Brigitte Ouvry-Vial

Yannick Estève

<1> Computers now offer opportunities to grasp, store, and explore huge corpora in a matter of weeks or months, and in a proportion ten years ago barely conceivable to scholars in the Humanities who would not have taken up the task of arranging such corpora with traditional archival and documentation tools, less committing themselves to a lifetime of work. Recent algorithms and hardware also allow computer scientists to develop new software tools based on machine learning and artificial intelligence, in order to emphasize inconspicuous links between data contained within very large archives, while such archives are composed by structured or non-structured digital documents.

<2> Beyond this obvious yet decisive and practical contribution, Informatics proves instrumental in the epistemological development of the Digital Humanities. New deliverables in the Humanities induce new research methods and concepts and require new interactions between scholars in the Humanities and researchers in Informatics beyond the level of technical assistance and mathematical development. This is specifically true for the case of archives, as both fields are involved, yet by separate means, in a converging process of restoring the original context, circumstances, and/or the experience, of the records considered.

<3> The background of this paper is today's potential (re)creation and (re)shaping of either established, forgotten, unexplored collections of historical documents and records, or of previously non-existing, ex-nihilo and newly invented archives, through computational methods and digital media. The paper considers archives as an object of study that has both greatly evolved and varied throughout the 20th century, in size, form, content, status, purpose, handling or processing altogether, e.g.moving from ancient, scholarly or patrimonial valuables to recent, mundane, personal or collective memorabilia; from physical to virtual matter; from archaism to futurism; from paper set to data or even metadata set.

<4> As new types and multimodal shapes of archives emerge it raises the needs to identify what archives are for scholars respectively in the Humanities and in Informatics.

<5> Archimorphosis thus intends to be a dialogue between such two researchers trying to bridge the gap between two distinct experiences of archives. While considering recently developed examples of work on audiovisual archival material or other kinds of digital set up within the broad area of Digital Humanities, they may converge in drawing crossovers and parallels, or at least raise shared issues that enlighten the changing status of archives today.

From archives to archive: reflection on an evolution

<6> Archives and archive are two distinct acceptations of the word that seem separated not so much by divergent concerns, definitions and even scientific objectives, rather than by academic and scientific methods of approach and practical purposes. Both the Humanities and Informatics resort to the plural "archives" to broadly designate series of documents assembled, variously classified and set aside for different purposes and future uses. Both also use the singular "archive," yet with a different meaning. In the Humanities, especially textual studies, the singular archive has an epistemological and ontological meaning referring to the origin of a work of art in the mind or the personal history of its author. It does not translate into identifiable material documents or features but rather designates the aesthetics and deep underlying specific endeavors in the Arts. From the computer science point of view, an archive refers to digital data stored on a physical medium in order to be preserved during a limited or unlimited period. Such archive can be composed by a simple digital document (text, image, audio, video, or binary raw), or by a huge collection of documents. During the last decade, with the Internet explosion, the development of social networks and the emergence of the cloud infrastructures, digital archive size has exponentially grown, and this tendency is still in progress. A noticeable aspect of the content of these archives is that they may be composed of heterogeneous and unstructured documents as well as homogeneous documents following a very strict format. Origins of such archives are multiple: general public, companies, artists, journalists, scientists who store their digital documents in order to preserve them from mishandling or material failure. These archives can contain documents initially produced under a digital form, but they can also be digitalized forms of material documents. The heterogeneousness and the massive quantity of documents stored in digital archives imply that specific tools must be developed and mastered in order to retrieve relevant information. Also, with new opportunities offered by computers to dematerialize, collect, explore and treat large corpora of information, written, visual and sound documents, that for long could not be grasped or visualized as an identifiable whole, even less processed, we now watch a parallel evolution in the conceptions of archives and archival curation [1].

<7> Collective studies and a current flow of work-in-progress papers describe a shift from a strict notion of conservation and management meant to preserve previously collected materials to a broader dynamic notion: [2] the term "digital curation" is increasingly being used for the actions needed to maintain digital research data and other digital materials over their entire lifecycle and over time for current and future generations of users. Implicit in this definition are the processes of digital archiving and digital preservation, but it also includes all the processes needed for good data creation and management, and the capacity to add value to data to generate new sources of information and knowledge of "creating," generating and assessing new sources of information and knowledge [3]. <8> More specifically, archives in the Humanities were for long seen as mostly unsorted, dusty yet valuable sets of papers, as tangible cultural heritage pertaining to sometimes forlorn pasts; they were essentially riveted on by highly erudite and skilled archivists mastering the techniques of authentication, classification, indexation, and devoted to making physical specimens accessible for potential (yet equally forlorn) future uses. As pointed out by Steven Marcus, "the Humanities as we think of them, the formal organized study of language and literature, philosophy and history, art and music did not exist in the late 19th century"; and for a long period of time afterwards," each(of these subjects was) considered an independent domain, properly organized as its own department of knowledge." [4] Thus the use of archives broadly meant resorting to Greek, Latin or Renaissance classics in order to provide a general cultural orientation and guidance, and was almost uniformly oriented toward understanding the past rather than grasping the specific background of works of more recent periods. <9> Later on, for most of the long 20th century, archives were still seen in the field of Humanities as primary sources of specific and almost encrypted documentation for scholarly inquiries in specialized single disciplines. While cultural studies in the 60ies and 70ies reshaped tendencies within the Humanities, the return of grand theory, cultural anthropology and "the general notion of the cultural or social construction of knowledge" [5] meant nevertheless that no trans-contextual validity was seen possible and archives were considered unethical, conservative sets of proofs that could not help establish the genuine sociocultural determination of thoughts and works. <10> Then came the pioneering cluster of "Humanities informatics" or "Humanities computing," essentially resorting to linguistics in the late 70ies and 80ies; and gradually archives have acquired, especially for the study of written culture at large, an almost reverse status: seen as a series of nondescript collections also open to multipurpose, profane and amateur uses, archives require an interdisciplinary approach mixing the whole realm of methodologies derived from the various branches of the Humanities including social sciences as well.

<11> Challenged by digital research and taken up on the growing interest for a revisited work of art, the restoration of its original context and circumstances of production, archives are not just providing authentic evidences of the past and the means of its understanding; they directly contribute the means of its visible recreation and exhibition (as in virtual museums for example) and the publishing, circulating or displaying of archives thus constitute a valid end-result of research per se. <12> It is not our purpose to dwell on the recent history of archival work nor of traditional versus digital data collection by scholars in the Humanities, but to reckon that the evolution of the Humanities as a field can be observed through the prism of the emblematic changes in the shape and functions of archival material, which grants the neologism archimorphosis a basic epistemic meaning.

