a Phalloidin GM13 p-tyr b p-tyr 23 116 96 PDGF Control Control EGF PDGF 52 35 28 Figure S1 Tyrosine phosphorylation induced by growth factors differs from that arising from the arrival of traffic at the Golgi complex. The Tyrphosphorylation response to growth factors was examined at both the morphological and biochemical levels. (a) Platelet-derived growth factor (PDGF) stimulates tyrosine phosphorylation at the PM, and particularly in ruffle-like structures, rather than in the Golgi area. Cells were serum-starved overnight and then treated with 1 ng ml -1 PDGF at 37 C for 5 min. F-actin was stained with Alexa488-phalloidin, and the cells were immunostained for the cis-golgi marker GM13 (red) and p-tyr (green), as indicated. Arrows, membrane ruffles. Images are representative of two independent experiments where at least 1 cells were analyzed. Scale bars, 1 µm. (b) Epidermal growth factor (EGF) and PDGF induce cellular tyrosine phosphorylation patterns different from those caused by the arrival of traffic. Cells were serum-starved for 48 h, treated with 1 ng ml -1 EGF or PDGF, as indicated, at 37 C for 5 min, and then homogenized. The p-tyr patterns were revealed by SDS-PAGE and immunoblotting, and they are different from those generated by the arrival of a traffic pulse at the Golgi complex (compare with Fig. 2a, b). Data are representative of three independent experiments. www.nature.com/naturecellbiology 1
a Yes HF HeLa NIH 3T3 62 d PC-IV Src Fyn 59 Lyn 56 53 PC-IV/GM13 b SFKs SFKs/Giantin 3 HF e p-sfks/gm13 HeLa FL p-sfks 3 c f p-tyr HF NRK HeLa g HF p-sfks NRK SFKs on the Golgi (% of total IF) 8 6 4 2 HF HeLa FL 116 96 52 35 28 3 3 3 3 h HeLa 4 C 32 C 4 C 32 C 4 C 32 C p-yes p-src/fyn p-lyn Figure S2 Although the SFKs repertoire is different across cell lines, traffic pulses induce similar phosphorylation patterns in all cells. Different cell lines have their own particular and complex SFK repertoire. (a) Western blotting of the SFKs Yes, Src, Fyn and Lyn (two isoforms) in, HF, HeLa and NIH 3T3 cell lysates. (b) Endogenous SFKs localize on the Golgi complex in HF, HeLa fibroblast-like (HeLa FL) and cells. The cells were grown under standard conditions, and processed as detailed in Methods. Staining was with an anti-sfk (red) and anti-giantin (green) antibodies. The right panels show the co-localization between SFK and giantin staining. Scale bars, 1 μm. (c) Quantification of data illustrated in b, showing the IF signal of the SFKs as means (±SE) (as assessed in 1 to 2 cells) in the Golgi area as a percentage of total cellular IF. p<.1 versus the other cell lines. p-sfk and p-tyr responses to traffic arrival at the Golgi complex. (d) HeLa cells secrete procollagen-iv (PC-IV) in a synchronisable fashion. For their synchronisation, HeLa cells were incubated for 3 h at 4 C, shifted at 32 C for 3 min in the presence of 5 µg ml -1 ascorbic acid, fixed and double-stained for GM13 (blue) and PC-IV (green). This experiment was carried out in triplicate. Scale bars, 1 µm. (e) The Golgi SFKs are activated by a PC-IV transport pulse in HeLa cells. The cells were treated as above and double-stained for GM13 (blue) and p-sfks (red). A strong increase in Golgi-located p-sfks is seen at 3 min, with respect to that at time. This experiment was carried out in triplicate. Scale bars, 1 µm. (f) The p-tyr patterns are similar in different cell lines exposed to a traffic pulse. HF, NRK, HeLa and cells were treated as in a and then homogenized. The p-tyr patterns of their lysates were analysed by immunoblotting with an anti-p-tyr antibody. All four cell lines show common traffic-arrival-dependent p-tyr patterns. confirming the generality of the p-tyr signalling response at the Golgi. (g) Western blotting of p-sfks in cells treated as in f. Traffic pulses induce SFK activation in all cell lines tested. The traffic-dependent SFK activation pattern varies across cell lines. (h) Western blotting of p-sfks in cells treated as in f. In high resolution gels of lysate from traffic-pulsed cells, a clear increase is seen in a -kda band that probably represents p-src and p-fyn and the slight increase seen in the 62-kDa band should represent p-yes. No lower bands were visible, indicating that Lyn activation is not controlled by traffic arrival in cells. Data shown in f, g and h are representative of at least two independent experiments. 2 www.nature.com/naturecellbiology
