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. 2018 May 9;8(1):7367.
doi: 10.1038/s41598-018-25668-2.

Morphoregulatory functions of the RNA-binding motif protein 3 in cell spreading, polarity and migration

J Pilotte  1 , W Kiosses  1 , S W Chan  1 , H P Makarenkova  1 , E Dupont-Versteegden  2 , P W Vanderklish  3
Affiliations

Morphoregulatory functions of the RNA-binding motif protein 3 in cell spreading, polarity and migration

J Pilotte et al. Sci Rep. .

Abstract

RNA-binding proteins are emerging as key regulators of transitions in cell morphology. The RNA-binding motif protein 3 (RBM3) is a cold-inducible RNA-binding protein with broadly relevant roles in cellular protection, and putative functions in cancer and development. Several findings suggest that RBM3 has morphoregulatory functions germane to its roles in these contexts. For example, RBM3 helps maintain the morphological integrity of cell protrusions during cell stress and disease. Moreover, it is highly expressed in migrating neurons of the developing brain and in cancer invadopodia, suggesting roles in migration. We here show that RBM3 regulates cell polarity, spreading and migration. RBM3 was present in spreading initiation centers, filopodia and blebs that formed during cell spreading in cell lines and primary myoblasts. Reducing RBM3 triggered exaggerated spreading, increased RhoA expression, and a loss of polarity that was rescued by Rho kinase inhibition and overexpression of CRMP2. High RBM3 expression enhanced the motility of cells migrating by a mesenchymal mode involving extension of long protrusions, whereas RBM3 knockdown slowed migration, greatly reducing the ability of cells to extend protrusions and impairing multiple processes that require directional migration. These data establish novel functions of RBM3 of potential significance to tissue repair, metastasis and development.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Localization of RBM3 to SICs in different cell types and plating conditions. Images of RBM3 (green), F-actin (red, phalloidin), and DAPI-stained nuclei (blue) in B104 cells (a–c) and HeLa cells (d–f) 30 minutes after plating onto glass, collagen, and fibronectin. For each cell type and substrate in panels a through f, upper right sub-panels show close-ups of F-actin distribution in regions (hatched yellow rectangles) at cell margins that contain SICs; sub-panels in lower right show the overlay of RBM3 with F-actin. (g–i) Images of B104 cells grown on fibronectin that were labeled for RBM3 and other SIC components by immunofluorescence, and for tRNA by FISH: cells were double labeled for (g) RBM3 (green) and the SIC component vinculin (red); (h) FUS (green) and F-actin (red); and (i) RBM3 (green) and tRNA-glycine (red).
Figure 2
Figure 2
Perturbation of RBM3 expression alters cell spreading and morphology. B104 cells were mock transfected (con), transfected with an siRNA to knock down RBM3 (si) or transfected with a CMV-based expression vector to overexpress EYFP-RBM3 (o/x), then replated 48 hours later onto fibronectin-coated glass cover slips. Cells were fixed at 60 minutes (a–f) and 120 minutes (g–l) post plating for imaging of RBM3 (green), F-actin (red, phalloidin), and nuclei (blue, DAPI). (a,c,e) Images of RBM3 and F-actin in B104 cells under control (a), RBM3 knockdown (c), and RBM3 overexpression (e) conditions at 60 minutes post plating. (b,d,f) Close-up images of areas delimited by hatched yellow boxes in panels a, c, and e. Whereas control and RBM3-overexpressing cells exhibit the beginnings of cell polarity, RBM3 knockdown cells adopted a highly rounded or polygonal morphology with prominent microspikes. (g,i,k) images of RBM3 and F-actin in B104 cells plated as in panels, a,c,e, but fixed at 120 minutes. At this timepoint, cells lacking RBM3 adopt markedly different shapes than control and RBM3-overexpressing cells. (h,j,l) close-ups of areas delimited within panels g, i, and k. Whereas control and RBM3-overexpressing cells were elongated in a polar morphology with growth cone-like protrusions containing RBM3 and F-actin, cells lacking RBM3 did not develop polarity, becoming rounded and highly spread. RBM3 overexpressing cells had a more monopolar appearance compared to control cells, but with similar growth cone-like protrusions containing F-actin and RBM3. (m) Bar graph of cell area measurements in control (con; n = 32), RBM3 knockdown (si; n = 30), and RBM3 overexpression (o/x; n = 18) conditions (*p < 0.0001, unpaired t-tailed t-tests). (n) bar graph summarizing the maximum lengths and widths of cells in control vs RBM3 overexpression conditions; group data were not significantly different.
