PML nuclear bodies: Assembly and oxidative stress-sensitive sumoylation

PML Nuclear Bodies (NBs) have fascinated cell biologists due to their exquisitely dynamic nature and their involvement in human diseases, notably acute promyelocytic leukemia. NBs, as well as their master organizer - the PML protein - exhibit multiple connections with stress responses. Initially viewed as a tumor suppressor, PML recently re-emerged as a multifaceted protein, capable of controlling numerous aspects of cellular homeostasis. NBs recruit many functionally diverse proteins and function as stress-regulated sumoylation factories. SUMO-initiated partner retention can subsequently facilitate a variety of other post-translational modifications, as well as partner degradation. With this newly elucidated central role of stress-enhanced sumoylation, it should now be possible to build a working model for the different NB-regulated cellular activities. Moreover, pharmacological manipulation of NB formation by interferons or oxidants holds the promise of clearing many undesirable proteins for clinical management of malignant, viral or neurodegenerative diseases.

PML Nuclear Bodies (NBs) have fascinated cell biologists due to their exquisitely dynamic nature and their involvement in human diseases, notably acute promyelocytic leukemia. NBs, as well as their master organizer -the PML protein -exhibit multiple connections with stress responses. Initially viewed as a tumor suppressor, PML recently reemerged as a multifaceted protein, capable of controlling numerous aspects of cellular homeostasis. NBs recruit many functionally diverse proteins and function as stress-regulated sumoylation factories. SUMO-initiated partner retention can subsequently facilitate a variety of other post-translational modifications, as well as partner degradation. With this newly elucidated central role of stress-enhanced sumoylation, it should now be possible to build a working model for the different NB-regulated cellular activities. Moreover, pharmacological manipulation of NB formation by interferons or oxidants holds the promise of clearing many undesirable proteins for clinical management of malignant, viral or neurodegenerative diseases.

A Brief History
The eukaryotic nucleus, which harbours a vast amount of genetic material and regulatory proteins, is highly organized. Not only do chromosomes occupy distinct, non-random territories, but many proteins that function in gene expression, DNA repair or RNA biogenesis are also stably compartmentalized or at least transit in distinct microenvironments called nuclear bodies. 1,2 The latter may enhance kinetics of enzymatic reactions in the nucleus that is otherwise devoid of membranes. 3,4 This nuclear organization is often compromised in cancers or viral infections, implying that normal cell function depends on precise organization of the nucleus. 5 PML Nuclear Bodies (NBs) are proteinaceous sub-nuclear compartments organized by the PML (ProMyelocytic Leukemia) protein. 6,7 These structures were initially discovered by electron microscopy and subsequently reobserved by immunofluorescence where autoimmune sera of primary biliary cirrhosis patients marked SP100, a prototypic PML NB resident protein. [6][7][8] Yet, PML NBs drew real attention only decades after with the observation that they were disrupted, in a treatment-reversible manner, in cells from patients suffering from Acute Promyelocytic Leukemia (APL). [9][10][11] This leukemia is driven by the PML/RARA oncogenic fusion protein, which results from a chromosomal translocation. 12 While it was widely accepted that altered retinoid signaling due to expression of the PML/RARA oncogene was the primary culprit in APL pathogenesis, [13][14][15][16] recent findings demonstrated that PML NBs actually drive therapy response through reactivation of p53 signaling and senescence. [17][18][19][20][21][22][23][24] A typical nucleus contains between 10-30 PML NBs. As observed by electron or super resolution microscopy, NBs are spherical structures of 0.1-1 mm in diameter. 7,[25][26][27] Immunoelectron or high resolution confocal analyses indicate that the PML protein forms the outer shell, whereas partner proteins are concentrated in the inner core. 26,[28][29][30] The outer PML shell is associated with the nuclear matrix, a loosely defined, insoluble nuclear scaffold that is resistant to nuclease and high salt extraction and thought to support nuclear organization and compartmentalization. 31,32 Over the course of many years, a growing number of functionally distinct proteins and enzymes were shown to be recruited into PML NBs, notably p53 and many of its regulatory enzymes. 6,23,33,34 Initially, the striking functional diversity of NB partner proteins made it difficult to assess NBs' biochemical function and physiological role. It is now generally accepted that NBs function promiscuously to fine-tune a wide spectrum of cellular activities, including stress response, senescence/apoptosis, DNA repair, anti-viral immunity and stem cell renewal. 