a b GM13 p-sfks 2 c VSVG d p-sfks IF intensity on the Golgi (AU) SFKs p-sfks PC-IV/GM13 PC-IV 2 175 125 75 25 * 32 C 4 C e IF intensity (AU) 12 1 8 4 2 ** -VSVG PC-IV +VSVG 2 1 2 2 4 min at 32 C min at 4 C Figure S3 Transfected Src localizes to the Golgi complex and is activated by traffic in cells. Transfected Src localizes to the Golgi complex and is activated by both PC-IV and VSVG traffic pulses. (a) cells secrete PC-IV in a synchronisable fashion. Cells were incubated for 3 h at 4 C, shifted to 32 C for 2 min in the presence of 5 µg ml -1 ascorbic acid, fixed and double-stained for GM13 (blue) and PC-IV (green). The ER staining is negligible probably because the anti-pc-iv antibody recognizes only the folded state of PC-IV. Images are representative of two independent experiments. Scale bars, 1 µm. (b, c) PC-IV and VSVG traffic pulses induce Golgi-SFK activation. cells were transfected with Src alone (b, traffic pulse here provided by endogenous PC-IV, see panel a) or with both Src and VSVG-GFP, c. During the traffic block (left panels), low levels of p-sfks were seen in the Golgi area. Twenty min after the block release to 32 C, PC-IV (panels a, b) and VSVG (panel c) have moved to the Golgi area and there are high levels of p-sfks in the Golgi area (b, c right panels). We also used a (phosphorylationinsensitive) anti-sfk antibody (SC-18) to examine the effects of traffic on total cellular SFKs (c, SFKs). The SFKs were clearly detectable in the Golgi area; however, their Golgi levels were unaffected by arrival of traffic. Data in b and c are representative of at least three independent experiments. Scale bars, 1 µm. (d) Quantification of p-sfk IF in the Golgi area from data illustrated in b and c as means (±SE) from at least five independent experiments, each assessing 1 to 2 cells. IF intensities are expressed as arbitrary units (AU). *p<.5, **p<.1, versus relative controls at time (ANOVA analysis). (e) To study the kinetics of traffic-dependent SFK activation and inactivation, we performed a time course of Golgi cargo loading and emptying with VSVG. A detailed analysis of p-sfks was carried out across several cellular compartments. The graph shows the quantification of the IF of VSVG (blue diamonds), Golgi p-sfks (pink squares) and p-sfks in the cytoplasm (red triangles) in cells following the 4-32 C traffic pulse protocol that was then followed by an ER transport block (4 C) for the indicated times. p-sfks increased in the Golgi area at 32 C; then, when the block of VSVG traffic was reinstalled (4 C), the VSVG and p-sfk levels in the Golgi area both gradually returned to basal levels over min. IF intensities (means ±SE) are expressed as arbitrary units (AU; see Methods). **p<.1,p<.1 versus relative controls at time (ANOVA analysis). Data are representative of at least three independent experiments, each assessing 3 to 4 cells. www.nature.com/naturecellbiology 3
a Dextran p-sfks p-sfks/gm13 Sar1GTP Control b c p-tyr p-sfks IF on the Golgi (AU) 12 1 8 4 2 * 4 C 32 C 4 C 32 C Control Sar1GTP 116 96 52 35 4 C 32 C Control 4 C 32 C CHX Protocol 1 4 C 32 C CHX Protocol 2 Figure S4 Traffic-induced tyrosine phosphorylation is not influenced by temperature shifts. As the traffic pulses involve temperature changes, we examined whether temperature per se influences the Golgi p-sfk response. (a) HeLa cells were incubated for 3 h at 4 C, and during the last hour of this they were microinjected with 1.5 mg ml -1 Sar1-GTP plus 1.2 mg ml -1 FITC-labelled dextran (marker), as Sar1-GTP microinjection blocks ER-to-Golgi transport. The cells were then shifted to 32 C for 3 min and double-stained for GM13 (blue) and p-sfks (red). Sar1-GTP prevented