The polysemiotic nature of subtitled films, whereby textual information (the subtitles) is combined with other audiovisual cues (the pictures and the soundtrack of the film) makes them very vulnerable as a form of translation, as well as a peculiar one: a consequence of the process of subtitling is that both the original (or source text) and the translation (target text) are presented simultaneously to viewers. The possibility of clashes between source and target texts is therefore very great, and incoherencies produced by the juxtaposition of visual referents from the source-language cultural sphere alongside textual referents originating from the target language are often commented upon by translation specialists. Whilst it is widely admitted that the use of cultural substitution – that is when a cultural reference in the source-text is replaced by another one in the target language – in subtitles should be resorted to with extreme caution, a coherent and consistent approach is also absolutely paramount in order to maintain viewers’ suspension of disbelief throughout the film. Looking at a corpus of films from the 1990s portraying predominantly African American characters, this paper analyses the ways in which the dialogues have been subtitled into French. While in most films subtitles display a tendency to neutralise the non-standard features of the original, some also reveal a great level of inventiveness and creativity: the juxtaposition in the subtitled films of features that are culturally-bound (whether to the source or to the target culture) produces a hybrid. Whilst the use of non-standard features from the target language in the subtitles can be justified by connotations they have in common with African American Vernacular English, the use of such features is not unproblematic and raises important issues.
©inTRAlinea & Pierre-Alexis Mevel (2014).
"On the Use of verlan to subtitle African American Vernacular English into French: transnational hybridity"
As the notions of domestication and foreignisation have become one of the dominant shibboleths of an increasing number of translation specialists, it is perhaps surprising that audiovisual translation, and specifically subtitled films, has seldom been discussed in the light of these two concepts brought to the fore of Translation Studies by Lawrence Venuti (1995). Subtitled films are semiotically very rich objects, and viewers are permanently reminded of their foreignness, both visually and auditorily: visually because of the text they have to read at the bottom of the screen when they are watching a subtitled film, and auditorily because of the foreign dialogue. The polysemiotic nature of subtitled films, whereby textual information (the subtitles) combines with other audiovisual cues (the film’s images and soundtrack) makes them a very vulnerable form of translation, as well as a peculiar one, to say the least. A peculiarity of subtitling is that both the original (or source text, henceforth ST) and the translation (or target text, henceforth TT) are presented simultaneously to viewers. The possibility of clashes between source and target texts is therefore very great, as is often commented upon by translation specialists who point out the incoherence resulting from the juxtaposition of visual referents from the source language (henceforth SL) cultural sphere with textual referents from that of the target language (henceforth TL). For instance, the use of features of African American Vernacular English (henceforth AAVE) to translate banlieue French in Mathieu Kassovitz’s La Haine (1995) and the systematic transposition of cultural references from the source culture to the target culture (which saw ‘Kronenbourg’ beer become ‘Bud’, ‘cinq francs’ become ‘two bits’, and characters’ names ‘Astérix’ and ‘Darty’ translated respectively as ‘Snoopy’ and ‘Walmart’) were commented on by a number of film critics who attributed the commercial failure of the film in the United States in part to poor subtitling: ‘a sloppy pastiche of black American slang hinders rather than helps an understanding […] of the film’ (an anonymous film critic, cited in Jäckel 2001: 227). The juxtaposition of a depiction of the daily lives of a trio of black-blanc-beur youths with American cultural references is indeed odd. In other words, because of the use of features of AAVE in the subtitles and of the systematic transposition of cultural references for the target (American) audience, American viewers were presumably led to believe that African American youths shared important similarities with youths from the French banlieues, only to be brought back to reality when particular themes in the films made it all the more conspicuous that the action and setting were indeed foreign. There are specific elements of the narrative that make American viewers aware of the foreign nature of the film: as a critic points out, ‘the media hysteria about a stray revolver in a housing project might appear touching to a society in which prepubescents tote Uzis, but not to American youths for whom the young men’s inability to drive would be “quaint”’ (Jäckel 2001: 233). Besides making an argument in favour of an extremely cautious use of cultural substitution in subtitles – that is when a cultural reference in the ST is replaced by another one in the TL – this also suggests that adopting an approach that takes into account the links between the source and target cultures is absolutely paramount in maintaining suspension of disbelief. Therefore, the context of culture-specific elements and the relationship between the source and target cultures are of ultimate importance when choosing a translation strategy.
We are now going to examine the challenges of subtitling from a theoretical perspective in order to crystallise the dynamics that govern the subtitling into French of non-standard features or varieties, such as AAVE.
Venuti’s original idea of foreignisation involves the use of the remainder which is defined as ‘the collective force of linguistic forms that outstrips any individual’s control and complicates intended meanings’ (Venuti 1998: 108). For Venuti, the remainder is not necessarily motivated by elements of the ST: the foreignness invoked by the remainder is not qualified, and although the remainder points towards the foreignness of that translation, it does not assert anything specific about the nature of this foreignness. This particular point can be addressed in the context of subtitled films: translators certainly have an impact on the content of subtitles, and their use of the remainder can express foreignness, precisely because this foreignness can in turn be supported by the other channels of the film (in other words, the other channels of a film can be used to orient the understanding of viewers in the TL towards a foreignness that is more specific), or it can clash with the subtitles. Venuti’s paradigm would seem to be relevant to the examination of the content of subtitles. There are, however, two important provisos: first, translators have to be cautious in their use of the remainder, for if they use archaisms or forms that have been under-used or that are not codified in writing, they might affect the ability of viewers to read the subtitles with sufficient speed. Secondly, and perhaps obviously, the use of words in the TL that have strong connections to a particular region or to a particular group of people might of course be problematic and trigger cognitive-narrative dissonances. In other words, the remainder used in subtitles should be compatible to some extent, with the images of the film, and this compatibility should pre-date its use. This is in fact an issue that is not specific to subtitled films, but to the foreignising approach in general. The main issue here is that the use of the remainder (archaisms or dialectal features) can be very problematic, precisely because dialectal forms of the TL are often bound to the target culture, and as such can clash with visual elements of a film that are bound to the source culture.
One of the main concerns with subtitled films stems precisely from the fact that foreignness is built from domestic material. Non-dominant discourses are used to create the impression of the foreign, and the foreign is thus built using local blocks of meaning, blocks that might consist in deliberate divergence from the standard but are still, ultimately, part of the target culture. When these blocks from the target culture are superimposed onto images in the form of subtitles, there is always the possibility that the two – images and subtitles – may clash, generating a failed bi-cultural object that culminates in a break of viewers’ suspension of disbelief. In the case of the French film La Haine, it is certainly odd for the American audience (those that frequent art houses and consume foreign films, and therefore presumably experience the foreign) to be faced with three protagonists who wander between their housing estate and Paris where hotdogs cost ‘two bits’, and who speak (in the subtitles) using certain forms of AAVE. The remainder, here (features of AAVE), does not fulfill its foreignising purpose, but rather the opposite. It literally dis-locates the film, it changes its locus – partially anyway – and renders it a cultural aberration that does not belong anywhere. In this particular context, the use of the remainder was also accompanied by a systematic transposition of cultural references to the target culture, as observed in the introduction of this paper. This dislocation of the film meant that the dissonance between the subtitles and the images was too great, it was too deliberate an attempt to make the unfamiliar become familiar, in the face of many indications of foreignness (images, soundtrack, subtitles).
Judging from this film, it would seem that the use of the remainder in film subtitles is doomed to failure, necessarily creating a schizophrenic object that inhabits two spaces at once. In what precise circumstances, then, is it possible to use the remainder in subtitles, without leading to two cultures clashing on screen? Cultures, far from being discrete entities, sometimes share a fair amount of overlap, can take inspiration from one another, and fuse. This contribution seeks to argue that in the event of such an overlap, it is possible to hint at the foreignness of the original by using a remainder that shares, to a more or less direct extent, associations with the culture of the ST. By playing on the overlap between two cultures, it is possible, through the use of linguistic devices from the TL, to evoke and even invoke, to awaken and bring to the fore meanings of the ST that might have been thought ‘lost’ in translation, precisely by taking advantage of the polysemiotic nature of films. We will see below that although the translators’ cultural transposition of La Haine proved unsuccessful (commercially and critically at least), such strategies are not necessarily condemned to failure. Whilst the use of African American slang to subtitle La Haine was generally deemed unsuccessful, I will argue that verlan can serve a valid purpose when used to translate AAVE.
A particularly noticeable feature used in the subtitles of some of the films portraying speakers of AAVE is verlan, which is often referred to by French linguists as a feature of banlieue French. The term banlieue French is used here to refer to the variety of French spoken mostly by teenagers and that developed initially in poor suburban areas of Paris before spreading to other urban areas. This variety of French has been described and discussed by several French sociolinguists, who have generally emphasised the link between its spatial and social dimensions (Armstrong & Jamin 2002; Gadet 1998; Jamin et al 2006; Lepoutre 1997; Liogier 2002; Trimaille 2004; Trimaille & Billiez 2006). Sociolinguists have given different names to this variety, each emphasising a particular characteristic: it has been referred to as ‘parler véhiculaire interethnique’ (Offord 1996: 109), ‘langage des jeunes des cités de banlieue’ (Lepoutre 1997: 153), ‘argot contemporain des cités’, ‘ parlers jeunes urbains’, ‘sociolecte urbain générationnel’, ‘français contemporain des cités’ (all found in Trimaille & Billiez 2007), ‘langage des jeunes’ or ‘français des cités’ (both in Liogier 2002). This diversity echoes the various labels used to designate AAVE, and accounts for a certain heterogeneity of practices as well. Variables such as age, ethnic origins and social networks have all been shown to be important criteria in the definition of banlieue French. Although there is an abundant literature on the subject, the terminological fuzziness also illustrates that banlieue French, as a variety, is particularly difficult to circumscribe. Gadet, for example, suggests that this variety is difficult to link primordially to a social environment (‘populaire’), to age (‘jeunes’) or to geographical areas (‘cités’ or ‘banlieues’): ‘Y a-t-il lieu d’opposer au français populaire traditionnel une “langue des jeunes”, objet difficile à nommer (langue des cités, des banlieues)?’ (Gadet 2003: 85) However, David Lepoutre, an ethnographer and sociolinguist, describes what he refers to as ‘le langage des jeunes des cités de banlieue’ (Lepoutre 1997: 153) in his book Cœur de banlieue, and explains that some of its features are particularly distinctive.