<13> Archimorphosis underlines the mutation in the medium of communication and display of archives, the consequent reshaping and re-evaluation of its status. Thanks to the potentials offered, the dematerialization of documents is counterbalanced by its visual reconstruction, albeit on screen; so that the issue is not so much the plastic, evolving granularity of the archival objects as its instability, its transitory status leading the document to a permanent metamorphosis and change in format, support, including its re-materialization through printed outputs. Thus a reflection of the metamorphosis of archives rejoins McKenzie's remarks in [START_REF] Francis | Bibliography and the Sociology of Texts[END_REF] on the editorial and bibliographical conflation he observes, the paradox he sees "in the ease with which new technologies now permit readers to reconstruct and disseminate texts in any form they wish, with few fully effective legal constraints" and how "such uncontrolled fluidity returns us to the condition of an oral society." [START_REF]O'Reilly 2005) included the customization of the user experience, by embracing on-the-fly Web page creation through AJX and other database driven Web technologies which provided an opportunity for users to actively query and select the available information on any given Website. By extension, such technologies also offered an additional opportunity for users to become active as content creators and contributors, which fundamentally altered the vertical alteration between Website providers and Website users. In turn, this potential for user participation and content creation also enabled the emergence of genuine horizontal collaboration between users, and for the formation of user communities[END_REF] As stated by D.Poulot in, [START_REF] Poulot | L'historiographie du patrimoine[END_REF] cultural heritage is linked to the representation of values forged by societies, to the ways in which they conceive transmission. In France, the importance of literary value explains why the evolution and construction history of the cultural heritage coincides with the history of texts. Similarly in art history, books set the hierarchies, and the choice of paintings and sculptures in museum collections follows the pattern of indications and quotations by ancient historians in their writings. "Reality has come to comply with writings." [START_REF] Thuillier | Leçon inaugurale[END_REF] It is only since the end of the 80ies [START_REF] Querrien | Pour une nouvelle politique du Patrimoine[END_REF] in France that the notion of cultural heritage has been reconsidered to include a multiplicity of collections both inert and living, objects of all kinds and all periods of times; the extension of the notion of archives to new media collections requesting new archival methods dates back to the 90ies, as shown by the recent interest for audio-visual archives and their still partial legitimacy. [START_REF] Guyot | Les Archives audiovisuelles, histoire, culture, politique[END_REF] Figure 1: 1920 children's book illustration <15> The digitalization of existing archives and the ex nihilo development of new digital collections or databases-each being seen as an "archive"originate new references, imply new concepts and purposes, new systems of organization and display, raising several questions about the status of documents, records or information at large, reconstructed under these new auspices. Among the variations on the theme are the archive as rediscovery, the archive as political act, the archive as argument, the archive as performance[…]. Obviously, digital tools provide a dynamic frame and context to retrieve and display formerly irretrievable objects and artifacts, but even more to exhibit and demonstrate the relationships between these objects. <16> The relationships exhibited can be simple; for example, connect a children's book illustration [START_REF] Neveu | Un nouveau patrimoine écrit en bibliothèque: les fonds de littérature jeunesse[END_REF] (cf. Figure 1) with the audio version of the popular nursery rhyme [https://biblogotheque.wordpress.com/]. <17> The relationships between archival pieces can also be indirect and less obvious, thus enhancing the impact of digital tools as seen in another example drawn from a different area of knowledge; the bulb in the attached photo, if flatly reproduced in a book, could be seen as a piece of contemporary art. [START_REF] Cuenca | Constitution d'un patrimoine scientifique et technique contemporain[END_REF] Yet it is a piece of industrial history which even a lengthy written explanation cannot properly reassess. <19> A more developed demonstration of relationships can be found in Theaville (http://www.theaville.org) a literary and musical research website (cf. Figure 3) and tool dedicated to dramatic parodies of French 18th-century opera [START_REF] Cethefi | Centre d'étude des théâtres de la Foire et de la Comédie-Italienne)[END_REF]. The website has two purposes: one is to reevaluate a vast array of musical works considered in the 18 th -c. as unofficial; another purpose is to illuminate the hidden and complex mechanisms underlying the composition of these works and the various levels of understanding they require. It does so by connecting music sheets, musical scores, popular versions derived from the parodies, and historic commentaries. The website is constantly edited, and has so far more than 260 parodies with its 2000 corresponding scores, variants, known recorded interpretations, original librettos, sources, links to other resources and archives, and of course explanations, notes, biographies and other works by the composers, etc. <21> The impact of NTIC is therefore especially noticeable on cultural heritage and for recently identified objects, artifacts or documents from the past that were not previously considered and valued as patrimony but became so in the wave of new patrimonial concerns and social sense of responsibilities leading to preserve testimonies of disappearing, or vintage identities, territories, etc. Numerous studies on cultural practices, interactivity and reading within context of saturated digital media and environments [START_REF] Bruns | Are we all producers now? Convergence and media audience practices[END_REF] also point out that the knowledge resulting from the consultation of digital archives is not so much induced by a scholarly and authorized preconception as it is deducted and reconstructed by the user. While each traditional reader confronts the printed and possibly on screen text with one's specific pace, mode and experience, the printed book still provides a strict rigid frame wherein reading direction and processes (from left to right in Western cultures, from top of the page to bottom, chapter by chapter from the start, etc.) are essentially guided by writing directions. Conversely, digital bodies of documents allow multidirectional approaches and all kinds of purposes, including random browsing, skipping, etc. While the resulting knowledge or content collected is far more unpredictable and hard to evaluate, the open system potentially attracts and empowers, albeit randomly, a wider and nonspecialized audience.

<22> Current developments and digital projects in the Humanities based on archives thus face at least a dual challenge. First, a legitimacy issue for archives of contemporary cultural artifacts: what is this fresh set of ephemera with short-term usefulness and popularity? Can it contribute to an understanding of the history of current social trends or living modes? Is this contribution comparable to that of ancient archives, the canonical value of which is guaranteed by time and sustainability? Secondly, and more central to this paper, a formal issue of nomenclature: with the development of multiple digital formats, collections of all sorts need to adjust to the computational or informational definition of archives as this definition undermines or sets aside previous historiographical hierarchies or curation methods and criteria.

Issues in the shift from material to digital archives <23> While Archives on Fire could convey a dramatic sense of no return, it is currently argued that the disintegration of traditional representations of archives and cultural heritage is counterbalanced by a correlated and renewed interest for a restoration of theory art, as well as for a reconstitution of the circumstances and means of its conception and production. Moreover, the metamorphosis of archives-the renewal of its form and function in Digital Humanities-correlates with two massive shifts: the movement of all data into digital form; and the creation of new modes of collaboration.

<24> The general reckoning of a change is obviously not a novelty and, since the beginning of the 21st century, scholars in the area of documentation studies have observed how new social and professional practices related to the web and new treatments of information induce a Fredericton notion of data, document, information, knowledge and of their relationships, a deconstruction of working methods, and a mutation in the architecture or structuration of documentation as well as openings to users acting as self-made documentarians. [START_REF] Chartron | Introduction à la première conférence de Document numérique et Société[END_REF] <25> As stated by D. Cotte, [START_REF] Broudoux | Introduction à la première conférence de Document numérique et Société[END_REF] one of the main "change[s] in paradigm" linked to the current generalization of digital documentation relies upon a series of shifts: first, confronting users with techniques and issues in the management of documents that were previously handled by specialists in the field; second, integrating documents or information objects into a broader setting based on computational calculation; thus requiring the users (both scholars and end-users) to rigorously manage, explore and exploit an essentially invisible matter compared to previous handling processes that required a different rationality and sorting out of visibledocuments.

<26> Not only does this shift induce a reconsideration of "old definitions and distinctions separating publishers, authors, and archivists as well as, occasionally, the separation of producers of scholarly material from its consumers" [START_REF] Moody | Publishing the Archive[END_REF], but it also increases the argumentative value of the resulting archive: "Once archived materials are digitized, possibilities for orchestrating access to them and building thematic (and even argumentmaking) connections with other materials increases exponentially. In structuring the archive, writing its metadata, and designing its <28> The models that shape the hermeneutics of information are at stake in this paper co-written by a researcher in Informatics and one in the Humanities resorting to different methods of interpretation: the first focuses on the deep structure or architecture of archives; the other is deprived of computational expertise but interested in the potentials of the technology as well as in the openings made to newcomers for a reticulation of archival documents into new patterns or arrangements and the subsequent display of unforeseen knowledge and meanings.

