Spatial control of Cdc42 signalling by a GM130-RasGRF complex regulates polarity and tumorigenesis

The Golgi pool of Cdc42 controls cell polarity

To study spatial Cdc42 activation patterns we used a Cdc42 Raichu probe ^{14, 15}. For all our measurements we used a probe containing the C-terminal domain of Cdc42 ¹⁵ (Cdc42-EM hereafter). This C-terminal domain targets the FRET reporter to different membranes including the plasma membrane and various cellular endomembranes such as the Golgi and endosomes (Supplementary Fig 1A) localizing as reported for endogenous Cdc42⁵. Cdc42-EM appeared in a juxtanuclear pool colocalizing with GM130, indicating its presence at the Golgi complex (Fig. S1B), thus allowing us to monitor Cdc42 activation at this cellular location where Cdc42 is active ⁶ (Fig. 1 A). Measuring FRET outside the Golgi-area or the plasma membrane did not reveal any specific signal, thus supporting the specificity of our FRET measurements (Fig. 1A). Silencing GM130 reduced the total cellular pool of Cdc42-GTP ¹⁶ (Fig. S1C), and reduced substantially the activity of Cdc42 at the Golgi (Fig. 1B). However, GM130 depletion did not affect the activity of this reporter at the plasma membrane indicating that it did not affect Cdc42 activation therein (Fig. 1C). Silencing GM130 inhibited 2D cell migration (Fig S1E) indicating that alteration of spatial Cdc42 signalling is relevant for directed cell motility. Moreover, the recruitment of aPKC to the leading edge of directionally migrating cells was also reduced (Supplementary Fig. 1F). To test for effects on polarity in a 3D cell culture model, we used Caco-2 cells, which are known to spontaneously polarize when plated in a 3D matrix, forming cysts with a single lumen. Previous work showed that alteration of Cdc42 activity (and thus of polarity) leads to the formation of multi-lumen (aberrant) cysts ^{17, 18}. Depletion of GM130 in Caco-2 cells led to an increase in the number of aberrant cysts, which is indicative of a polarity defect (Fig. 1D). These results suggested that regulation of spatial Cdc42 activity by GM130 was relevant for cell polarization in 2D and 3D cell culture. In contrast, GM130 depletion did not affect Golgi morphology (Supplementary Fig 2), ER-Golgi trafficking, Golgi-to-plasma membrane trafficking or coatomer recruitment to the Golgi (Supplementary Fig. 3A–C). Furthermore, the capability of the Golgi to nucleate microtubules was also unaffected (Supplementary Fig. 3D), in agreement with previous findings ¹⁹. Thus, the indirect impact of these processes on polarity could be excluded ⁸.

Directed trafficking causes asymmetric distribution of Cdc42

To obtain direct evidence for the role of the GM130-regulated Golgi pool of active Cdc42, we measured Cdc42 activity at the front and the rear of directionally migrating cells using the Cdc42-EM probe. Strikingly, Cdc42 activity exhibited an asymmetric distribution, being more active at the front compared to the rear of the cell (Fig. 1E, F). Silencing GM130 reduced the amount of activated Cdc42 at the leading edge, thereby breaking the asymmetric distribution of active Cdc42 (Fig. 1E, F). These results were confirmed by immunofluorescence staining of endogenous Cdc42-GTP (Fig. 1G). Sustained accumulation of active Cdc42 at the leading edge could be the consequence of an increased local activation. Alternatively, the Golgi might deliver Cdc42-GTP to the leading edge. If the latter mechanism was true, then blocking trafficking from the Golgi ought to diminish the amount of Cdc42-GTP at the leading edge. To investigate this point, directionally migrating cells in a wound closure assay were incubated at 20°C for one hour, which blocks exit from the trans-Golgi ²⁰. Measuring FRET reporter activity or staining for Cdc42-GTP revealed that blocking post-Golgi trafficking diminished the presence of GTP-Cdc42 at the cell front, thereby abolishing Cdc42-GTP asymmetry (Fig. 1E&F). Importantly, re-incubation of the cells at 37°C for one hour re-established Cdc42-GTP asymmetry with higher levels at the leading edge compared to the rear (Fig. 1F). The loss of Cdc42-GTP asymmetry in GM130 knockdown cells was likely due to abrogation of directed trafficking. Live cell imaging of GFP-VSVG tracks forming from the Golgi showed that while carriers formed readily in GM130-depleted cells, they were non-directed, contrarily to tracks in control cells, which mostly trafficked towards the leading edge (Fig. 1H). Of note, the directionality of post-Golgi trafficking was affected, whereas the global rate of post-Golgi trafficking was not (Supplementary Fig. 3B). Since, both GDP- and GTP-restricted Cdc42 is known to block post-Golgi trafficking ²¹ we conclude that GM130 depletion does not affect the GTP/GDP cycling of Cdc42. Taken together, the Golgi pool of active Cdc42 is relevant for cell polarization.

