Gillian Groeger

Catherine D Nobes
email: catherine.nobes@bristol.ac.uk

Kate Nobes

Cooperative Cdc42 and Rho signalling mediates ephrin-B triggered endothelial cell retraction

Keywords: Human Umbilical Venous Endothelial Cells MRCK: myotonic dystrophy kinase-related Cdc42-binding kinase Cdc42, EphB receptor, actomyosin contraction, MRCK, ROCK Biochemical

Cell repulsion responses to Eph receptor activation are linked to rapid actin cytoskeletal reorganisations, which in turn are partially mediated by Rho-Rho kinase (ROCK) signalling, driving actomyosin contractility. Here we show that Rho alone is not sufficient for this repulsion response. Rather, Cdc42 and its effector MRCK are also critical for ephrinB-induced cell retraction. Stimulation of endothelial cells with ephrin-B2 triggers rapid, but transient, cell retraction. We show that whilst membrane retraction is fully blocked by blebbistatin (a myosin-II ATPase inhibitor), it is only partially blocked by inhibiting Rho-ROCK signalling, suggesting there is ROCK-independent signalling to actomyosin contractility downstream of EphBs. We find that a combination of either Cdc42 or MRCK inhibition with ROCK inhibition completely abolishes the repulsion response. Additionally, endocytosis of ephrin-Eph complexes is not required for initial cell retraction but is essential for subsequent Racmediated re-spreading of cells. Our data reveal a complex interplay of Rho, Rac and Cdc42 in the process of EphB-mediated cell retraction/recovery responses.

Introduction

Eph receptors and their cell-surface anchored ligands, ephrins, regulate a wide variety of cell migration-related processes in developing and adult tissues [START_REF] Poliakov | Diverse roles of Eph receptors and ephrins in the regulation of cell migration and tissue assembly[END_REF][START_REF] Pasquale | Eph receptor signalling casts a wide net on cell behaviour[END_REF]. Signalling pathways triggered by Eph receptors and ephrins promote cell contact-dependent repulsion that prevents mixing of receptor-and ligand-expressing cells. The mechanisms underlying these cell repulsion responses are complex and not fully characterised but are known to involve cell:cell detachment by loss of high-affinity Eph-ephrin interactions, either due to proteolytic cleavage [START_REF] Hattori | Regulated cleavage of a contactmediated axon repellent[END_REF] or by their internalisation [START_REF] Marston | Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion[END_REF][START_REF] Zimmer | EphB-ephrinB bi-directional endocytosis terminates adhesion allowing contact mediated repulsion[END_REF], and concomitant increased contractility of Eph-expressing cells resulting in the withdrawal of cell extensions [START_REF] Wahl | Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase[END_REF][START_REF] Cheng | Cdk5/p35 and Rho-kinase mediate ephrin-A5-induced signaling in retinal ganglion cells[END_REF][START_REF] Riedl | Downregulation of Rap1 activity is involved in ephrinB1-induced cell contraction[END_REF]. Both of these component steps have been shown to involve activation of the Rho family GTPases, Rho (contractility) and Rac (Eph endocytosis), by Eph receptor recruitment and activation of guanine nucleotide exchange factors (GEFs), ephexin1 and Vav2 [START_REF] Shamah | EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin[END_REF][START_REF] Sahin | Eph-dependent tyrosine phosphorylation of ephexin1 modulates growth cone collapse[END_REF][START_REF] Cowan | Vav family GEFs link activated Ephs to endocytosis and axon guidance[END_REF].

Although Rho, acting through ROCK and MYPT1 phosphorylation, was initially established as the Rho GTPase family member associated with actomyosin contractility and cell retraction episodes [START_REF] Riento | Rocks: Multifunctional kinases in cell behaviour[END_REF], it has been demonstrated recently that another Rho family GTPase, Cdc42, acting through myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), can also drive cell contractility [START_REF] Dong | Cdc42 antagonizes inductive action of cAMP on cell shape, via effects of the myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) on myosin light chain phosphorylation[END_REF][START_REF] Zhao | PAK and other Rho-associated kinases--effectors with surprisingly diverse mechanisms of regulation[END_REF] and, indeed, cooperates with Rho-ROCK signalling in mediating cell invasion [START_REF] Wilkinson | Cdc42-MRCK and Rho-ROCK signalling cooperate in myosin phosphorylation and cell invasion[END_REF]. Cdc42 has been shown to be activated by Eph receptors but, rather than being linked to contractility, is proposed to regulate actin polymerisation during dendritic spine morphogenesis [START_REF] Irie | EphB receptors regulate dendritic spine development via intersectin, Cdc42 and N-WASP[END_REF]. Rac also appears not to regulate cell contractility downstream of Eph receptors, but it, and its exchange factor Vav2 mediate repulsion responses to ephrins by regulating endocytosis of ephrin-Eph complexes [START_REF] Marston | Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion[END_REF][START_REF] Cowan | Vav family GEFs link activated Ephs to endocytosis and axon guidance[END_REF].

In this study, we have investigated how Rho family GTPases and ephrin-Eph endocytosis regulate endothelial cell retraction and subsequent membrane re-spreading triggered by ephrin-B2. We show that the retraction response is entirely dependent on myosin-II ATPase activity and, in line with previous studies, is only partially dependent on Rho-ROCK signalling. Microinjection of dominant-negative constructs reveals that Cdc42-MRCK signalling plays a critical role in endothelial cell contractility triggered by ephrin-B2 and, in combination with ROCK inhibition, can entirely prevent Eph-mediated endothelial cell retraction. Interestingly, we find that blocking endocytosis of ephrin-Eph complexes, through expression of dominant-negative dynamin, had no effect on actomyosin driven cell retraction; rather, it inhibits cell re-spreading suggesting that removal of surface active Eph receptors is required to terminate actomyosin contractility signals prior to cell recovery from repulsion responses. Rac does not appear to play a role in the retraction event, but is necessary for the recovery of cells to a fully spread morphology. These findings suggest an important role for Cdc42 in repulsive signalling responses to activated Ephs.

Materials and methods

Reagents and expression plasmids

Antibodies for cell staining and Western blotting were anti-c-myc (9E10, Serotec, dilution 1:200), anti-dynamin (HUDY1, Upstate, dilution 1:50), anti-FLAG (M2, Sigma, dilution 1:50), anti-tubulin (YL1/2, Serotec, dilution 1:10,000), anti-phospho-MLC2 (ser19; New England Biolabs; dilution 1:1000) and anti-MLC2 (FL-172, Santa Cruz, dilution 1:500).

Anti-phospho-Eph receptor was described previously [START_REF] Marston | Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion[END_REF]. All secondary antibodies were from Jackson ImmunoResearch. All reagents were purchased from Sigma unless otherwise stated.

The following constructs were received as gifts: pcDNA3-dynamin1 (K44A Dyn; J.M.

Edwardson, Cambridge, UK) and pEfFLAG-CDC42BPA-isobK106M-FL (MRCKa kinasedead [KD]; C.J. Marshall, CRUK, London, UK).

Cell culture and microinjection

Human umbilical cord venous endothelial cells (TCS Cell Works) were cultured as published previously [START_REF] Marston | Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion[END_REF] and were used at 50-80% confluency. Cell nuclei were microinjected with DNA expression constructs (100-300 mg/ml) and an injection marker (FITC-, TRITC-or biotin-conjugated lysinated dextrans [Molecular Probes]) and cells allowed 1.5-4 h to express recombinant protein as indicated.