Verlan, according to Lepoutre (1997: 155-6), has a cryptic function: ‘Le verlan, comme tous les argots, est également un langage de fermeture, une langue du secret. Cette fonction cryptique du langage des rues s’exerce dans le cadre de l’école et plus largement dans les rapports avec les adultes’ [Like all forms of slang, verlan is also a closed language, a way of expressing whatever is secret. The cryptic function of street language thrives in schools and more generally in the relationship with adults]. Gadet (2003: 88) points out that verlan has ‘un renouvellement rapide’ [a fast-paced renewal], and that some words or expressions can be re-encrypted again and again, for instance ‘comme ça’ [kɔmsa] can become [sakɔm] and then [kɔmas] and even [askɔm] or [asmɔk]. Certain words in verlan have spread to other layers of the population or have become widely understood in France because of its widespread representation in the media (in particular for the purpose of caricaturing the young people who live in the banlieues) as early as the 1990s. Therefore, some words such as ‘meuf’ (femme [woman]), ‘teuf’ (fête [party]), or ‘keuf’ (flic [cop]) have lost their cryptic values, and can all be found in the Larousse dictionary.
What this means is that verlan is still very stigmatised today and yet prized by those who use it. Although some words have made it into mainstream French dictionaries, it still bears strong links with banlieue youths.
This feature is of particular interest to us here because some words of verlan are used, to a lesser or greater extent, in the subtitles of an important number of films portraying speakers of AAVE. ‘Keuf’ for instance is used only once in the subtitles of In Too Deep (Michael Rymer, 1999), but ‘meuf’ is used a number of times in White Men Can’t Jump (Ron Shelton, 1992). In other films, however, such as Boyz n the Hood (John Singleton, 1991), Menace II Society (Albert and Allen Hugues, 1993), and New Jack City (Mario Van Peebles, 1991), verlan in the French subtitles is often used. In Boyz n the Hood, ‘meuf’ is used ten times, ‘keum’ (mec [guy]) and ‘keuf’ are both used once. More interestingly from the perspective of a film, the word ‘keubla’ (verlan for ‘black’) is used in the opening line of the film. In Menace II Society, ‘meuf’ is used eight times, ‘tassepé’ (pétasse [floozy]) is used three times, and ‘teuf’ and ‘keuf’ are used twice each. ‘Beuh’ (herbe [grass]), ‘chelou’ (louche [weird]), ‘renoi’ (noir [black]), ‘pécho’ (chopper [catch]) are all used once. Again, ‘meuf’ and ‘teuf’ appear in the subtitles of the very opening exchanges of the film. Finally, in New Jack City, , ‘refré’ (frère [brother]) is used five times, while ‘feuj’ (juif [Jew]), ‘kepa’ (paquet [bag]) and ‘keuf’ are used once.
Vieillard-Baron (1996: 46) also states that ‘depuis trente ans, la ville de Chicago occupe une place mythique dans l’imaginaire des jeunes de banlieue. Elle exprime à la fois le rêve américain, l’exotisme, la relégation des ghettos et le grand banditisme avec Al Capone’ [For 30 years, the city of Chicago has enjoyed a legendary status in the imagination of suburban youths. It simultaneously embodies the American dream, exoticism, ghettoization, and organized crime through Al Capone.]. French youths draw their inspiration from their American counterparts for music, fashion, and also a certain idea of ghettoisation as highlighted by the two quotations above.
A piece of literature customarily evokes its culture through consequential and telling signals or details, typically parts or aspects of the culture that are saturated with semiotic significance and emblematic of the culture as a whole, both in terms of objective structure and subjective experience […] In this regard, such cultural elements within a literary work are metonymic evocations of the culture as a whole, including its material culture, history, economy, law, customs, values, and so on (Tymoczko 1999: 45).
Metonymy is widely used in films, and is a particularly useful rhetorical device, as viewers are given ‘comparatively limited time to identify and understand what is represented on screen’ (Di Giovanni 2007: 96), and culturally salient features (whether visual or verbal) are used for their intrinsic metonymic value. If we apply Monaco’s statement, then visual or verbal on-screen representations can be treated as metonymic evocations of a culture. In the films mentioned above, there are recurring elements, features and themes which appear to be used to represent a certain idea of African America in the films: loose-fitting clothes, baseball caps, sports, crime, violence and AAVE are all elements that contribute to creating or recreating stereotypical representations easily associated with African Americans. The subtitles, also presented ‘within the limits of the frame’ can yield the same power, as the words used in the translation can serve a metonymic purpose as well and can evoke a particular group in the target culture. And if a translation takes place between two cultures that share some contact or familiarity – as is the case in the films with American and French street cultures – then two assumptions can be made: first, that the cultural metonymies of the original may be accessible to viewers of the translated film, and secondly, that the cultural metonymies used in the subtitles may well exploit this familiarity and ‘bridge the gap’ between the two cultures for the viewers in the TL. The use of verlan in the subtitles suggests that translators perceive possible links between the source and the target culture and that because of the overlap between the two, which is also complemented by the other elements of the film (whether visual or acoustic), the use of a salient feature of the target culture such as verlan can help complement viewers’ understanding of a film.
Verlan is the only feature of banlieue French that is used in the subtitles, but it is a very salient one, and most importantly, one that is possible to put into writing. It would therefore be wrong to say that the subtitles are written in banlieue French, and more accurate to say that translators are relying on a particular feature, and on the associations that are stereotypically made with it, to trigger from viewers a certain indexing of characters.
This indexing is social, related to age, geography, and to a certain extent, ethnicity. Armstrong and Jamin (2002: 130) point out that banlieue French ‘is essentially a young working-class phenomenon’. By definition, banlieue French is also a (sub)urban variety. In the films mentioned above, verlan is exclusively used to subtitle the lines of characters who are young and from poor social backgrounds. In the films, only urban contexts are represented, and the level of social and ethnic isolation is always very important, both visually (the characters live in all-African American neighbourhoods) but also thematically (this relegation is often referred to by characters). Armstrong and Jamin (2002: 129), in their study of La Courneuve, highlight that ‘the majority of [banlieue French] features could not be allocated to any specific ethnic group, contrasting with the case of African American Vernacular English’, but although banlieue French is not specifically associated with black people, the social and ethnic relegation of the population who live in the French banlieues causes the variety to have strong associations with ethnic minorities.
The link between banlieue French and AAVE, because of these similarities and of the cultural overlap discussed above, is therefore very pertinent. But while the parallel between AAVE and banlieue French can be attractive, it is not unproblematic since one always runs the risk of making undesirable associations between the two and turning the subtitled films into hybrid objects.
Some verlan words like ‘teuf’, ‘keuf’ and ‘meuf’ are now in the Larousse dictionary. Words like ‘keubla’, ‘tassepé’, ‘feuj’ and ‘kepa’ (or any alternate spellings thereof) are not, which reflects their lack of integration in the French lexis. In other words, the former have all been codified, and to some extent, can be legitimately written, whereas codification has been ‘forced’ onto the latter words by the subtitlers, using certain tacit rules that appear to be used when it comes to verlanisation (for instance, the use of the letter ‘k’ rather than ‘qu’ with verlan words starting with the sound /k/). I would contend that words like ‘teuf’, ‘keuf’, and ‘meuf’ are recognised and understood by a larger part of the French public than ‘keubla’, ‘tassepé’, ‘feuj’, or ‘kepa’. If the codification of these words into writing is problematic (and not unanimous – ‘keubla’ can for instance be found spelt ‘kebla’, or ‘tassepé’ without the middle ‘e’), what is even more problematic is whether they will be understood by viewers or whether they will prevent them from engaging with the film.
The ‘Code of Good Subtitling Practice’, available on the webpage of the European Association for Studies in Screen Translation, states that in subtitles, ‘the language should be grammatically correct since subtitles serve as a model for literacy’ (Carroll & Ivarsson 1998). Whilst verlan is not grammatically incorrect, its use certainly challenges accepted models for literacy as well as a certain idea of the norm. Díaz Cintas and Remael (2007: 9) also suggest that subtitles have to be unambiguous and ‘semantically adequate’. Whilst it is difficult to establish precisely what ambiguity and semantic adequacy might be, and what particular elements in subtitles may be ambiguous for viewers, words in verlan – more particularly those that are less widely used – certainly run the risk of not being understood and of generating ambiguity. Naturally though, viewers can rely on other contextual clues to speculate on the meaning of those words, which overall, represent a very small portion of the dialogue. In addition, the opacity of verlan might even be desirable, in the same way that AAVE can potentially be opaque for non-speakers of AAVE.
In the subtitles of the films examined, the use of verlan is particularly salient because it is associated with a very specific culture, one that is local. The case of verlan, then, engages us in a particularly interesting analysis a posteriori: because it is so strongly associated with a particular age group (youths) from particular areas (the euphemistic banlieues) that are socially deprived, verlan runs the risk of triggering cognitive-narrative dissonances when used in film subtitles.