The GM130-Cdc42 crosstalk does not involve GEFs

Next we asked how the Cdc42 activity at the Golgi was regulated by GM130. One possibility is that GM130 recruited a Cdc42 GEF leading to local activation of this GTPase. If true, then this GEF must interact with GM130 and localize to the Golgi in a GM130-dependent manner (schematic in Fig. 2).

To identify GEFs that contribute to activate Cdc42 at the Golgi, we screened a library of 51 siRNAs against Rho family GEFs for effects on the Golgi pool of Cdc42 using the Cdc42-EM FRET probe. We focused primarily on the knockdowns that affected Cdc42 activity at the Golgi in a manner more conspicuous than GM130 depletion. While several GEF knockdowns decreased the FRET values at the Golgi, only three had an effect that was comparable to the effect of GM130, namely ARHGEF9, 11 and 12 (Fig. 3A). Depletion of GEF11 and 12 also resulted in reduced total cellular Cdc42-GTP (Supplementary Fig. 4B) supporting these FRET measurements. Depletion of any of these GEFs also resulted in an inhibition of directed migration as determined in a wound-healing assay (Supplementary Fig. 4C). In addition, polarization of the Golgi to the leading edge of migrating cells was also affected (Supplementary Fig. 4D). However, none of these GEFs interacted with GM130 in co-immunoprecipitation experiments, nor did they localize to the Golgi (Fig. 3B, C). We also failed to detect any evidence for an involvement of the GEF Tuba, as it did not localize to the Golgi (Fig. S5A, B), nor did it interact with GM130 (Supplementary Fig. 5C, D) contrarily to previous findings ¹⁶, and in agreement with others who failed to detect this GEF at the Golgi ^{22, 23}. In addition, neither Tuba depletion (Fig 3A, Fig S5E) nor its overexpression (Supplementary Fig. 5F) changed the activation of Cdc42 at the Golgi. Of note, none of the GEFs tested localizes to the Golgi (Oliver Rocks, personal communication). Therefore, the GM130-Cdc42 crosstalk was not mediated via recruitment of a GEF driving local activation of Cdc42.

The GM130-Cdc42 crosstalk involves RasGRF

Another explanation for the GM130-Cdc42 connection could be that GM130 was preventing the function of some Cdc42 inhibitor (schematic in Fig. 2). One possible candidate was the GEF RasGRF2, the RasGRF isoform expressed in epithelial cells, which has been recently shown to act as an inhibitor of Cdc42 ¹³. A marked association of endogenous GM130 and RasGRF2 could be detected in co-immunoprecipitation experiments (Fig. 4A). Moreover, in about 50% of the cells, RasGRF2 co-localized with GM130 at the juxtanuclear region, consistent with Golgi complex localization (Fig. 4B, C). RasGRF in addition also exhibited a localization pattern reminiscent of the ER, which is in agreement with previous work ²⁴. Noticeably, upon GM130 depletion, RasGRF2 juxtanuclear localization dropped strongly to less than 10% of the cells (Fig. 4B, C). Moreover we found that the interaction between these proteins was mediated by the C-terminal domain of GM130 and the Cdc25 domain of RasGRF2 (Supplementary Fig. 6A& B). These data suggested that GM130 is involved in localizing RasGRF to the Golgi complex.