activation of SFKs in the Golgi area, indicating that Golgi-SFK activation depends only on transport. Scale bars, 1 µm. (b) Quantification of data illustrated in a, as means (±SE) from four independent experiments, each assessing 1 to 2 cells. *p<.5, versus 32 C control without Sar1- GTP (Student s t-test). IF intensities are expressed in arbitrary units (AU). (c) Golgi p-sfk response in cargo depleted of HF cells. The cells were initially treated for 2 h with either vehicle (control) or CHX (protocol 1), to allow emptying of cargo from the secretory system; they were then incubated for 3 h at 4 C still in the presence of CHX, shifted to 32 C for 3 min, and then homogenized. The possible toxic effects of CHX were evaluated with CHX protocol 2: the cells were first incubated for 3 h at 4 C (to accumulate PC-I in the ER), and then treated with CHX for 5 h at 4 C; finally, they were shifted to 32 C (block release) for 3 min and homogenized. The p-tyr patterns were analysed by immunoblotting with an anti-p-tyr antibody. Data are representative of at least two independent experiments. For a summary of the other independent approaches that are relevant to the effects of temperature, see Supplementary Information, Text S2. 4 www.nature.com/naturecellbiology
a VSVG-GFP p-sfks KDEL-R KDEL-R sirnas Control b d 2 Control KDEL-R sirnas Src KDEL-R e c p-sfks IF intensity (AU) 14 12 1 8 4 2 Control KDEL-R sirnas Sec31 VSVG PDI 8 VSVG KDEL-R f 2 KDEL-R redistribution (% non-golgi/ total IF) 45 4 35 3 25 2 15 1 5 2 min at 32 C Figure S5 KDEL-R and chaperones in traffic-induced activation of Golgi SFKs. Silencing of the KDEL-R with sirnas impairs traffic-induced SFK activation in the Golgi area in cells. (a-c) Cells were transfected for 72 h with sirnas for the KDEL-R. During the final 24 h, they were transfected with VSVG-GFP and Src, then subjected to a VSVG traffic pulse, and stained for p-sfks. (a) Knock-down of the KDEL-R by the sirnas blocked SFK activation in the Golgi area. Scale bars, 1 µm. (b) Cells treated as in a, but not transfected with Src and VSVG-GFP, were analysed by Western blotting for Src and the KDEL-R. Data shown are representative of two independent experiments. (c) Quantification of data illustrated in a, (p-sfk IF intensity; means ±SE). IF intensities are expressed as arbitrary units (AU). p<.1, versus control (Student s t-test). Data are representative of two independent experiments, each assessing 1 to 2 cells. Of note, the morphology of the Golgi complex was not affected during this KDEL-R sirnas treatment (as assessed by giantin IF; data not shown). Non-targeting sirnas had no effects on KDEL-R levels when compared to untreated cells (data not shown). Chaperones exit the ER during a traffic pulse and induces the redistribution of the KDEL-R. (d) cells were infected with VSV and incubated for 3 h at 4 C, shifted to 32 C for 3 min (not shown) and 8 min, and processed for IF. During the block, the chaperone PDI was located in the ER, as was VSVG, while the ER exit site marker Sec31 was seen in numerous scattered puncta. Eight min after the block release, VSVG had moved from the ER to the Golgi area and its carriers were clearly visible (arrows in VSVG, 8-min panel). PDI was present in the same carriers (arrows, PDI 8-min panel); these did not contain Sec31 (arrows, Sec31 8-min panel). Thus 8 min after block release, these VSVG carriers are already uncoated and likely to be detached from the ER. Scale bars, 1 µm. (e) The KDEL-R redistributes to the periphery during a traffic pulse. In cells treated as in a, 2 min after the block release VSVG was concentrated in the Golgi area and the KDEL-R has partially redistributed back to the ER. Scale bars, 1 µm. (f) Quantification of data illustrated in e, showing the KDEL-R (means ±SE) outside the Golgi area (non-golgi IF) as a percentage of total cellular IF. p<.1 versus control min (Student s t-test). Data are representative of three independent experiments, each assessing 1 to 2 cells. www.nature.com/naturecellbiology 5
