Distributed under Creative Commons Cc-by 4.0 Vero Cells Harbor a Poly-adp-ribose Belt Partnering Their Epithelial Adhesion Belt

Poly-ADP-ribose (PAR) is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs) and degraded by poly-ADP-ribose-glycohydrolase (PARG). Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair). Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt). In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO). PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications. How to cite this article Lafon-Hughes et al. (2014), VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt.


INTRODUCTION
Poly-ADP-ribose (PAR) is a linear or branched polymer of up to 400 ADP-ribose units that binds (covalently or not) to target proteins. PAR is synthesized by poly-ADP-ribosepolymerases (PARPs) and the key catabolic enzyme is poly-ADP-ribose-glycohydrolase (PARG). PAR synthesis involves the cleavage of NAD + into ADP-ribose (monomers) and free nicotinamide (Virag & Szabo, 2002). A steady-state balance is maintained in normal cells regarding PAR synthesis and degradation. An excellent review on the roots and developments of PARylation research has been published recently (Virag, 2013).
A plethora of PARP inhibitors can be used to envisage the involvement of different members of PARP family in specific processes. These compounds display diverse binding and in vitro potencies towards PARP-1 and TNKS-1 (Table 1). Human PARG is expressed in alternative splice variants yielding isoforms that localize to different cell compartments (Bonicalzi et al., 2005;Bonicalzi et al., 2003;Ohashi et al., 2003). Cytoplasmic PARG accounts for most of the PARG activity in cells (Meyer-Ficca et al., 2004). Although most PARG activity would be cytoplasmic and most PARP family members can be detected in the cytoplasm, their role inside the nucleus has been better studied. PARP-1 (the single family member located exclusively in the nucleus), nuclear PARP-2 and -3 compete with histone deacetylases for NAD + consumption. Poly-ADP-ribosylation of chromatin-associated proteins usually correlates with increased histone acetylation, decreased DNA methylation and low chromatin compaction. Thus, PARylation may modulate gene expression and facilitate the access of DNA repair machinery to damaged sites (Tulin & Spradling, 2003). In fact, PARP-1, the most conserved and best studied PARP, plays a role in the recognition of DNA damage. Nevertheless, PARylation has also been Table 1 Potency of PARP inhibitors towards PARP-1 and TNKSs. Binding capacity expressed as ΔTm ( • C) according to Wahlberg et al. (2012), in vitro IC50 (correspondent citations in the right-most column). 3-AB, 3-aminobenzamide; OLA, Olaparib; PJ34; XAV, XAV 939. While OLA is considered a selective PARP-1 inhibitor, (which does also target PARP-2, -3 and -4; , PJ34 is a moderate potency inhibitor, displaying PARP-1 preference over TNKS-1, with a lower magnitude order and XAV 939 is relatively selective for TNKS-1 and -2, affecting their activity 169 times more than PARP-1 activity. The inhibitory concentrations in cell culture are always higher than in vitro. For example, 3-AB IC50 in vitro is 5 µM and in vivo, it is commonly used at a 5 mM concentration.
In the present work, we have described through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO). In fact, we detected PAR associated to the epithelial belt, in a location that has not been previously reported. We have used PARP inhibitors to demonstrate that the immunodetected signal associated to the epithelial belt is PAR and that if PAR synthesis is precluded, actin cytoskeleton as well as cell shape and cell adhesion are affected. Based on these data and previously reported information, TNKS-1 poses as a plausible candidate as the PARP responsible for PAR synthesis in the epithelial belt. However, the participation of other PARPs, particularly TNKS-2, cannot be ruled out. TNKS-1 knockdowns have proven to be unviable since mitosis and cell viability are dramatically affected (Vyas et al., 2013). Therefore, the mechanisms involved in PAR belt formation will be studied in the future by the implementation of other approaches.
Treatments were continuous and carried in duplicates, in parallel with a common (duplicate) control and the correspondent controls without primary antibodies.