Cells were stimulated with ephrin-B2-Fc (1 µg/ml; R+D Systems) or Fc (1 µg/ml; R+D Systems) pre-clustered (20 min) with goat anti-human IgG (5 µg/ml; Stratech). Cells were incubated with blebbistatin (100 mM; Tocris) for 30 min, or with Y-27632 (10 mM; Calbiochem) for 2 h, prior to, and also during, stimulation. Time-lapse movies were made using an inverted microscope (Zeiss Axiovert 200M) with a Zeiss, 40x, 0.6 NA lens, a digital camera (C4742-95, Hamamatsu) and Openlab (Improvision) software. Cells were maintained at 37 o C with 5% CO 2 during recording.

Immunocytochemistry

Cultured cells were fixed, permeabilised and quenched as published previously [START_REF] Marston | Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion[END_REF] and incubated with primary antibodies for 45 min, followed by appropriate secondary antibodies for 25 min. TRITC-or FITC-conjugated phalloidin was used to stain F-actin when required.

All antibody dilutions were in PBSA and incubations were at room temperature. Confocal images were collected using a Leica TCS-NT confocal attached to a Leica DM IRBE inverted microscope with a HCX, 63x, 1.32 NA, oil immersion lens using Leica Confocal Software and were subsequently handled with Adobe Photoshop.

To examine internalisation of ephrin/Eph complexes, two protocols were used. A) After fixation, cells were incubated with anti-goat-Cy3 antibodies to label external IgGephrin-B2-Eph complexes. Cells were then washed, post-fixed, permeabilised and incubated with anti-goat-FITC antibodies to label internalised IgG-ephrinB2-Fc. B) Alternatively, cells were acid washed (2 x 5 min) at 4 o C (0.2% BSA, 10 mM MES, 1 mM sodium pyruvate in DMEM (Gibco), pH=2) prior to fixation to remove any externally bound ephrin-B2-Fc before immunostaining.

Biotinylation and immunoblotting

The biotinylation method used was as published previously [START_REF] Martin | Activity-dependent endocytic sorting of kainate receptors to recycling or degradation pathways[END_REF]. Briefly, following biotinylation of cell surfaces at 4 o C for 10 min using EZ-Link NHS-SS-Biotin (Pierce), cells were maintained at 4 o C, and incubated in a Tris-Amine Buffer (25 mM Tris, 137 mM NaCl, 5 mM KCl, 2.3 mM CaCl 2 , 0.5 mM MgCl 2 , 0.143 g/L Na 2 HPO 4 ) for 10 min. Cells were stimulated as normal at 37 o C and, after the required internalisation time, any non-internalized biotin was removed from cell surfaces using a glutathione solution (75 mM NaCl, 75 mM NaOH, 1 mM EDTA, 0.1% BSA, 50 mM glutathione, pH=9) (2 x 20 min), which was neutralised by washing with a Tris-Glycine buffer (0.192 M glycine, 25 mM Tris). Cells were washed several times in TBS before lysates were prepared in a modified RIPA buffer (125 mM NaCl, 20 mM Tris-HCl, 10% glycerol, 1% NP40, 50 mM NaF, 200 mM Vanadate, 100 mg/mL PMSF, protease inhibitor cocktail [Roche]). Biotinylated proteins were precipitated using streptavidin-conjugated, agarose beads (Upstate) and were resuspended in Laemmli loading buffer and analysed by SDS PAGE and Western blot.

Analysis

ImageJ (version 1.33U; public domain software available from http://rsb.info.nih.gov/ij) was used to quantify results from Western blots. All graphs were drawn using Microsoft Excel.

Results and discussion

Previous studies have shown that ephrin-induced cell retraction and growth cone collapse is only partially blocked by inhibitors of Rho GTPase and its effector ROCK [START_REF] Wahl | Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase[END_REF][START_REF] Cheng | Cdk5/p35 and Rho-kinase mediate ephrin-A5-induced signaling in retinal ganglion cells[END_REF][START_REF] Harbott | A key role for Abl family kinases in EphA receptor-mediated growth cone collapse[END_REF], suggesting that additional parallel pathways regulate this component of the repulsion response.

To investigate signalling pathways underlying ephrin-regulated cell retraction, we have stimulated human umbilical vein endothelial cells (HUVECs) with soluble pre-clustered ephrin-B2-Fc. We show that non-confluent HUVECs display a dynamic, reproducible retraction-recovery response within minutes to ephrin-B2-Fc exposure (Fig. 1A and Supplemental Movie 1). Cells rapidly undergo plasma membrane retraction (within 10 min) but then subsequently, despite the continued presence of ephrin, recover to a spread state (within 35 min) by lamellipodial growth along the retraction fibres left behind (Fig. 1A andB). These early changes in cell morphology correlate with the initial phase of activation of Eph receptors as revealed by phosphorylation of Eph juxtamembrane tyrosine residues (Fig. 1C andD). Interestingly, although increased phosphorylation was observed after 5 min, maximal phosphorylation of Eph receptors was not until 40 min after stimulation (Fig. 1C andD), by which time most cells had recovered from their retracted state. This suggests that dephosphorylation of Eph receptors at the juxtamembrane sites is not a driver of, or even required for cell re-spreading.

Since the switch from retraction to re-spreading appears not to involve de-activation of Eph receptors, we investigated whether it requires Eph receptor internalisation. Previously, it has been shown that removal of ephrinB-EphB complexes at cell-cell contacts, through their internalisation, is required for cell separation during the repulsion response [START_REF] Marston | Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion[END_REF][START_REF] Zimmer | EphB-ephrinB bi-directional endocytosis terminates adhesion allowing contact mediated repulsion[END_REF]. More recent evidence suggests that endocytosis of EphA receptors may direct, rather than simply facilitate, ephrin-triggered repulsion responses, since Vav2 both regulates endocytosis of ephrin-Eph complexes and is necessary for growth cone collapse [START_REF] Cowan | Vav family GEFs link activated Ephs to endocytosis and axon guidance[END_REF]. Using both immunocytochemistry and biochemical techniques we have determined the temporal relationship between ephrin-Eph complex internalisation and membrane retraction/recovery events. HUVECs were stimulated with IgG pre-clustered ephrin-B2-Fc for various times as indicated, fixed in the absence of detergents and immunostained for ephrin-B2-Fc/IgG on the cell surface (red staining). Cells were then post-fixed, permeabilised and stained for internalised ephrin-B2-Fc/IgG (green).

