours, respectively. Data shown represents one experiment. Experiments were performed two times with similar results. Graph shows mean ± SD. Source numerical data are available in Source data.
Extended Data Fig. 6 CLASP and EB1 differentially localize on microtubules in response to axial confinement.
(a) Representative timelapse images of a 1205Lu cell stably overexpressing EB1-meGFP pre and post-axial confinement (unconfined and confined, respectively, 3 microns). (b) Temporal projection of EB1 comet tracks (42 seconds duration), pre and post-axial confinement. (c) Kymograph of regions marked in b. (white dashed line). (d) Quantitation of EB1 track displacement and (e) speed relative to confinement condition and time. EB1 comet speeds slow in response to confinement however EB1 remains associated with growing microtubule plus-ends. Measurement of EB1 track displacement and speed is from n = 60 individual tracks taken from an individual cell. Data shown represents one experiment. Experiments were performed two times with similar results. Data displayed as violin plot indicating minimum, first quartile, median, third quartile, and maximum, with width indicating frequency of values. d, and e, statistical analysis by Kruskal-Wallis one-way ANOVA test with Dunn’s multiple comparison. (f) Representative timelapse images of a 1205Lu cell stably overexpressing meGFP-tagged N-terminal fragment of CLASP1 (meGFP-CLASP1 N-term; TOG1 and TOG 2) pre- and post-axial confinement (3 microns). In the absence of confinement, meGFP-CLASP1 N-term localizes to the cytoplasm. Induction of confinement results in an association with microtubules (g), progressively increasing over time (24 min). (h) 3D kymographs of the cell in f. (i). 1205Lu cells stably overexpressing HaloTag-tagged C-terminal fragment of CLASP1 (HaloTag-CLASP1-C-term; SxIP-TOG3-CLIP-ID), labelled with Halo-JF549. HaloTag-CLASP1-C-term tracks microtubule plus-ends irrespective of axial confinement (pre-confinement 0’, and post-confinement 4’30”), similar to EB1 (j) 3D kymographs of cell in i. Images were gamma adjusted (0.3) to increase contrast of microtubule structures. Source numerical data are available in Source data.
Extended Data Fig. 7 Microtubule acetylation patterns in cells in confinement.
(a) Simplified graphical schematic of constriction microchannel patterns; Circular, continuous, and ratcheting. (b) Immunofluorescence of 1205Lu melanoma cells labelled for acetylated α-tubulin (AcK40, magenta), tyrosinated α-tubulin (white) and nuclei (DAPI, cyan) navigating through indicated microchannel designs. Scale bar 5 µm. Images are displayed as maximum intensity projections of z-stacks. Single channels are displayed as contrast inverted images, demonstrating regardless of design, acetylated-α-tubulin forms a polarized network that contrasts tyrosinated-α-tubulin. A rear cytoplasmic compartment can be seen which flanks the rear of the nucleus (dotted line), predominantly filled with stable acetylated tubulin (red arrow heads) forming the microtubule cushion. (c) Representative timelapse images of a 1205Lu cell endogenously tagged for tubulin (eGFP-α-tubulin; orange hot) and coexpressing a nuclear market (SnapTag-H2B-JF646; cyan hot) undergoing nuclear occlusion and transmigration. (d) Maximum intensity projections of inverted greyscale tubulin images, orthogonal sections demonstrate the presence of a microtubule cage surrounding the nucleus. (e) 3D-Structured Illumination microscopy of acetylated tubulin (AcK40, magenta) and tyrosinated α-tubulin (Tyr, white) immunolabelled 1205Lu cells, nuclei labelled with DAPI, undergoing confined migration. Cells are expressing alternative CLASP1 and 2 hairpins from Fig. 4b.
Extended Data Fig. 8 CLASP-depleted cells exhibit aberrant nuclear morphologies and hyper contractility.