7,27,[35][36][37] How are such distinct tasks achieved at the biochemical level? Post-translational modifications (PTMs) dynamically tune protein function and stability and NBs were rapidly associated to several PTMs, and in particular to the SUMO (Small Ubiquitinlike MOdifer) protein. Indeed, SUMOs provide a glue for NBs, favoring partner recruitment, sequestration, and post-translational modifications. NBs serve as highly dynamic hubs onto which multiple stress signals converge. In response to oxidative stress (ROS, Reactive Oxygen Species), NBs promote partner sumoylation. 26 This may directly regulate partner function or serve as a pivot that cross-regulates other PTMs. This not only provides a mechanistic basis for the diversity of cellular functions that can be modulated by PML NBs, but also explains why absence of PML results in altered response to multiple stresses in pml ¡/¡ animals. 38 The Oxidant-Sensitive PML Protein The PML protein is the master organizer and scaffold of PML bodies. 39 Nuclei of cells from pml ¡/¡ mice do not possess NBs, and these structures are disrupted in APL cells that express the PML/RARA oncoprotein which interferes with the function of wild type PML in a dominant negative manner. 10,11 Ex vivo knock-down of PML is also sufficient to abrogate NB formation, while knock-down of other partner proteins has no major effects on NB number or gross structure. Although different PML isoforms exist due to alternative splicing, all these isoforms assemble into NBs when expressed in a pml ¡/¡ context, though with different trafficking and dynamics. 40,41 Earlier studies have shown that oxidation-driven intermolecular disulphide cross-linking promoted association of some nuclear proteins, such as polyoma large T antigen or poly ADP-ribose polymerase, to the nuclear matrix. 42,43 Subsequently, it was proposed that the majority of sulfhydryls in the nuclear matrix were oxidized and engaged in disulphide links. 44 PML is a zinc finger protein. Aside from 2 N-terminal zinc fingers, referred to as B-boxes, it contains a RING-finger that is found in various ubiquitin E3 ligases. 6,10 This N-terminal distal RING domain, with adjacent B-boxes and an a-helical coiled coil (CC) together form the RBCC motif, which characterizes a protein family called TRIM (TRI-partite Motif), 45 many of which are SUMO or Ubiquitin E3 ligases. 46 PML is cysteine-rich, and several groups have shown that it is a redox-sensitive protein: matrix-associated PML protein is highly oxidized and forms disulphide crosslinked covalent multimers that self-organize into the NB outer shell. 26,47,48 Indeed, oxidation drives NB formation in vivo or ex vivo, 26 while antioxidants disrupt basal NB formation. 47 The cysteines involved in intermolecular PML multimerization remain to be identified.

Role of SUMO in PML NB Biogenesis
Like the related ubiquitin peptide, SUMO may be reversibly and dynamically conjugated to its targets on specific lysine residues that exist in the context of a particular consensus sequence motif. [49][50][51] Covalent attachment of SUMO may modulate protein stability, conformation, enzymatic activity, subcellular localization, solubility, as well as interactions with other proteins, particularly through binding with SIMs (SUMO Interacting Motifs). PML can be SUMO conjugated on 3 target lysines (K65, K160 and K490) and also carries a SIM. 10,52,53 Nuclear matrix-associated PML is heavily sumoylated and arsenic or other oxidants can drastically induce PML sumoylation. 28,47 These results initially suggested that SUMO may be the glue holding the matrix-associated PML shell together through intermolecular PML SUMO-SIM interactions. [54][55][56] This simple model was challenged by recent studies that reported stepwise dissection of NB biogenesis, according to which, PML is sumoylated only once oxidized and nucleated into NBs 26,28,47 ( Fig. 1). Consistently, abrogation of the major SUMO-acceptor lysine K160 on PML does not impair NB formation and a PML isoform that lacks the SIM still forms nuclear bodies. 28,39,41,57,58 Even a PML mutant lacking all 3 SUMO acceptor lysines and its SIM still forms morphologically normal NBs in pml ¡/¡ MEFs. 26 Finally, extinction of SUMO expression abrogates recruitment of Stepwise depiction of PML NB biogenesis. NB biogenesis follows a non-stochastic path during which initial nucleation of the PML NB mesh provides a docking platform for subsequent partner recruitment. In the first step, oxidation-induced PML cross-linking via covalent disulphide bonds triggers PML oligomerization and formation of the NB shell that becomes associated with the nuclear matrix. This initial seeding step reflects cellular redox status. In the second step, multimeric PML mesh recruits UBC9, so that the NB shell then becomes heavily sumoylated. In the third step, SUMO-conjugated PML recruits SIM-containing partners via non-covalent SUMO/SIM interactions. This is followed by in situ partner sumoylation by UBC9 in the NB inner core (step 4), which leads to partner sequestration through enhanced SUMO/SIM interactions. Among the recruited partners, many are enzymes, which may favor other post-translational modifications, including RNF4-mediated ubiquitination and proteasome-mediated degradation.
