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. 2015 Nov 9;35(3):344-57.
doi: 10.1016/j.devcel.2015.10.005.

Global Analysis of mRNA, Translation, and Protein Localization: Local Translation Is a Key Regulator of Cell Protrusions

Faraz K Mardakheh  1 , Angela Paul  2 , Sandra Kümper  2 , Amine Sadok  2 , Hugh Paterson  2 , Afshan Mccarthy  2 , Yinyin Yuan  3 , Christopher J Marshall  2
Affiliations

Global Analysis of mRNA, Translation, and Protein Localization: Local Translation Is a Key Regulator of Cell Protrusions

Faraz K Mardakheh et al. Dev Cell. .

Abstract

Polarization of cells into a protrusive front and a retracting cell body is the hallmark of mesenchymal-like cell migration. Many mRNAs are localized to protrusions, but it is unclear to what degree mRNA localization contributes toward protrusion formation. We performed global quantitative analysis of the distributions of mRNAs, proteins, and translation rates between protrusions and the cell body by RNA sequencing (RNA-seq) and quantitative proteomics. Our results reveal local translation as a key determinant of protein localization to protrusions. Accordingly, inhibition of local translation destabilizes protrusions and inhibits mesenchymal-like morphology. Interestingly, many mRNAs localized to protrusions are translationally repressed. Specific cis-regulatory elements within mRNA UTRs define whether mRNAs are locally translated or repressed. Finally, RNAi screening of RNA-binding proteins (RBPs) enriched in protrusions revealed trans-regulators of localized translation that are functionally important for protrusions. We propose that by deciphering the localized mRNA UTR code, these proteins regulate protrusion stability and mesenchymal-like morphology.