(a) Temporal colour-coded projection of nuclear outlines of a control (non-targeting) and CLASP2-depleted cell undergoing nuclear constriction. (b) Instantaneous cell speeds during active and post nuclear constriction phases; comparing control (non-targeting) and CLASP2-depleted cells (CLASP2 shRNA). Instantaneous speed measurements during active constriction stage were n = 876 individual frame-frame measurement values for control from 57 individual cells, and n = 1162 values for CLASP2 shRNA from 40 individual cells. Instantaneous speed measurements during post constriction stage were n = 1257 individual frame-frame measurement values for control from 57 individual cells, and n = 906 values for CLASP2 shRNA from 40 individual cells. Data shown represents one experiment. Experiments were performed two times with similar results. Data displayed as violin plot indicating minimum, first quartile, median, third quartile, and maximum, with width indicating frequency of values. Statistical analysis by Kruskal-Wallis test with Dunn’s multiple comparison test. (c) Quantitation of nuclear circularity traces of control (non-targeting) and CLASP-depleted cells showing lower nuclear circularity values as cells enter and exit constrictions. Graph shows mean circularity values with shaded 95% C.I. Nuclear circularity measurements during confined migration represent n = 57 individual cells for control, n = 54 individual cells for CLASP1 shRNA, and n = 40 individual cells for CLASP2 shRNA. Data shown represents one experiment. Experiments were performed two times with similar results. (d) Nuclear NLS intensity quantitation demonstrating major and minor nuclear envelope rupture events (magenta arrows) during confined migration. Graph shows mean with shaded 95% C.I. n = 36 individual cells for control, n = 23 individual cells for CLASP1 shRNA, and n = 25 individual cells for CLASP2 shRNA. Data shown represents one experiment. Experiments were performed two times with similar results. (e) Timelapse images of CLASP-depleted (CLASP2 shRNA) cells coexpressing membrane (LCK-mScarlet-I) and cortical (eGFP–LifeAct) markers undergoing nuclear constriction in microchannels. Insets are temporal colour-coded projections (1 frame/sec for 1 min) of actin bleb dynamics at the cell surface. CLASP2 depletion increases bleb size. (f) Enlarged inset of timelapse imaging of bleb formation and recovery in a control cell. Membrane blebs (LCK, black) form from breaks in the actomyosin cortex (5 s) and are recovered by subsequent cortical repair (LifeAct-magenta; 10–20 seconds). Kymograph origin of Fig. 6b. (g) CLASP2 depletion increases bleb size compared to control (non-targeting). Measurements of individual bleb sizes from control n = 107 blebs and CLASP2 shRNA n = 203 blebs taken from an individual cell, per condition. Data shown represents one experiment. Experiments were performed two times with similar results. Data displayed as violin plot indicating minimum, first quartile, median, third quartile, and maximum, with width indicating frequency of values. Statistical analysis by two-tailed Mann-Whitney Test. (h) Analysis of bleb protrusion and retraction velocity. Velocity measurements of control individual blebs undergoing protrusion n = 87 or retraction n = 82 CLASP1 shRNA n = 77 bleb protrusions and n = 91 retractions. CLASP2 shRNA n = 107 bleb protrusions and n = 92 retractions. Bleb measurements were taken from an individual cell per condition. Data shown represents one experiment. Experiments were performed two times with similar results. Data displayed as violin plot indicating minimum, first quartile, median, third quartile, and maximum, with width indicating frequency of values. Statistical analysis by Kruskal-Wallis test with Dunn’s multiple comparison test. (i) Representative timelapse images of 1205Lu cells expressing MLC-mTurquoise2 (myosin) undergoing nuclear translocation in collagen 3D hydrogels. Source numerical data are available in Source data.
Extended Data Fig. 9 CLASP2-reinforced microtubules are required to coordinate contractility in confined cells.