Overexpression of RasGRF1 or 2 blocked activation of Cdc42 at the Golgi as determined using the Cdc42-EM probe, in a manner dependent on the GEFs DH domain (Fig. S6C). Importantly, co-knockdown of GM130 and RasGRF2 restored Cdc42 activation levels at the Golgi (Fig. 1A). The involvement of RasGRF in the GM130-Cdc42 crosstalk was further supported by the finding that GM130 depletion did not affect the levels of GTP-Cdc42 in CHL cells which naturally lack all RasGRF isoforms (Fig. 4D). Therefore, we conclude that RasGRF is an essential mediator in the GM130-Cdc42 crosstalk.

Regulation of the GM130-RasGRF complex

We hypothesized that GM130 could sequester RasGRF, thereby preventing it from binding and inhibiting Cdc42. Thus, under conditions that activate Cdc42, GM130 would sequester RasGRF to prevent it from inhibiting Cdc42. To test this hypothesis, we determined whether the GM130-RasGRF complex is affected by serum-starvation, a condition under which Cdc42 is not activated. Under these conditions, GM130 did not co-immunoprecipitate with RasGRF (Fig. 5A), contrary to cells in steady-state cultured under complete conditions (Fig. 3). Treatement with 10% FCS for 10 min resulted in re-formation of the GM130-RasGRF complex (Fig. 5A). If RasGRF were free under serum-starved conditions, then we would expect the Golgi pool of Cdc42 to be unresponsive to GM130 depletion. Cdc42 at the Golgi responded to serum treatment in control siRNA transfected cells, while GM130 depleted cells did not respond to this treatment (Fig. 5B). Of note, the FRET values at the Golgi were similar in control and GM130 silenced serum-starved cells, further supporting the notion that this pool becomes GM130-independent under serum-starved conditions (Fig. 5B).

If GM130 acts to protect Cdc42 from inhibition by RasGRF, then depletion of GM130 ought to increase the association between RasGRF and Cdc42. Co-immunoprecipitation experiments demonstrated that this was the case: the amount of RasGRF in association with Cdc42 increased in GM130 knockdown cells compared to control cells (Fig. 5C).

Finally, we tested whether RasGRF is relevant for the GM130-Cdc42 crosstalk in the context of polarity. This was the case, as co-depletion of RasGRF2 and GM130 rescued the polarity defects imposed by GM130 depletion (Fig. 3F&G).

The GM130-RasGRF complex controls Ras activity

Our data indicated that GM130 acts as a repressor of RasGRF. Consistent with this model (Fig. 6A), knockdown of GM130 would release RasGRF, allowing it to inhibit Cdc42. However, RasGRF is also a well-known activator of Ras. Thus, in agreement with our model, loss of GM130 should also result in an increase of Ras activation. To test this prediction, we performed a knockdown of GM130 and measured Ras activation using a pulldown assay. Depletion of GM130 increased the levels of GTP-loaded Ras and co-knockdown of RasGRF abolished this effect (Fig. 6B), indicating that the increase in Ras activity was dependent on RasGRF. We also tested whether signalling downstream of Ras was modulated at the level of ERK1/2 in response to mitogen treatment. Knockdown of GM130 increased ERK1/2 activation in response to serum stimulation and this effect was again dependent on RasGRF (Supplementary Fig. 7A). To test whether the effect on ERK1/2 signaling is biologically relevant, we depleted GM130 in PC12 cells. These cells are a standard model for ERK-dependent cell fate decisions ²⁵. Higher and more sustained ERK1/2 signalling increases the likelihood that PC12 cells shift from proliferation to differentiation, which is evident by the emergence of neurites. Depletion of GM130 resulted in elevated and sustained ERK1/2 activation (Supplementary Fig. 8A) and accordingly the percentage of cells that underwent differentiation was significantly increased (Supplementary Fig. 8B&C). Thus, the effects of GM130 on Ras signalling are relevant for ERK-dependent cell fate decisions.