partners such as SP100 and DAXX, whereas it does not interfere with formation of the PML NB shell. 26,28,39 Collectively, these observations rule out the proposal that the structure of the NB outer shell is maintained through SUMO/SIM interactions. Although dispensable for PML NB nucleation, SUMOs nevertheless plays a key role in subsequent steps of NB biogenesis. An ever-growing number of structurally and functionally diverse proteins, referred to as partners, reside in or are recruited temporarily into PML NBs. These include transcription factors, DNA repair proteins, enzymes involved in post-translational protein modifications, oncogenes and viral proteins. A unique feature that is common to all partners is their ability to undergo sumoylation and to display one or more SIMs. 26 This raises the possibility that either partner SIM or SUMO-conjugated lysine(s) initiates NB-association, and/or, inversely, that recruitment onto NBs may promote partner sumoylation. Deletion of SIM sequence consistently impairs partner NB-association, implying that partner SIM may interact with sumoylated PML to initiate and enforce recruitment. 26,[59][60][61] Indeed, mutation of PML K160 does not interfere with the formation of matrix-associated NB, but completely abrogates partner recruitment. These observations therefore suggest that PML NB assembly is not a stochastic process, but follows an ordered path, which is initiated by an initial seeding (nucleation) step: formation of the PML outer shell (Fig. 1). According to this model, ROS-regulated, disulphidelinked PML multimers associate with the nuclear matrix as empty shells, providing a seeding platform on which partner proteins can subsequently be recruited. Matrix-associated PML becomes heavily sumoylated, likely reflecting its direct binding to UBC9 through its RING domain and sumoylation in trans. 48,62 Sumoylated PML then recruits partners through polarized noncovalent interactions with their own SIMs. The role of PML SIM may be to fine-tune this process, as it is unessential for NBbiogenesis or partner recruitment, but may contribute to partner retention. Moreover, sequences adjacent to PML SIM undergo multiple PTMs (see below), which may constitute another regulatory mechanism. Whether there is a hierarchical order to recruitment of different partners is currently unclear. It is possible that recruitment of certain partners (i.e. those with multiple SIMs) may quench SUMO-conjugated PML K160s, impairing recruitment of others partners. Exchange rate and diffusion of recruited partners at NBs are significantly impaired compared to those in the nucleoplasm, but are still substantially higher than that of NB-associated PML, supporting the notion that NB shell provides a stable scaffold for dynamic association of partners. 26,41 PML can also be subjected to multiple post-translational modifications, particularly phosphorylation and acetylation, which might further regulate NB biogenesis and partner recruitment. [63][64][65] In response to DNA damaging stress, multiple serine residues on PML are phosphorylated. The latter was proposed to facilitate DNA damage-induced apoptosis. For example, phosphorylation of S8, S36 and S38 by HIPK2 was proposed to enhance PML sumoylation and was implicated in apoptosis in response to DNA damage. Phosphorylation of PML S117 by CHK2 can similarly regulate cell death in response to gamma irradiation. 66,67 PML phosphorylation by ATR upon DNA damage regulates PML sub-nuclear localization, as well as p53 stability. 68 Interestingly, PML SIM is surrounded by several serine residues (S505/518/527/530) that can be phosphorylated, particularly in response to EGF/ERK activation. Phosphorylation of these residues may enhance PML sumoylation. 69 Finally, CK2-dependent PML phosphorylation at multiple sites triggers proteasomal degradation of PML. 70 Importantly, increased CK2 activity was directly correlated with reduced PML levels in lung tumor samples. Some CK2 target amino acids are located in PML SIM and phosphorylation of SIMs may modulate interactions with SUMO2/3. 71 PML acetylation on K487 and K515 was also associated with enhanced sumoylation, as well as with efficient induction of apoptosis. 72 Finally, similar to PML, both SUMO1 and SUMO2/3 can be acetylated on specific residues (K37 and K33, respectively). Although SUMO acetylation does not interfere with conjugation onto PML, it regulates NB dynamics and can impair partner recruitment, possibly through abrogation of PML-SUMO/partner SIM interactions. 73 In any case, the astonishing variety of modifications that target the PML protein likely contribute to the extreme dynamics of NBs and their association to partners, including the different SUMO paralogs. 74