Figure 3
Figure 3
RBM3 promotes migration and regulates migration mode. B104 cells were transfected with EGFP alone (con), EGFP plus RBM3 siRNA to knockdown RBM3 (si), or a construct encoding an EYFP-RBM3 fusion protein (o/x). After 48 hrs, cells were replated onto fibronectin-coated coverslips and transferred to a homeostatic chamber for live imaging of cell movement. (a) Representative images of cell migration paths during live imaging of EGFP and EYFP-labeled B104 cells in the three treatment conditions. Path color indicates temporal progression: start = white; end = red. (b) Graph of the average path speeds displayed by B104 cells in the three treatment conditions (n = 33, con; n = 57, si; n = 90, o/x; *p < 0.05, **p < 0.0001; ***p < 0.001 2-tailed t-tests). (c) Relative frequency distribution of path speeds in the three treatment conditions; knockdown and overexpression of RBM3 significantly skewed the distribution of path speeds (con vs si, p < 0.05; con vs o/x, p < 0.0005; si vs o/x, p < 0.0005, Kolmogorov-Smirnov test). (d) Frames from Supplementary Movie S2 showing filopodia-based, mesenchymal–like migration of highly polarized cells in the control condition (con). Long extensions with growth cone-like ends (hatched circle) are evident in the control (and RBM3 overexpression) condition, but not after RBM3 knockdown (si). Two highly polarized cells (arrowhead and arrow) are seen migrating away from each other (e) Frames from Supplementary Movie S5 illustrating the morphology of cells in the knockdown condition as they migrate; some remain rounded (hatched rectangle) with continued blebbing. Another cell can be seen switching from a slightly elongated state with short protrusions, to a rounded, blebbing state (arrow).
Figure 4
Figure 4
Reduction of RBM3 impairs wound healing. Scratch wounds were created in confluent cultures of 3T3 fibroblasts and wound closure was imaged in real time under homeostatic conditions. (a and b) Representative images of wounds and superimposed traces of fibroblast migration into the wound area in control (a, blue) and RBM3 knockdown (b, red) conditions. (c) Graph of the extend of wound closure as a function of time in the control (con) and RBM3 knockdown (si) experiments shown in panels a and b. (d) Graph summarizing group data on wound closure in terms of area reclaimed, normalized to scratch length, in control and RBM3 knockdown conditions (con, n = 4; si, n = 3; *p < 0.05 1-tailed t-test).
Figure 5
Figure 5
Dynamic localization of RBM3 in differentiating primary myoblasts. (a) Image of F-actin (red, phalloidin), RBM3 (green) in myoblasts fixed shortly after plating (1 hr) onto fibronectin. SICs are clearly visible along the edges of recently adherent cells as F-actin-delimited outpouchings of membrane. RBM3 is concentrated within SICs. (c) DAPI-stained nuclei in cells from Panel a. (b and d) Blowup of upper and lower margins of cell from the top-right portion of panel A. SICs (arrowheads) can be seen clearly with a peripheral F-actin ring surrounding an RBM3-rich core. (e) Image of a myoblasts partially differentiated into a myotube. At this stage, RBM3 is present in membranous blebs devoid of F-actin that are known to mediate myoblast migration. (f) Image of a fully differentiated satellite cell. RBM3 relocalizes from membrane protrusions to the cytoplasm and nucleus (nuc). (g) Images of a myotube explant at 24 hrs in culture in which activated satellite cells migrating along the myotube border (dashed outline) co-express MyoD (red) and RBM3 (green). (h) Images of dividing satellite cells along the border of a myotube that co-label for histone H3 phosphate (H3-P, red) and RBM3 (green).
Figure 6
Figure 6