Expression of GM130 in colon cancer

Loss of polarity is a frequent feature of epithelial tumours and is associated with dysplasia and increased metastatic potential ^{3, 4}. Our results indicated that GM130 regulates polarity by modulating the Golgi pool of active Cdc42 and, in addition, Ras activity. Therefore, we asked whether GM130 might be associated with the malignant phenotype. To investigate this possible connection, we used two strategies. First, we analysed GM130 expression in a colon cancer progression TMA comparing normal colon mucosa (29 cases), adenoma (16 cases), and adenocarcinoma (109 cases) (Fig 7A). While the expression of GM130 only differed marginally in normal colon and adenoma, the expression in carcinoma was reduced markedly (Fig. 7B–D). We next extended this analysis in samples from 16 patients, present on TMA, with matched normal and cancer tissues. We compared the scored intensity of GM130 expression and found that in 14 out of 16 individual patients the expression of GM130 was reduced in tumours compared to the matched normal tissue (Fig. 7E&F). The down-regulation of GM130 in cancer was further confirmed by an analysis of 11 matched normal and cancer colon tissues from a different source (Supplementary Fig. 9). In this case, the intensity of the GM130 staining was measured and healthy tissues were compared with the matching cancerous tissues. In agreement with the previous results, GM130 was down-regulated in the cancerous tissue in 9 out of 11 patients (Supplementary Fig. 9A–C).

In support for a role of GM130 loss in tumour progression, we found that knockdown of GM130 in Caco-2 cells led to a loss of E-cadherin expression (Supplementary Fig. 9D), a phenotype that is often considered to be linked to tumour progression and a loss of epithelial identity and is in line with the notion that loss of polarity is a typical phenomenon in EMT ²⁶. We could show that the effect of GM130 on E-Cadherin was not cell line specific, as the depletion of GM130 also caused loss of E-Cadherin in another epithelial cell line (Supplementary Fig. 9D).

Cell Culture and transfection

HeLa, HEK293, HepG2, MCF7 cells were purchased from ATCC and were cultured in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% FCS and 100 U/ml penicillin/streptomycin. RPE-1 cells were grown in DMEM/F12 supplemented with 10% FCS and 100U/ml penicillin/streptomycin. MDA-MB-231 were cultured in RPMI supplemented with 10% FCS and 100 U/ml penicillin/streptomycin. PC12 cells were cultured in RPMI supplemented with 10% HS, 5% FCS and 100U/ml penicillin/streptomycin. Caco-2 cells were cultured in minimal essential medium supplemented with 20% FCS and 100 U/ml penicillin/streptomycin. To generate stable cell lines, cells were transduced with lentiviruses (produced according to the protocols of Trono’s lab) and transduced cells were either selected with antibiotics or sorted via FACS. Transfection of siRNA was performed using HiPerFect (Qiagen) or Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions. The siRNAs used for the GEF screening were purchased from Thermo Fisher Scientific. Control siRNA was purchased from Thermo Fisher Scientific (ON_TARGETplus Control pool – Cat. Nr. D-001810-10-20). The siRNAs against GM130 were from Thermo Fisher Scientific (ON-TARGETplus smart pool – Cat. Nr. L-017282-00 – indicated in the text as siRNA1) and from QIAGEN (SI04235147 - indicated in the text as siRNA2 – and SI00429191, indicated in the text as siRNA3). The siRNA against RasGRF2 was from Qiagen (SI04329941). The siRNA against TUBA was from QIAGEN (SI04220237 – used in Fig 1B). Plasmid transfection was performed using Fugene6 (Promega) according to the manufacturer’s instructions. Cells were typically transfected 24 h prior to the experiment. PC12 cells were transfected with FugeneHD according to the manufacturer’s instructions.