Biochemical Consequences of Partner Recruitment
PML influences basal sumoylation of SP100, and PML overexpression in yeast has an overall effect on global sumoylation profile. [75][76][77] As mentioned above, PML directly binds UBC9 62 . UBC9 is indeed recruited into PML NBs, particularly upon arsenic exposure and oxidative stress. 26 The concentration of UBC9 and PML partners (all of which are UCB9 substrates) results in their hyper-sumoylation in situ. 26,78 NB inner core may also provide a favorable redox environment for thiol enzymes involved in sumoylation. As proposed for certain TRIM family proteins, PML may also have an intrinsic SUMO E3 ligase activity, and therefore, facilitate direct transfer of UBC9-bound SUMO onto substrate proteins. 76,90 The global cumulative effect of NBinduced partner sumoylation is clearly detectable in whole extracts of cells exposed to oxidative stress. In contrast, sumoylation of non-partner proteins, such as RanGAP1, is unaffected by oxidative stress. Thus, ROS-induced NB formation and subsequent UBC9 recruitment promotes in situ partner hyper-sumoylation within NB inner cores 26 (Fig. 1). Note that the relationship between oxidative stress and cellular sumoylation is likely to be multifactorial, since oxidative stress can also inhibit sumoylation by cross-linking the catalytic cysteines on SUMO E1 and E2 (UBC9) enzymes. 79 PML NBs may prevent the E1/UBC9 crosslinks by buffering excess ROS. High levels of oxidative stress also inactivate SUMO proteases (SENPs), enhancing global sumoylation 80,81 Thus, although PML NBs play a central role in redox regulation of global cellular sumoylation, future studies are required to build a fully integrated view.
Arsenic is a metalloid element, well known for its ability to promote cellular oxidation, as well as to induce apoptosis most notably in leukemic cells. 82 Through multiple complex mechanisms, arsenic perturbs cellular redox balance, inducing formation of ROS. 83 Interestingly, arsenic can also directly bind PML, as demonstrated both in vitro and in vivo 47,48 Arsenic binding further enhances PML/UBC9 interaction in vitro, possibly through conformational alterations. 48 In vitro, it coordinates with 3 conserved cysteines in either or both of the zinc fingers (C60/C77/C80 and C72/C88/C91, respectively), forming both inter-and intra-molecular arsenic-sulfur bonds, likely displacing the zinc atoms in the process. Cysteines C212 and C213 were also directly implicated in covalent arsenic binding. 47 Arsenic binding promotes PML oligomerization, both through formation of inter-or intra-molecular arsenic-cysteine links. Mutation of either the zinc finger cysteines or the C212/213 motif not only impairs arsenic binding, but also affects PML matrix association, basal NB formation and critically arsenic response. 47 Future studies should further delineate the arsenic/PML interactions, as they clearly underlie therapeutic response and cure of APL upon arsenic therapy (see below). 84 PML NBs were once envisioned as simple depots where cellular or viral proteins were sequestered or stored. 85,86 Yet, a more complex view has recently emerged wherein sumoylated partners may be modified by other NB-associated enzymes, particularly enzymes controlling various post-translational modifications. In this more integrated view, NBs become sites integrating multiple post-translational modifications of a large group of proteins in response to stress, particularly oxidative stress. This PML-dependent simultaneous conjugation and post-translational modification of a whole class of proteins is reminiscent of the role of sumoylation in DNA-damage response, where sumoylation of the group, rather than any individual protein, is critical to sustain an efficient DNA repair. 87 One particularly well-explored modification is partner ubiquitination following sumoylation. Indeed, upon prolonged ROS exposure, NBs recruit RNF4, a SUMO-targeted E3 ubiquitin ligase which preferentially poly-ubiquitinates a growing number of sumoylated proteins 26,57,88 (see below). RNF4 recruitment correlates with decay of initial hyper-sumoylation on some recruited partners, which reflects their ubiquitination and degradation. 26 Yet, other NB-associated posttranslational modifications have been described (phosphorylation by HIPK2, acetylation by CBP) although partner ubiquitination is probably the best described one. 23,63,89 It remains to be determined whether PML has an intrinsic ubiquitin E3 ligase activity.