(a) 1205Lu cells expressing the ratiometric GEF-H1 FRET reporter in 2D. Depolymerization of microtubules with nocodazole results in an increased FRET signal (yellow) (b) quantitation of a and (c) magnified regions from a. Measurements of n = 3 individual cells treated with DMSO, n = 4 cells treated with 3.3 µM Nocodazole. Data shown represents one experiment. Experiments were performed two times with similar results. Data displayed as mean ± SD. (d) GEF-H1 localization on microtubules in 1205Lu cells migrating in collagen undergoing a nuclear transmigration event. Rear FRET signal increase (yellow) precedes nuclear transmigration. (e) GEF-H1 localization on microtubules in control (non-targeting) and CLASP1-depleted cells. (f) Enlarged insets from areas outlined in control cell front compartments in e (purple boxes) demonstrating changes in membrane blebbing in the front compartment during nuclear constriction phases. (g) eGFP-RhoA expressing 1205Lu cell migrating in collagen undergoing two consecutive nuclear transmigration events. eGFP-RhoA distribution accumulates at the rear of cells (yellow).
Extended Data Fig. 10 Modelling nuclear transmigration forces.
(a) Gradual shrinking of the rear microtubule cushion (grey line) and simultaneous increase of the Rho up-regulation factor (rear contractility multiplier (orange line) after initiation of microtubule cushion disassembly. (b) Velocity of cell (grey) and nucleus (blue) for the simulation in Fig. 6c and Supplementary Video 21 depicting a control cell. The initiation of the Rho burst (red line) accelerates both cell and nucleus which finally leads to the sudden passage of the nucleus through the constriction. (c) Velocity of cell (grey) and nucleus (blue) for the simulation without the microtubule cushion in Fig. 6d and Supplementary Video 21. (d) Disrupting the LINC complex via expression of DN-KASH2 (dominant negative) does not inhibit nuclear passage time in 1205Lu cells. Representative images of 1205Lu cells in constriction microchannels expressing either a KASH2-ext or DN-KASH2 construct. (e) Analysis of nuclear passage times in KASH2-ext and DN-KASH2 expressing cells. Data points represent individual nuclear passages. n = 159 (KASH2-ext) and 209 (DN-KASH2) individual cells pooled from two independent experimental replicates. Statistical analysis by two-tailed Mann-Whitney test. (f) Representative images of control (DMSO) and myosin inhibited (Blebbistatin) 1205Lu cells migrating in confinement. (g) Blebbistatin treatment inhibits cell migration, decreasing cell entry to microchannels. n = the mean number of cells per field of view (FOV) entering microchannels. n = 19 and 20 FOV for control (DMSO) and (-) blebbistatin treatments, respectively. (h) Blebbistatin treatment results in delayed nuclear passage time. n = 41 and 8 cells for control (DMSO) and (-) blebbistatin treatments, respectively. Panel g and h data displayed as violin plot indicating minimum, first quartile, median, third quartile, and maximum, with width indicating frequency of values. Data shown represents one experiment. Experiments were performed two times with similar results. g, h statistical test by two-tailed Mann-Whitney Test. (i) We use a heuristic to identify occlusion of the passage between the two micropillars (grey) and to find the boundary points between front and rear (see below). This allows to split the volume of the cytoplasm V_0^C into rear and front components V^R and V^F. (j) Sensitivity analysis of the cell response to variations in the threshold pressure at which microtubule (MT) disassembly is triggered. A higher threshold requires more time for the rear pressure to build and trigger the Rho burst and therefore delays transmigration. (k) Time until transmigration vs., gap size in simulations. CLASP1 KD cells only transmigrate when the gap size is large. j and k LOESS regression and 95% confidence intervals (by Bootstrap method) indicated by shaded regions, computed using the R-package spatialeco. Source numerical data are available in Source data.
Supplementary information
Supplementary Information
Supplementary Table 1. Mathematical parameters.
Supplementary Data 1
Mathematical model.
Supplementary Video 1
Microtubules form a dynamic cage in cells moving through complex 3D environments. Timelapse LSFM of 1205Lu CRISPR-CAS9-edited cells expressing meGFP–α-tubulin (orange) embedded into 2.5 mg ml−1collagen I hydrogels housed within a custom-made sample holder. Collagen I was labelled and visualized using CNA35-mScarlet-I (cyan). Microtubules surround the nucleus in a single migrating cell, dynamically reorganizing as the cell routes through collagen I fibres. Images were acquired every 30 s for a total duration of 2 h, at each time interval 126 Z-slices were acquired at 0.4-µm steps totalling 50.4 µm. Images are maximum intensity projections. Scale bar, 10 µm.