Plasmids

Cdc42-GFP-wt (nr. 12599), Cdc42-GFP-TN (nr. 12601), Cdc42-GFP-QL (nr. 12600) were from Addgene (Klaus Hahn’s Laboratory⁶). RasGRF1-HA, RasGRF2-FLAG, RasGRF1-ΔPH-DH, RasGRF1ΔCdc25, RasGRF2-ΔDH and RasGRF2-ΔCdc25 were described previously¹³. myc-GM130-ΔN690 was cloned using pcDNA 3.1 myc-His as backbone pL-Venus-Cdc42 was cloned using a modified version of pLVTHM as backbone. RAICHU-Cdc42-EM and RAICHU-Cdc42-PM were kind gifts from Dr. Michiyuki Matsuda (Kyoto University, Japan). The RUSH-MannII-GFP expressing HeLa cells, as well as the VSVG-GFP-RUSH plasmid were a kind gift from Dr. Franck Perez³⁹. Temperature-sensitive VSVGts045-GFP was a kind gift from Dr. Kai Simons. Flag tag ARHGEF9, 11 and 12 were a gift from Dr. Oliver Rocks. Lentiviral packaging plasmids were bought from Addgene (psPAX2 nr. 12260, pMD2.G nr. 12259 and pLVTHM nr. 12247, from Didier Trono’s laboratory). GM130 GIPZ shRNA was bought from Thermo Scientific (Clone ID: V3LHS_313361). GM130 rat shRNAs (RSH050521 nr 1 to 4) and Control shRNAs (MSH029723-CH1) were bought from Genecopoeia. pmTurquoise2-Golgi was from Addgene (nr. 36205 ⁴⁰).

Pull-down experiments

To pull down active Cdc42 cells were lysed in buffer (25mM HEPES pH7.5, 150mM Nacl, 1% Nonidet-P-40, 10mM MgCl₂). Purified GST-tagged effector domain of PAK1 immobilized on GSH-sepharose beads was added to the lysate that was pre-cleared by centrifugation at 21,000xg for 5 min at 4°C. After 1 h incubation at 4 °C, beads were washed twice followed by boiling in sample buffer to elute bound material.

To precipitate active Ras, the same procedure was used with the exception the purified GST-tagged Raf1-RBD immobilized to GSH-sepharose beads were added to the lysate.

ERK1/2 activation assays

250,000 HeLa or HEK293 cells were plated in 35mm dishes and transfected with siRNA at the same time (HiPerFect from Qiagen). After 72h cells were serum-starved for at least 2 h. Cells were subsequently stimulated with 10% FCS followed by lysis in ice cold MAPK buffer (150mM NaCl 20mM TRIS-HCl pH7.5, 1% Triton X-100) supplemented with protease and phosphatase inhibitors. The amount of phosphorylated ERK1/2 was determined by immunoblotting.

PC12 differentiation

PC12 cells were plated at low density on Collagen-coated coverslips. 24 h later, cells were transfected with plasmids encoding either control shRNA or GM130 shRNA. Cells were then serum starved overnight. Cells were then stimulated with 10% NGF and 1% HS for 48h, then coverslips were stained and fixed. Cells with neurites as long as the body length were scored as ‘differentiated’. Due to the presence of GFP in the plasmid encoding for the shRNAs, we were able to count only cells effectively transfected. To measure P-ERK intensity, cells were stimulated as described above for 0, 30 or 90 minutes and then stained.

