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Rab6 regulates cell migration and invasion by recruiting Cdc42 and modulating its activity

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Abstract

Rab proteins are master regulators of intracellular membrane trafficking, but they also contribute to cell division, signaling, polarization, and migration. The majority of the works describing the mechanisms used by Rab proteins to regulate cell motility involve intracellular transport of key molecules important for migration. Interestingly, a few studies indicate that Rabs can modulate the activity of Rho GTPases, important regulators for the cytoskeleton rearrangements, but the mechanisms behind this crosstalk are still poorly understood. In this work, we identify Rab6 as a negative regulator of cell migration in vitro and in vivo. We show that the loss of Rab6 promotes formation of actin protrusions and influences actomyosin dynamics by upregulating Cdc42 activity and downregulating myosin II phosphorylation. We further provide the molecular mechanism behind this regulation demonstrating that Rab6 interacts with both Cdc42 and Trio, a GEF for Cdc42. In sum, our results uncover a mechanism used by Rab proteins to ensure spatial regulation of Rho GTPase activity for coordination of cytoskeleton rearrangements required in migrating cells.

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Acknowledgements

We acknowledge the NorMIC Oslo imaging platform (Department of Biosciences, University of Oslo), Catherine Anne Heyward, and Frode Skjeldal for technical assistance, Hesso Farhan and Salim Ghannoum (Institute of Basic Medical Sciences, University of Oslo) for assistance and access to the IncuCyte ZOOM instrument. We thank Luis Hodgson (Albert Einstein College of Medicine, USA), Keith Burridge (University of North Carolina, USA), and Jaap D. Van Buul (University of Amsterdam, The Netherlands), for the kind gift of the pTriEX Cdc42 constructs, pEGFP-C3 Cdc42 plasmids, and pEGFPC1-Trio respectively. We thank Guillaume Jacquemet (University of Turku, Finland) for providing the FiloQuant plugin for Fiji/ImageJ, Giorgio Scita (IFOM, Milan, Italy) and Fabio Giavazzi (University of Milan, Italy) for advice on PIV analysis. We are grateful to Bruno Goud (Institut Curie, Paris, France) for critically reading the manuscript. The financial support of the Norwegian Cancer Society [Grants 5760850 to C.P. and 4604944 to O.B.], the Research Council of Norway [grants 239903 to C.P., 230779 to O.B., and through its Centre of Excellence funding scheme, Project Number 179573], and a Mayent-Rothschild- Institut Curie Award to OB is gratefully acknowledged.

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Author notes

  1. Katharina Vestre and Ingrid Kjos contributed equally to this work.

Authors and Affiliations

  1. Department of Biosciences, University of Oslo, Oslo, Norway

    Katharina Vestre, Ingrid Kjos, Noemi Antonella Guadagno, Marita Borg Distefano, Federico Fenaroli, Oddmund Bakke & Cinzia Progida

  2. Centre for Immune Regulation, University of Oslo, Oslo, Norway

    Katharina Vestre, Ingrid Kjos, Noemi Antonella Guadagno, Marita Borg Distefano, Oddmund Bakke & Cinzia Progida

  3. Department of Physics, The NJORD Centre, University of Oslo, Oslo, Norway

    Felix Kohler

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  1. Katharina Vestre
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Contributions

KV, IK, NAG, MBD, FK, and CP performed the experiments and analyzed data. FF helped with setting up the Zebrafish xenotransplantation model. CP conceived and supervised the project. IK and CP wrote the manuscript with input from all the authors. CP and OB procured funding.

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Correspondence to Cinzia Progida.

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Movie 1

: Cdc42 is transported towards the cell periphery in Rab6-positive vesicles. U2OS cells co-transfected with GFP-Rab6 and mCherry-Cdc42 were imaged using a spinning-disk confocal microscope at 1 second intervals. A vesicle positive for both GFP-Rab6 and mCherry-Cdc42 moving towards the cell periphery is tracked over time. Scale bar: 10 µm (AVI 2233 kb)

Movie 2: Rab6-positive vesicles are transported towards filopodia.

U2OS cells transfected with GFP-Rab6 and labeled with SiR-actin, were imaged with a Total Internal Reflection fluorescent (TIRF) microscope. (AVI 188 kb)

Supplementary figure 1: Rab6 silencing promotes cell migration in RPE-1 cells.

(a) RPE-1 cells treated with siRNA control, siRNA Rab6 #1 or siRNA Rab6 #2 were scratch-wounded and imaged every 15th minute for 24 h. Representative images of (T0) and 24 h after scratching are shown. Scale bar: 300 µm. Quantification of single-cell speed (b) and directness (c) is represented as mean ± s.e.m.; n>230 cells from five independent experiments. (d) Cell lysates from each of the indicated samples were subjected to Western blot analysis with antibodies against Rab6 and tubulin (as a loading control). (e) The intensities of the bands were quantified using densitometry, normalized against the amount of tubulin, and plotted relative to the intensities obtained in cells transfected with siRNA control. The values represent the mean ± s.e.m. for five independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (paired Student’s t test). (f) Representative track plots of the single-cell migration distances. Individual tracks are shown so that each starts at the origin (distance 0). (g) Velocity vector fields of particle image velocimetry analysis (PIV). Scale bar: 100 µm (TIFF 1719 kb)

Supplementary figure 2: Rab6 depletion promotes cell migration independently from cell proliferation.