Confocal microscopy and image analysis
Single images or image stacks were recorded with an Olympus FV300 with a Plan Apo 60 ×/1.42 NA oil immersion objective or a Leica TCS SP5 II confocal microscope with a Plan Apo 63×/1.4 NA (or a Plan Apo 100×/1.4 NA) oil immersion objective, with or without digital zoom. To assure signal specificity, original images were taken under the same conditions as reference images of cells not labeled with primary antibodies, at the same confocal session. ImageJ free software was used for image processing (including brightness/contrast adjustment and Gaussian blur filtering).

Untreated VERO cells harbor different nuclear and peripheral PAR polymers
Poly-ADP-ribose was detected in nuclear and peripheral localizations using the BD anti-PAR antibody. These signals were detected after 10% TCA or 4% PFA fixation. Given that TCA causes protein precipitation, a stronger background was detected in the absence of primary antibody; therefore, PFA was selected for subsequent experiments (Fig. S1).
Since it has recently been demonstrated that at least one member of the ecto-ARTC family can catalyze the synthesis of short lineal PAR chains on the extracellular side of the plasma membrane (Morrison et al., 2006), we decided to check whether the detected epitope was intracellular or extracellular. Hence, immunolocalization was performed avoiding the permeabilization step (in parallel to the routine protocol). In the absence of permeabilization, neither the nuclear nor the peripheral PAR signals were detected (Fig. S2), demonstrating the intracellular nature of the epitope.
Immunostaining with different primary antibodies in parallel yielded apparently conflicting results. For example, nuclear PAR was detected with polyclonal rabbit BD or chicken Tulip anti-PAR antibodies (Fig. 1), but not with Tulip monoclonal H10 clone antibody. According to the respective data sheets, the three antibodies were generated against PAR of unknown length and branching conjugated to methylated BSA. Nevertheless, the latter antibody has known specificity for long PAR chains (above 20 residues; Kawamitsu et al., 1984) and has been widely used to monitor the nuclear response to DNA damage, which is mainly PARP-1 dependent (Vodenicharov et al., 2005;Gagne et al., 2008). PARP-1 synthesizes long branched chains (Hottiger et al., 2010). Coherently, DNA damage response proteins such as p53 or XPA form complexes mainly in the presence of long PAR chains (Fahrer et al., 2007). In fact, while short PAR chains (16-mer) do not interact with XPA and form a single complex with p53, long PAR chains (55-mer) promote the formation of a complex with XPA and three specific complexes with p53 (Fahrer et al., 2007).
PAR belt was detected with BD rabbit anti-PAR antibody (#551813) but not with Tulip chicken anti-PAR antibody (#1023) (Fig. 1), suggesting again the existence of a differential structure of both PAR polymers. Interestingly, this is not the first report of differential recognition of PAR polymers by antibodies. For example, 16B antibody, which has a preference for branching regions, recognizes just 50% of PAR polymer detectable by H10 (Kawamitsu et al., 1984). Although this phenomenon is more likely to occur with monoclonal antibodies, it seems to be also true for some polyclonal antibodies. In any case, this PAR would correspond to short-chain polymer (up to 20-mer), not recognizable by H10, as expected under the hypothesis that belt PAR is an oligomer (up to 20 units) synthesized by TNKS-1.