However, we have shown that French street culture has strong ties with its African American counterpart, which is absolutely crucial to our study. The link between the two cultures is a strong and significant one, and is constantly re-asserted in the media by artists, through clothing, parallels made between American inner cities and French cités. They are so deeply connected that a layperson might think that they are, from a French perspective, very similar. Whilst representations of African America are ubiquitous in France, the opposite is far from true. I argue here that it is possible, through the use of verlan in the subtitles, to trigger associations with French street culture and also, by proxy to its overarching ‘mother’ culture. In the end, the use of verlan to translate AAVE can benefit from networks of representations and stereotypes that associate French street culture and African Americans to trigger connotations for viewers in the TL, connotations which go beyond mere domestication. The use of verlan may actually serve a purpose that can be called foreignising (I qualify this statement below).
It is worth noting that whilst the English subtitles of La Haine were singled out as problematic, French film critics rarely discuss the quality of the translation (whether dubbing or subtitling) of films in their reviews. In the vast majority of cases, it is in fact impossible to determine whether the critics have seen a film in a dubbed or subtitled version. Whilst the use of features of AAVE to translate banlieue French was deemed a hindrance, the use of verlan to translate AAVE has not been commented upon. The use of verlan is relevant precisely because French street culture is in many ways subordinate (for lack of a better word) to its American counterpart, because it draws so much from it – in terms of streetwear, sports, and arts – and because it draws its inspiration and momentum from it. Whilst French teenagers in the cités (those we have described as the primary users of verlan) certainly hold their American counterparts in high regard, the opposite is not true, or certainly not to the same extent, as most Americans teenagers would be ignorant of the French reality, being considerably less exposed to it. American youths know little about French rap music or sports culture, and have little interest in French cités. A careful assessment of the cultural dynamics at play is therefore necessary, should translators want to use features that are connotated geographically or socially in the TL. The important difference with Venuti’s theory is that the choice of verlan in the TL is clearly motivated by the ST. Verlan metonymically invokes French street culture as well as African America, and the images and soundtrack, rather than clashing with verlan, provide a context for its understanding. The meaning of verlan is channeled by the other semiotic systems of the film: they make verlan make sense. This is only possible in a situation where the overlap between the source and target cultures is appropriate, and, crucially, shared by viewers.
et en route pour le resto.
Bouge ton cul, hockey man.
sauf pour M. le gymnaste.
Qu’est-ce qu’il dit, le péquenaud?
qu’il a un accent de péquenot.
Est-ce que j’ai l’air d’une bonniche?
The examples in this table illustrate that when cultural references are not retained in the French subtitles, they are not transposed to the target culture either. In all these examples, a cultural reference in the original has been made more generic in the subtitles – Keith Sweat, an African American singer and his lyrics become ‘vieux refrain’, a Sizzlers restaurant becomes ‘resto’, and ice hockey superstar Wayne Gretzsky becomes a somewhat surprising ‘hockey man’. In White Men Can’t Jump, Billy is compared first to American gymnast Cathy Rigby (‘M le gymnaste’) and then to the fictional character Opie Taylor from a small community in North Carolina (‘péquenaud’). In The Wood (Rick Famuyiwa, 1999), references are made to Willy Nelson, a country music singer (‘péquenot’) and to Alice, the housekeeper (‘bonniche’) of the Brady family in the American show The Brady Bunch. In all these examples, translators have clearly picked up on one particular characteristic of each reference. In none of these cases have references been transposed to the target culture. Verlan, then, represents the only significant effort to adapt culturally-bound target culture elements.
This analysis indicates that Venuti’s paradigm, if used in the context of films, needs elaboration. Since the use of the remainder in the TT is not necessarily linked with any singular properties that may be found in the ST, in The Translator’s Invisibility Venuti does not carry out the type of cultural assessment that we are suggesting in this paper. According to his analysis, unlike ‘fluent’ translations, the translation that ‘releases the remainder’ (Venuti 1998: 10) opens itself to the incursion of the foreign, ‘the substandard, and the marginal’ (Venuti 1998: 11), and this in spite of the fact that the idea of the foreign is built with domestic material. Venuti (1998: 11) elaborates: ‘Cultivating a heterogeneous discourse […] does not so much prevent the assimilation of the foreign text as aim to signify the autonomous existence of the text behind (yet by means of) the assimilation process of translation’. This is perhaps never more correct than in the case of subtitled films: the autonomous existence of the foreign text (film) is not in doubt, as outlined above. What is more crucial here is the expression of the marginal and of the substandard (in the words of Venuti), specifically through the use of verlan. The use of this heterogeneous discourse (verlan), although eminently assimilating, does not and cannot domesticate or foreignise the source film on its own. Rather, it signifies the non-standard qualities of the dialogue in the source film. The difference with Venuti’s theory is that the use of verlan in the TL is motivated precisely by the relationship between source and target cultures, and relates to the variation and to the non-standard qualities found in the original.
Díaz Cintas and Remael note that good translators somehow manage to ‘suggest’ variation in subtitles. Whilst their assumption is quite vague and the authors do not explain exactly how translators can manage to ‘suggest’ particular information to viewers, we would like to offer the explanation that the use of verlan in French subtitles, when juxtaposed with images of African American youths, can aptly ‘suggest’ the variation of the original.
The assessment that translators make to decide whether some forms of the remainder can be used in the TT involves looking at a film as a whole – and not merely at the original dialogue in an atomised way, translating item-for-item, word-for-word. Translators analyse the culture(s) portrayed in the film and decide whether features of the TL can trigger the right associations from viewers and give them access to the meanings of the ST. Because of the constraint of reduction and the presence of the other channels in subtitles, pieces of information are discarded (because they are deemed superfluous, irrelevant or semiotically redundant), while others are selected (because they are deemed meaningful or salient) and undergo some level of reorganisation. Subtitling therefore involves a de-atomisation of the ST: translators must look at the film in its entirety and in its complexity before they are able to make a judgment and decide whether a particular use of the remainder makes sense from a cultural perspective.
‘Suggesting’ the right associations for viewers in the TL requires knowledge of the networks of representations that are intertwined in subtitled films in order to work out the meanings that viewers will understand. While this may sound commonplace in translation and translation theory, it has deeper implications in audiovisual translation. The semiotic hybridity of films implies that meanings are constantly the subject of a negotiation between the different channels: images portraying African American youths, over which subtitles are presented, can ‘channel’ meanings and have the power to privilege one or more interpretations over others. The foreignness inherent to subtitles, the other channels of the films, and the fact that cultural references are not transposed to the target culture invite viewers to consider the film as foreign. Because of this we would like to argue that Venuti’s domestication/foreignisation paradigm does have some operational relevance in the context of subtitled film: the subtitling mode is a constant reminder of foreignness, but the content of subtitles can use features like verlan which are in a way domesticating because they are so deeply rooted in the target culture. However, when juxtaposed with the image of the foreign (and with the subtitles as a further reminder of foreignness), viewers can match the socio-cultural features associated with the remainder with the socio-cultural properties of the original, and in those cases only can translators make use of the domestic to express the foreign. In the case of subtitled films, the use of domesticating forms can serve a foreignising purpose and create a specific Other, and say something of the nature of the Other’s foreignness.
In this context, the task of translators is most particular: it obviously involves a willingness not to standardise the dialogue, but also and more crucially, a great deal of empathy to determine what associations viewers will be making. Translators have to draw on a pool of widely acknowledged (and therefore hopefully more recognisable) stereotypes to construct and express difference. There is a tension – almost a discomfort – in the practice of audiovisual translation: whilst we have acknowledged that translation is always-already ideological, translation seems to require from translators a combination of specialist – and to a certain extent elitist – skills, and the use of lay persons' assumptions. In relying on recognised stereotypes, translators run the risk of perpetuating those very stereotypes. For example, the use of verlan may well bear social and geographical connotations that are appropriate in the representation of African America, but the themes tackled in the films discussed here (such as racism, substance abuse, and violence) may well be associated with speakers of verlan (i.e. people who use verlan are violent and so on) and perpetuate negative perceptions of certain social groups. The use of verlan, whilst very interesting from a translational perspective because of the parallel it exploits with African America, also means that negative stereotypes associated with speakers of verlan can be attributed to the characters portrayed. In other words, alongside the socio-geographical information, negative stereotypes are also ‘carried over’ in the translation process.
It can be argued here that this is also the case with such media of representation as films: films that claim to realistically portray the experiences of African Americans in the United States somehow pigeonhole them to a limited set of stereotypes. Ghettoisation, drugs, and violence are the only leitmotivs, and it is no wonder, then, that speakers of AAVE are often associated with negative stereotypes, such as poor education and aggressive behaviour. AAVE is not sub-standard, but rather is a rule-governed system that is learnt and known by its speakers. AAVE is often thought of as somehow inferior to English, and the characters portrayed in the films discussed in this paper who use features of AAVE are systematically perpetuating negative stereotypes. Paradoxically, the films that portray speakers of AAVE were said to be presenting a more realistic representation of African America but appear to be themselves perpetuating to some extent the stereotypes that plague the perception of AAVE and its speakers. Whilst translators may well be aware of issues connected to prestige and of the social status of languages, it is very difficult – perhaps impossible – to ignore these issues and find a middle ground in the form of translations that do not reinforce these poor perceptions. If the films could be accused of perpetuating negative stereotypes about African Americans, then some might argue that the subtitles ought to do the same. What the subtitles do, however, is perpetuate stereotypes about the French banlieues (more specifically about speakers of verlan) and reassert the link between American inner cities and French banlieues.