Ephrin-Eph complex endocytosis is relatively slow compared with the cell retraction response and most internalised ephrin/IgG appears around 20-45 min after initial exposure to ephrin-B2-Fc (Fig. 2A). These data are corroborated by surface biotinylation studies that also show the time-course of phosphorylated-Eph (p-Eph) internalisation (Fig. 2B and2C). In these experiments all surface proteins were biotinylated for 10 min at 4 o C, washed and returned to 37 o C for various times in the presence of ephrin-B2-Fc to allow endocytosis. Biotinylated proteins remaining on the surface after incubation were debiotinylated by incubating with glutathione buffer at 4 o C leaving only the endocytosed proteins biotinylated. The amount of biotinylated p-Eph was determined by binding cell lysate to streptavidin-agarose beads and Western blotting with anti-p-Eph. These experiments show that very little p-Eph is internalised within 10 min of ephrin-B2 stimulation (Fig. 2B and2C), the time at which we observe maximal cell retraction (Fig. 1B). Moreover, most internalisation of activated Eph receptors is between 25-40 min after ephrin-B2 exposure when we observe withdrawn lamellipodia subsequently extending along retraction fibres. These experiments address the temporal relationship between ephrin-B2 triggered repulsive signalling and endocytosis of activated Eph receptors. They show that internalisation of activated Eph receptors coincides with the recovery of retracted cells rather than the retraction phase itself.

Next we tested whether the cell re-spreading response is dependent on internalisation of ephrin-Eph complexes by expressing a dominant-negative mutant form of dynamin, dynamin-1 (K44A). We show that expression of dominant-negative dynamin inhibits internalisation of soluble ephrin-B2 (Fig. 3A). Importantly, blocking dynamin activity had no effect on cell retraction triggered by ephrin-B2, but did abrogate subsequent cell re-spreading (Fig. 3B and3C). Time-lapse microscopy shows that dominant-negative dynamin expressing cells remain contracted for up to 60 min after stimulation with ephrin-B2-Fc (Fig. 3C and Supplemental Movie 2). These movies demonstrate, however, that contracted cells are able to produce small, localised ruffles but were unable to re-spread lamellipodia. In order to control whether dominant-negative dynamin may be acting by inhibiting actin filament polymerisation required for cell spreading, we tested whether dynamin-1 (K44A) interfered with lamellipodial extension after cell trypsinisation, and found that dynamin-1 (K44A) expressing cells adhere and spread by extending lamellipodia similarly to uninjected controls after re-plating (data not shown). These experiments show that endocytosis of ephrin-Eph complexes is not necessary for ephrin-B2 induced cell retraction in HUVECs, but rather is required for the subsequent recovery of cells to a fully spread morphology. In line with our results, Mann et al., [START_REF] Mann | B-type Eph receptors and ephrins induce growth cone collapse through distinct intracellular pathways[END_REF] showed that internalisation of EphB receptors is not necessary for collapse of Xenopus retinal growth cones. By contrast, Vav2-dependent internalisation of EphA receptors is necessary for collapse of retinal growth cones [START_REF] Cowan | Vav family GEFs link activated Ephs to endocytosis and axon guidance[END_REF] indicating that there may be differences between EphB and EphA signalling pathways that control internalisation and retraction, in ways that are not cell type specific.

Many cellular contraction events are actomyosin-dependent and can be blocked by the pharmacological agent blebbistatin, which inhibits myosin-II ATPase activity [START_REF] Kovacs | Mechanism of blebbistatin inhibition of myosin II[END_REF][START_REF] Straight | Dissecting temporal and spatial control of cytokinesis with a myosin II inhibitor[END_REF].

HUVECs treated with blebbistatin (100 µm) lose their stress fibres but remain well spread with membrane ruffles (Fig. 4B). At 10 min post ephrin-B2-Fc stimulation, when we generally see most cell retraction, blebbistatin completely blocked the retraction response (Fig. 4D).

These data demonstrate that rapid retraction triggered by ephrin-B2 is strictly dependent on myosin activity and increased actomyosin contractility. We next tested the effect of inhibiting Rho-ROCK signalling on ephrin-induced endothelial cell retraction. Inhibition of ROCK with Y-27632 (10 µm) had the predicted effect of removing actin stress fibres (Fig. 4C), as did expression of C3 transferase (data not shown). In line with previous studies of ephrin-induced collapse of retinal ganglion cell growth cones and rounding of PC-3M cells [START_REF] Wahl | Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase[END_REF][START_REF] Cheng | Cdk5/p35 and Rho-kinase mediate ephrin-A5-induced signaling in retinal ganglion cells[END_REF][START_REF] Harbott | A key role for Abl family kinases in EphA receptor-mediated growth cone collapse[END_REF], endothelial cell retraction was only partially inhibited by Y-27632 or C3 transferase. Pretreatment of cells for 2h with Y-27632 (10 µm) or microinjection of C3 transferase (20 µg/ml; 2h expression time) attenuated the retraction response by about 50% (Fig. 4D) compared with a greater than 90% suppression by blebbistatin. The blebbistatin-sensitive, but Y-27632/C3insensitive, component of ephrin-triggered cell contractility was most clear at 10 min (Fig. 4D). Actomyosin-driven cell retraction involves phosphorylation of myosin-II light chain kinase (MLC) [START_REF] Amano | Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase)[END_REF]. We find that MLC was phosphorylated 2 min after stimulation of cells with ephrin-B2, reached a peak at 5-10 min, and then returned to control levels at 20-60 min (Fig. 4E andF). Taken together, these results show that endothelial cell retraction rapidly triggered by ephrin-B2-Fc is strictly actomyosin-dependent, but that there is Rho/ROCKindependent signalling to this actomyosin contractility.

Next we investigated the involvement of other Rho family GTPases in endothelial cell retraction triggered by ephrin-B2. We injected dominant negative expression constructs (pRK5mycN17Rac and pRK5mycN17Cdc42) to inhibit the activity of Rac and Cdc42 and again examined the response to pre-clustered ephrin-B2-Fc stimulation. Expression of dominant negative Rac barely altered the cell retraction phase, but cells remained retracted and did not subsequently re-spread (Fig. 5A). A sustained retraction phase is not unexpected because Rac regulated lamellipodia extension is known to be required for cell spreading from a rounded morphology [START_REF] Price | Activation of Rac and Cdc42 by integrins mediates cell spreading[END_REF]. By contrast, expression of N17Cdc42 significantly reduced the retraction response. At 10 min post ephrin-B2 exposure, when control injected cells display maximum retraction, expression of N17Cdc42 attenuated the retraction response by about 50%. However, by 20 min the retraction response of N17Cdc42 injected cells was similar to control-injected cells suggesting that Cdc42 signalling to actomyosin contraction is a component of the initial phase of Eph signalling to cell contractility. Recent studies have demonstrated that Cdc42, like Rho, can regulate actomyosin contractility, through its effector MRCK [START_REF] Leung | Myotonic dystrophy kinase-related Cdc42-binding kinase acts as a Cdc42 effector in promoting cytoskeletal reorganization[END_REF], to regulate cell invasion and nuclear movements during cell polarisation [START_REF] Wilkinson | Cdc42-MRCK and Rho-ROCK signalling cooperate in myosin phosphorylation and cell invasion[END_REF][START_REF] Gomes | Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells[END_REF]. MRCK, like ROCK, phosphorylates MLC. We show that expression of dominant negative MRCK (MRCKa kinase-dead [KD]) inhibits cell retraction (Fig. 5A). To determine whether the Cdc42-MRCK signalling pathway may interact in a parallel, non-redundant role with Rho-ROCK signalling to regulate the cell retraction response, we combined N17Cdc42 and MRCKa KD microinjection experiments with the addition of Y-27632 and, in both cases, this resulted in the inhibition of retraction levels to those obtained with blebbistatin (Fig. 5B andC). Taken together, these data imply that Rho-ROCK and Cdc42-MRCK signalling pathways play complementary/additive roles in the retraction of HUVECs in response to EphB activation.