<29> Such a standpoint (illustrating the current intersection of hard and human sciences induced by Digital Humanities) may lead to many questions, including: how to bridge the knowledge gap in computational techniques required to develop consistent digital projects in the Humanities; how to accurately translate research purposes or hypotheses into digital patterns; and conversely how to render computational findings graspable for further observations and research developments in the Humanities. Obviously, these questions will not be explicitly raised here, even less answered, but they implicitly suggest an evolution from the previous ancillary role of Informatics (as a technical yet fundamental support to some areas of research in the Humanities) to a co-understanding and coconstruction of digital projects that imply a greater computational literacy for users in the Humanities. This is especially true for the choice and use of metadata, whether structural or descriptive, to support the identification and categorization of data within documents in databases. While inserting metadata and descriptive elements requires a technical expertise, which scholars in the Humanities do not master and find strenuous, defining and listing the metadata requires a global and complementary conception and design both by the informatician, developer or expert in documentation science, and by the scholar in the Humanities who is the likely initiator and end-user of the database.

<30> Digitization of existing archives makes more accessible the information they contain, by the use of relevant algorithms, which allow users from all over the world to remotely request specific queries through the Internet, and allow them to navigate quickly between documents inside an archive. An instance of such digitization of existing archives is illustrated by the Venice Time Machine project. This international project aims to construct a large digital open access database from the archives stored at the State Archives in Venice (Archivio di Stato). Beyond the important efforts to digitize historical Venetian documents by using recent technologies, [START_REF] Albertin | X-ray spectrometry and imaging for ancient administrative handwritten documents[END_REF] one purpose of this project consists in assisting the transcription of digitized texts by analyzing and comparing handwritten sequences of characters in order to detect recurrent patterns. These patterns are then processed manually in order to assign them to words or word sequences. Named entities (proper names or location names for instance) are then detected among these words and a named entity linking [START_REF] Hachey | Evaluating entity linking with Wikipedia[END_REF] is processed, producing a semantic graph of the documents included in the archives. This semantic graph can be perceived as a global semantic structure of the archive, one that is useful to navigate inside the archive.

<31> Pure digital archives (in opposition to archives of digitized documents) are also processed similarly. Even if by nature a digitization process is not required on such data, algorithms applied in order to extract relevant information from these archives are very similar to the ones used to process digitized versions of old material resources. Moreover, the Venice Time Machine project can be compared to the European EUMSSI project, [START_REF] Grivolla | EUMSSI: a Platform for Multimodal Analysis and Recommendation using UIMA[END_REF] which aims to develop technologies to detect, extract, and aggregate data present in multimedia archives (video, image, audio, text, social content) composed of unstructured information. The EUMSSI project's ambition is to produce tools able to assist journalists in exploring multimedia archives and contents from social networks (tweets for instance). In addition to textual documents, this project focuses on image, audio and video documents that imply the use of pattern recognition algorithms and automatic speech recognition systems. However, all the collected clues are then stored in a database associated with a search platform.

<32> Through these two examples (the Venice Time Machine project and EUMSSI) one can see that traditional archives or more recent ones can now be processed and explored remotely inside large collections of data in real time, allowing the user access to an amount of information impossible to imagine only a few decades ago. Moreover, these algorithms and these semantic representations of the archives may also emphasize links between events, people or locations that were not easily detectable without such numeric tools.

Information is on fire

<33> How can digital archives both convey an accurate transmission of past circumstances and documents while at the same time reconstruct those into new sequences through tools of mapping or indexing meant for new modes of reading and new uses? Is this digital representation a minor or radical alteration of the form and function of archives? Can objects from the cultural heritage be reformatted without a change in purpose and informational value? Are computational methods and codes "light" tools for the adaptation and transfer of documents from the print culture era into a new display of old artifacts? Or does it drastically reshape information for the purpose of a new cultural logic, defined by technological rules and imposing a distinct set of norms and values [START_REF] Watterson | Beyond Digital Dwelling: Practice-Based Solutions for Interpretive Visualization in Archaeology[END_REF]?

<34> It seems important to better understand how automatic data mining works in order to propose answers to these questions. Data mining consists of the automatic extraction of knowledge from huge amounts of digital documents. Such knowledge can, for instance, be related to named entities recognition (as defined above), and often focuses on linking different documents stored in the same archive or stored from different ones. This allows us to extract a structure of the data contained in archives, depending on a similarity measure, as presented in Figure 4,in which each link represents a co-occurrence in the same document of person names represented as nodes.

<35> State-of-the-art methods for automatically clustering, linking and classification of textual documents commonly use a vector approach, also known as the vector space model. [START_REF] Salton | A vector space model for automatic indexing[END_REF] In this approach, a vector represents a document. For instance, if one assumes that 10,000 different non-function words are sufficient to analyze a set of textual data, each document will be represented with a 10,000-dimensional vector.

<36> Classically, each dimension of this vector corresponds to a word (or a predefined sequence of words), and its numeric value is computed from the frequencies of this word in the targeted document and in other documents of the archive. The classical method to compute such values is called Term Frequency-Inverse Document Frequency (TF-IDF). This calculation is simple, and is related to word frequencies.

<37> Formally, let us consider the number, N, of words, w, in a given vocabulary, V.

<38> In this framework, each dimension of a TF-IDF vector, v⃗, stands for a word: dimension i is for the i th word in the vocabulary V, denoted wi.

For each textual document, d, in the database, D (the database can be an archive), a TF-IDF vector, v⃗(d), can be computed. <42> Representing textual documents with vector allows us to handle documents through the vector algebra paradigm. So, in order to evaluate similarities between textual documents, similarity measures of vectors are computable. Different similarity measures between two vectors are possible, like Euclidian distance or cosine similarity. Euclidian distance can be expressed as:

while cosine similarity can be expressed as:

which is the ratio between the scalar product (also known as the dot product) of the two vectors and the product of their magnitude.

<43> Both similarities are illustrated in Figure 5, where d1 and d2 refer to two documents. This figure shows also that using a n-dimensional vector representation of a document means that this document is represented as a point in a n-dimensional space. Figure 5 is a trivial case where n=2.

Actually, clustering a large set of documents in regards of their textual content can be seen as clustering clouds of points; and several mathematical approaches are known to process this problem, [START_REF] Christopher D Manning | Introduction to information retrieval[END_REF]like the k-Nearest Neighbors algorithm [START_REF] Yang | A comparative study on feature selection in text categorization[END_REF] or the Hierarchical Cluster Analysis. [START_REF] Kaufman | Finding groups in data: an introduction to cluster analysis[END_REF] Figure 5: Euclidian distance and cosine similarity between two vectors representing two documents <44> These examples illustrate how projecting documents into a vector representation is useful to apply automatic analysis based on a mathematical approach. This is also a transformation of symbolic information (words and texts) into numeric information, and similar approaches can be applied to pictures, audio, or video. Since computation power and available data increase exponentially, data mining becomes more and more precise and semantic extraction more and more powerful. [START_REF] Quoc | Distributed representations of sentences and documents[END_REF] These tasks are part of artificial intelligence, [START_REF] Bengio | Scaling learning algorithms towards AI[END_REF] which evaluates extremely quickly nowadays. Currently, the deep learning paradigm, [START_REF] Bengio | Learning deep architectures for AI[END_REF] which exploits artificial multi-layers of neural networks, allows researchers to make important advances in these applicative fields, with the ambitious objective of creating an artificial intelligence comparable to human intelligence.