a VSVG-KDEL-R ONO SU6656 Control b Control VSVG 15 nm GM13 1 nm 32 C 4 C (min at 4 C) VSVG 15 nm GM13 1 nm SU6656 VSVG 1 nm GM13 15 nm VSVG 15 nm COP I 1 nm VSVG 15 nm COP I 1 nm VSVG 1 nm MannII 15 nm NRK Figure S6 SFKs are not required for retrograde, Golgi-to-ER, trafficking of the KDEL-R, whereas they are needed for intra-golgi transport of VSVG. (a) KDEL-R recycling does not require basal SFK activity in cells. The retrograde, Golgi-to-ER, pathway was monitored using a chimeric construct that expressed a KDEL-R and VSVG fusion protein. As with wild-type KDEL-R, this protein cycles between the ER and the Golgi complex at 32 C, resulting in a prominent Golgi complex localization (panels ). On changing temperature to 4 C (panels ), the VSVG component of the chimera unfolds and becomes trapped in the ER, resulting in a rapid shift in the distribution of the chimera from the Golgi complex to the ER (control) 15. When the same treatment was repeated in the presence of the SFK inhibitor SU6656 (5 µm), the chimera still shifted from the Golgi to the ER after min at 4 C, indicating no impairment of Golgi-to-ER trafficking under SFK inhibition (SU6656 panel). As a positive control, we used the PLA 2 inhibitor ONO-RS- 82 (1 µm), which has been shown to block Golgi-to-ER trafficking 16. Data are representative of at least two independent experiments. Scale bars, 1 µm. (b) EM analysis shows that SFK inhibitors block intra-golgi transport of VSVG. and NRK cells were infected with VSV and kept at 4 C for 3 h, with addition of vehicle (Control) or 5 µm SU6656 for the last 3 min. The temperature block was released (32 C) for min, and the samples were processed for cryo-immunogold labelling with anti-vsvg (filled arrowheads) in combination with either GM13, COPI (empty arrowheads) or mannosidase II (MannII), as indicated. Data are representative of at least three independent experiments. Scale bars, 2 nm. Morphometric analysis carried out on NRK cells 1 h after the 4 C block release of VSVG transport revealed that the mean number of Golgi cisternae per stack was 5.5, with a mean length of 552 nm in control cells; in SU6656-treated cells, these increased to 7. and 159 nm, respectively. Together, these data indicate that SU6656 inhibits cargo transit from the medial-trans cisternae to the TGN. We also surveyed the distribution of Golgi machinery proteins in SU6656-treated cells, here used as markers. We noticed that the locations of GM13 and COPI were altered. The functional significance of these alterations is unclear but they certainly indicate a disorganization of the trafficking machinery and may provide potentially useful indications for future molecular studies. 6 www.nature.com/naturecellbiology
Figure S7 Regulation of intra-golgi trafficking in SYF cells. (a) SFK-inhibitors impairs transport of VSVG in SYF cells. The cells were infected with VSV and kept at 4 C for 3 h, with vehicle (control) or SKI-1, (1 μm), for the final 45 min. The block was then released (32 C) for 9 min, and the total (green) and PM (red) VSVG was revealed with an antibody against the extracellular domain of VSVG. (b) Quantification of VSVG transport in SYF cells treated as in a with SKI-1, and with PP1, PP3, SU6656 (1 μm) and PP2 (2 μm). VSVG transport was inhibited by all of the SFK blockers tested, but not by the inactive analogue of PP3. The further SFK inhibitors were added either for the final 45 min of the 4 C block (SU6656) or for the full 3 h 4 C block (PP1, PP2, PP3). These data thus strongly indicate that SYF cells express at least one member of the SFKs, the activity of which is required for membrane transport. Data are means (±SE) from three independent experiments, each assessing 1 to 2 cells. The VSVG IF intensities on the PM are expressed as arbitrary units (AU). p<.1, versus control (ANOVA analysis). (c) Presence of various members of SFKs in SYF cells. PCR for all of the SFKs suggested the presence of Fgr, Hck (not shown) and Lck (not shown) in SYF cells, with the presence of Fgr and Hck confirmed by further analysis, as described