NB-associated Protein Degradation and Nuclear Protein Quality Control
The first reported substrates of ROS/NB/SUMO-dependent degradation were PML itself, and the oncogenic PML/RARA fusion. 28 Arsenic-enhanced PML or PML/RARA sumoylation promotes their RNF4-mediated ubiquitination and degradation and this underlies the therapeutic effect of arsenic in APL. 57,88 While SUMO-dependent PML degradation initially appeared as an exception in the ubiquitin/proteasome field, it is becoming clear that the PML/SUMO/RNF4 axis constitutes a general mechanism to destroy NB-targeted proteins in stressed cells. 26,90,91 Various exogenous factors, such as heat or oxidative stress cause protein misfolding or aggregation, but also hyper-sumoylation. [94][95][96] Thus, ROS/NB-facilitated hyper-sumoylation could provide a rapid first line protection against protein aggregation. Proteins irreversibly damaged could then be channelled into the RNF4/Ubiquitin pathway, and eventually degraded by the nuclear proteasome which accumulates around PML NBs 28,97 The mechanistic basis of how misfolded proteins are recognized by PML needs further exploration. 90,98 The coiled coil domain of PML was proposed to mediate protein-protein interactions and could recognize similar domains naturally found in aggregation prone poly-glutamine stretches. 90 Interestingly, similarly to PML, SUMOs are also essential for mediating cellular response and survival under various stress conditions. 92,99-102 Critically, the neurological phenotype of animals with PolyQ proteins appears significantly worse in pml ¡/¡ background. 90 A hallmark of neurodegenerative diseases is the formation of inclusion bodies containing aggregates of misfolded proteins (i.e., Huntingtin, ataxin). Often, such intranuclear inclusions show strong association with molecular chaperons, ubiquitin, proteasome, as well as PML NBs, implying the existence of a coordinated cellular response to counteract aggregation-induced neuronal dysfunction, the pace of which is accelerated in PML absence. 90,[103][104][105][106] Future studies should clarify whether perturbed redox control, a characteristic of neurons undergoing aggregation-induced degeneration, drives NB-formation in these cells.

PML NB Formation Is Drug-inducible
PML gene expression can be transcriptionally induced by interferons, which results in both an increase in size and number of NBs. 107 Interferons are cytokines secreted from virus-infected cells to initiate innate immune response. In certain settings, they may have anti-proliferative activity through induction of senescence. Interestingly, PML has well-documented anti-viral and anti-proliferative functions, implying that some of interferons' downstream effects may depend on induction of NB formation (see below). 35,108,109 A large pool of PML has a diffuse localization in the nucleoplasm and may even be found in the cytosol. 40 As describe above, arsenic rapidly induces redistribution of nucleoplasmic PML toward NBs, enhancing NB size. 47,110 Critically, non-arsenical oxidants such as paraquat can effectively induce PML multimerization and NB formation, both in cultured cells and in vivo. 26,47 Thus, in addition to increasing the level of PML expression by interferon, arsenic or other oxidants can further enforce NB-formation. The maximal level of partner sequestration can be obtained by the combination of interferon and arsenic 28,111 (Fig. 2).