<45> Are we now closer or further away from each other than we were at the beginning of this paper? Are we collecting and working on archives with similar purposes and objectives? Probably so, as one seeks to imitate and reproduce human intelligence while the other seeks a comprehensive and evidence-based reconstruction of activities and artifacts. The various processes of Digital Humanities are also an understandable academic response to societal changes, especially in terms of information and knowledge dissemination. And because scholars in the Humanities have the responsibility to look at and produce deeper understandings of past and present, Digital Humanities is a rebranding of a long tradition of scholarship. It is as well a progression of qualitative and quantitative methods through computational methods applied to problems in the Humanities. New archives bear evidence of at least two principles and requests for digitally assisted research in the Humanities: building and making new things and new methods of meanings; and fostering encounters with new and old objects, texts, humans, and non-humans. New archives also engage readers to read outside the traditional boundaries of reading and create the circumstances for new modes of exploratory reading to take place. Thus, scholars in the Humanities seem committed to the powerful tradition of the book: while creating new forms of structured data and new modes for exhibiting archival materials, they still discuss the theoretical and operational changes that may occur "when we think of archives and collections as data aggregations in need of publishing," and state that "The overarching question is: how do new forms constitute something more than straightforward repositories, publications in wheelwrights?" [31] <46> Conversely, the example of documents projected in vector representation highlights a reversal, albeit a humanist commitment, to the "counter-mythology which has affirmed the demands of the world, against those of the book." [32] A counter-mythology which McKenzie sees at work in the novel The Name of the Rose, where Eco reconstructs "the ingeniously ordered, but labyrinthine, Alexandrian archive, only to deconstruct it again in the old and fearful symbolisms of the library as a furnace. Fire consumes the books." To that apparent, yet bookish counter-mythology, McKenzie opposes Marlowe's Faustus constructing his own versions of selected texts in which he seeks "only a single sense dictating a fixed fate" [33] and yet to his despair experiences variant reading. Pushing the metaphor only slightly further, yet not until it breaks, one could relate Eco's closed-book system library to the traditional model of archival interpretation; and Faustus's encounter with the "openness to interpretation, the paradox that texts are both closed and open, fixed and flexible, defined by one context only to be redefined in others" to the digital model of archival interpretation. At a distance from the fact that in both fictions, and in numerous others, books or entire libraries are being burned (and sometimes readers, too), we could say that through digital media, archives may be quasi-literally on fire (burned with flames) and consumed. At the same time, the mutual questioning at the background of this paper (as well as the whole Journal's theme) suggests that the figurative meanings of the expression seem to apply both to archives and to information itself, which is on fire, that is to say: attractive and doing very well!

Notes

[1] The Genesis of data curation and its co-evolution with digital curation is depicted. Carole Palmer et al., "Foundations of data curation: the pedagogy and practice of purposeful work with research data," Archives Journal Issue 3 [2013],http://www.archivejournal.net/.

[2] Neil Beagrie, "Digital Centre," Learned publishing 17, no.1 (2004): 7-9.

[3] C. Palmer, N. M. Weber, T. Munoz, A.H. Renear, Archive Remixed, Archive Journal, Issue 3, Summer 2013: " As the data accessible in digital environments continue to increase at a rapid pace, sound collection development and description will be essential to presenting data for exploration and discovery. In conjunction with searching capabilities, researchers will greatly benefit from the ability to browse dense and cohesive layers of data sources that not only anticipate researchers' known needs, but also allow them to effectively navigate through and interpret extensive bodies of openly accessible data." Ibid.

[4] Steven Marcus, "Humanities from classics to cultural studies: notes toward the history of an idea," Daedalus 135, no.2 (2006): 15-21.

[5] Ibid.

Figure 2 :

 2 Figure 2: A bulb: piece of industrial history or piece of contemporary art?

Figure 3 :

 3 Figure 3: Theaville: a literary and musical research website

Figure 4 :

 4 Figure 4: Automatic document linking and visualization


Quentin Deveuve

Kevin Lesage

Thomas Mouveaux

Mathieu Gissot
email: mathieu.gissot@pasteur-lille.fr

The Toxoplasma gondii inhibitor-2 regulates protein phosphatase 1 activity through multiple motifs

Keywords: Toxoplasma, phosphatase, PP1, inhibitor-2

come

The Toxoplasma gondii inhibitor-2 regulates protein phosphatase 1 activity through multiple motifs.

INTRODUCTION

Toxoplasma gondii is a unicellular eukaryote that infects around a third of the human population worldwide. This parasite belongs to the Apicomplexa phylum which contains other deadly parasite such as Plasmodium (the causative agent of malaria) and Cryptosporidium (responsible for cryptosporidiosis). T. gondii is of clinical importance especially in pregnant women. The first infection can potentially cause severe illness and death in the developing fetus. Moreover, the opportunistic nature of this parasite can lead to encephalitis in immunocompromised individuals and ocular diseases in immunocompetent or immunosuppressed patients [START_REF] Weiss | Toxoplasmosis: A history of clinical observations[END_REF]. T. gondii is acquired primarily through the ingestion of sporulated oocysts containing sporozoites, which are shed by the definitive host (felids) or by ingestion of cystscontaminated meats. In the gastrointestinal tract, the sporozoites or bradyzoites present in the cysts are released and can invade the intestinal epithelium where they differentiate in rapidly dividing tachyzoites. Those tachyzoites disseminate and proliferate during the acute stage of the infection before differentiating in bradyzoites, which encyst in several organs such as muscle tissue or in the central nervous system establishing a chronic infection [START_REF] Blader | Lytic Cycle of Toxoplasma gondii: 15 Years Later[END_REF]. Thus T. gondii has a complex life cycle which is characterized by multiple differentiation steps that are essential for its survival in both human and definitive feline host.

Several studies have demonstrated the importance of phosphorylations by protein kinases during the life cycle of T. gondii, from invasion [START_REF] Sugi | Use of the kinase inhibitor analog 1NM-PP1 reveals a role for Toxoplasma gondii CDPK1 in the invasion step[END_REF] to egress [START_REF] Moudy | The loss of cytoplasmic potassium upon host cell breakdown triggers egress of Toxoplasma gondii[END_REF]. In T. gondii tachyzoites, a few protein kinases have been shown to play a role in cell division during endodyogeny [START_REF] Suvorova | A novel bipartite centrosome coordinates the apicomplexan cell cycle[END_REF] [START_REF] Berry | The conserved apicomplexan Aurora kinase TgArk3 is involved in endodyogeny, duplication rate and parasite virulence[END_REF] [START_REF] Morlon-Guyot | The Toxoplasma gondii calcium-dependent protein kinase 7 is involved in early steps of parasite division and is crucial for parasite survival: Functional dissection of T. gondii CDPK7 protein[END_REF]. However, very little is known about protein phosphatases (PPs) and their regulators in the parasite. In other eukaryotes, the protein phosphatase type 1 (PP1), is considered as one of the major phosphatase involved in the control of many dephosphorylation steps. This serine/threonine phosphatase, which exhibits a high degree of conservation throughout evolution, is known to be involved in numerous essential cellular processes including protein synthesis, actin organization, transcription and cell cycle progression [START_REF] Ceulemans | Functional diversity of protein phosphatase-1, a cellular economizer and reset button[END_REF]. It is now known that PP1 localization, activity, and/or specificity are controlled by a large number of interacting-proteins [START_REF] Peti | Strategies to make protein serine/threonine (PP1, calcineurin) and tyrosine phosphatases (PTP1B) druggable: Achieving specificity by targeting substrate and regulatory protein interaction sites[END_REF]. Functional and structural studies revealed that interactions between PP1 and its regulators are mediated by specific binding sites that are conserved throughout evolution, including the so called RVxF and SILK motifs [START_REF] Wakula | Degeneracy and Function of the Ubiquitous RVXF Motif That Mediates Binding to Protein Phosphatase-1[END_REF] [START_REF] Huang | Characterization of the Inhibition of Protein Phosphatase-1 by DARPP-32 and Inhibitor-2[END_REF]. So far, about 200 regulators have been described in humans [START_REF] Hendrickx | Docking Motif-Guided Mapping of the Interactome of Protein Phosphatase-1[END_REF] while 100 proteins potentially interact with PP1 in Plasmodium [START_REF] Hollin | Analysis of the interactome of the Ser/Thr Protein Phosphatase type 1 in Plasmodium falciparum[END_REF].