Specific PARP inhibition alters microfilament distribution
In VERO cells, PARP-1 is exclusively nuclear (data not shown). The nuclear PARPs inhibitor OLA (250 nM, 6 days; Narwal, Venkannagari & Lehtio, 2012) depleted nuclear PAR without affecting belt PAR (Fig. S3). Treatments with other PARP inhibitors (5 mM 3AB for 24 h, 80 µM PJ34 up to 7 h, 25 µM XAV 939 up to 24 h) applied on the established monolayer had no effect on the PAR belt (data not shown). Taken together, these results suggest that belt PAR is characterized by extremely high stability.
To confirm PAR identity, we evaluated the influence of PARP inhibitors (5 mM 3-AB or 80 µM PJ34) on cellular PAR synthesis. To that end, inhibitors were added at the moment of cell seeding (Fig. 2, green). 3-AB slightly affected PAR while PJ34 showed a stronger effect, particularly on peripheral PAR. The combined treatment yielded a result similar to PJ34 alone. 3-AB is a non potent PARP inhibitor with detectable binding to PARP-1 but undetectable effect on TNKS-1 (Wahlberg et al., 2012;Riffell, Lord & Ashworth, 2012 ,  Table 1). PJ34 binds both nuclear PARPs and TNKS with higher affinity than 3-AB, but the IC50 for PARP-1 is nearly 30 times lower than that reported for TNKS-1. Thus, it affects PARP-1 activity preferentially, but inhibits TNKS-1 and-2 in a non-negligible proportion.
3-AB displayed a border-line effect on PAR and actin distribution (Figs. 2C, 2H, 2M). PJ34 induced a deeper blocking effect on PAR belt synthesis (Figs. 2D, 2I, 2N) and the strongest effect was obtained with the combined drugs (Figs. 2E, 2J, 2O). Notice that in spite of diminished belt synthesis, there has been no diminution in cellular density. However, cell shape appeared semi-rounded in some cases and cell area is diminished, reflecting incomplete spread out of the cells. The vehicle, DMSO, affected neither PAR nor the actin belt (Figs. 2B, 2G, 2L).
To sum up, PARP inhibitors affected not only the PAR belt but also the distribution of actin filaments, suggesting the existence of a physical direct or indirect interaction of PAR with the actin cytoskeleton.

Peripheral PAR colocalized with cortical actin and vinculin in the epithelial belt
VERO epithelial cells present adhesion belts separating apical and basal domains, with cortical actin filaments anchored to the belts. To analyze PAR localization in more detail, confocal stacks of cells immunostained for PAR and co-stained with phalloidin (Fig. 3) or co-immunostained to detect vinculin (Fig. 4), were used. Figure 3 highlights the fact that PAR is associated to submembrane domains only in the proximity of a neighbor cell. PAR distribution in the intercellular limits, novel to our knowledge, punctuated lines in intercellular not fully formed contact regions (Figs. 3D-3F, double arrows) and present as a single punctuated line in completely joined cells (Fig. 3C, single arrows) but absent in membrane/cortical domains without neighbor cells (Fig. 3, arrowheads). PAR was located at the place where cortical filaments were anchored, as evidenced by the unequal filament direction between both sides of the intercellular limit/adhesion belt. PAR seemed to be a partner of cortical actin filaments. Z-stacks revealed the existence of a structure that we called the "PAR belt", with a height of around 1 to 1.5 µm (up to 4 slices every 0.5 µm). The clear-cut presence of PAR in intercellular junctions (arrows) but not associated to the plasma membrane in neighbor-free domains (arrowheads) is illustrated in Fig. 3 orthogonal views (G-R). The yellow lines indicate the cutting planes.
Vinculin is an actin-binding protein that displays a dual localization: basal and apical, related to cell-matrix focal adhesions and to ZO-1 positive tight junctions in the epithelial belt, respectively (Maddugoda et al., 2007). Interestingly, while focal adhesion vinculin is not PARylated (Fig. 4, arrowheads), a colocalization of PAR (in green) and vinculin (in red) is observed at the apical position correspondent to the epithelial belt (Fig. 4, arrows).

During actin cytoskeleton disruption, PAR accompanied actin
In order to test the physical association of PAR to the actin cytoskeleton in this particular localization, we induced microfilament disassembly through cytochalasin D (2 and 20 µM,  30 min) treatment. While in control cells the actin/PAR belt could clearly be observed (Figs. 5A-5D, arrow), after 30 min of low-dose cytochalasin D treatment (2 µM) the actin belt was fractured. In some cells, belt microfilaments could still be observed (Figs. 5E-5H, arrows), although they looked non-continuous and tensionless; in other cells, the actin/PAR belt had disappeared arrowheads). After exposure to a stronger treatment (20 µM cytochalasin D), no filamentous structure remained. Instead, there was a punctuated actin pattern with some precipitates (Figs. 5I-5L, arrow) colocalizing with PAR. Therefore, belt PAR accompanied actin microfilaments during their structural loss.