(a) U2OS cells treated with either siRNA control, siRNA Rab6 #1, siRNA Rab6 #2, or treated with the same siRNAs and afterwards transfected with GFP-Rab6 were scratch-wounded and imaged for 24 h. The graph represents the mean from three independent experiments of the relative wound density (%) as function of time. ***P < 0.001 compared to control (two-way repeated measures ANOVA followed by Tukey’s post test for t=24h). (b) Cell lysates from each sample were subjected to Western blot analysis with antibodies against Rab6 and tubulin (as a loading control). (c) U2OS cells treated with either siRNA control, siRNA Rab6 #1 or siRNA Rab6 #2 were scratch-wounded and imaged in medium containing Cytotox Green reagent to label dying cells green. Cell death during migration was quantified by dividing the number of green objects at each timepoint with the number of green objects at time 0. The graph represents the mean from three independent experiments. (d) U2OS cells treated with either siRNA control, siRNA Rab6 #1 or siRNA Rab6 #2 were scratch-wounded and incubated in medium (with or without serum as indicated) containing the modified thymidine analogue Edu. The graph shows the percentage of Edu-positive cells after 24 h, relative to Edu-positive control cells in medium containing serum. The data represents the mean ± s.e.m from three independent experiments. (e) HeLa cells treated with either siRNA control, siRNA Rab6 #1, or siRNA Rab6 #2 were scratch-wounded and imaged for 24 h. Representative images of (T0) and 24 h after scratching are shown. Scale bar: 300 µm. (f) Quantification of the relative wound density (%) as function of time for control cells and Rab6-depleted cells. Data represents the mean of three (siRNA #1) or two (siRNA #2) independent experiments. *P < 0.05; ***P < 0.001 compared to control (two-way repeated measures ANOVA followed by Tukey’s post test for t=24h). (g) Cell lysates from each sample were subjected to Western blot analysis with antibodies against Rab6 and tubulin (as a loading control). (TIFF 809 kb)

Supplementary figure 3: Role of Trio in Rab6-dependent Cdc42 activation and cell migration.

(a) Lysates from U2OS cells transfected with control siRNA, siRNA Rab6 #1, siRNA Trio, siRNA Rab6 #1 and siRNA Trio, or siRNA DOCK10 were mixed with beads coupled to GST–PAK–PBD to pull down the active forms of Cdc42 and analyzed by western blot. (b) Quantification of the levels of active Cdc42 were normalized to the amount of tubulin and plotted relative to the intensities of GTP-bound Cdc42 in the control sample. The graphs represent the mean ± s.e.m. normalized to the control of at least three independent experiments. *P < 0.05; **P < 0.01 (paired Student’s t test). (c) U2OS cells treated with either siRNA control, siRNA Rab6 #1, siRNA Trio, siRNA Rab6 #1 and siRNA Trio, or treated with siRNA Trio and afterwards transfected with GFP-Rab6, were scratch-wounded and imaged for 24 h. The graph represents the mean from at least three independent experiments of the relative wound density (%) as function of time. ***P < 0.001 compared to control (two-way repeated measures ANOVA followed by Tukey’s post test for t=21h). (TIFF 309 kb)

Supplementary figure 4: Rab6 and filopodia.

(a) HeLa cells treated with siRNA control or siRNA Rab6 #1 were fixed and stained with DAPI and rhodamine-conjugated phalloidin. The lower insets show magnifications of the boxed areas. Scale bar: 20 µm. (b) Quantification of the number of filopodia per 100 µm cell membrane length. The graph represents the mean ± s.e.m.; n>100 cells from four independent experiments. *P < 0.05 (paired Student’s t test). (c) U2OS cells transfected with GFP-Rab6 and labeled with SiR-actin, were imaged with a Total Internal Reflection fluorescent (TIRF) microscope. Right panels: arrows in the insets point to Rab6-positive vesicles moving towards filopodia. Scale bar: 10 µm. (TIFF 698 kb)

Supplementary figure 5: Rab6 knockdown promotes spread of cancer cells in zebrafish embryos following xenotransplantation.

(a) H1299 cells transfected with control siRNA or siRNA Rab6 #2 were stained with QDs Q-Tracker 655 and injected into the otic vesicle (red circle) of 2-dpf zebrafish wild-type embryos. Images were taken soon after the injection (T0) as well as 24 and 48 h later. In contrast to the control cells (left panels) that remained confined to the injection area, cell silenced for Rab6 (right panels) showed higher ability to migrate outside from the otic vesicle, both at 24 h and at even more at 48 h (arrows). Magnification of the boxed areas are shown in the inset on the right of each panel. Scale bar: 100 µm. (b) Quantification of cell migration was performed by measuring the area occupied by the cells at 24 h and 48 h after injection and was normalized to the area occupied by the cells in the otic vesicle at T0. The graph represents the mean ± s.e.m. from n>15 embryos. *P < 0.05 (paired Student’s t test). (c) H1299 cells transfected with control siRNA or siRNA Rab6 #2 for 24 or 48 h were subjected to western blot analysis using antibodies against Rab6 and tubulin (as a loading control). (TIFF 477 kb)

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Vestre, K., Kjos, I., Guadagno, N.A. et al. Rab6 regulates cell migration and invasion by recruiting Cdc42 and modulating its activity. Cell. Mol. Life Sci. 76, 2593–2614 (2019). https://doi.org/10.1007/s00018-019-03057-w

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