below: the total SYF RNA was reverse transcribed with a random hexamer. Amplification of 25 ng of cdna was carried out for 35 cycles. Ten μl of the PCR products was analyzed by electrophoresis on 2% agarose gels. Controls are represented by amplification of Fyn (knocked out in SYF cells), and two parallel samples (Fyn and Fgr) containing everything but the reverse transcriptase (-RT). M, molecular weight standards. Sequence analysis of the purified PCR bands confirmed the identity of the amplicons as murine Fgr and Hck (not shown). (d) Finally, we confirmed the presence of Hck and Fgr in SYF cells by Western blotting. Lane 1, wild-type SYF cells; lane 2, transfected SYF cells. (e) Endogenous Hck localizes to the Golgi complex in SYF cells. SYF cells were grown under standard conditions, fixed and stained with anti-hck and anti-gm13 antibodies. Endogenous Hck staining mainly co-localized with GM13, although it is also present in the ER, at the PM, and on some unidentified cytoplasmic structures. Altogether, these data and other published results 17 show that SYF cells contain at least three members of the SFKs, and that a SFK is involved in supporting intra-golgi trafficking. www.nature.com/naturecellbiology 7
a VSVG b KDEL-R KDEL-R D193N VSVG IF intensity on PM (AU) 1 14 12 1 8 4 2 KDEL-R KDEL-R D193N 15 c VSVG-GFP KDEL-R M KDEL-R M D193N d VSVG IF intensity on PM (AU) 14 12 1 8 4 2 KDEL-R M Src KDEL-R D193N M Src KDEL-R M Src c.a. ** KDEL-R D193N M Src c.a. Src Src c.a. Figure S8 DN KDEL-R blocks intra-golgi trafficking in cells, a block reversed by active Src. (a) cells were transfected with KDEL-R-GFP (KDEL-R) or DN KDEL-R-D193N-GFP (KDEL-R-D193N), and infected with VSV. They were then subjected to the 4-32 C VSVG traffic pulse and examined by IF microscopy. The KDEL-R and KDEL-R-D193N (green) and VSVG (red) are shown at the end of the temperature block () and after 15 and min at 32 C (block release). The effects of the KDEL-R- D193N mutant are very similar to those of the SFK inhibitor SU6656 (see Fig. 6a, b): ER-to-Golgi trafficking was not affected (15 min), whereas the arrival of VSVG at the PM was inhibited ( min). Scale bars, 1 µm. (b) Quantification of data illustrated in a, as means (±SE) from two independent experiments, each assessing 1 to 2 cells, showing the arrival of VSVG at the PM min from temperature block release. IF intensities are expressed as arbitrary units (AU; see Methods). **p<.1, versus KDEL-R (Student s t-test). (c) The transport block imposed by the DN KDEL-R is overcome by overexpression of constitutively active Src. cells were transfected with VSVG-GFP, Myc-tagged KDEL-R (KDEL-R M ) or KDEL-R-D193N-Myc (KDEL-R-D193N M ), and wild-type Src (control; Src) and constitutively active Src (Src c.a.). The cells were subjected to the 4-32 C VSVG traffic pulse and examined under IF microscopy. KDEL-R M, KDEL-R-D193N M (red) and VSVG (green) are shown min after release of the temperature block. The block of VSVG transport to the PM seen in KDEL-R-D193N-transfected cells was rescued by overexpression of constitutively active Src, where VSVG arrival at the PM was restored; this was not the case for wild-type Src transfection. Scale bars, 1 µm. (d) Quantification of data illustrated in c, with VSVG-GFP on the PM determined by integrating the VSVG IF excluding the Golgi and the nuclear areas. (b, d) Data are representative of at least two independent experiments, each assessing 3 to 4 cells. The VSVG IF intensities (means ±SE) are expressed as arbitrary units (AU; see Experimental Procedure). **p<.1, KDEL-R-D193N plus constitutively active Src versus plus wildtype Src; p<.1, versus respective controls (ANOVA analysis). 8 www.nature.com/naturecellbiology