Supplementary Video 2
Contrasting microtubule organization in 2D versus 3D migrating cells. Spinning-disc confocal microscopy of 1205Lu CRISPR-edited cells expressing meGFP–α-tubulin (orange) migrating on 2D glass coverslips and migrating within 2.5 mg ml−1 collagen I hydrogels. Collagen I was labelled and visualized using CNA35-Halotag-JF549 (cyan). Images of 2D cell migration was acquired every 1 min for a total duration of 2 h and 9 min. Images of 3D cell migration was acquired every 5 min for a total duration of 2 h, at each time interval, three Z-slices were acquired at 3-µm spacing, totalling 9 µm. Z-slices were maximum intensity projected. Scale bar, 10 µm.
Supplementary Video 3
A microtubule cage assembles in cells undergoing constricted migration. Rotating 3D projection of 3D-SIM of cells undergoing constricted migration in microchannels, fixed and stained for acetylated (magenta) tyrosinated (grey) α-tubulin and nuclei (cyan). The microtubule cage structure can be seen to envelope the nucleus, lining the entire cell. Notably, in areas where the cell experiences active constriction, microtubules organize in parallel bundles surrounding the nucleus (mid). During early constriction timing (early) acetylated-α-tubulin is predominately localized to a posterior microtubule pool behind the nucleus (opposing the direction of migration) and interspaced between breaks of tyrosinated-α-tubulin. Z-scroll through the rear compartment demonstrating density and curvature of microtubules posterior to the nucleus (24–26 s). Scale bar, 5 µm.
Supplementary Video 4
CLASP depletion results in cell rupture during confined migration. The 1205Lu CRISPR-CAS9-edited cells coexpressing meGFP–α-tubulin (grey) and SNAP-tag-H2B-JF549 (magenta). Control cells (left, non-targeting) successfully navigate constrictions between micropillars by deforming the nucleus and cell body. CLASP1-depleted cells (right) rupture. Images were acquired at 10-min intervals for a total duration of 9 h. Scale bar 10 µm
Supplementary Video 5
CLASP-dependent cell death precedes annexin V positivity. Spinning-disc confocal microscopy of a CLASP2-depleted 1205Lu CRISPR-CAS9 edited cell expressing meGFP-α–tubulin attempting to navigate constriction microchannels. CLASP2 depletion results in microtubule depolymerization and cell death (cytoplasmic condensation) before membrane positive annexin V–Alexa Fluor 647-positive staining. Images were acquired at 2-min intervals for a total duration of 670 min. Scale bar, 5 µm.
Supplementary Video 6
CLASP1 dynamics are altered during confined migration. Timelapse Airyscan laser-scanning confocal microscopy of 2xmNeonGreen-CLASP1-expressing cells. During nuclear mid-constriction CLASP1 exhibits stable association with microtubules at the rear behind the nucleus. The dynamic pool in front of the nucleus can be seen associating with fast growing plus-ends. Images were acquired at 1-s intervals for a total duration of 2 min. Scale bar, 2 μm.
Supplementary Video 7
EB1–eGFP-expressing cells demonstrate local microtubule growth in confined migration. Timelapse Airyscan laser-scanning confocal microscopy of 1205Lu cells expressing EB1–eGFP undergoing confined migration. During nuclear mid-constriction EB1 dynamically associates with microtubules. Images were acquired at 1-s intervals for a total duration of 6 min. Scale bar, 5 µm.
Supplementary Video 8
Axial confinement results in microtubule depolymerization in CLASP-depleted cells. Timelapse spinning-disc confocal images of non-targeting control and CLASP1-depleted 1205Lu CRISPR-CAS9-edited cells expressing meGFP–α-tubulin undergoing compression (3 µm). Inverted to enhance contrast. Scale bar, 10 µm.