What does this study reveal about domestication and foreignisation as general concepts for translation? According to Venuti, the use of the remainder is not motivated by specific elements of the ST, and as a result the foreignness is one that disturbs dominant discourses. It is a foreignness that is not revelatory of the nature of the original. This study addresses this particular point and suggests that foreignisation can be qualified. The use of the remainder (such as verlan) can, in some cases, go beyond hinting at the foreign nature of the original and can actually explore this foreignness. Foreignisation as a concept needs to see its scope broadened: beyond a disturbance of the dominant discourses, it can also serve more specific purposes. Naturally, this is done through the use of domestic material (which, as Venuti rightly pointed out, is always the case with translation), and foreignisation remains essentially reliant on the use of this material. One of the main issues that translators face when they are translating non-standard varieties is the illustration of specific connotations of the original in the translation. The use of verlan to translate AAVE reveals that this can be achieved to some extent when two cultures share strong ties that are widely recognised. However, such solutions are far from universal and are only possible in very limited cases. It is therefore important to explore the cross-cultural potential of non-standard features used in the TL. Relations between the source and target cultures need to be explored case by case in order to find out how much overlap there is between the two, and how familiar viewers in the TL are with the source culture. While French viewers can be expected to be quite familiar with African American culture (either through media exposure or even by proxy through exposition to French street culture), American viewers do not benefit from the same level of exposure to French street culture. Consequently, the use of verlan to subtitle AAVE can trigger associations with the United States, but the use of AAVE to subtitle banlieue French cannot, and instead brings the film back ‘home’ to American viewers in a way that is deeply problematic.
Subtitlers work under strict time constraints, and their research into how much is shared by the source and target cultures is bound to be limited. Their assessment relies on systems of representations and existing stereotypes, and their translation decisions are informed by these assessments. This might in itself be considered problematic insofar as it means that translation can confirm existing stereotypes, in a negative way, and there seems to be an irreconcilable tension between relying on stereotypes to establish characters quickly on the one hand, and perpetuating those stereotypes on the other.
Subtitled films offer the possibility of familiarising oneself with foreign languages and cultures: first the soundtrack retains intonation and pronunciation patterns that would be replaced through dubbing, and in parallel, the images bring viewers into contact with ‘mannerisms and behaviours of other cultures (gesticulation, way of dressing, interpersonal relationships, geographical spaces)’ (Díaz Cintas & Remael 2007: 15). And whilst this possibility of having direct access to the original is part of the reason why subtitling is such a vulnerable form of translation, it has also been described by specialists of subtitling as one of the most positive aspects of subtitling (D’Ydewalle & Pavakum 1992; Koolstra & Beentjes 1999). It is because of the foreignness already present that the idea of foreignisation is so much more relevant in the context of subtitled films: the subtitles can make use of the domestic tools and values described by Venuti specifically for the purpose of the foreign, because the foreign is already there and is complemented by the subtitles.
Whilst linguists are well aware that linguistic varieties are not hierarchically organised, it is possible that because of the mechanics described above, the use of the remainder in translation reinforces the status of the standard as the perceived superior variety. In films, as in literature, linguistic forms have high metonymic values, and stand for whole networks of representations. As we have seen, Venuti’s concepts – domestication and foreignisation – prove very useful when considering the cultural dynamics at play in interlingual subtitling. Because of the nature of films, however, the level of cultural proximity between features in the dialogue of the original and the subtitles should be looked at carefully before translation is attempted. While Venuti argues that most translations are transparent and translators invisible, subtitles are anything but transparent. Consequently, their foreignising form can potentially be benefited from, and subtitles (both in terms of their form and their content), can work together with images to constitute a system, which despite having domesticating elements, serves a purpose that is ultimately foreignising.
Armstrong, Nigel, and Mikaël Jamin (2002) “Le français des banlieues: uniformity and discontinuity in the French of the Hexagon” in French In and Out of France: Language Policies, Intercultural Antagonisms and Dialogues, Kamal Sahli (ed.), Bern, Peter Lang: 107-36.
Bachmann, Christian, and Luc Basier (1984) “Le verlan: argot d’école ou langue des Keums?”, Mots 8: 169-87.
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Cheneau, Véronique (1992) Le Verlan: étude phonologique, MA diss., Université de Paris VIII, France.
D’Ydewalle, Géry, and Ingrid Gielen (1992) “Attention allocation with overlapping sound, image, and text” in Eye Movement and Visual Cognition: Scene Perception and Reading, Keith Rayner (ed.), New York, Springer-Verlag: 415-27.
Di Giovanni, Elena (2007) “Disney Films: reflections of the Other and the Self”, Culture, Language and Representation,4: 91-109.
Díaz Cintas, Jorge, and Aline Remael (2007) Audiovisual Translation: Subtitling, Manchester, St Jerome.
Gadet, Françoise (1998) “Des fortifs aux técis: persistence et discontinuities dans la langue populaire”, in Linguistic Identities and Policies in France and the French-Speaking World, Dawn Marley et al (eds), London, AFLS/CILT: 11-26.
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Mével, Pierre-Alexis (2008) “Traduire La Haine: Banlieues et sous-titrage” Glottopol 12 (Pratiques langagières dans le cinéma francophone), Michaël Abecassis (ed.), URL: http://www.univ-rouen.fr/dyalang/glottopol/telecharger/numero_12/gpl12_14mevel.pdf (accessed 30 September 2014).
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 The use of American references in the subtitles is also potentially misleading for American viewers who might believe that French banlieue youths actually share all those cultural references with American youths (i.e. the use of ‘beer’ in the subtitles instead of ‘bud’ might at least have prevented American viewers from thinking that French youths actually drink Budweiser) (see also Mével 2008 and 2010) The possibility of such ‘mismatches’, whereby there are discrepancies for viewers that are a direct product of the juxtaposition of the different channels of the film, may incidentally be one of the reasons why interlingual subtitles display a tendency to neutralise non-standard features and cultural references.
 This is an important difference between films and literature, and the latter may have to rely on paratextual material to achieve a similar effect.
 It should be noted that the use of ‘jeunes’ in those labels is metonymic as well as euphemistic, ‘jeunes’ referring to a socially stigmatised category rather than to an age group. The same is true about the use of ‘quartiers’, which often means ‘quartiers défavorisés’ ou ‘quartiers sensibles’, and about ‘banlieues’ which does not designate all banlieues, but rather poor neighbourhoods of the type sometime referred to as ‘grands ensembles’.
 See among others Bachmann & Basier (1984), Bagemihl (1988, 1989), Cheneau (1992), Kerleroux (1982), Lefkowitz (1987, 1989, 1991), Lefkowitz & Weinberger (1991), Mela (1988, 1991), Paul (1985), Plénat (1992, 1995) Villoing (1992), Walter (1984), Weinberger & Lefkowitz (1991).
 The opening line of C’est ton destin (1990), a song by French comedians Les Inconnus is still often heard today: ‘Eh les keufs, et les meufs, dans le RER, la banlieue, c’est pas rose, la banlieue c’est morose’ [Hey cops, hey broads, in the RER, suburbs are no fun, suburbs are moribund].
 http://www.esist.org/ [accessed on 30th April 2012].
 The Code of Good Subtitling Practice also provides rules that aim to avoid ambiguity in subtitles. For instance, ‘simple syntactic units should be used’, and ‘the text must be coherent’ (Carroll & Ivarsson,1998).
 Again, this is not to say that all the young people in the banlieue use verlan, or that verlan is exclusively used in the banlieues by unemployed young people of various ethnic origins. Rather, the banlieues are treated, by various specialists of verlan, as the birthplace of verlan, where the most recent innovations take place. Therefore the inhabitants of the banlieues are considered the primary innovators and users of verlan.
Dr Pierre-Alexis Mével is currently a lecturer in Translation Studies at the University of Nottingham, and is course director of the MA in Translation Studies. He teaches Translation Theory and Audiovisual Translation at both undergraduate and postgraduate level. His experience as a freelance translator working with both subtitles and websites has given him the practical background he needed to complete his doctoral thesis at the University of Nottingham in 2011. He has a particular interest in the representations of non-standard varieties on screen, and on the theoretical underpinnings of juxtaposing two linguistic varieties on screen. This raises particular issues relating to the formation of identities, about their cultural porosity, and the transferability of culturally bound features and the nature of their adaptation in another culture.


Telomeres cap chromosome ends and protect the genome. We studied individual telomeres in live human cancer cells. In capturing telomere motions using quantitative imaging to acquire complete high-resolution three-dimensional datasets every second for 200 seconds, telomere dynamics were systematically analyzed.
The motility of individual telomeres within the same cancer cell nucleus was widely heterogeneous. One class of internal heterochromatic regions of chromosomes analyzed moved more uniformly and showed less motion and heterogeneity than telomeres. The single telomere analyses in cancer cells revealed that shorter telomeres showed more motion, and the more rapid telomere motions were energy dependent. Experimentally increasing bulk telomere length dampened telomere motion. In contrast, telomere uncapping, but not a DNA damaging agent, methyl methanesulfonate, significantly increased telomere motion.
New methods for seconds-scale, four-dimensional, live cell microscopic imaging and data analysis, allowing systematic tracking of individual telomeres in live cells, have defined a previously undescribed form of telomere behavior in human cells, in which the degree of telomere motion was dependent upon telomere length and functionality.
Telomeres, essential for protecting chromosome ends [1–3], consist of tandem telomeric DNA repeats bound by multiple proteins that collectively 'cap' the telomere (reviewed in ). A minimum length of telomeric repeats is necessary to support this protective function. Telomeric repeats are replenished by the cellular ribonucleoprotein enzyme, telomerase [2, 5]. Most human cancer cells have high telomerase activity. In contrast, normal human cells have naturally limited levels of telomerase that can lead to telomere shortening over time. Many aspects of telomeres remain incompletely understood, especially their dynamic properties and behavior over time. At one extreme, over the decades of human lifespan, telomeres generally shorten, with rates and extents that have been associated with disease progression and risk [6–9]. Here we report new findings on telomere behavior at the other end of the timescale spectrum: the dynamics of individual human telomeres in living cells analyzed at 1-second time resolution.