The Interferon Connection and Medical Implications
Interferon not only enhances NB formation, but also partner recruitment 26,107 (Fig. 2). Interestingly, SUMO expression is also massively upregulated by interferons, through a recently elucidated microRNA based mechanism, 112 resulting in a global enrichment of SUMO conjugates. Although, unlike arsenicinduced sumoylation, interferon-enhanced sumoylation does not strictly depend on PML, PML does redistribute interferoninduced SUMO peptides toward NB partners. Indeed, while PML enhances SP100 sumoylation upon exposure to interferon, sumoylation of RanGAP1 is favored in interferon-treated cells only when PML is absent. 26,112 This points to the existence of a highly coordinated interferon-responsive sumoylation network where PML NBs and SUMOs are induced altogether, greatly facilitating modifications (or degradation) of partner proteins (Fig. 2). Notably, several PML NB partners, such as SP100, are themselves also interferon-inducible, and many of these function in anti-viral immunity. 113,114 This implies that NB/ interferon-enhanced sumoylation of host or viral factors may restrict viral fitness and replication. In line with this, not only SUMOs have been shown to have intrinsic anti-viral/pathogenic function, but also both PML and SUMOs are required for interferons' anti-viral activity against a variety of viruses. 112,[115][116][117][118][119] Critically, many viruses have evolved mechanisms that either interfere with cellular sumoylation and/or disrupt PML NBs. 117,119,120 Interferon, when used together with arsenic, induces proteasome-mediated degradation of the HTLV-1 oncoprotein Tax, in a PML-and SUMO-dependent fashion 121 (Sahin et al, manuscript submitted). Importantly the combination of interferon and arsenic has unambiguous clinical benefit in HTLV-1 infected Adult T-Cell Leukemia (ATL) patients, where remissions can be achieved in the absence of chemotherapy. 122,123 Spinocerebellar ataxia (SCA1) is caused by accumulation of ataxin 1, a poly-glutamine stretch containing protein, which is degraded by the PML/SUMO/RNF4 system. 90 Interferon treatment can induce clearance of ataxin 7 from the brain in a PMLor SUMO-dependent manner, resulting in clinical benefit. [124][125][126] Various other poly-glutamine proteins, such as Huntingtin, were also shown to be cleared in PML NBs. 90 Overall, these findings support a novel and exciting notion whereby utilization of PMLand SUMO-inducing agents (i.e. interferon) may be an effective strategy in clinical management of some neurodegenerative pathologies. The role of arsenic should also be investigated in these models, as it could further accelerate mutant protein degradation. Yet, its effect on oxidative stress, the primary cause of neuronal death in many neurodegenerative diseases, remains a concern and should be evaluated.
Other NB-associated factors, notably p53, may be involved in mediating the effects of interferons on senescence. Together with p53, p21 and PAI-I, PML is a master regulator of senescence. 23,127,128 PML controls p53 signaling and is a crucial mediator of Ras-induced p53-mediated senescence, as well as a downstream p53 target. 18,19,23,[127][128][129][130] The physiological importance of this PML/p53/senescence axis is highlighted by APL cure where therapy-induced PML NB reformation drives leukemia eradication through p53 activation. 24 NB-mediated p53 sumoylation may activate a p53/senescence axis through in situ acetylation by CBP or MOZ, or phosphorylation by HIPK2, or other indirect mechanisms. 23,89 Indeed, p53, as well as many of its regulatory enzymes (MDM2, HIPK2, CBP) are localized to PML NBs and can be sumoylated, and PML-dependent sumoylation of multiple p53 regulators may fine-tune p53 activation. Critically, the sumoylated form of p53 was proposed to contribute to interferon effects on senescence. 131 In various settings, interferon-triggered cancer cell apoptosis or senescence involves PML and p53. [132][133][134][135][136] Similarly, both PML and sumoylation of DAXX were shown to be critical for interferon-triggered apoptosis in B cells. 137 Thus, the PML interferon crosstalks are not limited to antiviral effects. Collectively, pharmacological harnessing of PML NBs holds the promise of being a feasible and effective strategy to eliminate a growing number of undesirable toxic, viral or oncogenic proteins (Fig. 2). NBs' clinical implication, therefore, largely exceeds APL where they were already conclusively implicated in the cure of the disease. Figure 2. NB formation and downstream functions can be pharmacologically manipulated. Various external stimuli, in particular redox and interferon signaling, converge onto NBs, resulting in context-dependent pleiotropic functions. Arsenic enhances NB formation both through direct PML binding and ROS generation. Interferons increase PML transcription, as well as SUMO abundance. Both drugs enhance partner recruitment onto NBs and favor in situ sumoylation. Drug-induced, NB-facilitated partner sumoylation may be clinically harnessed to trigger PML/SUMO/RNF4-dependent clearance of toxic proteins, or to initiate NB-regulated senescence or apoptosis.