In T. gondii, phosphatase inhibitors were used as a proxy to measure the biological role of phosphatases activity. In particular, the use of okadaic acid, an inhibitor of PP2A and PP1, lead to a decreased invasion of the treated tachyzoites suggesting an important role of PP1 in T.

gondii biology [START_REF] Delorme | A role for Toxoplasma gondii type 1 ser/thr protein phosphatase in host cell invasion[END_REF]. Moreover and similarly to Plasmodium falciparum, only one gene encoding TgPP1 has been identified [START_REF] Daher | A Toxoplasma gondii leucine-rich repeat protein binds phosphatase type 1 protein and negatively regulates its activity[END_REF]. In this study, the authors identified a protein (TgLRR1) homolog to the yeast sds22 protein, known to regulate the yeast PP1 activity [START_REF] Hong | Sds22p is a subunit of a stable isolatable form of protein phosphatase 1 (Glc7p) from Saccharomyces cerevisiae[END_REF]. TgLRR1 belongs to the leucine-rich repeat protein family and interacts with TgPP1 in vitro leading to the inhibition of its activity [START_REF] Daher | A Toxoplasma gondii leucine-rich repeat protein binds phosphatase type 1 protein and negatively regulates its activity[END_REF].

Other interactors are also conserved and well described in the literature. For example, a small protein called inhibitor-2 (I2) was shown to play an important role in cell cycle regulation of yeast and human [START_REF] Tung | Regulation of chromosome segregation by Glc8p, a structural homolog of mammalian inhibitor 2 that functions as both an activator and an inhibitor of yeast protein phosphatase 1[END_REF] [START_REF] Wang | Phosphatase inhibitor-2 balances protein phosphatase 1 and aurora B kinase for chromosome segregation and cytokinesis in human retinal epithelial cells[END_REF]. Likewise, an homolog of this protein was identified in P. falciparum [START_REF] Fréville | Plasmodium falciparum encodes a conserved active inhibitor-2 for Protein Phosphatase type 1: perspectives for novel anti-plasmodial therapy[END_REF]. This study suggested an essential role for PfI2 for normal growth of the parasite [START_REF] Fréville | Plasmodium falciparum encodes a conserved active inhibitor-2 for Protein Phosphatase type 1: perspectives for novel anti-plasmodial therapy[END_REF].

In the present study, we report the molecular and functional characterization of the T. gondii ortholog of PfI2, designated TgI2. We show that TgI2 encompasses conserved motifs involved in the interaction and modulation of TgPP1 activity.

MATERIALS AND METHODS

Molecular cloning of TgPP1 and TgI2

The full length coding region of TgPP1 and TgI2 were amplified by PCR using the primers P1-P2 and P3-P4 (Table S1). PCR products were then subcloned in a pJET1.2/blunt vector (Thermofisher) and verified by sequencing for the absence of any modification introduced by Taq polymerase. For the expression of TgPP1 and TgI2, pET28a (Novagen) and pGEX 6p3 (GE Healthcare) expression system were used respectively. Restrictions sites are mentioned in Table S1.

To obtain the TgI2 mutant constructs, we performed a PCR-based site-directed mutagenesis strategy using the pGEX 6p3-TgI2 as template, the primers P5-P6 (TgI2K6A) or P7-P8 (TgI2W37A) or P9-P10 (TgI2R121A/K122A) and Phusion polymerase (NEB). The PCR conditions consisted of 30s at 98°C followed by 16 cycles at 98°C (5s), 55°C (15s) and 72°C (7min). The parental DNA plasmid was then digested with DpnI. Mutated plasmids were verified by sequencing for the replacement of lysine 6, tryptophan 37, arginine 121 and lysine 122 by an alanine and then used for the expression of mutant TgI2 recombinant proteins.

Recombinant proteins expression

Protein expression was carried out in the Escherichia coli BL21 strain. For the expression of TgPP1, culture was induced overnight at 16°C in the presence of 0.5mM isopropyl β-D-1 thiogalactopyranoside (IPTG) and 1mM MnCl2. Cells were harvested in sonication buffer (20mM Tris, 150mM NaCl, 1% Triton X-100, lysozyme 1mg/ml, 1mM phenylmethylsulfonyl fluoride (PMSF). 6His-tagged TgPP1 proteins were purified according to manufacturer's instructions by Ni 2+ chelation chromatography (Macherey-Nagel). The extract was prepared using a buffer containing 20mM Tris HCl (pH 7.5), 150mM NaCl, 20mM Imidazole and 1mM

MnCl2 and loaded on a 1 ml nickel-NTA (Nitrilotriacetic acid) resin column (Macherey Nagel).

Elution steps were performed with a buffer containing 20mM Tris HCl (pH 7.5), 500mM NaCl, 400mM Imidazole and 1mM MnCl2. The imidazole-eluted proteins were dialyzed against the following buffer: 20mM Tris HCl (pH 7.5), 500mM NaCl and 1mM MnCl2. For the expression of the different forms of TgI2 (wild-type and mutated), cultures were induced at 37°C for 3h in the presence of 0.5mM IPTG. GST-tagged proteins were then purified according to manufacturer's instruction using glutathione-agarose beads (Macherey Nagel). Elution steps were performed with a 10mM glutathione, 50mM Tris HCl (pH 8) buffer. Under these conditions, the purity checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Instant Blue TM (Expedeon) was >80%.

Prior to phosphatase activity assays, GST-tagged proteins have been incubated in a buffer containing 50mM Tris HCl (pH 7), 150mM NaCl, 1mM EDTA and 1mM dithiothreitol (DTT) following the washing steps. After addition of 7 units of PreScission protease (GE Healthcare), the mixture is incubated for 6h at 4°C allowing the cleavage of the glutathione S-transferase (GST).

GST pull down assays

Purified TgI2-GST recombinant protein (wild-type or mutated) were bound to glutathioneagarose beads 2h at 4°C on a rotating wheel and washed with a binding buffer containing 20mM HEPES (, 20mM Tris HCl (pH 7.5), 500mM NaCl, 0.2mM EDTA, 1mM MnCl2, 0.1% Triton X-100, 1mM PMSF and 25µg of bovine serum albumin (BSA). Ten µg of TgPP1-6His recombinant protein were incubated with the TgI2-GST or GST bound to glutathione-agarose beads in binding buffer for 2h à 4°C on a rotating wheel. After 5 washes with binding buffer, proteins were eluted in SDS-PAGE buffer and loaded on a denaturing 15% polyacrylamide gel.

Separated proteins were then blotted to nitrocellulose. Blots were revealed with anti-His and anti-GST antibodies. Horseradish peroxidase labeled anti-mouse or anti-rabbit immunoglobulins (1:5000) were used as secondary antibodies followed by chemiluminescence detection (Pierce).

TgPP1 activity assay

The activity of recombinant TgPP1 with p-nitrophenyl phosphate (pNPP) as substrate was assayed as previously described [START_REF] Daher | A Toxoplasma gondii leucine-rich repeat protein binds phosphatase type 1 protein and negatively regulates its activity[END_REF]. Initial experiments were performed to determine the optimal conditions for TgPP1 activity. The activity was assessed in a reaction containing 50mM Tris-HCl (pH 7.5), 50mM HEPES, 100mM NaCl, 1mM DTT, 1mM MnCl2 and 0.1mM Brij35. The reaction was started by adding 5mM of pNPP. After incubation 1h at 37°C, phosphatase activity was determined spectrophometrically at 405nm.

To investigate the role of TgI2 recombinant proteins on TgPP1-6His activity, various concentrations of proteins were added to TgPP1 recombinant protein and preincubated 30min at 37°C before testing the TgPP1 activity. GST was used as control.