Supplementary Information Supplementary Information, Text S1. Subcellular distribution of p-sfks in traffic-pulsed cells under immunoelectron microscopy. The detailed Golgi distribution of p-sfks induced by traffic was analysed by immunoelectron microscopy (immuno-em). In quiescent cells (at 4 C), p-sfks were distributed throughout the cytosol and in various organelles, including the Golgi complex (Fig. 2g, h). During trafficking, p-sfks increased markedly on the Golgi complex, but not elsewhere (Fig. 2h), in agreement with the immunofluorescence (IF) data. In the Golgi, the p-sfks were mostly on the cisand trans-most cisternae and (to a lesser extent) on the cis- and trans-golgi networks (CGN and TGN). These structures accounted for 65% of the total labelling (approximately equally distributed between the two poles). The rims of the cisternae and vesicles accounted for the remaining 35%, while the core of the stacks were nearly devoid of labelling. Supplementary Information, Text S2. Golgi-SFK activation on the Golgi is not restricted to HF cells and is exclusively dependent on trafficking. Virtually all cells secrete one or more forms of procollagen 1, 2, and might therefore show activation of Golgi-SFKs under traffic synchronization protocols. We thus examined the Golgi p- SFK response to traffic (by IF) in other cell lines, including NRK fibroblasts (which secrete PC-I), HeLa and cells (which secrete PC-IV, albeit in variable amounts) (see Supplementary Information, Fig. S2d and S3a) 3, 4. In HeLa cells, the basal Golgi p-sfk signal was faint at 4 C, but increased during the PC-IV pulse in %-7% of cells (Supplementary Information, Fig. S2e). In NRK and cells, however, the p-sfk signal was too low to be readily assessed on the Golgi in most cells, even during trafficking (not shown), possibly because the amount of SFKs present on the Golgi complex in these cells is lower than in other cell lines (see Supplementary Information, Fig. S2c for details). We thus looked at Tyr phosphorylation patterns in cell lysates of NRK and cells, potentially a more sensitive assay than IF in single cells. Indeed, traffic-induced p-tyr bands were detectable in lysates from, NRK and HeLa cells (Supplementary Information, Fig. S2f) and their patterns were similar to those in HF cells. There was also a similar, albeit not identical, increase in p-sfks (Supplementary Information, Fig. S2g). For instance, Yes and Src (and, or Fyn), but not Lyn, were activated in and HF cells (Supplementary Information, Fig. S2h and Fig. 2c, respectively), while Yes, Fyn and possibly Lyn were activated in HeLa cells (not shown). Moreover, adding to the variability, Src was abundant in cells and scarce in HeLa 1
cells, with Fyn showing the opposite (see Supplementary Information, Fig. S2a). Thus the repertoire and activation by traffic of the SFKs varies across cell types. We also sought to overcome the limitations of the single-cell IF-based assay in cells by transfecting these cells with Src and the synchronizable cargo VSVG, as this should increase the size and reproducibility of the traffic pulse. Indeed, when these cells were subjected to a traffic pulse, the arrival of VSVG at the Golgi complex was accompanied by a large increase in the Golgi p-sfk signal (Supplementary Information, Fig. S3c-e). Similar results were obtained in VSV-infected cells. When cells were transfected with Src but not VSVG (i.e. where the main cargo was PC-IV; Supplementary Information, Fig. S3a, b, d), the p-sfk increase was visible but smaller, presumably reflecting the smaller cargo input. The same protocols were used in other cell lines (i.e. NRK, NIH 3T3 and Vero cells); these behaved like cells, confirming that this activation of SFKs on the Golgi complex by traffic in several cell lines (not shown). We also carried out control experiments to ensure that the Golgi p-sfk response is not induced or influenced by the 4-32 C temperature shift itself. First, we microinjected HeLa cells with a recombinant GTP-locked mutant of the small GTPase Sar1 (Sar1-GTP), a construct that blocks cargo exit from the ER 5, and then subjected these cells to the 4-32 C shift. This treatment completely prevented the p-sfk response on the Golgi complex (Supplementary Information, Fig. S4a, b). Secondly, we depleted the cells of cargo with cycloheximide (CHX), thus preventing them from generating