The dynamics of the nuclear contents are complex and determined by multiple contributing factors. First, the diffusion of non-interacting particles experimentally inserted into the nucleoplasm is much slower compared with their diffusion in water . The apparent diffusion coefficient of these free inserted particles has strong size-dependence, beyond that predicted by Stokes' Law [10, 11]. This deviation from the values expected from Stokes' Law has been attributed to the characteristic free space between structural elements. In the specific case of a polymer molecule, the diffusion within entangled concentrated polymers is described as snake-like reptation motion confined by a tube formed by the other polymer molecules . Reptation is orders of magnitude slower than diffusion in dilute solutions. Secondly, the dynamics of chromatin within the dense nucleus are even further slowed down, probably due to chromatin architecture and its specific interactions. Chromosomal dynamics include oscillatory-like fast motions as well as apparently diffusive motions. Labeled chromatin sites move with seconds timescale oscillations , as well as showing constrained diffusion [11, 13, 14]. This motion is highly variable for different regions within the same nucleus or even the same chromosome ([15, 16], and references cited therein). This variability has been related to interactions with nuclear structures (nuclear envelope , nuclear pores, nucleoli , promyelocytic leukemia (PML) and Cajal bodies [19–21], nuclear matrix, DNA-associated proteins, telomeres [14, 22]) and to the conformation of the chromatin itself (centromeres , heterochromatin, DNA unwinding at highly expressed genes, histone acetylation , DNA methylation and cell cycle-related chromosome condensation ). In this study, we focused on short timescale, apparently diffusive, motion of the telomeres. We did not attempt to explain theoretically the diffusion coefficient values measured. Rather, we concentrated on differences found between telomeres and their relation to other measured properties (notably position and size) and to controlled perturbations.
A recently developed microscopy platform was employed. This platform (and variants of it) is applicable for a variety of cell-biological observations. Here we applied it to analyze rapid telomere dynamics, in order to compare the motions of telomeres of different known lengths and functionality. As the present study focused on short timescales, where the motion is described closely by diffusion laws, the motions are expected to reflect telomere interactions relatively directly. We report that in living cells, telomeres exhibit novel, heterogeneous rapid motions that directly reflect impaired telomere functionality.
Telomeres were visualized using two of the major telomeric binding proteins, TRF1 or TRF2, fluorescently labeled as green fluorescent protein (GFP) fusion proteins (Figure 1A) and expressed at low levels. The GFP tag moiety per se expressed in cells does not confer motility, as GFP-histone H2B shows very little movement in nuclei . For these analyses, we focused on a bladder carcinoma cell line, UMUC3, because it has relatively short telomere lengths (ranging from around two to five kilobase pairs), typical of human tumor cells. Stably transfected clonal lines were generated. For minimal perturbation of telomere properties, the clonal lines with the lowest expression levels of the fusion GFP-telomeric protein were chosen for telomere dynamics analyses (Figure 1B, A in Additional file 1). Individual telomeric signals within a nucleus had the expected large variation in their intensities and spot sizes. These parameters were reflective of the relative magnitudes and known intra-cell variability  of telomere lengths in cancer cells, as seen in both the UMUC3 cell line (Figure 1C, Additional File 1) and the other cell lines analyzed. Several criteria, described below and throughout the paper and discussed below, established that the fluorescent spots analyzed were bona fide telomeres. First, the cell lines analyzed in the present study were telomerase-positive cancer cell lines and not cell lines with alternative lengthening of telomeres (ALT), in which ALT-associated PML bodies occur [27, 28], and in which some PML body dynamics have been reported [14, 29]. The total numbers of fluorescence points per nucleus were counted and fitted well with the number of chromosome ends, and were independent of the expression level of the tagged telomeric proteins. Colocalization experiments using pair-wise combinations of three different telomeric proteins, TRF1, TRF2 and hRAP1, also showed the expected patterns of colocalization and intensity correlations expected for individual telomeres (Figure 1C).
Visualization of telomeres in live cultured cancer cells. (A) A schematic of lentivector expressing green fluorescent protein (GFP)-tagged TRF1 or TRF2. The vector map is referenced in the Methods section. (B) Western blotting analysis of UMUC3 cells stably expressing GFP-TRF1 or GFP-TRF2 using antibodies against TRF1 or TRF2. Clonal lines with the lowest average expression (indicated by red numbers) were chosen for comparison with the endogenous level of TRF1 or TRF2 for telomere dynamic analysis. (C) Indirect immunofluorescence of clonal UMUC3 cell lines expressing either GFP-TRF1 (upper row) or GFP-TRF2 (lower row). Complete colocalization was observed from GFP-TRF1-expressed telomeres stained with anti-TRF2 antibody in fixed cells in the upper row. Lower row: immunofluorescence images of GFP-TRF2-expressed telomeres stained with anti-hRap1 antibody also showed telomere colocalization. (D) A schematic of the data collection regime for a representative UMUC3 nucleus. One complete three-dimensional (3-D) stack of 8 μm deep (an entire UMUC3 nucleus) with 0.5 μm focal spacing (17 sections) was acquired every second. (E) Representative examples of projection images from one complete 3-D stack of GFP-TRF1-expressed UMUC3 nucleus that was acquired every second for 200 consecutive seconds (T, time in seconds). The photobleaching curve is shown in D in Additional file 1.
High-resolution, three-dimensional (3-D, xyz) spatial image datasets were acquired within rapid timeframes (100 ms for a stack of 17 consecutive focal planes with 0.5 μm focal spacing, encompassing the entire 6–8 μm high nucleus, every second for 200 seconds) using an optical microscope platform (OMX) (Figures 1D and 1E, Additional file 2). By analyzing local motion over such short times, we avoided the complexities of reaching the constrained motion thresholds found in yeast, Drosophila and mammalian nuclei [11, 13, 18], and could apply diffusion equations to quantify the telomere motion tracks as a function of time.
Two separate software programs (Track4D and TrackIt4D) were custom written to analyze the image datasets for large numbers of cells. Typically, per UMUC3 nucleus, 80 or more spots were resolved, of which 40 to 60 unambiguous telomeres could be tracked for extended periods within each total 200-second data collection timeframe (see C-E in Additional file 1). The total numbers of identifiable telomeres were consistent with those expected if the great majority of fluorescent spot signals are from single telomeres. Hence in these cells, unlike telomeres in yeast [30, 31], it is unlikely that in general, multiple telomeres cluster into single spots. For the experiments reported here, the complete four-dimensional (4-D) datasets (3-D and time) for every nucleus were displayed and analyzed in six different ways and are shown as appropriate throughout this paper in one or more of the following ways: (1) as movies (Additional files 3, 4, 5); (2) as kymographs – plots showing the movement of each individual telomere as its position in the xy-plane as a function of time T (see A and B in Additional file 6, Additional files 7 and 8); (3) as a trajectory of each telomere, showing its position in 3-D space as a function of time for 200 consecutive seconds (see E in Additional file 1, C in Additional file 6, Additional files 7 and 8); (4) as the end-to-end (E2E) distance in 3-D space as a function of time (each line in the plot tracks the distance of an individual telomere at time T seconds away from its original starting point at time 0) (see D in Additional file 6, Additional files 7 and 8); (5) as the cumulative path distance traveled by a telomere between time 0 and time 200 seconds (see E in Additional file 6, Additional files 7 and 8); (6) as the average E2E distance for each telomere for the time interval traveled (see F in Additional file 6, Additional files 7, 8, 9). E2E measurements were used to calculate D, the effective diffusion coefficient, according to Einstein's diffusion equation, E2E = (6DT)1/2 (T is the time in seconds). Hence telomere motion was quantified for every telomere and the population of telomeres, both in a single cell and in multiple cells.
In unperturbed UMUC3 cells within a single live cell nucleus, individual telomeres moved independently and showed a large variability of motion (Figure 2A–D, A in Additional file 10, Additional file 3). As shown in the plots of cumulative path (Figure 2B, E in Additional file 6, Additional files 7 and 8, A in Additional file 11, B in Additional file 12, E in Additional file 13, D in Additional file 14), over the 200-second measurement period, for all telomeres tracked, the accumulated path maintained the same line slope: the fast-moving telomeres kept moving fast, and the slower telomeres kept moving slowly. Hence, there was no indication of any change in the trend of motion that might have been attributed to photobleaching effects.
Heterogeneous motions of telomeres. Telomeres tracked using green fluorescent protein (GFP)-TRF1 are shown as representatives. GFP-TRF2 telomeric tracks are shown in A in Additional file 10. (A) Tracking of telomere motion by the software program Track4D, shown as end-to-end (E2E) distance in three-dimensional (3-D) space. An individual telomere has moved in space away from its 0 time-point position. Hence this displays motion of each telomere as a function of time T (seconds), and its distance (μm) from its starting point in the first timeframe. The effective diffusion coefficient, D, was calculated from these E2E measurements. (B) TrackIt4D tracking of telomere motion as demonstrated by plotting of the cumulative path distance traveled between time 0 and time T (seconds). (C) Tracking from kymographs (3-D tracks projected on to a xy plane) of a live UMUC3 cell showing one representative of a calm telomere and a jittering telomere. (D) Telomere motion statistics calculated from average E2E distance over sub-intervals. All distances traveled by the same telomere over time intervals of the same length were averaged together and plotted. Telomeres tracked for 200 seconds were plotted for intervals up to 100 seconds. (E) Telomere motion quantified as a histogram of D values, N = around 400 telomeres.