In summary, the data presented demonstrate that Rho and Cdc42 function in concert to mediate ephrin-B2 induced cell retraction. Whilst blocking Rho-ROCK signalling with C3 transferase and Y-27632 and Cdc42-MRCK signalling with dominant negative constructs, only partially inhibited ephrin-triggered cell contractility, blocking both signalling pathways mirrored the absolute inhibition achieved with blebbistatin, a myosin II ATPase inhibitor. The recovery to a fully spread morphology, on the other hand, requires functioning Rac and the internalisation of ephrin/Eph complexes. The removal of activated Eph receptors from the membrane may be vital to enable cells to reset their signalling machinery and to continue migrating after a temporary Eph-mediated contact inhibition response.

Figure 1 .

 1 Figure 1. Retraction-recovery response of HUVECs to ephrin-B2-Fc. A) Time-lapse movie stills (pre-clustered ephrin-B2-Fc added at 0 min; arrows indicate regions of cell retraction; see also Supplemental Movie S1). B) Cell retraction in response to pre-clustered ephrin-B2-Fc or control Fc (n=5, with >100 cells counted /condition). C, D) Time-course of phosphorylation of Eph receptors (pEph) in response to ephrin-B2-Fc (n=3; means ± S.E.M). Scale bar = 30 µm.

Figure 2 .

 2 Figure 2. EphB-ephrinB2 internalisation coincides with cell recovery, and not with cell retraction. A) Schematic shows extracellular ephrin-B2-Fc (red) and intracellular ephrin-B2-Fc (green). Confocal images show that most ephrin-B2-Fc internalisation occurs between 20 and 45 min. B, C) Surface biotinylation experiments to assay time-course of internalisation of active Eph receptors (n=3; means ± S.E.M). Scale bar = 20 µm.

Figure 3 .

 3 Figure 3. Internalisation of ephrin-B2-Fc is necessary for cell re-spreading. A) Cells expressing K44A Dyn (green) do not internalise EphB-ephrinB2 complexes (red); acid washing the cells immediately prior to fixation removed external ephrin-B2-Fc, therefore red staining represents internalised ephrin-B2-Fc. B) Ephrin-B2 triggered retraction in cells injected with K44A Dyn, or with control injection marker (dextran) (n=3-5, >30 cells/condition; means ± S.E.M). C) Time-lapse stills showing that K44A Dyn injected cells do not re-spread lamellipodia after ephrinB2-Fc stimulation (see Supplemental Movie S2). Scale bar=30 mm.

Figure 4 .

 4 Figure 4. Ephrin-B2-Fc induced retraction is myosin II-dependent but only partially blocked by Rho/ROCK inhibition. A-C) Treatment of HUVECs with blebbistatin (100 µm) or Y-27632 (10 µm) results in the loss of actin stress fibres. D) Quantification of cellular retraction in response to ephrin-B2-Fc in the presence of blebbistatin and Y-27632 (n=3, >100 cells/time point) and in cells injected with C3 transferase (n=8, with >40 cells/experiment). E and F) Phosphorylation of myosin light chain II (MLC) during the retraction response (n=6; means ± S.E.M). Scale bar = 40 µm.

Figure 5 .

 5 Figure 5. Cdc42 and Rho pathways cooperate in ephrin-B2-Fc induced retraction. A) Time-course of ephrin-B2-induced retraction in cells injected with injection marker (dextran), N17Rac, N17Cdc42 or MRCKa KD (n=3-7). B) Quantification of the combined effects of

Biochemical Journal Immediate Publication. Published on 14 Feb 2007 as manuscript BJ20070146 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 14 Feb 2007 as manuscript BJ20070146 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

We thank Chris Marshall, Mike Olson and Stefan Martin for reagents and discussion, Iwan Evans for help with the manuscript preparation and Paul Martin for comments on the manuscript. This work was funded by the Medical Research Council. C.D.N is an MRC Senior Research Fellow.

Supplemental Material:

Supplemental Movie S1: Stimulation of HUVECs with pre-clustered ephrin-B2-Fc triggers transient membrane retraction (see also Fig. 1A). Movie commences 5 min before addition of ephrin-B2-Fc (marked by a single white frame) and covers 46 min (140 frames).

Supplemental Movie S2: Dominant-negative dynamin expression in HUVECs blocks respreading of cells after retraction triggered by ephrin-B2-Fc (see also Fig. 3C). Movie commences 3h after microinjection of K44A dynamin. Single white frame indicates time of stimulation with ephrin-B2-Fc. Movie corresponds to 66 min (199 frames).


Ching Wan Chan

Youn-Bok Lee

James Uney

Andrea Flynn

Jonathan H Tobias

Michael Norman
email: m.r.norman@bris.ac.uk

A novel member of the SAF (scaffold attachment factor)-box protein family inhibits gene expression and induces apoptosis

Keywords: SAF-box, oestrogen, nucleus, transcription, apoptosis SAF, Scaffold attachment factor, SLTM, SAF-Like Transcription Modulator, S/MAR, Scaffold/Matrix attachment regions, SERM, selective oestrogen receptor antagonist, EYFP, enhanced yellow fluorescent protein, SFC, splicing factor compartment, STA, staurosporine

The SLTM protein contains a SAF Box DNA binding motif, an RNA binding domain, and shares an overall identity of 34% with SAFB1 (also known as SAF-B, HET or HAP).

Here, we show that SLTM is localised to the cell nucleus but excluded from nucleoli, and to a large extent it co-localises with SAFB1. In the nucleus, SLTM has a punctate distribution that does not co-localise with serine/arginine (SR) proteins. Over expression of SAFB1 has been shown to exert a number of inhibitory effects, including suppression of oestrogen signalling. Although SLTM also suppressed the ability of oestrogen to activate a reporter gene in MCF-7 breast cancer cells, inhibition of a constitutively active -galactosidase gene suggested that this was primarily the consequence of a generalised inhibitory effect on transcription. Measurement of RNA synthesis, which showed a particularly marked inhibition of [ 3 H]uridine incorporation into mRNA, supported this conclusion. In addition, analysis of cell cycle parameters, chromatin condensation and cytochrome c release showed that SLTM induced apoptosis in a range of cultured cell lines. Thus inhibitory effects of SLTM on gene expression appear to result from generalised down-regulation of mRNA synthesis and initiation of apoptosis consequent upon over expressing the protein. While indicating a crucial role for SLTM in cellular function, these results also emphasise the need for caution when interpreting phenotypic changes associated with manipulation of protein expression levels.

Introduction

Scaffold attachment factor B (SAFB1, also known as SAF-B, HET or HAP) was originally isolated on the basis of its ability to bind Scaffold/Matrix attachment regions (S/MARs) of genomic DNA [START_REF] Renz | Purification and molecular cloning of the scaffold attachment factor B (SAF-B), a novel human nuclear protein that specifically binds to S/MAR-DNA[END_REF]. Such sequences are present throughout eukaryotic genomes and are thought to play a role in organising chromatin structure and regulating gene expression [START_REF] Boulikas | Chromatin domains and prediction of MAR sequences[END_REF]. SAFB1 contains a number of domains that may be responsible for functions such as tethering components of the nuclear matrix to S/MARs. These include an amino terminal SAF Box DNA binding motif [3;14], an RNA binding domain [START_REF] Birney | Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors[END_REF] and a carboxy terminal sequence rich in arginine and glutamic acid residues that has been shown to interact with components of the transcriptional and translational machinery [2;18].