Supplementary Video 9
Axial confinement induces CLASP re-localization. Live-spinning-disc confocal timelapse images of 1205Lu cells expressing 2xmNeonGreen–CLASP1 undergoing compression (3.7 µm) and release cycles. Upon release of compression, CLASP re-localizes to +TIPs. Upon re-compression, CLASP binds along the lattice. Inverted to enhance contrast. Scale bar, 5 µm.
Supplementary Video 10
CLASP1 localization and dynamics are altered upon axial confinement. Timelapse spinning-disc confocal microscopy of 1205Lu cells CRISPR-CAS9 edited to express 2xeGFP–CLASP1 axially confined (3.7 μm). CLASP1 associates with dynamic microtubule plus-ends in the absence of axial confinement (0–1 min 40 s). Upon application of axial confinement (1 min 50 s) CLASP1 begins to stably binds to microtubules. Images were acquired at 10-s intervals for a total duration of 31 min. Scale bar, 5 μm.
Supplementary Video 11
CLASP1 stable microtubule binding is reversible upon axial confinement release. Timelapse spinning-disc confocal microscopy of 1205Lu cells CRISPR-CAS9 edited to express 2xeGFP–CLASP1 axially confined (3.7 μm). CLASP1 associates stably with microtubule under axial confinement. Upon release of axial confinement (14 s) CLASP1 begins to revert to dynamic plus-end microtubule association. Images were acquired at 1-s intervals for a total duration of 3 min and 9 s. Scale bar, 5 μm
Supplementary Video 12
Dynamic EB1 microtubules continue to grow upon axial confinement. Timelapse Airyscan laser-scanning confocal microscopy of 1205Lu cells stably expressing EB1–eGFP undergoing axial confinement (3.7 μm). EB1 associates with fast growing dynamic plus-ends of microtubules at baseline (0–40 s). Upon application of axial confinement (42 s) cells distend and blebbing is induced. EB1 continues to associate with dynamic microtubule plus-ends even under axial confinement for 30 min. Images were acquired at 2-s intervals for a total duration of 30 min. Scale bar, 5 μm.
Supplementary Video 13
Stable microtubules that associate at the rear during confined migration are disrupted by CLASP depletion. 1205Lu cells stably overexpressing β-tubulin–Halotag-JF585 and the live-cell acetylated-α-tubulin marker-StableMARK (Kif5b-(1-560)–eGFP). During control cell confined migration, StableMARK microtubules associate with microtubules at the rear upon nuclear occlusion. CLASP depletion results in a loss of stable rear microtubules, denoted by the loss of β-tubulin and StableMARK fluorescence. Images were acquired at 2-min intervals for a total duration of 2 h. Scale bar 5 μm.
Supplementary Video 14
CLASP depletion results in cortical blebbing. Nested, timelapse spinning-disc microscopy of membrane (LCK-mScarlet-I, grey inverted) and F-actin (eGFP–LifeAct, magenta inverted) dynamics in non-targeting control and CLASP1-depleted 1205Lu cells migrating within constriction microchannels. Control cells exhibit blebbing at the rear cortico-membrane region during early- and mid-confinement stages. CLASP1-depletion evokes uncontrolled blebbing at both the anterior and posterior cortico-membrane regions. Every hour, for a total of 24 h, nested acquisition of images were acquired at 1-s intervals for a total duration of 1 min. Scale bar, 5 µm.
Supplementary Video 15
CLASP depletion disrupts the proximal enrichment of myosin during confined migration. Timelapse spinning-disc microscopy of non-targeting control and CLASP1-depleted 1205Lu CRISPR-edited cells expressing meGFP–α-tubulin (grey), and lentivirally transduced with mTurqoise-MLC (myosin, cyan heatmap LUT) and SNAP-tag-H2B-JF549 (magenta heatmap LUT). Control cells exhibit polarized proximal enrichment of myosin during early to mid-confinement stages. CLASP1-depletion perturbs polarized proximal myosin enrichment during confinement stages. Images were acquired at 15-min intervals for a total duration of 24 h. Scale bar, 10 µm.