Antibodies and immunofluorescent labels

Rabbit monoclonal anti-Cdc42 (dilution 1:300 – Cat. Nr. 2466), rabbit polyclonal ERK1/2 (dilution 1:1000 – Cat. Nr. 4695) and rabbit polyclonal against phospho-ERK1/2 (dilution 1:1000 – Cat. Nr. 9101) were purchased from Cell Signalling. Mouse monoclonal GTP-Cdc42 was purchased from NewEast Biosciences (dilution 1:50 - Cat. Nr. 26905). To stain for GTP-Cdc42, Image-iT, FX Signal Enhancer from Invitrogen was used according to manufacturer’s instructions. Mouse monoclonal anti-GM130 antibody was from BD-Biosciences (IF dilution: 1:1000 – WB dilution 1:250 – Cat. Nr. 610823). Rabbit monoclonal anti-GM130 was from cell signalling (dilution 1:500 – Cat. Nr. 12480). Rabbit monoclonal anti-RasGRF (WB dilution 1:200 – IF dilution 1:50 – Cat. Nr. Sc-863) and Mouse monoclonal E-Cadherin antibody (dilution 1:500 – Cat. Nr. 21791) were purchased from Santa-Cruz. Rabbit polyclonal anti-Giantin antibody was from Covance (dilution 1:1000 – Cat. Nr. PRB-114C). Monoclonal ANTI-FLAG^® M2-Peroxidase (HRP) was from Sigma (dilution 1:2000 – Cat. Nr. A8592). Mouse monoclonal anti-HA 12CA5 antibody was from Roche (WB dilution 1:4000 – IF dilution 1:500 – Cat. Nr. 11583816001). Mouse monoclonal anti-γ-tubulin (dilution 1:1000 – Cat. Nr. Ab11317), Rabbit polyclonal anti TGN46 (dilution 1:1000 – Cat. Nr. Ab50595) were from Abcam, Mouse monoclonal anti-GFP (dilution 1:1000 – Cat. Nr. 11814460001) was from Roche. AlexaFluor647 Phalloidin (Cat. Nr. A22287) and AlexaFluor488 concanavalin (Cat. Nr. C11252) and AlexaFluor647HPA (Cat. Nr. L32454) were from Invitrogen. For immunofluorescence staining cells were grown on glass coverslips. To stain for RasGRF, cells were fixed on ice with 4% formaldehyde for 10 min followed by permeabilization with PBS containing 0.05% triton X100 for 3 min at RT. To stain for β-COP cells were fixed with methanol-acetone (50/50) for 2 min on ice. For all other stainings, cells were fixed with 4% paraformaldehyde for 15 min at RT followed by permeabilization with PBS containing 3% BSA and 0.2% triton X100.

Microtubule nucleation assay

RPE-1 cells were plated on coverslips and transfected with siRNAs using HIPerFect. After 72 h, 0.1 mM HEPES pH 7.35 was added to the cells and the cells were then transferred on ice for 40 minutes. Then cells were put back at room temperature and fixed after 0, 3 and 4 minutes at room temperature in fixation/permeabilization buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0, PFA 2% in buffer CB, 0,025% glutaraldheide, 0,2% Tx-100). Immunostaining was then performed using antibodies against GM130, TGN46 and Tubulin.

Caco2 Cysts

Caco-2 cells were plated in 35mm dishes. 24 h later they were transfected with siRNAs using Lipofectamine 2000. After an additional 24 h cells were trypsinized and an additional siRNA transfection using Lipofectamine 2000 was performed. Cells (6×10⁴) were re-suspended in 60% DMEM + 40% Matrigel (previously thawed overnight on ice). 100ul of this solution was plated into 8-wells chambers (BD-biosciences), incubated at 37°C for 30 minutes and, once solid, Matrigel was covered with 400ul of DMEM. Media was changed every other day. On the 4^th day of culture cysts were fixed with 300ul of paraformaldehyde (4%) 30 min at RT, washed with PBS, permeabilized with 0.5% triton X-100 for 10 min and washed with PBSG (PBS containing 7.5mg/ml glycine). Non-specific staining was blocked with 300ul of blocking buffer (7.7mM NaN₃, 0.1% BSA, 0.2% Triton X-100, 0.05% Tween20) and afterwards the 8-wells chambers were incubated overnight with the primary antibodies diluted in PBS containing 3% BSA. On the next day, cells were washed with blocking buffer and secondary antibodies + fluorescent phalloidin were added for 2 h at RT. Cells were washed again with blocking buffer and the plastic support was removed. A glass coverslip was put on the top of the matrigel and the samples were imaged.