A protein that subsequently proved to be identical to SAF-B was identified independently and named Heat shock protein 27 -Estrogen response element-TATA box binding protein (HET) due to its ability to bind and modulate transcription of the HSP-27 promoter [START_REF] Oesterreich | Novel nuclear matrix protein HET binds to and influences activity of the HSP27 promoter in human breast cancer cells[END_REF]. Subsequent studies showed that over expression of this protein suppressed the transcriptional activity of the oestrogen receptor, augmenting the antagonistic effects of the selective oestrogen receptor antagonist (SERM) tamoxifen in breast cancer cells [START_REF] Oesterreich | Tamoxifen-bound estrogen receptor (ER) strongly interacts with the nuclear matrix protein HET/SAF-B, a novel inhibitor of ER-mediated transactivation[END_REF]. This inhibitory effect appears to be explained, in part, by recruitment of the N-CoR co-repressor [13]. Over expression of SAFB1 is also associated with reduced rates of cell proliferation and multinuclearity [START_REF] Townson | HET/SAF-B overexpression causes growth arrest and multinuclearity and is associated with aneuploidy in human breast cancer[END_REF]. Conversely, deletion of SAFB1 in mouse embryonic fibroblasts leads to spontaneous immortalisation and lack of contact inhibition [START_REF] Dobrzycka | Disruption of Scaffold Attachment Factor B1 Leads to TBX2 Up-regulation, Lack of p19ARF Induction, Lack of Senescence, and Cell Immortalization[END_REF]. Loss of heterozygosity at 19p13.2-3, the site at which HET has been localised in the human genome, has been observed in breast cancer cells, suggesting that it may act as a tumour suppressor gene [START_REF] Oesterreich | High rates of loss of heterozygosity on chromosome 19p13 in human breast cancer[END_REF]. A third protein which proved to be identical to SAFB1 was isolated independently by Weighardt et al. [START_REF] Weighardt | A novel hnRNP protein (HAP/SAF-B) enters a subset of hnRNP complexes and relocates in nuclear granules in response to heat shock[END_REF]. This protein, initially designated HAP, was detected using a two-hybrid screen to identify proteins interacting with heterogenous nuclear ribonuclear protein (hnRNP) A1.

Interestingly, the sub-nuclear distribution of HAP was found to be markedly altered in response to heat-shock. More recently, a closely related protein, SAFB2, was isolated and SAFB1 was proposed as an alternative name for SAFB/HET/HAP [START_REF] Townson | SAFB2, a new scaffold attachment factor homolog and estrogen receptor corepressor[END_REF].

A novel gene that we described (GenBank Accession Number AF462146) also encodes a protein containing a SAF Box DNA binding motif and an RNA binding domain [START_REF] Colley | MET, a novel stimulator of estrogen-induced transcription isolated from mouse bone marrow[END_REF]. This gene was initially named Met, but has now been re-named SAF-Like Transcription

Experimental

Plasmids and adenoviral vector

Plasmids expressing full length HA tagged SLTM cDNA (pSLTM-HA), HA tagged SAFB1 (pHA-SAFB1) and EYFP-tagged SLTM (pEYFP-SLTM) were kindly provided by Dr Shane Colley. To construct an adenoviral vector, SLTM was cloned into the plasmid pXcX.CMV (Microbix Biosystems Inc). Recombinant AdSLTM-HA virus was generated by homologous recombination in human embryonic kidney (HEK) 293 cells grown to high titer, then purified by double caesium chloride density gradient centrifugation as described [START_REF] Graham | Methods for construction of adenovirus vectors[END_REF].

Viral titre, determined by plaque assay, was 2 10 9 plaque-forming units per ml. As a control, adenovirus lacking a transgene (Ad0) was used.

Cell culture and transfection

Cell lines were obtained from the European Collection of Cell Cultures (ECACC), and maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum (GibcoBRL), 2mM glutamine and penicillin-streptomycin. Cells were transfected using Fugene 6 transfection reagent (Roche).

Confocal microscopy

Cells were washed with PBS and fixed in 2% paraformaldehyde for 20min at room temperature before permeabilization with 0.1% Triton X in PBS for 5mins. Non-specific protein binding was then blocked with 1% (w/v) bovine serum albumin in PBS for 20mins before incubation with anti-HA mouse monoclonal antibody (Sigma, 1:5000 dilution) or mouse anti-SC-35 primary antibody (Sigma, 1:1000 dilution) at 4 o C overnight. After washing in 1% BSA/PBS, anti-mouse antibody linked to Cy3 (Jackson Immunology) was added for 1.5hrs at room temperature in the dark. Coverslips were then mounted on glass microscope slides using DAKO Fluorescent Mounting Medium (DAKO Corporation). Confocal images were obtained using a Leica TCS SP scanning laser microscope equipped with a Kr/Ar laser using a 63x oil immersion objective. EYFP was detected using 510nm excitation and a 530nm emission filter set. Cy3 was visualized using 568nm excitation and 590nm emission filters. Digital images were acquired using Leica TCSNT confocal software.

Reporter assays MCF-7 cells were seeded in 48-well plates at 3x10 4 cells per well. The following day, cells were transiently transfected with either 250ng of pERE-tk-luc reporter plasmid [START_REF] White | Environmentally persistent alkylphenolic compounds are estrogenic[END_REF], or 60ng pSV--galactosidase control vector (Promega) per well, plus pSLTM-HA or pHA-SAFB1 plasmids using Fugene 6 transfection reagent. Where necessary, pCR3.1 plasmid was used to maintain equal quantities of DNA for transfection. Following overnight incubation, cells were washed and cultured in serum-free medium (phenol red-free DMEM/F-12 mix (GibcoBRL) containing 0.1mg/ml BSA and 10 g/ml apo-transferrin) for a further 6 hrs. Cells were subsequently exposed to 10 -8 M 17 -oestradiol (E 2 ) for 18hrs prior to cell lysis. Reporter gene activity was measured using the Luciferase Assay System (Promega) in a Microtitre Plate Luminometer (Dynex). -galactosidase activity was measured following the addition of chlorophenol red-ß-D-galactopyranoside substrate (Roche). Assays were performed in triplicate and the data presented are representative of at least 3 independent experiments.

RNA synthesis

To measure [ 3 H]uridine incorporation into total RNA, HeLa cells (3x10 4 ) were seeded in 96-well plates. Cells were either transfected with 0.1µg empty plasmid or pSLTM-HA and incubated for a further 48 hours, or treated with staurosporine (STA) for 3hrs. [ 3 H]uridine (1µCi/well) was then added for 1 hour before the cells were lysed and transferred to Printed Filtermats (Wallac) for determination of radioactivity. To measure [ 3 H]uridine incorporation into mRNA, HeLa cells (1.5x10 5 ) were seeded into 60mm dishes before being treated with STA as above or transfected with either 2µg of pcDNA3 (empty plasmid) or pSLTM-HA.