a cargo pulse, and subjected them to the same temperature shift. Again, no p-sfk and p-tyr responses were detected. Note that CHX does not by itself affect secretory function nor the ability of cells to produce the p-tyr response, as shown by specifically designed control experiments (Supplementary Information, Fig. S4c). We also summarize here other independent approaches that are relevant to the effects of temperature and are described in the main body of the text. First, Golgi SFKs are activated in Vero cells upon synchronized retrograde transport of a modified Shiga toxin B (STB) fragment engineered to bear a KDEL tetrapeptide on its C-terminus (STB-KDEL). This toxin enters the cells by endocytosis. and the cells also undergo an upward 19-37 C temperature shift instead of the classical downward 4-32 C. Moreover, and more importantly, Golgi-SFK activation is seen about 1 h after the temperature shift, concomitant with the arrival of STB-KDEL at the Golgi, which also rules out possible acute effects of this temperature shift (Fig. 3a, b). Second, Golgi SFKs are activated in cells upon overexpression of an artificial KDEL- R ligand (HRP-KDEL) or the receptor itself, without any temperature shift (see Fig. 3c-e). 2
Third, there are the series of experiments that follow microinjection of an anti-kdel-r antibody and transfection of a DN KDEL-R (see Fig. 4), and the transfection of KDEL-R-directed sirnas (see Supplementary Information, Fig. S5) This collective evidence leaves little doubt that the Golgi p-sfk response is due to activation of the KDEL-R and not to temperature-change effects. Collectively, the above data show that traffic pulses from the ER to the Golgi complex induce the phosphorylation and activation of SFKs on the Golgi itself, and SFK-dependent phosphorylation of other proteins. Supplementary Information, Text S3. Traffic pulses carry enough chaperones to activate the KDEL-R and the Golgi p-sfk response. To determine whether a traffic pulse carries enough chaperones to activate the KDEL-R p- SFK-response, we first showed the presence of the chaperone disulphide isomerase (PDI) in traffic carriers leaving the ER during a 4-32 C VSVG traffic pulse, in agreement with previous observations (Supplementary Information, Fig. S5d) 6-8. Then, we examined the effects of a VSVG traffic pulse on the KDEL-R: this induced recycling of some 3% of the KDEL-R from the Golgi complex to the ER (Supplementary Information, Fig. S5e, f). Since the recycling of the KDEL-R is due to its binding to KDEL ligands (the chaperones are the only known natural KDEL ligands), this indicates that chaperones bind a sizeable portion of the Golgi KDEL-R during a traffic pulse. Finally, we sought to determine the roles of chaperones in activation of Golgi SFKs by overexpressing the abundant chaperone BiP. When expressed at medium levels (2-3-fold over basal), BiP induced a partial redistribution of the KDEL-R and of cis-golgi markers from the Golgi complex to the ER (so BiP was binding to the Golgi KDEL-R), and at the same time the Golgi p- SFK signal increased (2.5 ±.3 fold; n = 5). Very high BiP expression levels caused instead the complete dispersal of the cis-golgi and loss of the Golgi p-sfk signal (not shown). Thus, overexpressed BiP can escape the ER retention mechanisms, and like other KDEL ligands (see Fig. 3), it can induce recycling of the KDEL-R and also induce the Golgi p-sfk response (albeit less effectively than a traffic pulse). More work is needed to establish whether all of the chaperones can activate the Golgi SFKs and whether other components can mimic the chaperones or modulate their effects. 3