The mean and median of these UMUC3 cell telomeric D values fell in the general range for diffusion coefficients previously reported for chromatin segments in internal chromosome regions [11, 13, 32] (see A and B in Additional file 6, Additional files 7 and 8). Notably, the histogram based on around 400 telomeres exhibited a left-skewed distribution of D values with its mode at around 2.3 ± 0.6 × 10-4 μm2/second (Figure 2E). We estimated the error bars in such histograms using a non-parametric statistics bootstrap procedure (see B in Additional file 10). When the histograms were plotted with a log scale for the D values, the distribution still showed asymmetrical skewing (shown in C in Additional file 10 for the data in the histogram in Figure 2E). This indicates that the skewing seen in Figure 2E does not describe an underlying additive or multiplicative random process. Based on the modal value, we arbitrarily defined the sub-set of telomeres having an average D value of less than 2.3 × 10-4 μm2/second as 'calm' telomeres (moving less than 0.37 μm during 100 seconds tracking). Within the same nucleus, other telomeres, defined as 'jittering' telomeres, moved back and forth rapidly and irregularly (some having D > 10-3 μm2/second, corresponding to their moving on average more than 0.77 μm during 100 seconds) (Figure 2C and 2E, Additional file 3).
We asked how the variability of telomeric motion, and in particular that of the more rapidly moving telomeres, compared with the mobility of another class of heterochromatic regions. To mark such internal regions along chromosome arms in the same cell background, we analyzed live UMUC3 cells transiently transfected with HP1α, a non-histone protein that binds to certain heterochromatin regions . This produced a punctate fluorescence pattern (Figure 3A), consistent with the reported localization of HP1 proteins to heterochromatic regions in human HeLa cells [13, 18, 34]. Immunofluorescence staining of the CFP-HP1α protein revealed that the majority of the spots did not colocalize with telomeres (Figure 3A). Hence we could distinguish telomeric signals from this class of chromosome-internal signals. Analysis of the dynamics of these HP1 heterochromatin loci was carried out as for telomeres. Consistent with reports on heterochromatin regions in yeast, Drosophila and mammalian models (D values of 0.05–1.3 × 10-4 (m2/second)) reviewed in  and ; see A and B in Additional file 6, Additional files 7 and 8), the HP1α heterochromatin foci were less motile than the telomeres in the same UMUC3 nucleus (Figure 3B), with significantly smaller and more uniform effective diffusion coefficients (D values: mode around 1 × 10-4 μm2/second) (Figure 3C, A in Additional file 11). Thus, notably, the modal D values for telomeres were twice as high as for the HP1α-containing heterochromatic class of internal positions on chromosomes. However, most striking was the much greater range of telomeric motions compared with the HP1α-positive spots; some individual telomeres had diffusion coefficients over 10 times greater still (see B in Additional file 11). The possibility that the fastest telomeric fluorescence spots were free telomeric protein particles was ruled out because free particles with comparable sizes have D values in nuclei thousands of times greater than those measured here for the fastest-moving telomeres  (see A in Additional file 15). These and other findings described below excluded the possibility that the telomeric fluorescence signals came from free protein aggregates. Therefore, based on these findings, and those described below, the widely heterogeneous motion is not a property shared with the specific HP1α-containing class of heterochromatic regions at internal positions on chromosome arms. However, variability in motion of internal chromosomal loci has been reported in a study examining the relative motions of two fluorescently marked loci within a nucleus . These studies (which were done with a time-point resolution of 1 minute instead of the 1-second resolution in the present work) showed that the motions of the chromosome internal loci examined were more constrained when they were in proximity to the nucleolus or nuclear periphery .
Characterization of heterochromatin and telomere motion. (A) Colocalization of telomeres using green fluorescent protein (GFP)-TRF1 with heterochromatin stained by CFP-HP1α shows that most telomeres do not colocalize with the heterochromatin foci in UMUC3 cells. (B) Track4D analysis of heterochromatin motion in three-dimensional (3-D) space shows that the UMUC3 cell heterochromatin moves with much less motion than UMUC3 cell telomeres. (C) Histogram of D values for heterochromatin motion, N = around 400 heterochromatin spots. Heterochromatin hardly moves, and D values for heterochromatin fall in the range reported (mode around 1 × 10-4 μm2/second). (D) 3-D trajectory image of tracked telomeres in a UMUC3 nucleus. The enlarged view shows the motion path of a representative jittering telomere and its non-directional movement and telomeric motion conforms to a 'constrained non-directional walk' description. (E) A scatter plot of diffusion coefficients of telomeres against distance from the periphery shows that there is no significant association of telomere movement with the nuclear periphery, and no correlation between distance to the periphery and motion characteristics.
The telomere motion can be described as 'a constrained non-directional walk'. Unlike non-constrained free diffusion, which predicts a linear relationship between the square of the distance traveled by a particle, the mean square distance (MSD), and the time duration T [32, 36], the MSD curve in the internal regions of a chromosome generally reaches a plateau of T > 200 seconds in yeast and mammalian cells [11, 18]. This is interpreted as 'constrained diffusion'. However, our telomeric analyses, performed for only 200 seconds, were done before the E2E distances (around 0.37 μm) reached any evident chromosome territory confinement limit, estimated to be 0.6 μm in mammalian and other eukaryotic nuclei [11, 32]. Therefore, below 200 seconds, the assessment of telomere movement by the effective diffusion coefficient is justified (see A in Additional file 16). The movement of individual telomeres was also clearly not directional (Figure 3D). In yeast, telomeres and centromeres are tethered at or near the nuclear envelope  and reviewed in Gasser , with obvious implications for telomere motility. As mentioned above, a study on the motion of non-telomeric loci in mammalian cells (examined with a 1-minute time resolution) found that these motions were more constrained when the loci were close to the nuclear periphery . In contrast, no attachment of telomeres to the nuclear periphery has been reported in mammalian cells. Consistent with this, we observed that telomere motion was statistically no lower at the peripheral region of a nucleus than deep in the same nucleus (Figure 3E). This is shown by inspection of the nuclear localization of individual telomere trajectories, approximating the nucleus as an ellipsoid and using the Mahalanobis distance from the center to define proximity to the nuclear periphery (see B in Additional file 16). Hence, jittering and calm telomeres may occur randomly throughout the nucleus.
The higher rates of motion (jittering) of individual telomeres required energy. A brief azide treatment (6 mM for 30 minutes) dramatically and reversibly reduced the motion of the faster telomeres, while the position of the modal D value stayed the same (Figure 4A, A-C in Additional file 12). Various potential spurious contributions to motility, such as nuclear volume changes, nuclear drift, or rotation, were ruled out as described in B in Additional file 12; see also Additional file 4.
Telomere movement can be altered experimentally. (A) Azide treatment of UMUC3 cells shows that jittering telomere movements require energy. Washing away azide partially restores the movement of jittering telomeres. (B) Southern blotting (insert in right panel) shows that UMUC3 telomeres were extended by expressing an extra telomerase RNA (WT-hTER) in cells, which dampens the telomere movement (right panel). The empty vector does not change the telomere motion (middle panel). (C) 47A MT-hTer, which induces DNA damage foci colocalizing with telomeres (see E in Additional file 14), increases telomere motility in UMUC3 cells (middle panel), whereas the general DNA damaging drug methyl methanesulfonate does not (right panel). (D) A summary of histogram analysis of parental telomeres, longer telomeres under WT-hTER treatment, uncapped telomeres under 47A MT-hTer treatment, or heterochromatin (N = around 400 telomeres or heterochromatin spots). The Additional file 18 insert shows that the Komolgorov-Smirnov non-parametric comparison between histograms is statistically significant. (E) A model for the novel form of the dynamic telomere behavior reported here: short telomeres and uncapped telomeres have higher motility than longer, functionally capped telomeres or other internal regions of chromosome arms. The motility of each mobile chromosomal entity is indicated by the length/strength of the arrow.
Two types of observations suggested that the more rapid telomere motion was related to telomere shortness. First, in three other human cancer cell lines with different mean telomere lengths, we noted that the longer the average bulk telomeric DNA length, the lower the telomere motions (see A and B in Additional file 13). This relationship between mean telomere spot signal intensity and telomeric DNA length within (see below) and across different cell types provided additional corroborative evidence that the signals were bona fide telomeres. Second, comparing telomere motions within each single UMUC3 nucleus, there was an inverse relationship between telomere fluorescence intensity and motion: short telomeres, identified as those with lower intensity values, contributed the majority of the jittering telomeres (see C and D in Additional file 13). Finally, plotting the entire data for a collection of 20 different parental UMUC3 cells in a clonal sub-line expressing GFP-tagged TRF1 shows an inverse linear relationship between the effective diffusion constant (D) of a telomere with signal intensity, across the spectrum of telomeric signal intensities (except at the extreme upper signal intensity boundaries) (see Additional file 17). These data showed that in these UMUC3 cells, a two-fold decrease in telomere length (as shown by the telomere signal intensity) corresponded to a 2.7-fold increase in effective diffusion coefficient.
We tested whether there was a causal relationship between telomere shortness and higher telomere motion. To change the average bulk telomere length with the minimal perturbation possible while keeping the same cell background, we stably expressed telomerase RNA (WT-hTER) above its endogenous level in UMUC3 cells . As shown, this extends telomeres moderately, by three to four kilobase pairs, in these cells (Figure 4B). Telomeres became less motile and more homogeneous in their movement than in the parental cells or cells that had received the control empty vector. The quantitative changes in motility were evident from two different parameters: both the modal D value and the number of highly motile telomeres decreased (see E-G in Additional file 13). The great majority of the telomeres (as shown by the position of the modal peak) became shifted to the lower D value when the great majority of the telomeres in the UMUC3 cells had been shifted to a longer telomeric DNA tract length (the mean telomere DNA length seen in the Southern blot in Figure 4B). The cell doubling time was the same in all these cells (parental, vector-control, and WT-telomerase RNA overexpressing cells), as reported previously . Together, these results ruled out the possibility that a shift in the cell population cell cycle could account for the difference seen. Hence, shifting the bulk telomere population length upwards dampened telomere motility.