After 48hrs, cells were exposed to 20µCi [ 3 H]uridine/dish for 1hr before being collected, lysed and RNA extracted using the RNeasy Mini Kit. Poly(A)+ RNA was then isolated using the PolyATract kit (Promega) and radioactivity measured using a Wallac 1409 DSA Liquid Scintillation Counter.

Cell cycle analysis

For cell cycle analysis, cells (3x10 5 ) were seeded into each well of a 6-well plate and incubated overnight. The cells were co-transfected with pEYFP-C1 and either pcDNA3.1 (empty plasmid), or pSLTM-HA (1 g of each plasmid). The cells were then incubated overnight before serum starvation in serum-free medium for 24 hours. The cells were collected, fixed in 70% ethanol for 45 min at

Results

Cellular localization of SLTM protein

Previous studies have shown that SAFB1 is expressed with a punctate distribution in the cell nucleus [18;31]. To investigate the subcellular distribution of SLTM, confocal microscopy was used to examine HeLa cells transfected with plasmids expressing EYFP or the EYFP-SLTM chimera. In cells transfected with pEYFP-C1 alone, EYFP staining was evenly distributed throughout the cells, while cells transfected with pEYFP-SLTM showed a punctate, nuclear localization pattern excluded from nucleoli (Fig. 1a). A similar distribution pattern was observed in MCF-7 cells (data not shown). In order to determine whether the distribution of SLTM coincided with that of SAFB1, HeLa cells were co-transfected with pEYFP-SLTM and pHA-SAFB1. 48 hours later, the cells were fixed, permeabilized and stained for the SAFB1 protein using an anti-HA mouse monoclonal antibody. As shown in SLTM is clearly different from that of SFCs. Initial studies showed that a partial SAFB1 sequence co-localised with SC-35 [START_REF] Nayler | SAF-B protein couples transcription and pre-mRNA splicing to SAR/MAR elements[END_REF], but a subsequent study showed that speckles associated with intact SAFB1, like those formed by SLTM, are distinct from SC-35 speckles [START_REF] Weighardt | A novel hnRNP protein (HAP/SAF-B) enters a subset of hnRNP complexes and relocates in nuclear granules in response to heat shock[END_REF].

Transcriptional activity of the oestrogen receptor in cells over expressing SLTM

In the light of previous data showing that SAFB1 suppresses oestrogen-induced transcription [START_REF] Oesterreich | Tamoxifen-bound estrogen receptor (ER) strongly interacts with the nuclear matrix protein HET/SAF-B, a novel inhibitor of ER-mediated transactivation[END_REF], we decided to test the possibility that SLTM might exert a similar effect. MCF-7 cells were co-transfected with an oestrogen response element-luciferase reporter construct, pERE-tk-luc, along with plasmid expressing SLTM. Co-transfected cells were subsequently exposed to oestrogen or vehicle and the activation of reporter genes compared.

Luciferase reporter expression was induced four fold in MCF-7 cells exposed to oestrogen compared with untreated controls (Fig. 2a). Co-transfection with increasing amounts of SLTM resulted in reduced luciferase activity, the oestrogen effect being completely reversed when the highest amount (200ng) of plasmid was used.

Constitutively active -galactosidase vectors are widely used to control for transfection efficiency. Surprisingly, we found that SLTM also caused a profound inhibition of -galactosidase activity when MCF-7 cells were co-transfected with SLTM expression plasmid (Fig 2b). Nevertheless, inhibition of -galactosidase is less than that of luciferase, so that correction of luciferase results for -galactosidase showed an apparent inhibition of the ability of oestrogen to activate reporter by SLTM (Fig 2c).

Over expression of SLTM results in a generalised inhibition of transcription.

Given the nuclear localization of SLTM, together with evidence from previous studies showing that SAFB1 interacts with RNA pol II [START_REF] Nayler | SAF-B protein couples transcription and pre-mRNA splicing to SAR/MAR elements[END_REF], we postulated that SLTM might produce a generalised inhibition of mRNA transcription. Therefore, we tested the effect of SLTM on were transfected with SLTM (Fig 4 ) showed a clear increase in the pre-G1 population. To ascertain whether the effect of SLTM on the cell cycle was limited to MCF-7 cells, we also investigated its effect on MC3T3 cells (a mouse calvarial fibroblast cell line), Ros (rat osteosarcoma), HepG2 (human hepatocarcinoma), HeLa (human cervical adenocarcinoma) and SF (human fibroblast, primary culture) cell lines. For these experiments, 3x10 5 cells from each line, seeded in 6-well plates, were transfected with 1µg of pEYFP-C1 or pEYFP-SLTM as described before. From Table 1, it can be seen that SLTM induces significant apoptosis in all of these cell lines, albeit to different extents.

As additional confirmation of the ability of SLTM to induce apoptosis, HeLa cells (2x10 5 ) were seeded into 60mm dishes, incubated overnight and then transfected with either 1µg of pEYFP-C1 or pEYFP-SLTM. After incubation for a further 24 hours, cells were serum starved for 24 hours before being fixed and stained with Hoechst. Fluorescent microscopy was performed and 300 fluorescent cells per dish were counted. As shown in Figs. 5a and5b, pEYFP-SLTM transfected cells correspond to cells with the classical features of apoptosis, shown by Hoechst staining. Cell counts of the pEYFP and pEYFP-SLTM transfected cells (Fig. 5c) show SLTM increases the apoptotic index from 3+1% (SD) to 20+2% (SD) (p<0.001), consistent with results obtained by cell cycle analysis. In order to provide further confirmation of an apoptotic process, and also to avoid possible artefactual results caused by transfection, we carried out additional studies using an adenoviral vector expressing SLTM.

Discussion

In this study we describe some properties of the protein encoded by the SLTM gene. The proteins most closely related to SLTM appear to be SAFB1 [START_REF] Renz | Purification and molecular cloning of the scaffold attachment factor B (SAF-B), a novel human nuclear protein that specifically binds to S/MAR-DNA[END_REF] and SAFB2 [START_REF] Townson | SAFB2, a new scaffold attachment factor homolog and estrogen receptor corepressor[END_REF], which share similar structures to SLTM, including a SAF Box, an RNA binding domain and a region rich in glutamine and arginine residues. SAFB1 is a nuclear protein, whereas SAFB2 is also found in the cytoplasm [START_REF] Townson | SAFB2, a new scaffold attachment factor homolog and estrogen receptor corepressor[END_REF]. The compartmentalised architecture of the nucleus is increasingly recognised, with nuclear factors which may be highly mobile or localised to distinct compartments. Both SAFB1 [18;31] and SLTM have a punctate, nuclear distribution that is excluded from nucleoli, and to a large extent the two proteins co-localise (Fig 1b), suggesting that they share similar functions. Splicing factor compartments (SFCs) are well-characterised nuclear structures that are thought to represent storage sites for splicing factors [START_REF] Lamond | Nuclear speckles: a model for nuclear organelles[END_REF]. Because there is evidence that SAFB1 functions as a splicing factor [START_REF] Nayler | SAF-B protein couples transcription and pre-mRNA splicing to SAR/MAR elements[END_REF], and given the similarity between SLTM and SAFB1, we were interested to determine whether or not SLTM colocalises with SFCs. Although the speckled distribution pattern of SLTM resembles that of SFCs, we found no co-localization of SC-35 (a splicing factor localised to SFCs) and SLTM (Fig 1c). Initially, it was thought that SAFB1 co-localised with SC-35 [START_REF] Nayler | SAF-B protein couples transcription and pre-mRNA splicing to SAR/MAR elements[END_REF], but these studies were performed with a C-terminal fragment of SAFB1 and a subsequent study with the complete protein showed that the distribution of SAFB1, like SLTM, is quite distinct from that of SC-35 speckles [START_REF] Weighardt | A novel hnRNP protein (HAP/SAF-B) enters a subset of hnRNP complexes and relocates in nuclear granules in response to heat shock[END_REF]. It will be important to determine whether SAFB1 and SLTM identify novel nuclear compartments, or whether they correspond to previously described nuclear structures such as paraspeckles, Cajal bodies or promyelocytic leukaemia bodies [10;26].