RBM3 regulates myotube formation in vitro. Satellite cells maintained under differentiating conditions were transfected with RBM3 siRNA (si), an EYFP-RBM3 expression construct (o/x), or subjected to mock transfection (con). (a–c) Images of myoblasts in the indicated RBM3 expression conditions. siRNA-mediated knockdown of RBM3 (b) impaired myotube formation from differentiating cells, whereas RBM3 overexpression (c) enhanced it. (d–g) Graphs summarizing the length (d) and area (e) of formed myotubes, the percentage of all cells incorporated into myotubes containing at least 2 nuclei (mean ± SEM; n = 43, 14, 17 for con, si, o/x, respectively; *p < 0.05, 1-tailed t-test) (f) and the mean nuclei count (g) of all cells in each RBM3 expression condition (n = 135, 127, 43 for con, si, o/x, respectively; *p < 0.05, 1-tailed t-test).
Figure 7
Figure 7
RBM3-regulated changes in cell morphology involve RhoA-ROCK signaling. (a) Western blots showing expression levels of the indicated proteins at 0, 60, and 120 minutes after replating of B104 cells maintained in control (con), RBM3 knockdown (si), and RBM3 overexpression (o/x) conditions. A large increase in RhoA is observed at all timepoints in RBM3 knockdown cells, along with tubulin and a modest increase in vinculin, an SIC component. CRMP2, rhotekin, and CDC42 were downregulated in the knockdown condition. In cells overexpressing RBM3, RhoA was initially downregulated. (b) Bar graph of changes in RhoA, CRMP2 and vinculin at 2 hrs in RBM3 knockdown vs control cells (n = 4 experiments; *p < 0.05, 1-sample t-test), normalized to eIF6 (present blots) or β-actin (additional experiments, Supplementary Fig. S11). (c–f) Images of RBM3 (green), F-actin (red, phalloidin), and nuclei (blue) in B104 cells under control (con) and RBM3 knockdown conditions (si), with and without the ROCK inhibitor Y27632 (100 μM). Inhibition of ROCK rescued cell polarity in RBM3 knockdown B104 cells (scale bar = 10 μm). (g,h) Graphs summarizing cell body areas (g) and major process lengths (h) at 2 and 24 hrs post replating in the following treatment conditions: control cells (con: n = 17 at 2 hrs; n = 23 at 24 hrs), RBM3 knockdown cells (si: n = 22 at 2 hrs; n = 20 at 24 hrs), control cells treated with Y27632 (con + Y27632: n = 15 at 2 hrs; n = 25 at 24 hrs), RBM3 knockdown cells treated with Y27632 (si + Y27632: n = 12 at 2 hrs; n = 15 at 24 hrs; *p < 0.05, **p < 0.005, ***p < 0.0005; t-tailed t-test). Cells in which RBM3 was knocked down, but not treated with Y27632, did not have processes to measure.
Figure 8
Figure 8
RBM3-associated changes in cell morphology involve CRMP2, a substrate of ROCK. (a–d) Images showing rescue of cell polarity and spreading in RBM3 knockdown B104 cells by transfection with a FLAG-CRMP2 construct: (a) FLAG-CRMP2 (green); (b) nuclei (DAPI, blue); (c) F-actin, (phalloidin, red); (d) overlay. (e) Enlargement of area highlighted by yellow box in d. Arrows in panels a, d and e show FLAG-CRMP2 expressing B104 cells, which have rescued polarity.
Figure 9
Figure 9
Model of RBM3’s influences on cell morphology and migration. (a) Schematic of the morphological trajectories taken by B104 cells under control, RBM3 knockdown, and RBM3 knockdown plus ROCK inhibitor conditions. Replated B104 cells initially exhibit SICs and a rounded morphology. Whereas control cells go on to adopt a bi- or multi-polar shape, cells lacking RBM3 lose polarity and undergo exaggerated spreading. Inhibition of ROCK during replating of RBM3 knockdown cells rescues polarity but enhances process elongation. (b) Diagram of proposed mechanisms by which RBM3 regulates cell polarity and spreading. Knockdown of RBM3 results in parallel changes in translation and a RhoA-ROCK-CRMP2 pathway. Elevated RhoA expression activates ROCK, a kinase involved in the transition from a mesenchymal to an amoeboid state. Reduced levels of CRMP2, a microtubule binding protein that is inhibited by ROCK phosphorylation, contributes to loss of polarity. (c) Schematic of hypothesized effects of RBM3 expression level on transitions between mesenchymal and amoeboid states. High levels of RBM3 lock cells into a highly polar, mesenchymal like mode of migration. Low RBM3 may favor an amoeboid state, but still be permissive to transitions between amoeboid and mesenchymal modes of migration, so-called MAT and AMT, respectively. This is may enhance metastasis of cancers by enhancing migration mode flexibility.
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