Supplementary Video 16
Rear myosin accumulation progresses confined migration in 3D collagen I hydrogels. Spinning-disc confocal microscopy of 1205Lu cells expressing mTurquoise-MLC (black) embedded into 2.5 mg ml−1 collagen I hydrogels. Myosin dynamically reorganizing and enriches at the rear as a cell migrates within collagen I hydrogels. Images were acquired every 2 min for a total duration of 5 h, at each time interval 11 Z-slices were acquired at 4.5455 µm steps totalling 50 µm. Images are maximum intensity projections. Scale bar, 10 µm.
Supplementary Video 17
Timed GEF-H1 activation upon microtubule depolymerization precedes nuclear transmigration. Timelapse spinning-disc microscopy showing ratiometric FRET:donor images of non-targeting control and CLASP1-depleted 1205Lu cells expressing the inducible GEF-H1-FLARE191 biosensor (black-purple-yellow heatmap LUT). Active GEF-H1 (high FRET/Donor ratio) can be visualized by yellow LUT colour and inactive (low FRET:donor ratio) GEF-H1 in black-purple LUT colours. Active GEF-H1 polarizes proximally in control cells during early to mid-constriction stages. CLASP1 depletion disrupts the proximal polarization of active GEF-H1 causing cells to fail constricted migration and rupture. Images were acquired at 10-min intervals for a total duration of 24 h. Scale bar, 10 µm.
Supplementary Video 18
Timed GEF-H1 activation progresses confined migration in 3D collagen I hydrogels. Spinning-disc confocal microscopy of 1205Lu cells expressing the inducible GEF-H1-FLARE191 biosensor (black-purple-yellow heatmap LUT) embedded into 2.5 mg ml−1 collagen I hydrogels. GEF-H1 activation (right video high FRET:donor ratio) dynamically localizes at the rear as a cell migrates within collagen I hydrogels. Donor fluorescence only channel maximum intensity projection (left video). Images were acquired every 5 min for a total duration of 2 h 5 min, at each time interval 11 Z-slices were acquired at 3.1818-µm steps totalling 34.99 µm. Images are maximum intensity projections. Scale bar, 10 µm.
Supplementary Video 19
A timed RhoA burst at the cell-rear progresses nuclear transmigration during confined migration in microchannels. Timelapse spinning-disc confocal microscopy of eGFP–RhoA (black-purple-yellow heatmap LUT) dynamics in non-targeting control and CLASP1-depleted 1205Lu cells. A polarized accumulation of proximal RhoA (yellow LUT colours) before nuclear translocation is associated with successful transmigration between constriction microchannels. CLASP1 depletion perturbs the polarized accumulation of RhoA and cells instead accumulate RhoA at both anteriorly and posteriorly leading to a static phenotype. Images were acquired at 10-min intervals for a total duration of 24 h. Scale bar, 10 µm.
Supplementary Video 20
A rear RhoA-burst in cells migration within 3D collagen I hydrogels progresses confined migration. Timelapse spinning-disc confocal microscopy of 1205Lu cells expressing eGFP–RhoA embedded in 2.5 mg ml−1 collagen I hydrogels. RhoA polarization and accumulation can be seen at the rear of cells undergoing confined migration (35 min) and resolves once cell has undergone nuclear transmigration. Images were acquired at 5-min intervals for a total duration of 4 h 55 min, at each time interval five Z-slices were acquired at 3-μm steps totalling 15 μm. Images are maximum intensity projections. Scale bar, 5 μm.
Source data
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A single file containing all source data, with clearly named tabs for each figure or extended data figure.
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Unmodified gels from Extended Data Fig. 1.
Source Data
Unmodified gels from Extended Data Fig. 2.
Source Data
Unmodified gels from Extended Data Fig. 3.
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Ju, R.J., Falconer, A.D., Schmidt, C.J. et al. Compression-dependent microtubule reinforcement enables cells to navigate confined environments. Nat Cell Biol 26, 1520–1534 (2024). https://doi.org/10.1038/s41556-024-01476-x
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DOI: https://doi.org/10.1038/s41556-024-01476-x