Further clues to the precise function of SAF proteins might be provided by protein/protein interactions. SAFB1 has been reported to interact with a surprisingly wide range of proteins. These include SR [START_REF] Nayler | SAF-B protein couples transcription and pre-mRNA splicing to SAR/MAR elements[END_REF] and hnRNP splicing proteins [2;31], the oestrogen receptor [START_REF] Oesterreich | Tamoxifen-bound estrogen receptor (ER) strongly interacts with the nuclear matrix protein HET/SAF-B, a novel inhibitor of ER-mediated transactivation[END_REF] and several other nuclear receptors, including the Peroxisome Proliferator-Activated Receptor, the Farnesoid X receptor, the ROR, the Vitamin D receptor, steroidogenic factor 1 and liver receptor homolog 1 [START_REF] Debril | Scaffold attachment factor B1 directly interacts with nuclear receptors in living cells and represses transcriptional activity[END_REF]. In addition, SAFB1 has also been reported to interact with RNA pol II [START_REF] Nayler | SAF-B protein couples transcription and pre-mRNA splicing to SAR/MAR elements[END_REF], P2P-R [START_REF] Scott | Functional potential of P2P-R: a role in the cell cycle and cell differentiation related to its interactions with proteins that bind to matrix associated regions of DNA?[END_REF], SRPK1 [START_REF] Nikolakaki | Cloning and characterization of an alternatively spliced form of SR protein kinase 1 that interacts specifically with scaffold attachment factor-B[END_REF], ZO-2 [START_REF] Traweger | The tight junction protein ZO-2 localizes to the nucleus and interacts with the heterogeneous nuclear ribonucleoprotein scaffold attachment factor-B[END_REF], CHD1 [START_REF] Tai | CHD1 associates with NCoR and histone deacetylase as well as with RNA splicing proteins[END_REF] and cJun [START_REF] Debril | Scaffold attachment factor B1 directly interacts with nuclear receptors in living cells and represses transcriptional activity[END_REF]. Evidence for specific transcriptional regulatory functions of SAFB1 has been accumulated by Oesterreich et al.

Thus SAFB1 was found to exert a negative regulatory effect on the hsp 27 promoter [START_REF] Oesterreich | Novel nuclear matrix protein HET binds to and influences activity of the HSP27 promoter in human breast cancer cells[END_REF], while both SAFB1 and SAFB2 were found to inhibit expression of oestrogen reporter genes [23;28]. In addition, GST pull-down experiments were used to show that the oestrogen receptor interacts with both SAFB1 [START_REF] Oesterreich | Tamoxifen-bound estrogen receptor (ER) strongly interacts with the nuclear matrix protein HET/SAF-B, a novel inhibitor of ER-mediated transactivation[END_REF] and SAFB2 [START_REF] Townson | SAFB2, a new scaffold attachment factor homolog and estrogen receptor corepressor[END_REF]. More recently, this group has shown that recruitment of a co-repressor, N-CoR, is in part responsible for the inhibitory effect of SAFB1 on transcription by the oestrogen receptor [13]. The presence of RRMs in SLTM is also compatible with studies showing that RNA [START_REF] Lanz | Distinct RNA motifs are important for coactivation of steroid hormone receptors by steroid receptor RNA activator (SRA)[END_REF] and RRM proteins [START_REF] Norris | A negative coregulator for the human ER[END_REF] can regulate transcriptional activity of the oestrogen receptor. For these reasons, we were interested to determine whether or not SLTM would exert a similar effect on oestrogen signalling. Unexpectedly, results with reporter genes led us to conclude that the inhibitory effects of SLTM on oestrogen signalling are primarily a reflection of its ability, when over expressed, to produce a generalised inhibition of transcription. Thus, although oestrogen reporter results corrected for transfection efficiency by measurement of -galactosidase suggested that oestrogen signalling was inhibited by SLTM (Fig 2c), this is likely to result primarily from the shorter half-life of luciferase as compared to -galactosidase. Several previous reports have cautioned against the potential for misinterpretation of reporter assays corrected by use of internal standards [1;8;12], and conditions that affect transcription and/or translation clearly require particular care. As shown in Figs 3a andb, over-expression of SLTM in HeLa cells has little effect on incorporation of radioactive uridine into total RNA, but incorporation into poly(A) + RNA is strongly inhibited. Because efficiency of transfection for these cells is about 50%, it would appear that mRNA synthesis is almost completely inhibited. Similar results were obtained when cells were exposed to staurosporine, suggesting that the decreased transcription associated with over expression of SLTM might be linked to activation of an apoptotic pathway. This possibility was confirmed by Hoechst staining of HeLa cells transfected with plasmid expressing SLTM, and further confirmed by DAPI staining together with cytochrome c release in cells transduced with adenovirus expressing SLTM. We have also found similar effects when SAFB1 is over expressed (results to be reported separately).

Apoptosis may be induced as a response to blockage of transcription via several possible mechanisms, including induction of a p53 response, a relative decrease in antiapoptotic factors, and aberrant accumulation of proteins in the nucleus [START_REF] Ljungman | Transcription -guarding the genome by sensing DNA damage[END_REF]. Although the function of SAF family proteins remains poorly understood, the presence of DNA and RNA binding domains, as well as a carboxy terminus capable of interacting with multiple transcription factors, suggests that a precise balance in the stoicheiometry of SLTM relative to other components of the transcriptional apparatus is critical. The apoptotic response to over expression of SLTM that we observed in every cell type tested (Table 1) could reflect such a requirement for accurate regulation of SLTM expression. Further studies will be required, however, to confirm that apoptosis resulting from over expression of SLTM is the direct result of decreased transcription, rather than diminished transcription being a consequence of an apoptotic process that is activated by some other mechanism.

In conclusion, SLTM is a nuclear protein with a distribution similar to that of SAFB1 (but unlike that of SAFB2, which is also found in the cytoplasm [START_REF] Townson | SAFB2, a new scaffold attachment factor homolog and estrogen receptor corepressor[END_REF]). The SLTM protein contains a putative SAF Box and RNA binding domains, and overall displays significant homology with SAFB1 and SAFB2, which have both been reported to inhibit oestrogen signalling [START_REF] Townson | SAFB2, a new scaffold attachment factor homolog and estrogen receptor corepressor[END_REF]. Rather than regulating expression of specific genes, however, changes in the expression level of SLTM have profound effects on mRNA transcription in general and eventually lead to apoptosis. Although over expression is a widely used technique to identify protein function, these results serve as a reminder that disturbing the balance in levels of protein expression may give misleading results.