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Figures

Figure 1
Figure 1
Filter-Based Analysis of Protrusions in MDA-MB231 Cells (A) Confocal 3D rendering of MDA-MB231 cells forming protrusions through 3-μm transwell filters. Cells were stained with CellTracker-CMFDA (green) 24 hr prior to seeding. The collagen coated filter was stained prior to seeding by CellTrace Far Red DDAO-SE (blue). White arrow, cell body; red arrow, protrusions. Axes scales are in μm. (B) Transwell separated protrusions are actin-rich, but lack a nucleus. MDA-MB231 cells protruding through 3-μm transwell filters were fixed and stained with phalloidin-Alexa Fluor-488 (green) and Hoechst (red). Confocal images were taken from the top (right image) and bottom (left image) of the filter. The filter was visualized by transmitted light (blue). Scale bar, 10 μm. (C) Formation of protrusions is dependent on RAC1 and CDC42. Control, RAC1, or CDC42 siRNA transfected MDA-MB231 mKate-CAAX cells (red) were seeded on collagen-coated 3-μm transwell filters and time-lapsed as they formed protrusions through the pores. Confocal images were taken from the bottom of the filter. Collagen coated filters were stained by CellTrace Far Red DDAO-SE (blue) prior to seeding. Scale bar, 10 μm. (D) Protrusions formed through transwell filters are enriched in VASP, PXN, ZYX, and F-actin. Cross-section side views of confocal 3D renderings of MDA-MB231 cells protruding through 3-μm transwell filters. Cells were fixed and immuno-stained with antibodies against indicated markers (green), or phalloidin for F-actin staining (red). DAPI was used for nuclear staining (blue). Dashed line marks the position of the filter. Protrusions are marked as (P) and the cell-bodies are marked as (B). Scale bar, 10 μm. (E) Quantification of the protrusion (P)/cell body (B) fluorescent intensities for indicated markers from (D). At least three independent tiled scans, each formed from nine fields of view, were used. All error bars are SD. (F) Protrusion diameters of MDA-MB231 cells in 3D pepsinized collagen-I are comparable to transwell protrusion diameters. MDA-MB231 mKate-CAAX cells (red) were seeded on 3D collagen-I gels and imaged 2–4 hr after seeding by confocal microscopy. Scale bar, 10 μm. (G) Protrusion width and length measurements from 92 cells (as in F) are displayed as histograms. A total of 172 individual protrusions were measured. Average protrusion width in 3D collagen-I is 3.1 μm (red line). Average protrusion length in 3D collagen-I is 16.5 μm (red line). (H) Transwell protrusions are stable for a variety of time lengths, with a median stability of 2 hr and 5 min. The stability of protrusions was assessed from nine time-lapse movies of protrusion formation through 3-μm transwell filters (see also Movie S1). A total of 136 protrusions from 85 cells were timed over a 5-hr period, and the results are displayed as a histogram. Red line marks the median stability (125 min). (I) 3D protrusions are stable for a variety of time-lengths, with a median stability of 2 hr and 42 min. The stability of protrusions was assessed from four time-lapse movies of protrusion formation in 3D collagen-I (see also Movie S2). A total of 200 protrusions from 99 cells were timed over a 10-hr period. The results are displayed as a histogram. Red line marks the median stability (162 min).
Figure 2
Figure 2
Localized Translation, but Not mRNA Targeting, Significantly Contributes toward the Proteome Asymmetry between Protrusions and the Cell Body (A) Schematic representation of SILAC protrusion proteomics analysis; the H/L ratios are the measures of protrusion/body protein distributions. (B) Proteome distribution between protrusions and the cell body. Log2 SILAC ratios from reciprocal SILAC mixtures of protrusion and cell body fractions (Data S1A) were plotted against each other. CC, Pearson’s correlation coefficient between the two pairs. P value of the correlation is displayed on the graph. (C) Messenger-RNA distributions between protrusions and the cell body. Log2 of protrusion/cell body FPKM ratios from two replicate experiments (Data S1C) were plotted against each other. CC, Pearson’s correlation coefficient between the two replicates. P value of the correlation is displayed on the graph. (D) Protein distributions between protrusions and the cell body do not correlate with mRNA distributions. Averaged Log2 of protrusion/cell body protein ratios were plotted against averaged Log2 of Protrusion/Cell body mRNA ratios. CC = Pearson’s correlation coefficient. P-value of the correlation is displayed on the graph. (E) Schematic representation of pSILAC protrusion proteomics analysis. H/M ratios for each protein are the measures of protrusion/body translation rates. (F) Translation rate distributions between protrusions and the cell body. Log2 of H/M SILAC ratios from reciprocal pSILAC mixtures of protrusion and cell body fractions (Data S1D) were plotted against each other. CC, Pearson’s correlation coefficient between the two pairs. P value of the correlation is displayed on the graph. (G) Protein distributions between protrusions and the cell body significantly correlate with translation rate distributions. Averaged Log2 of