Supplementary Information, Text S4. SFK activation on the Golgi complex is required for steady-state trafficking. The traffic synchronization conditions used to reveal this Golgi signalling pathway involved the accumulation of cargo in the ER at 4 C, followed by rapid release of large amounts of cargo to the Golgi complex. This could result in ER stress and in Golgi overload. It has been shown previously that under similar conditions (VSVG accumulation in the ER at 4 C), the ER stress response is not activated 9. Nevertheless, a possible concern would be that the Golgi-SFK response to the arrival of traffic could come into play only under these synchronization conditions, and not during physiological steady-state trafficking. We addressed this experimentally, by asking two questions: first, can steady-state trafficking elicit a p-sfk signal on the Golgi complex, and second, are the activities of SFKs needed to sustain steady-state trafficking through the Golgi complex? For the first, we used HF and HeLa cells, where endogenous p-sfks are detectable by IF. Under conditions of non-perturbed trafficking at 37 C (Fig. 7a-c), p-tyr and p-sfk signals were visible in the Golgi area. When trafficking was arrested by the 4 C block, the Golgi p-tyr and p- SFK labelling dropped to background levels (Fig. 7a-c); when this block was lifted, the Golgi p-tyr and p-sfk signals rebounded to higher values (Fig. 7a-c). This indicates that activation of SFKs on the Golgi complex is also associated with steady-state trafficking. To examine the second question, i.e. whether activation of SFKs is needed for steady-state trafficking, we analysed the effects of the SFK blocker SU6656 on PC-I transport in non-perturbed HF cells. Here, the PC-I present in the Golgi complex in transit towards the PM is detectable by IF (Fig. 7d, time ). Thus, if SU6656 blocks intra-golgi but not ER-to-Golgi trafficking (as with traffic pulses), SU6656 should result in accumulation of PC-I in the Golgi area. Indeed, in HF cells treated with SU6656, PC-I increased in the Golgi area, gradually reaching high levels (Fig. 7d, e), although after a few hours the cargo-filled Golgi complex appeared to partially recover its export of cargo. Similar experiments were performed in cells expressing low levels of VSVG-GFP, where the VSVG in the Golgi complex during steady-state transport was barely visible (Fig. 7f, time ). Also in these cells, SU6656 induced a gradual accumulation of VSVG in the Golgi area (Fig. 7f, g), confirming that exit of cargo from the Golgi complex was inhibited. The converse was to treat HF cells with CHX to block synthesis of PC-I, and then to monitor the exit rate of the PC-I present in the secretory system at the time of the block. In SU6656-treated cells, PC-I exited the Golgi complex much more slowly than in control cells, again consistent with inhibition of intra-golgi trafficking (not shown). Similar results were obtained in other procollagen-secreting cells (NRK cells), and in cells where trafficking was monitored using VSVG () (not shown). 4
We also investigated whether steady-state secretion of a soluble cargo, albumin, is affected by SFK inhibition 1. Albumin secretion in HepG2 cells was markedly reduced by 5 µm SU6656 applied for 3 min (not shown; shorter exposures to SU6656 were less effective 1, 11 ). We thus conclude that the Golgi-SFK signalling circuit also operates during steady-state trafficking. Supplementary Information, Text S5. SYF cells evolved salvage mechanisms to replace the ablation of Src, Yes and Fyn as a trafficking regulator. Complex cellular functions, such as proliferation and motility, are controlled by robust regulatory networks within which one pathway is often dominant over the others, but not unique 12, 13. Moreover, inhibition of the dominant pathway can lead to a functional switch towards the subordinate ones. This appears to be the case for SYF embryonic fibroblasts, where the ubiquitous Src, Fyn and Yes SFKs have been genetically ablated and trafficking appears to be supported by other members of the SFKs, which are normally absent in fibroblasts (see Supplementary Information Fig. S7). The expression of these kinases in these cells is probably supported by adaptive mechanisms designed to compensate for the lack of Src, Yes and Fyn, and that thus sustain trafficking (as well as other essential SFK-dependent functions) 14. 5
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