Confocal microscopy image analysis and FRET

Microscopy was performed on a LeicaSP5 confocal laser-scanning microscope equipped with a Ludin chamber. Most imaging was performed using the 63X objective (1.4 NA). Images were converted into TIFF files and analysed using ImageJ. FRET was performed using the donor-recovery after acceptor photobleaching method using the FRET-AB wizard of the Leica software. Acceptor (YFP) fluorescence was bleached at 100% laser intensity assuring a minimal extent of bleaching by 75%. To analyse FRET at the Golgi, we chose a region in the juxtanuclear region where the reporter accumulated. To test whether we could recognize the Golgi correctly when looking at the reporter, we initially performed experiments where we colocalized the reporter with Golgi markers and found that we could always recognize the Golgi accurately. To measure FRET at the plasma membrane, we chose random regions at the plasma membrane of cells grown up to 80% confluency. Briefly, cells were divided into 4 regions (quadrants of a Cartesian Coordinates Plane) and FRET was measured every time in one different region (the first cell was measured in the first quadrant, the next cell in the second quadrant, and so on). The only limitation we imposed was to avoid regions overlapping with neighbor transfected cells. ImageJ was used to process the images. Briefly, the process “subtract background” was used on every image. If needed to improve visibility, brightness and contrast of the images were corrected (linearly) using the “adjust” menu of ImageJ. Images coming from the same experiment were all modified the same way to leave unaltered the information. Merged images were created with the appropriate function of ImageJ, after correcting the single channel images as described above.

GEF screening

FRET measurements at the Golgi were performed in HEK293 cells expressing the Cdc42-EM probe. We calculated the CI95 of GM130 knockdown vs. control cells according to:

Any value below the CI95 (red line in Fig. 2) was considered a hit. Our previous measurements of control and GM130 knockdown cells were used to quantitate the CI95 with the formula described above. 30–45 cells were measured for each condition from two-three independent experiments. The rationale for measuring at least 30 cells was based on our calculation that 18 data points (i.e. cells) are necessary to distinguish a population that differs from the control by 80% of the GM130 effect.

FRET of directional migrating cells

HeLa cells were plated on glass coverslips and transfected with siRNA (HIPerfect). After 48 h, cells were transfected with plasmids encoding Cdc42-EM or Cdc42-PM FRET probes (Fugene6). After 72 h, a wound was made using a 200μl pipette tip and cells were allowed to migrate at 37°C for 6 hours. Then cells were either fixed or incubated for 1 h at 20°C and subsequently fixed. To measure FRET, we selected the region of the cells directly facing the wound and named it “front”. For regions designated as “rear” we chose regions from the side of the cell opposing the wound. Up to 3 different regions were measured in the “rear” and averaged. We did not detect any dependency on where we measured as long as the plasma membrane was opposing the wound. We avoided measuring FRET in regions overlapping with neighbor transfected cells.

Video acquisition of VSVG-GFP vesicles

HeLa cells were plated on glass coverslips and transfected with siRNA (HIPerfect). After 48 h, cells were transfected with plasmids encoding for the VSVG-GFP-RUSH (Fugene6). After 72 h, a wound was made using a 200μl pipette tip and cells were allowed to migrate at 37°C for 4 hours. Then imaging was started and biotin added to release the VSVG construct from the ER. Images were acquired every 1.5 sec.

Golgi orientation, cell migration and aPKC recruitment assay

For Golgi orientation assays, cells were plated on glass coverslips. After 72 h, a wound was made manually using a 200μl pipette tip. Cells were stained with antibodies anti γ-tubulin and anti-giantin immediately after wounding and after 6 hours. For assays measuring aPKC recruitment to the leading edge, cells were fixed 1 h after wounding.

For migration assays, cells were transfected with siRNA, and trypsinized after 48 h and plated into Ibidi® chambers placed on glass cover slips. This insert consists of two chambers separated by a 600 μm wall. Removal of the insert generates a wound with a defined width of 600 μm. 12000 cells were plated into each chamber. After 6h, the IBiDi insert was removed and cells were allowed to migrate at 37°C for 24 h. Then, cells were fixed and stained with concanavalin-alexa 488.