  4 o C, stained with propidium iodide by incubation at 4 o C for 1 hour, and the cell cycle profile analysed by flow cytometry. The method of fixation in this instance was modified to a shorter incubation period as EYFP is known to leak out of permeabilized cells. Cytochemical detection of apoptosis. Induction of apoptosis in HeLa cells transfected with plasmid expressing SLTM was assessed by Hoechst staining. HeLa cells were seeded into 60mm dishes and transfected with 1µg of pEYFP-C1 or pEYFP-SLTM. The cells were then incubated overnight before being transferred to serum-free medium for 24 hours. Next, cells were fixed and stained with Hoechst and assessed by fluorescent microscopy. In HeLa cells transduced with adenovirus expressing SLTM, nuclear morphology was assessed by fixing cells with 4% w/v paraformaldehyde and nuclear staining for 15 minutes at room temperature with 100ng/ml DAPI (4',6-diamidino-2-phenylindole). For release of mitochondrial cytochrome c, cells grown on cover-slips were washed once with PBS, fixed for 5 minutes in ice-cold methanol and immediately re-hydrated in PBS. Primary antibodies (rabbit anti-cytochrome c from Becton Dickinson and mouse anti-HA from Sigma) were diluted to working concentrations in PBS containing 1% w/v BSA. After incubation overnight at 4 C, cover slips were washed three times with PBS. Secondary antibodies (FITC-conjugated anti-mouse IgG goat antibody and TRITC-conjugated anti rabbit IgG swine antibody (both from Sigma) were diluted to the recommended final concentration in PBS containing 2% BSA. After 1 hour at room temperature, cover slips were washed three times in PBS before being mounted in Vectastain ABC (Vector Laboratories, CA). Microscopy was performed with a Leica TCS confocal laser apparatus.

Fig 1b ,

 1b Fig 1b, SLTM co-localises with SAFB1, but co-localization is not complete, suggesting that the functions of the two proteins may not be identical.

[ 3 H

 3 ]uridine incorporation into RNA. These experiments were performed in HeLa cells using a transfection protocol which resulted in transfection of 50 to 60% of cells (results not shown).There was very little effect on incorporation into total RNA (representing predominantly ribosomal RNA), consistent with the exclusion of SLTM from nucleoli (Fig3a). When incorporation into Poly(A) + RNA (representing mRNA) was measured, however, there was a strong inhibition to 47+14% (p < 0.005) of control values (Fig3b). When account is taken of transfection efficiency, this result suggests a near total inhibition of mRNA synthesis in cells over-expressing SLTM. Preliminary results suggested that these inhibitory effects might indicate induction of apoptosis by over expression of SLTM. For comparison, we therefore repeated these experiments after exposing cells for 3hrs to different concentrations of staurosporine (STA), a kinase inhibitor derived from Steptomyces which has a well-characterised ability to induce apoptosis. As shown in Fig.3c, incorporation of uridine into mRNA was again more severely inhibited than total cellular incorporation of uridine. At an STA concentration of 100nM the effect was similar to that produced by expression of SLTM, with total incorporation of [ 3 H]uridine being decreased to 87.9+3.6% and incorporation into mRNA reduced to 55.6+3.3%.These results led us to investigate in greater detail the possibility that the inhibition of RNA synthesis caused by SLTM was associated with induction of apoptosis.Over expression of SLTM induces apoptosis in cultured cells.Plasmids encoding HA-tagged SLTM cDNA were used to assess whether the protein altered the cell cycle profile of MCF-7 cells. Transfection efficiencies of MCF-7 cells are relatively low. Therefore, to allow identification of transfected cells, a plasmid encoding enhanced yellow fluorescent protein (pEYFP-C1, Clontech) was co-transfected with plasmid expressing SLTM. Using a gate that only accepted fluorescent cells, the cell cycle profile of the selected cell population was then analysed. A typical experiment in which MCF-7 cells

  As with Hoechst staining of HeLa cells transfected with plasmid expressing SLTM, nuclear condensation and fragmentation were clearly demonstrated by DAPI staining of cells transduced by adenovirus expressing SLTM, but not by the Ad0 empty virus (Fig 6a). Further, extensive release of cytochrome c was apparent in cells transduced with virus expressing SLTM, but not in non-transduced cells (Fig 6b).

Biochemical

  Journal Immediate Publication. Published on 13 Jul 2007 as manuscript BJ20070170 © 2007 The Authors Journal compilation © 2007 Biochemical Society

FIG. 1 FIG. 2

 12 FIG. 1 Cellular localization of SLTM expression. a) HeLa cells were grown on coverslips and transfected with either pEYFP-C1 or pEYFP-SLTM, fixed and viewed by confocal microscopy. b) Co-localization of EYFP-SLTM with HA-SAFB1. HeLa cells were transfected with pEYFP-SLTM and pHA-SAFB1. The cells were fixed and stained with anti-HA antibody before viewing under the confocal microscope. The figure shows the distribution of SLTM, SAFB1 and an overlay of both images. c) SLTM does not co-localise with SFCs. Distribution of SLTM compared with that of SC-35, an SR protein. HeLa cells were grown on coverslips and transfected with pEYFP-SLTM, fixed, permeabilized, and stained for SC-35. (Scale: white bars = 5µm)

Fig 3 Fig 4

 34 Fig 3 Inhibition of transcription in cells over expressing SLTM. (a) Total RNA synthesis. HeLa cells were transfected with 0.1µg of either empty plasmid or pSLTM-HA, and incubated for 48hrs. The cells were then exposed to [ 3 H]uridine (1µCi/well) for 1hr before being lysed, harvested, and radioactivity measured. (b) mRNA synthesis. HeLa cells were

Fig 5 Fig 6

 56 Fig 5 Induction of apoptosis in HeLa cells by SLTM, assessed by Hoechst staining. HeLa cells were seeded into 60mm dishes and transfected with 1µg of pEYFP-C1 or pEYFP-SLTM. The cells were incubated overnight and then transferred to serum free medium for 24 hours. Next, cells were fixed and stained with Hoechst and assessed by fluorescent microscopy. a) Fluorescence of cells transfected with pEYFP-SLTM, b) Hoechst staining of sam field shown in panel a (Scale: Bars = 5µm), and c) Change in apoptotic index of HeLa cells after expression of SLTM, assessed by cell counting. This experiment was performed in triplicate (mean + SD) and repeated 3 times. The results shown are those of a typical experiment.

  Fig 2a

  Fig 2b

  Fig 2c

Fig 3 a

 3 Fig 3

Fig 3 c

 3 Fig 3

Fig 4 FLH2

 4 Fig 4

Biochemical Journal Immediate Publication. Published on 13 Jul 2007 as manuscript BJ20070170 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

This work was supported by grants from the Medical Research Council and the Wellcome Trust. CWC was supported by the government of Singapore. We wish to thank Dr Mark Jepson and Alan Leard for generous assistance with confocal microscopy, and Dr Niall Kerr for numerous technical discussions. The pERE-tk-luc reporter plasmid was kindly provided by Malcolm Parker.