Parasite tissue culture and manipulation T. gondii strain RHΔKu80 tachyzoites were propagated in vitro in human foreskin fibroblasts (HFF) using Dulbeccos's modified Eagles medium supplemented with 10% fetal calf serum (FCS), 2mM glutamine, and 1% penicillin-streptomycin (100Units/µL of penicillin and 100µg/µL of streptomycin). T. gondii tachyzoites were grown in ventilated tissue culture flasks at 37°C and 5% CO2. Transgenes were introduced by electroporation into tachyzoites of T. gondii strains and stable transformants were selected by culture in the presence of 2µM pyrimethamine. Clonal lines were obtained by limiting dilution.

Antibodies

The anti-TgENO2 rabbit, anti-TgAlba1 mouse and anti-TgMIC1 mouse antibodies were used at 1:1000 dilution. The anti-HA rabbit antibody (Cell signaling) was used at 1:500 in ImmunoFluorescence assay (IFA) and in Western blots.

Immunofluorescence Assay and confocal imaging

Intracellular tachyzoites were fixed with 4% paraformaldehyde in PBS for 15min, followed by two PBS washes. Parasites were permeabilized with 0.1% Triton X-100 in PBS containing 0.1% glycine for 10min at room temperature. Samples were blocked with 3% FCS in the same buffer and the primary antibodies were added on parasites in the same buffer for 1h at room temperature. Secondary antibody coupled to Alexa-488 or to Alexa-594 diluted at 1:1000 were added together with DAPI (4',6-diamino-2-phenylindole) for nucleus staining. Confocal imaging was performed with an LSM880 microscope (Zeiss) and a Plan Apochromat objective (Plan-Apochromat 63x:1.40 Oil DIC M27, Zeiss).

Statistical analysis

The Student t-test was performed to assess the statistical significance of the results.

RESULTS

Toxoplasma gondii I2 protein encompasses conserved motifs.

In order to expand our knowledge of TgPP1 regulators repertoire in T. gondii, we searched the ToxoDB database [START_REF] Gajria | ToxoDB: an integrated Toxoplasma gondii database resource[END_REF] for a homolog of the Inhibitor-2 (I2) protein, a well know regulator of PP1 in numerous organisms. We performed a Basic Local Alignment Search Tool (BLAST) analysis [START_REF] Altschul | Basic local alignment search tool[END_REF] using the PfI2 protein sequence and found a protein whose sequence showed 31 % identity on the full length sequence. We aligned the TgI2 sequence with those of the human and P. falciparum homologs (Fig. 1A) using the MultAlign software [START_REF] Corpet | Multiple sequence alignment with hierarchical clustering[END_REF]. Using the alignment shown in Fig. 1A and the location of known PP1 interacting motifs present on the human and PfI2 proteins we identified three putative PP1 binding motifs in the TgI2 sequence (Fig. 1, boxed). In PfI2, only two motifs were identified and shown to have an impact on its binding to PfPP1 [START_REF] Fréville | Plasmodium falciparum encodes a conserved active inhibitor-2 for Protein Phosphatase type 1: perspectives for novel anti-plasmodial therapy[END_REF], while three functional motifs are found in the human I2 protein [START_REF] Bollen | The extended PP1 toolkit: designed to create specificity[END_REF]. By sequence homology and according to the alignment shown in Fig. 1A, we found in the TgI2 sequence a first motif, 3 VIGK 6 , potentially corresponding to a variation of the sequence of the SILK motif consensus ([SG]ILK). The second motif, 33 KHLTW 37 , matched the consensus sequence for the RVxF motif ([RK]x [VI]x [FW]) with the exception of the valine or isoleucine position. In the human I2, this particular residue is also changed (Fig. 1A) with no impact on the binding properties of this RVxF motif variant [START_REF] Huang | Characterization of the Inhibition of Protein Phosphatase-1 by DARPP-32 and Inhibitor-2[END_REF]. Finally, we found a third motif ( 117 FELKRKQHY 125 ) that matches the consensus sequence for the FKK/HYNE motif (FxxR/KxR/KxHY).

To better understand the distribution of these PP1 binding motifs among eukaryotic homologs of TgI2, we gathered the sequence of these proteins in apicomplexan parasites, kinetoplastids, plants and few Opisthokonts and identified their putative PP1 binding motifs (Fig. 1B). Surprisingly, TgI2 is the only apicomplexan I2 protein to encompass a SILK-like motif, which is found in most Opisthokonts (Human, mouse or Drosophila) but not in plants (Arabidopsis) or Kinetoplastids (Trypanosoma and Leishmania) homologs of the I2 protein. (Fig. 1B).

Toxoplasma gondii I2 protein interacts with TgPP1 through the RVxF motif.

To test the ability of the TgI2 protein to bind TgPP1, we produced both recombinant proteins with a GST and 6His-tag respectively (Fig. S1A andB). We also produced mutated forms of the TgI2 protein for which each putative motif was mutated (Fig. S1C). To investigate the respective role of each motif, we produced a combination of each mutation, yielding to a total of 7 mutants for the TgI2 protein. We also checked the purity of each TgI2 mutants and proceeded to a GST-pull down assay (Fig. S1D). In a binding assay, we incubated TgPP1-6His with wild-type or each TgI2-GST mutant coupled to Glutathione-Agarose beads and washed them extensively. We then performed a Western-blot on the eluates of each binding-assay using an anti-6His antibody to identify the TgPP1 protein. We used a Western-blot with an anti-GST antibody to verify the quantity of each TgI2 protein that was originally on the beads. Using this protocol, we showed that the wild-type TgI2 protein is able to bind to TgPP1 (Fig. 2A -lane 3) while the GST alone did not yield detectable TgPP1 signal (Fig. 2A -lane 2). We also showed that the mutation of SILK-like or HYNE motifs of TgI2 protein (Fig. 2A -lanes 4 and 6, respectively) had little change in its TgPP1 binding capacity. In contrast, the mutation of the RVxF motif exhibited a drastic effect, with the TgPP1 signal level close to the background (Fig. 2A -lane 5). The combinatorial mutation of both HYNE and SILK-like resulted in similar results than those obtained with the WT TgI2 (Fig. 2A -lane 8) suggesting that the RVxF motif is the major motif for TgPP1 binding.

While the interaction with TgI2 encompassing any mutation combined with the mutated RVxF resulted in small but close to the background levels of TgPP1 (Fig. 2A -lane 7 and 9), the interaction with the TgI2 protein mutated for the three motifs produced no detectable TgPP1 (Fig. 2A -lane 10). These results suggest that both SILK-like and HYNE motif may contribute to the TgPP1 binding and are therefore secondary binding motifs.

We repeated this experiment three times and measured the intensity of the TgPP1 band for each interaction. We also measured the quantity of TgI2 for each lane in order to normalize for the quantity of TgI2 protein that was initially on the beads. We therefore calculated a ratio of the intensity of the TgPP1 band over the intensity of the GST-tagged protein that reflects the ability of each GST-tagged protein to bind to TgPP1 (Fig. 2B). These ratio calculated for the mutant lacking the SILK-like and/or HYNE motif indicate that they bind to TgPP1 in a similar manner as the WT TgI2. In contrast, the mutants lacking the RVxF motif show a ten-fold reduction of their TgPP1-binding capacity while the GST and the triple mutant lack the ability to bind to TgPP1 (Fig. 2B). Therefore, these results indicate that the RVxF motif is a major contributor to the TgPP1 binding ability while the HYNE and SILK-like motifs only show a residual contribution.

TgPP1 is an active phosphatase inhibited by okadaic acid.