Coimmunoprecipitations

1×10⁶ cells were plated in 10cm² cell culture dishes. After 24 h, cells were transfected. After an additional 24 h, cells were lysed in IP buffer (20mM HEPES pH 7.3, 200mM KCl, 0,5% Triton X-100), then centrifuged for 15 min at 20’000xg. The supernatant was incubated with protein G sepharose beads coupled to the appropriate antibodies. For anti-HA immunoprecipitation commercial anti-HA beads (Sigma) were used. After 2 h incubation at 4°C, beads were recovered by centrifugation, washed and the bound material was eluted by boiling in sample buffer.

RUSH assay to study ER-Golgi trafficking

The RUSH assay was performed as described previously ³⁹. Briefly, 250,000 HeLa cells stably expressing RUSH-ManII-GFP were plated in 35mm cell culture dishes and transfected with siRNA (using HiPerFect from Qiagen). After 72 h, cells were either fixed directly or were treated with 40 μM of biotin for 1 h followed by fixation in 4% paraformaldehyde at RT.

RT-PCR

Total RNA was isolated from cells by using the Qiagen RNeasy kit. cDNA was prepared from 100ng total RNA by using the high capacity cDNA reverse transcription kit (Applied Biosystems) following manufacturers’ instructions. Q-PCR was performed by using Fast SYBR green PCR MasterMix (Applied Biosystems). RT-PCR were run on 7900HT Fast RT-PCR system (Applied Biosystems). Expression of each gene was normalized to the expression of ubiquitin and β-2-microglobulin. Specific primers for RasGRF2 were described in Jacinto et al. ⁴¹. Primers for TUBA, ARHGEF9, ARHGEF11 and ARHGEF12 were designed using PrimerExpress®.

Immunohistochemistry

Three-μm slides cut from three different tissue microarray block containing a total of 229 different formalin-fixed, paraffin-embedded samples from colorectal cancers (n=133), tubulovillous adenomas (n=27) and normal colonic mucosa (n=69) were used (duplicate cores for each samples). The project was approved by the ethical committee of the IEO. Samples were rehydrated through xylene and graded alcohols. Antigen retrieval was accomplished using 1 mM EDTA, 0.05% Tween. After peroxidase block with 3% H₂O₂ for 5 min, and block with 2% goat serum in PBS for 1 h, samples were incubated with primary antibodies (Mouse monoclonal anti-GM130 antibody, BD-Biosciences) in a 1:50 dilution for 60’ min at RT in 2% goat serum. Immunocomplexes were visualized by the EnVision^™+ HRP Rabbit (DAKO, K4011), and acquired with the Aperio ScanScope system. Duplicate cores for every sample were evaluated and then the scores were averaged and considered as weak expression (average score 0.5 – 1), moderate expression (average score 1.5 – 2), strong expression (average score 2.5 – 3), or as negative if they scored 0. IHC data for GM130 expression were available only for 29 normal colonic mucosa, 16 tubulovillous adenoma and 109 colorectal cancer samples, since in some cases, individual cores detached from the slides during the manipulations.

11 matched colon cancer samples were analysed at the University of Regensburg according to the following procedure: two-μm slides cut from a tissue microarray block containing 60 different formalin-fixed, paraffin-embedded samples from colorectal cancers (n=20) and matched normal tissues (n=40) were used. Immunohistochemistry of GM130 was carried out in a BenchMark ULTRA (VENTANA) autostainer according to the manufacturer’s instructions. Briefly, retrieval was done using a citrate buffer (pH 6.0) for 44 min. The anti-GM130 antibody was used in a 1:100 dilution and incubated for 32 min. The slides were detected using the ULTRA View DAB detection kit (VENTANA). Negative controls without primary antibody were included in each experiment.

Statistical analysis

The data were analysed using Student T Test, Chi Square test or ANOVA test for multiple comparison in GraphPad Prism. All IF images are representative of at least 3 independent experiments. Western blots evaluations and IF evaluations are averages of at least 3 independent experiments. For TMA analysis differences between experimental groups were examined for statistical significance using the nonparametric comparisons for each pair using Wilcoxon Method (Figure 7B) or Pearson Chi Square test (Figure 7C). Where applicable, data are expressed as average ± SD. TMA data analysis were performed using JMP 10.0 statistical software (SAS Institute, Inc).