Even with adequate telomerase, as in the human cancer cells analyzed here, telomeres cycle between capped and uncapped states as a normal part of their maintenance . It was proposed that such temporarily uncapped, often shorter, telomeres are those acted on by telomerase [3, 41], thereby replenishing their length and restoring their ability to be capped. We hypothesized that the greater motility of shorter telomeres in the experimental settings described above could reflect such a partially uncapped status. We therefore tested whether uncapping of telomeres by an independent means could cause increased motion. We induced rapid and irreversible telomere uncapping by an independent means: expressing a mutant-template telomerase RNA (MT-hTer). Such MT-hTers cause telomerase to add terminal mutant repeats, which are designed to be unable to bind sequence-specific protective telomeric proteins. MT-hTers were shown to rapidly de-protect (uncap) telomeres and accumulate colocalizing DNA damage foci on telomeres [38, 39, 42]. Notably, MT-hTers uncap telomeres without detectable shortening of the rest of the telomeric DNA tract or depletion of bulk telomeric proteins TRF1 or TRF2 from the telomeres [38, 39, 42]. Such expression of an MT-hTer ('MT-hTer 47A') in UMUC3 cells greatly increased the fraction of jittering telomeres, as seen by kymograph analyses, their expanded trajectories and large E2E and D values (Figure 4C, A-F in Additional file 14). We eliminated the possibility that this exaggerated telomere movement induced upon telomere uncapping is a result of the general DNA damage response state of these cells. This was done by examining telomere motions in cells treated with a DNA alkylating agent, methyl methanesulfonate (MMS), which produces comparable numbers of DNA damage foci that do not colocalize with telomeres (see E in Additional file 14). Under MMS treatment, telomeres remained as restrained in their movement as the controls (Figure 4C). Measurement of nuclear volumes upon these treatments established that cell/nuclear volume shrinkage or expansion did not occur, excluding this contribution to changes in telomere motions. Hence, uncapping of telomeres by MT-hTer 47A increases telomere motion; in contrast, treatment with MMS did not.
The ability of the OMX to collect 4-D live datasets with high time resolution (every second for a short timeframe of 200 seconds) has allowed examinations of large numbers of data points for statistically robust analyses on telomere dynamic behavior. This enabled evaluation of the apparent telomere diffusion coefficient without the complications of long-term constraint diffusion imposed by the restriction of chromosomes to limited spatial nuclear domains. In this setting, we have discovered sub-sets of telomere dynamics within human cancer cell nuclei. Previous work has shown energy dependence of the motions of PML bodies in live human cancer cells, but such previous analyses have not addressed the rapid high-resolution motions of validated telomeres [14, 29].
A novel finding is that chromosome end motion is highly heterogeneous and inversely proportional to telomere length. Furthermore, experimentally uncapping telomeres, but not the general DNA damage caused by MMS treatment, greatly increased telomeric motions (Figure 4D, Additional file 18). It is possible that the telomeric uncapping resulting from incorporation of mutant repeats causes the increased telomeric motion and that DNA damage at other genomic locations in general does not. Alternatively, MMS damage may engage a different DNA response pathway from that elicited by telomere uncapping.
Even in untreated cells there was a wide range of telomere motility, with the shorter telomeres more motile on average than longer ones in the same nucleus. Experimentally increasing the telomerase activity level, with concomitant lengthening of the telomeres, damped down their movement and reduced the number of jittering telomeres in the population. Although it could be conceived that longer telomeres could allow more binding sites for (unspecified) protein attachments, such load addition to just a few kilobase pairs of telomere length would be insignificant considering the size of the chromosome. We therefore propose the model (Figure 4E) that short and, therefore, uncapped telomeres, or telomeres uncapped by mutant repeat addition, with resultant loss of protection, become more motile and move at a greater speed, which enables them potentially to explore and possibly sample a larger volume in the nucleus. The greater motility could occur during or upon uncapping as a result of telomeres detaching from a nuclear sub-structure, or active (and hence potentially energy-dependent) decompaction of a relatively large sub-telomeric (heterochromatic) region, hence altering the elasticity of this region and allowing greater freedom of telomere movement. The data also are consistent with the possibility that telomeres may not specifically attach to an immobile structure in the nuclear matrix, since one specific class of internal arm regions of chromosomes moved slower than telomeres.
Telomeres and their replenishment machinery including telomerase have elicited interest as possible targets for use in anti-cancer therapies . Therefore, understanding the basic behavior of single telomeres in living cells will be an important underpinning of any use of such potential therapeutics. Furthermore, the critically short telomeres in non-cancerous cells without sufficient telomerase eventually become uncapped, triggering cellular senescence and apoptosis, which are thought to contribute to aging and mortality (reviewed in Shay and Wright ). Tracking the very rapid, seconds-scale time events at telomeres in cells in real time will thus provide more understanding of the heterogeneity of the dynamics of this complex biological system.
The examination and systematic analysis of telomere dynamics at 1-second time intervals for 200 consecutive seconds reported here has shown that chromosome ends show distinct movements that are different from chromosome-internal HP1α heterochromatin regions. Specifically, individual telomeres within a single live cell nucleus displayed wide heterogeneity in their degrees of movement over short time intervals, and quantitative analyses show that such telomere motion consists of a 'constrained non-directional walk'. The class of chromosome-internal heterochromatin regions examined in this study showed less, more homogeneous motion. Short telomeres and experimentally 'uncapped' telomeres had higher motility than longer telomeres. Conversely, experimentally extending telomere length lowered telomere motility. Hence, integrating physical/mathematical and biological approaches has shown new properties of telomeres reflective of telomere functionality. The systems developed here to accurately and statistically track telomeres should be generally applicable to any punctate chromatin (or cellular) foci in live cells.
The N-terminus of the TRF1 and TRF2 cDNAs were fused in-frame with enhanced GFP (Clontech, Mountain View, CA, US). The C-terminus of the TRF1 and TRF2 cDNAs were fused with internal ribosome entry site-hygromycin (IRES-hygromycin; Clontech, Mountain View, CA, US). The resultant fusion constructs were sub-cloned into the pHRCMVGFPWSin18 vector under the CMV promoter (courtesy of Dr Didier Trono) . The CFP-HP1α was a kind gift from Dr Tom Misteli . WT-hTER and 47A MT-hTer in lentiviral vectors were generated as in Li et al . Briefly, WT-hTER was polymerase chain reaction-cloned from human genomic DNA and sub-cloned into the Blg II/Sal I site in pIU1-T7 plasmid (generously provided by Dr Edouard Bertrand). MT-hTer was then generated by site-directed mutagenesis to create an hTer with the RNA template sequence 3'-CAAA CCCAAA C-5' 47A-MT-hTer. Both the WT-hTER and 47A MT-hTer were cloned into the pHRCMVGFPWSin18 lentiviral vector under the IU1 promoter (see A in Additional file 14).
Lentivirus was generated as described previously . Briefly, 5 μg of pMD.G plasmid (virus envelope plasmid), 10 μg of pCMVDR8.91 (virus packaging plasmid), and 15 μg of the lentivector (GFP-TRF1, GFP-TRF2, lentiviral empty vector, WT-hTER, or 47A MT-hTer) were cotransfected into amphotropic 293T cells using the Fugene 6 transfection method (Roche, Indianapolis, IN, US). Virus was harvested 48 and 72 hours post-transfection and filtered through 0.45-μm filters. Virus titers were calculated in 293T cells by counting the number of GFP-expressing foci divided by the dilution factor (106-107 transduction units/ml viral titer is achieved). For virus infection, culture cells were incubated with culture medium-diluted virus supernatant supplemented with 8 μg/ml of polybrene (Sigma, St Louis, MO, US) for 8 hours. A virus titer of 20 transduction unit/cells were used for a > 95% infection efficiency. GFP color starts to appear after 13 hours of infection and low expression monoclonal cell population stables were purified with hygromycin (200 μg/ml) (Invitrogen, Carlsbad, CA, US) or puromycin (2 μg/ml) (Sigma, St Louis, MO, US) selection for 5 days. Weak expression stables were chosen for Western analysis and imaging analysis. For cell growth measurement and cell cycle analysis, 2 × 104 cells were reseeded in triplicates in six-well plates 48 hours after virus infection, and the cell numbers were counted every day or cells harvested for propidium iodide (Molecular Probes, Carlsbad, CA, US) staining and fluorescence activated cell sorting analysis.
Clonal lines with the lowest average expression from Western blotting analysis were chosen for comparison with the endogenous level of TRF1 (60 to 65 kilobase pairs) or TRF2 (66 kilobase pairs) on 10% or 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis gels for telomere dynamic analysis. For the low-expressing stable lines used in these analyses, the cell proliferation rates, morphology and cell cycle distribution (around 80% of the cells were in G1 in these cultures) were verified as showing no significant differences from the parental line (see A in Additional file 1). CFP-HP1α plasmid was transiently transfected into UMUC3 cells using Fugene 6 transfection and cells were imaged 36 hours later.
Mammalian cancer cell lines were used in these analyses, as cell lines are known generally to retain the genomic characteristics of the primary tumor . For all analyses, we used cell lines that do not contain PML bodies to observe systematically telomere motion in live cells. PML bodies are free nuclear particles that are not part of the chromosomes, and contain telomeric proteins and other telomeric components [27, 28].