protrusion/cell body protein ratios from the two reciprocal SILAC pairs were plotted against averaged Log2 of protrusion/cell body pSILAC ratios. CC, Pearson’s correlation coefficient. P value of the correlation is displayed on the graph. (H) RNA-binding proteins and ribosomal components are enriched but not locally synthesized in protrusions. The 2D-annotation enrichment analysis data from Data S1E was plotted with each data point representing a protein category. Most protein categories exhibit a correlative regulation of their protein distributions and relative translation rates, for example all protrusion-enriched actin-related categories (blue), and all protrusion-depleted organelle-related categories (red), but an anti-correlative behavior is observed for RNA-binding and ribosomal protein categories (green), with their relative translation rates significantly lower than their relative protein amounts. (I) Removal of RNA-binding and ribosomal protein categories from (G) significantly increases the correlation between protein distributions and translation rates from 0.30 to 0.47. CC, Pearson’s correlation coefficient. P value of the correlation is displayed on the graph. See also Figure S1.
Figure 3
Figure 3
Local Translation Is Needed for Protrusion Stabilization (A) Widespread translation occurs in protrusions. MDA-MB231 cells seeded on 3-μm transwells for 2 hr were treated with Puromycin (10 μg/ml) for 10 min to label nascent proteins. Equal amounts of isolated protrusion and cell body fractions were then resolved by SDS-PAGE and blotted for puromycinylation, VASP as a protrusion marker, H2AX as a cell body marker, and GAPDH as loading control. (B) Protrusions initiate but retract in translation-inhibited cells. MDA-MB231 mKate-CAAX cells were seeded on 3-μm transwells in presence or absence of cycloheximide (10 μg/ml), fixed at indicated times, and analyzed by confocal microscopy. Images show protrusions at the bottom of transwell filters. Red, cell membranes; blue, filter. Scale bar, 50 μm. See also Movie S3. (C) Quantification of protrusions from (B) (n = 4). The significant p values are stated above the bar graph. Error bars are SD. (D) Translation inhibition switches cell morphology from protrusive to round in 3D. MDA-MB231 cells were seeded on top of 3D collagen-I gels for 4 hr and treated with cycloheximide (10 μg/ml) for indicated times before being fixed and imaged. Scale bar, 50 μm. See also Movie S4. (E) Quantification of protrusive versus round morphologies from (D) (n = 3). Significant p values are stated above each bar graph. Error bars are SD. (F) Local inhibition of translation in protrusions. MDA-MB231 cells were seeded on 3-μm transwells for 2 hr. Emetine (1 μg/ml) was then added to the bottom chamber for 5 min as in Figure S2F. Transwells were then washed and treated with Puromycin (10 μg/ml) for 10 min to label nascent proteins before lysis. Equal amounts of isolated protrusion and cell body fractions were then resolved by SDS-PAGE and blotted for puromycinylation, VASP (protrusion marker), H2AX (cell body marker), and GAPDH (loading control). The emetine treatment specifically inhibits translation in protrusions. (G) Inhibition of translation in protrusions destabilizes protrusions. MDA-MB231 mKate-CAAX cells were seeded on 3-μm transwells for 2 hr before being treated with emetine (1 μg/ml) or vehicle as in (F). The cells were then either fixed immediately (0 min), or left for 1 hr (60 min) before being fixed and analyzed by confocal microscopy. The images show protrusions at the bottom of the filter. Red, cell membranes; blue, filter. Scale bar, 50 μm. See also Movie S5. (H) Quantification of protrusions from (G) (n = 10). The significant p values are stated above the bar graph. Error bars are SD. See also Figure S2.
Figure 4
Figure 4
mRNA Enrichment Does Not Determine Local Translation Rates in Protrusions (A) mRNA distributions between protrusions and the cell body negatively correlate with translation rate distributions. Averaged Log2 of protrusion/cell body mRNA FPKM ratios from two replicate experiments were plotted against averaged Log2 of protrusion/cell body pSILAC translation ratios. CC, Pearson’s correlation coefficient. P value of the correlation is displayed on the graph. (B) Most categories of proteins show a negative correlation between their mRNA localization and local translation. The 2D-annotation enrichment analysis data from Data S2A was plotted with each data point representing a protein category. Most actin cytoskeleton-related categories (blue) show high relative local translation rates but their mRNA is not highly enriched in protrusions while mitochondrial (purple) and RNA-binding/ribosomal (green) protein categories show high relative mRNA enrichment but low local translation rates in protrusions. mRNAs for ER and other organelle categories (red) are enriched and translated more in the cell body. (C) Specific 3′ UTR elements are associated with higher local translation rates in protrusions. Data of averaged Log2 of protrusion/cell body pSILAC translation ratios were annotated for known UTR elements from UTRdb (Grillo et al., 2010) (see also Data S2B), and subjected to 1D annotation enrichment analysis. Six UTR elements were significantly enriched in locally translated mRNAs as listed. Size, the number of mRNAs annotated with each element in the dataset; Benj. Hoch. FDR, Benjamini Hochberg false detection rate for enrichments; median, median of the averaged Log2(protrusion/cell body) pSILAC translation ratios for each UTR category.