EGTA or XAV 939 disturbed PAR belt synthesis, affecting the actin cytoskeleton, cell shape and cell adhesion
We reasoned that in a condition in which TNKS-1 was not recruited to the epithelial belt, peripheral PAR would not be synthesized. It is well established that extracellular Ca 2+ chelation hampers cell adhesion. More recently, it has been shown that TNKS-1 is recruited from the cytoplasm to the lateral plasma membrane upon formation of E-cadherin-based cell-cell contacts in renal epithelial cells, and the recruitment depends on extracellular calcium ion (Yeh et al., 2006). Thus, we depleted extracellular calcium with EGTA (3 mM). Under this condition, cell roundness and diminished cell adhesion leading to reduced and irregular cell density were observed (although not reflected in the photographs because empty fields were not photographed). Concomitantly with cell roundedness, PAR diminution was observed (Figs. 6B, 6E, 6H), as expected under our hypothesis.
EGTA chelation is a very unspecific treatment. Thus, we next exposed cells from the moment of seeding to XAV 939, an inhibitor which exhibits a strong preference for TNKSs over other PARPs (Table 1; Wahlberg et al., 2012;Riffell, Lord & Ashworth, 2012). Like with EGTA, a decrease in cell density was repeatedly observed (Figs. 6C, 6F, 6I). As the time interval was short (just 5 h), this cannot be explained by a reduction in the number of cell cycles, but by diminished cell attachment. This effect (Figs. 6C, 6F, 6I) is stronger than the one observed with PJ34 + 3AB (Figs. 2E, 2J, 2O) which had reduced cell spreading area without lessening the number of monolayer-constituting cells. There were a plethora of cell shapes including round and binucleated cells. Finally, while in control populations it was difficult to find an isolated cell pair (a confluent monolayer was almost everywhere), in XAV-treated populations cell pairs were frequent, but many times the PAR belt junction was incomplete. To sum up, XAV 939 displayed a strong effect on the cell junction regions, with diminished cell attachment, increased roundness and partial loss of PAR/actin belt.

CONCLUSIONS
In the present work we have shown for the first time the existence of a PAR belt associated to the actin cytoskeleton and colocalizing with the anchorage protein vinculin. Vinculin associates to the E-cadherin complex. Thus, it is expected that PAR interacts with several members of the complex. Although our data fits the reported vinculin/alpha-catenin co-immunoprecipitation with anti-PAR antibodies (Gagne et al., 2008;Gagne et al., 2012), in our system, given the resolution of confocal microscopy, we did demonstrate that the cell junction apparatus (not necessarily nor exclusively vinculin) is PARylated.
Actin cytoskeleton disruption affects the PAR belt whereas the interference with PAR belt synthesis leads to actin cytoskeleton, cell shape and cell adhesion changes.
The graded effects obtained with 3AB, PJ34 and XAV 939 indicate a probable involvement of TNKSs in PAR belt synthesis. We cannot formally exclude TNKS-2 as the responsible enzyme. Parsimony favors the hypothesis of TNKS-1 involvement in PAR belt synthesis, since (1) In VERO cells, belt PAR is not detected with an antibody targeting long PAR chains (TNKS-1 synthesizes short chains); (2) in another renal epithelial cell line (MDCK), TNKS-1 is localized at the epithelial belt and its activity is required for cell-cell adhesion (Yeh et al., 2006). Demonstrating that TNKS-1 is responsible for the observed PARylation is a difficult task. The generation of a mammalian cell TNKS-1 knockdown has been attempted (Vyas et al., 2013), but it resulted in an unviable cell line, affecting the whole cell and rapidly leading to cell death, precluding a clear dissection of the underlying mechanisms. Though TNKS-2 participation cannot be ruled out, it should be pointed out that its functionality as a PARylating enzyme has not been proven up to date. The evaluation of the detailed mechanisms involved in PAR belt synthesis as well as the identification of the individual components of the E-cadherin/actin complex which are PARylated, will be the subject of future research. Further work will also be necessary to analyze the existence of the PAR belt in other epithelial cells to fully characterize the biochemical differences among nuclear and belt PAR, and to study the functional implications of the PAR belt in different systems.