The endothelial bladder carcinoma cancer cell line UMUC3 was purchased from the American Type Culture Collection (ATCC), (#CRL-1749). We observed that the UMUC3 karyotype was stable over the course of this and other studies in our laboratory [39, 47], consistent with the near-triploid karyotype and modal chromosome number of 80 described for this line by the supplier (ATCC). The HeLa cell line (cervical adenocarcinoma cancer cell line) was purchased from ATCC (#CCL-2). H1299, an epithelial lung carcinoma cancer cell line, was purchased from ATCC (#CRL-5803). Cells were cultured under standard cell culture conditions (37°C, 100% relative humidity, 5% CO2) in Dulbecco's modified Eagle's medium (Gibco, Langley, OK, US) supplemented with 10% fetal bovine serum (Gibco, Langley, OK, US), 2 mM L-glutamine (Gibco, Langley, OK, US), 100 U/ml penicillin and streptomycin (Gibco, Langley, OK, US). LOX cells (a human amelanotic melanoma cancer cell line) were cultured in RPMI medium (Gibco, Langley, OK, US) with the same supplements as above. Clonal cells expressing either GFP-TRF1 or GFP-TRF2 from each cancer cell line were generated by picking a single cell colony. HEPES (N-2-hydroxyethylpiperazone-n-2-ethanesulfonic acid) buffer (pH 7.4) was added to cell culture medium at a final concentration of 20 mM to avoid the need for carbon dioxide gas before imaging.
ATP depleting medium was made in culture medium, supplemented with 50 mM 2-deoxy-D-glucose (Sigma, St Louis, MO, US) and 6 mM sodium azide (Sigma, St Louis, MO, US). After washing away azide, cells were allowed to rest for 30 minutes before imaging again (azide wash). The methylating agent MMS (Sigma, St Louis, MO, US) was added at a concentration of 0.01% for 1 hour before imaging and immunofluorescence. WT-hTER lentivirus was used to infect the UMUC3 cells for 20 days, and analyzed for telomere length before imaging. 47A MT-hTer lentivirus was used to infect the cells for 3 to 6 days before imaging and immunofluorescence.
For immunofluorescence, cells were fixed in chilled 4% paraformaldehyde, permeabilized with 0.5% NP-40 in phosphate buffered saline (PBS), blocked in 5% bovine serum albumin (BSA), incubated with the primary antibody in 5% BSA in PBS, washed, and then incubated with either Alexa fluor 568 or Alexa fluor 488-conjugated secondary antibody (either anti-mouse or anti-rabbit against the primary antibody) (Molecular Probes, Carlsbad, CA, US) in 5% BSA in PBS, washed, and finally in 0.15 μg/ml 4',6-diamidino-2-phenylindole (DAPI) before mounting. Indirect immunofluorescence showed the expected complete colocalization of telomeric signals from GFP-TRF1 with anti-TRF2 antibody or GFP-TRF2 with anti-hRap1 antibody (Figure 1C). For HP1α plasmid immunofluorescence, no DAPI staining was performed.
For immunoblotting, cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS, supplemented with a complete protease inhibitor cocktail from Roche, Indianapolis, IN, US). Lysates were quantitated using Bradford assay (BioRad) and 15 μg of protein were mixed with SDS sample buffer (0.08 M Tris (pH 6.8), 2.0% SDS, 10% glycerol, 0.1 M dithiothreitol, 0.2% bromophenol blue) before boiling for 5 minutes and then subjected to immunoblotting with the antibodies.
The following antibodies were used for indirect immunofluorescence analyses and Western blotting: mouse monoclonal antibodies TRF2 (#611200, BD, Franklin Lakes, NJ, US), β-actin (#A5441, Sigma, St Louis, MO, US), rabbit polyclonal antibodies 53BP1 (#A-300-273A-3, Bethyl, Montgomery, TX, US), ATM pS-1981 (#AF1655, R&D systems), Rap1 (#A300-306A, Bethyl, Montgomery, TX, US), γ-H2AX pS-139 (#NB100-384, Novus Biologicals, Littleton, CO, US) and TRF1 (#Ab1423, Abcam, Cambridge, MA, US).
A 32P-labeled telomeric (CCCTAA)3 probe was purified using the microsystem spin column (Bio-Rad) and hybridized to HinfI and RsaI-digested human genomic DNA at 37°C overnight. DNA samples were extracted from cells using a genomic DNA purification kit (Gentra, Minneapolis, MN, US) and separated on a 0.65% agarose gel. Southern results were exposed with a phosphor-imager.
Cells were seeded on to a glass-bottomed cell culture dish (Bioptechs, Butler, PA, US) for OMX microscopy. This microscope is described in Schermelleh et al . A ×60/1.25 water-immersion objective (Olympus) was used to collect 3-D stacks of 17 focal sections (an entire nucleus of 6–8 μm) with 0.5 μm focal step, once a second for 200 seconds. OMX requires 100 milliseconds (the fastest reported imaging speed to date) to acquire one complete 3-D live cell nucleus stack. Cells were exposed for 5 milliseconds to excitation light from a 500 mW 488 nm laser attenuated to 10% transmission with a neutral density filter. The total duration of timelapse imaging is limited by photobleaching (at the end of such data collection, fluorescence intensity is roughly half) (see D in Additional file 1). The high resolution 4-D data analysis allows the development of the system to accurately and statistically track any punctate chromatin foci including telomeres reported here.
Telomere tracking requires a robust image processing algorithm to localize the fluorescent spots in noise-limited minimal exposure images. We applied a 3-D Wiener filter with telomere modeled by a Gaussian with full-width half-height of 2.5 pixels. The filtered image was then subjected to a non-linear thresholding procedure based on intensity and local contrast threshold criteria for each pixel and its six 3-D neighbors. This algorithm reliably segmented telomere spots throughout the duration of the experiment despite bleaching.
3-D contiguous components analysis provided for each telomere a set of quantitative parameters, including local background, center of 'mass' (weighted by the background-subtracted fluorescence intensities), position of maximum intensity, volume, the number of boundary pixels, and the background-subtracted integrated intensity. Due to the high and inhomogeneous background, the local background was determined from repeated dilation of each telomere segment to define the asymptotic value for the averaged intensity out of the segments as the background value. The parameters are then used to reject non-telomere spots (volume, intensity and surface-to-volume outliers) and time-track telomere motion. For dense time-sampling, tracking is simply achieved by searching for the closest telomere spot in the preceding and following time points.
We corrected for nucleus drift or rotation during the live imaging, which might have confounded interpreting telomere mobility (see E in Additional file 1).
For each telomere track (each line in the Track4D plot tracks an individual telomere, but colors of telomere tracks are random between plots), we calculated the 3-D distance to position at time zero (E2E) and the accumulated root-mean-square distance between consecutive times. The effective diffusion coefficient D was calculated from E2E using Einstein's diffusion equation, E2E = √(6DT) (T is the time in seconds). D is based on E2E distances > 0.3 μm, and sampling at 150 nm steps (1/6 of the microscope resolution in z).
The TrackIt4D (tracks analysis) program calculated the accumulated path distance (PATH). It also generates a histogram plot for the D values of each single nucleus. An accumulated histogram was generated based on around 400 telomeres or heterochromatin spots. The distance that a cell nucleus has drifted during imaging is indicated by a line near the bottom of the x-axis (timescale). Drifts are corrected when quantifying telomere motion.
Average E2E was calculated independently. Telomere foci were first sampled above the background noise using the custom-written Python software and telomere mobility was tracked, calculated (Additional file 9) and plotted. By averaging E2E for all pairs with a given time difference, there is less noise. Briefly, telomere motion statistics were calculated from average E2E distance over sub-intervals. The rate of timelapse imaging was 1 second in all cases. All distances traveled by the same telomere over time intervals of the same length were averaged together and plotted. Telomeres tracked for N seconds were plotted for intervals up to N/2 seconds (N is any number). Global nucleus motion was estimated from the drift in the average spots position and subtracted from all spots. Rotational motion was found negligible for the tracking times used.
Trajectory images were created and plotted on top of original nucleus images after TrackIT4D tracking. The association of each telomere to the nuclear periphery was approximately assessed by viewing the nucleus as an ellipsoid and using the Mahalanobis distance from the center to define proximity to the nuclear periphery. Nuclear volumes were estimated by drawing the nuclear periphery contour with polygons and evaluation of the area and volume of the nucleus in all optical sections using the Priism software suite.
Error was estimated by statistical bootstrapping. Bootstrapping of histograms of D values for non-parametric statistical comparison was done by repeatedly and randomly splitting the histograms of 400 telomeres into two sub-groups of 200 telomeres each, and averaging mean and standard deviation for the mode or the Komolgorov-Smirnov (K-S) comparison test between them. Diffusion coefficient histograms were accumulated for about 20 nuclei (>400 telomere tracks) for each experimental condition. For skewed, certainly non-normal distributions, K-S statistics have to be used. The difference between histograms was scored by the K-S comparison test. Error bars were estimated from statistical bootstrap. A K-S score of more than three times the error (standard deviations) from bootstrapping procedures is considered significant.
We are grateful to Drs Yuri Strukov, Wallace Marshall, Jennifer Fung, Baojie Li and Jack Griffith for helpful and stimulating discussions. We thank Eric Branlund, Sebastian Haase and Maura Clancy for technical assistance. We also thank Drs Tom Misteli and Shang Li for sharing plasmids and technical advice. This work was supported by grants from the NIH to JWS (GM25101) and EHB (CA96840). Support to EHB was also provided by the Bernard Osher Foundation and to JWS by the Sandler Program in Basic Sciences. XYW was supported by a postdoctoral fellowship from the Susan G Komen Foundation.
XYW constructed the fusion telomere protein-fluorescent protein gene constructs, performed cellular transformations and cell cultures, image collection and analyses and participated in drafting and writing the manuscript. PMC designed the analytical approaches, performed image acquisition and image analyses and participated in writing the manuscript. ZK conceived the analytical methods, wrote the software used for telomere motion analyses and participated in writing the manuscript. LX constructed the fusion telomere protein-fluorescent protein gene constructs, trained XYW in the cellular systems and some of the methods used in this study, and participated in the interpretation of results. JWS conceived and designed the OMX microscope and high time-resolution approach and participated in writing the manuscript. EHB conceived the biological study and experimental designs, interpreted the results, and participated in writing the manuscript. All the authors read and approved the manuscript.