Figure 5
Figure 5
Specific Categories of RBPs Are Enriched in and Functionally Important for Protrusions (A) Sm domain-containing RBPs are significantly enriched in protrusions. Log2 of SILAC-quantified protein ratios were plotted against each other with all Sm domain proteins marked in red. P value for the annotation enrichment is displayed on the graph. (B) KH domain-containing RBPs are significantly enriched in protrusions. Log2 of SILAC-quantified protein ratios were plotted against each other with all KH domain proteins marked in red. P value for the annotation enrichment is displayed on the graph. (C) RNase PH domain-containing RBPs are significantly enriched in protrusions. Log2 of SILAC-quantified protein ratios were plotted against each other with all RNase PH domain proteins marked in red. P value for the annotation enrichment is displayed on the graph. (D) List of all significant hits from RNAi screening of protrusion-enriched RBPs in MDA-MB231 mKate-CAAX cells, their depletion effect on protrusions (inhibition or enhancement), as well as their depletion effect on 3D invasion into collagen-I. First p value column is for the t test significance of the change in protrusions. Second p value column is for the t test significance of the change in 3D invasion index.
Figure 6
Figure 6
Exosome Core Is Enriched in Protrusions and Is Required for Protrusion Stability, Mesenchymal-like Morphology, and 3D Migration (A) Exosome core subunits, but not catalytic subunits, are enriched in protrusions. Log2 of SILAC-quantified protein ratios were plotted against each other with exosome core subunits marked in red and catalytic subunits in green. All nine exosome core subunits were present more in protrusions than the cell body with eight showing at least 2-fold enrichment, while both expressed catalytic subunits show a strong enrichment in the cell body. (B) Western blot analysis of exosome core and catalytic subunits enrichments in protrusion and cell body fractions of MDA-MB231 cells. Equal amounts of isolated protrusion and cell body fractions were resolved by SDS-PAGE and blotted for EXOSC5, RRP6, DIS3, as well as VASP (protrusion marker), H2AX (cell body marker) and GAPDH (loading control). While EXOSC5 shows a strong enrichment in protrusion fraction, RRP6 and DIS3 show enrichment in the cell body. (C) Immunofluorescence analysis of exosome core and catalytic subunits localizations. MDA-MB231 mKate-CAAX cells seeded on top of collagen-I coated coverslips were fixed and stained with indicated antibodies. A fraction of EXOSC5 localizes to the leading edge of the cells, while RRP6 and DIS3 are mostly nuclear. (D) RNAi-mediated depletion of exosome core subunits inhibits protrusions. MDA-MB231 mKate CAAX cells were transfected with indicated siRNAs. After 72 hr, the cells were seeded on top of transwell filters for 4 hr before being fixed and analyzed by confocal microscopy. Images show protrusions at the bottom of the filter. Red, cell membranes; blue, filter. Scale bar, 50 μm. See also Movie S6. (E) Quantification of protrusions from (D) (n = 10). Significant p values are stated above each bar graph. Error bars are SD. (F) Exosome core depletion switches MDA-MB231 cell morphology from protrusive to round. MDA-MB231 cells were transfected with indicated siRNAs before being seeded on top of a thick 3D collagen-I matrix 72 hr post-transfection. Cells were imaged 24 hr after seeding. Scale bar, 50 μm. See also Movie S7. (G) Quantification of the percentage of protrusive versus round cells from F (n = 3). Significant p values are stated above each bar graph. Error bars are SD. (H) Inhibition of 3D invasion by knockdown of four different exosome core subunits. MDA-MB231 cells were transfected with indicated siRNAs. After 72 hr, the cells were allowed to invade through a collagen matrix. Cells were labeled 24 hr prior to invasion analysis by CellTracker-CMRA (orange). Z stack images were taken at 5-μm intervals and put together serially from 0 μm to 105 μm. Scale bar, 200 μm. (I) Quantification of 3D invasion analysis from (H) (n = 6). Significant p values are stated above each bar graph. Error bars are SD. See also Figure S3.
Figure 7
Figure 7
Two-Step Model for Regulation of Protrusion Formation by Local Translation Protrusions are initiated by the action of actin regulators such as Rho-GTPases, which promote actin polymerization via ARP2/3 complex and formins. Once initiated, ribosomes and RBPs in complex with their target mRNAs are transported to protrusions where they mediate local synthesis of more actin-associated proteins, resulting in protrusion stabilization and growth. Local translation rates in protrusions are defined, not by mRNA enrichment alone, but via specific mRNA cis-regulatory UTR elements and trans-acting RBPs enriched in protrusions.
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