To verify if the TgPP1-6His protein we produced was active in our experimental conditions, we performed a colorimetric phosphatase assay using a small phosphorylated peptide. Using the same quantity as starting material, we compared the TgPP1 activity to that of a commercial rabbit PP1 (Fig. 3A). These data showed a robust phosphatase activity increased with concentration of the PP1 protein. TgPP1 activity is undistinguishable from that of the rabbit PP1. When TgPP1 incubation buffer lacked MnCl2, a cation that is indispensable for the PP1 phosphatase activity, we found that the activity measured was independent from the concentration of TgPP1 added, indicating that TgPP1 activity is dependent on the presence of MnCl2.

To ensure that the phosphatase activity measured in the previous assay was due to a PP1-like phosphatase, we incubated TgPP1 and PfPP1, as a control, in increasing concentration of okadaic acid and measured their phosphatase activity (Fig. 3B) [14]. We found that both TgPP1 and PfPP1 phosphatase activity were inhibited by okadaic acid. We measured an 80 % inhibition of their activity without inhibitor at the highest concentration of okadaic acid.

Therefore, the TgPP1 produced in our experimental condition shows a measurable phosphatase activity.

TgI2 acts as an inhibitor of TgPP1 phosphatase activity through its RVxF and SILK motifs.

To test the regulatory function of TgI2 on TgPP1, we performed a phosphatase activity assay in presence of increasing concentration of wild-type TgI2. While the same concentrations of GST alone were not able to induce any significant change in TgPP1 (Fig. S2), increasing concentration of wild-type TgI2 led to a 50 % inhibition of TgPP1 activity (Fig. 4A).

To test the effect of each PP1 binding motif on the inhibitory activity of TgI2, we incubated TgPP1 with increasing concentration of mutated TgI2. First, we tested TgI2 mutated for the 3 binding motifs and found that this fully mutated version of TgI2 is unable to significantly alter the TgPP1 activity (Fig. 4B).

In good concordance with the binding-assay, the TgI2 protein mutated for the SILK-like (Fig. 4C) or the HYNE (Fig. 4D) motif alone, kept their ability to inhibit TgPP1 activity.

Surprisingly, the single mutation of the RVxF motif was not sufficient to abolish the inhibitory effect of TgI2 on TgPP1 activity (Fig. 4E) although the inhibition level was decreased compare to the wild-type TgI2, suggesting that the SILK-like and/or the HYNE motif are able to convey the inhibitory function of TgI2. To identify whether both SILK-like and HYNE motifs are necessary for the TgI2 inhibition of TgPP1 activity, we used the TgI2 mutants where two out of three motifs are mutated. The TgI2 protein encompassing only the unmutated HYNE motif was unable to inhibit TgPP1 activity, suggesting that this motif alone is not sufficient for the inhibitory function of TgI2 (Fig. 4F). These results indicate that the SILK-like motif alone may be able to mediate the inhibition of the TgPP1 activity. This was confirmed when using a TgI2 mutant for both HYNE and RVxF motifs, which contained the SILK-like motif alone (Fig. 4G).

Indeed, this mutant TgI2 protein was able to inhibit the TgPP1 activity (Fig. 4G), indicating its functional role in the TgPP1 activity inhibition.

Finally, we verified whether the TgI2 protein mutated only in the RVxF motif was able to inhibit the TgPP1 activity. In this assay, the TgI2 protein mutated for both HYNE and SILKlike motif was able to significantly inhibit the TgPP1 activity (Fig. 4H), as suggested by the binding-assay.

TgI2 is located both in the nucleus and the cytoplasm of tachyzoites.

To gain more information about the function of TgI2 in the biology of the parasite, we produced an epitope-tagged TgI2-HA strain. We used this strain in IFA and colocalized TgI2-HA with T. gondii cellular markers. First, we used a nuclear marker (TgENO2) [START_REF] Ferguson | Evidence for nuclear localisation of two stage-specific isoenzymes of enolase in Toxoplasma gondii correlates with active parasite replication[END_REF]and performed an IFA on the TgI2-HA strain. The Fig. 5A shows the colocalization of these proteins and demonstrate that the signal from the HA-tag is localized in the nucleus as well as the cytoplasm. This was further confirmed by the quantitative fluorescence intensity plot that shows that the HA signal is distributed in the nucleus and cytoplasm compartments (Fig. 5B). To confirm this hypothesis, we used a cytoplasmic marker (TgAlba1) [START_REF] Gissot | Toxoplasma gondii Alba proteins are involved in translational control of gene expression[END_REF] in IFA together with an anti-HA antibody. In Fig. 5C, we show a strong codistribution of the signal given by the HA-tag and the cytoplasmic marker. This was further confirmed by the quantitative fluorescence intensity plot showing that most of the HA signal is localized in the cytoplasm but also in the nucleoplasm (Fig. 5D). To further describe TgI2 localization, we performed a costaining using a microneme marker (TgMIC1). Micronemes are secretory organelles that reside at the apical end of the parasite. We showed that TgI2-HA does not codistribute with micronemes (Fig. S3).

DISCUSSION

T. gondii genome encodes a gene homolog to the human inhibitor-2 gene. The resulting protein sequence, TgI2, presents motifs that have been found in other I2 proteins to be important for both interaction and regulation of the protein phosphatase 1 catalytic subunit. Surprisingly, while most apicomplexan parasites only present two potential interaction motifs (Fig. 1B, RVxF and HYNE), TgI2 protein sequence may encompass three potential motifs (SILK-like, RVxF and HYNE). The presence of the SILK-like motif is supported by the alignment of the TgI2 and HsI2 protein (Fig. 1A). However the sequence of the SILK-like motif in TgI2 does not correspond to the motif consensus sequence [SG]ILK. The importance of the TgI2 SILKlike motif is revealed by the ability of the TgI2 protein mutated for the two other motifs (RVxF and HYNE) to inhibit the TgPP1 activity (Fig. 4G).The distribution of the motif across species may argue for a later acquisition of the SILK domain in Opisthokonts. The presence of the SILK-like domain in the distantly related eukaryote T. gondii but not in other Apicomplexan parasites may argue for an independent acquisition and selection of this domain in T. gondii.

Other proteins in T. gondii display such a structure/function convergence to their mammalian orthologs. For example toxofilin, a protein able to bing G-actin, was shown to exhibit two sequences similar to sequences present in several G-and F-actin-binding proteins, while the third appears to be specific to toxofilin [START_REF] Jan | The toxofilin-actin-PP2C complex of Toxoplasma: identification of interacting domains[END_REF]. Although the SILK-like domain of TgI2 plays a minor role in the interaction with TgPP1, it has an important contribution in the inhibitory activity of this protein (Fig. 4G). The SILK domain was shown to display similar feature when mutated in the HsI2 protein, with a 500 fold decrease in activity after the Lysine mutation [START_REF] Park | Domains of phosphatase inhibitor-2 involved in the control of the ATP-Mg-dependent protein phosphatase[END_REF][START_REF] Huang | Characterization of the Inhibition of Protein Phosphatase-1 by DARPP-32 and Inhibitor-2[END_REF]. Similar to what was observed for the HsI2, the SILK-like domain has a little role in the interaction with PP1c but is crucial for its activity [START_REF] Huang | Characterization of the Inhibition of Protein Phosphatase-1 by DARPP-32 and Inhibitor-2[END_REF]. This apparent discrepancy between binding and inhibition ability through the SILK domain of the I2 proteins may be due to the experimental condition used to assess TgPP1 activity where binding affinity is not the limiting factor for TgPP1 activity inhibition. The interactions at the SILK domain involve residues 12-17 (KGILK) of the HsI2 protein that bind primarily to a surface created by amino acids 50-59 in the PP1c subunit [START_REF] Hurley | Structural Basis for Regulation of Protein Phosphatase 1 by Inhibitor-2[END_REF]. Structure-function studies have found that the two amino-acids (I 14 and L 15 ) of the SILK domain are critical for the binding of surface formed by the PP1c E 54 and E 56 [START_REF] Hurley | Structural Basis for Regulation of Protein Phosphatase 1 by Inhibitor-2[END_REF]. Mutagenesis of either the I2 I 14 and L 15 or the PP1c E 54 and E 56 decreased dramatically the PP1c inhibition [START_REF] Huang | Characterization of the Inhibition of Protein Phosphatase-1 by DARPP-32 and Inhibitor-2[END_REF][START_REF] Connor | Cellular Mechanisms Regulating Protein Phosphatase-1: a key functional interaction between inhibitor-2 and type 1 protein phosphatase[END_REF]. In the TgI2 protein, the Isoleucine of the SILK domain is conserved while the Leucine is changed to a Glycine. This exchange seems to be neutral for the capacity of the TgI2 SILK-like domain to inhibit the TgPP1 activity, suggesting that there is a flexibility in this amino acid position for the activity of the I2 protein. However, our mutagenesis experiments suggest a crucial involvement of the conserved Lysine (K 6 ) in the T. gondii 3 VIGK 6 (SILK) motif, since its mutation is sufficient to abolish the SILK-mediated inhibitory activity of TgI2 on TgPP1 (Fig. 4). It has been suggested that the Lysine in the GILK motif (K 16 ) of the HsI2 protein is responsible for the proper orientation of the protein when interacting with PP1c therefore enabling the interactions contributed by the I 14 and L 15 [START_REF] Hurley | Structural Basis for Regulation of Protein Phosphatase 1 by Inhibitor-2[END_REF]. Our study confirms the importance of the terminal Lysine of the SILK-like motif for the activity of TgI2.

The RVxF motif is shared by more than 70 % of the protein binding to PP1c [START_REF] Hendrickx | Docking Motif-Guided Mapping of the Interactome of Protein Phosphatase-1[END_REF]. However, its consensus sequence [HKR]

[ACHKMNQRSTV][V][CHKNQRST][FW]
defined by Meiselbach et al [START_REF] Meiselbach | Structural Analysis of the Protein Phosphatase 1 Docking Motif: Molecular Description of Binding Specificities Identifies Interacting Proteins[END_REF].would not include the KSQKW sequence, a well-established RVxF variant, present in the HsI2 sequence [START_REF] Hurley | Structural Basis for Regulation of Protein Phosphatase 1 by Inhibitor-2[END_REF]. This sequence was nonetheless shown to be crucial for binding and activity of the HsI2 [START_REF] Huang | Characterization of the Inhibition of Protein Phosphatase-1 by DARPP-32 and Inhibitor-2[END_REF]. The most remarkable change is the replacement of the Valine by a Glutamine which was shown to bind to the PP1 [START_REF] Hurley | Structural Basis for Regulation of Protein Phosphatase 1 by Inhibitor-2[END_REF] TgI2 33 KHLTW 37 does match the consensus sequence described earlier with the exception of the Valine that is changed to a Leucine. As for the HsI2 or PfI2 (where the Valine is replaced by an Isoleucine) and based on our mutagenesis and binding assay, this change is not sufficient to prevent the binding of TgI2 to TgPP1.

Therefore, the consensus sequence for the RVxF motif should include other amino-acids at the third position as suggested earlier [START_REF] Hurley | Structural Basis for Regulation of Protein Phosphatase 1 by Inhibitor-2[END_REF]. The RVxF motif of HsI2 was found to bind at least the D 166 in HsPP1c within the RVxF binding groove [START_REF] Hurley | Structural Basis for Regulation of Protein Phosphatase 1 by Inhibitor-2[END_REF].

Therefore, the SILK and RVxF motif are predicted to bind different region of the HsPP1c protein. This would explain the complementarity of both SILK and RVxF motifs for the inhibitory activity of TgI2. Since the RVxF groove on HsPP1c is the site of binding of multiple proteins [START_REF] Egloff | Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1[END_REF], the binding affinity may need to be greater than other site of the I2 protein (e. g. the SILK motif) to accommodate the competition.

The HYNE motif of the HsI2 was found to directly interact with the HsPP1c catalytic domain [START_REF] Hurley | Structural Basis for Regulation of Protein Phosphatase 1 by Inhibitor-2[END_REF], therefore we anticipated that TgI2 HYNE mutants will show a defect in their inhibitory activity. Moreover, this region of the sequence is highly conserved between HsI2, PfI2 and TgI2. In the assays presented here, the TgI2 protein mutated for the HYNE motif behaved as the wild-type TgI2 protein. This suggests that the HYNE motif has only a minor contribution in the activity of TgI2. Mutagenesis of the HYNE motif on the PfI2 protein resulted in a partial loss of the inhibitory activity of this protein [START_REF] Fréville | Plasmodium falciparum encodes a conserved active inhibitor-2 for Protein Phosphatase type 1: perspectives for novel anti-plasmodial therapy[END_REF], suggesting a smaller contribution than the PfI2 RVxF motif. However, site directed mutagenesis and deletion of the HYNE motif of the HsI2 protein showed that it was not involved in inhibition of HsPP1c Activity is represented as a % of the phosphatase activity measured without inhibitor.

Experiment was repeated three times with duplicate measurement.

  Fig. Legends

Fig. 1 A

 1 Fig.1

Fig. 2 :

 2 Fig. 2: TgPP1 binding assay using different mutant TgI2 proteins.

Fig. 3 :

 3 Fig. 3: TgPP1 is an active phosphatase responsive to okadaic acid inhibition.

Fig. 4 :

 4 Fig. 4: TgPP1 activity is inhibited by TgI2 through different motifs.

Fig. 5 :

 5 Fig. 5: TgI2 is localized at the cytoplasmic and nuclear compartments.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Jamal Khalife for critically reading the manuscript. The authors also thank Dr. Géraldine Tellier for her help setting up the TgPP1 protein purification and the BioImaging Center Lille for access to instruments. This work was supported by Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM) and a grant from the French National Research Agency (ANR) [grant number ANR-13-JSV3-0006-01 to MG]. The funding bodies had no involvement in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

 [START_REF] Huang | Characterization of the Inhibition of Protein Phosphatase-1 by DARPP-32 and Inhibitor-2[END_REF]. Therefore, depending on the number of PP1 binding site on the protein, the HYNE motif may not be relevant for the activity of I2 homologs.

TgI2 was found to localize at both the nuclear and cytoplasmic compartments as shown for the PfI2 protein [START_REF] Fréville | Plasmodium falciparum encodes a conserved active inhibitor-2 for Protein Phosphatase type 1: perspectives for novel anti-plasmodial therapy[END_REF]. TgPP1 localization is similarly distributed in both compartments [START_REF] Daher | A Toxoplasma gondii leucine-rich repeat protein binds phosphatase type 1 protein and negatively regulates its activity[END_REF] which suggests that they may interact in both. We found that the PXTP motif is conserved in the TgI2 protein ( 58 PPTP 61 ). This motif is known to be phosphorylated during mitosis therefore increasing HsI2 localization to the centrosome [START_REF] Leach | Phosphorylation of Phosphatase Inhibitor-2 at Centrosomes during Mitosis[END_REF]. Phosphopeptides were mapped for the T. gondii proteome but no phosphopeptides were recovered for this motif. Instead, two phosphorylated residues were uncovered for TgI2 (S67 and S77) in a region that is poorly conserved. Although, we cannot exclude that phosphorylation of the PXTP motif occurs in T. gondii, there is no evidence for its participation in TgI2 localization.

We show here that the TgI2 protein is a potent inhibitor of TgPP1 phosphatase activity. TgI2 SILK-like and RVxF motifs are critical for regulating the activity of TgPP1, a feature that is common with the higher eukaryotes inhibitor-2 protein.