Asbestos, carbon nanotubes and the pleural mesothelium: a review of the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma

The unique hazard posed to the pleural mesothelium by asbestos has engendered concern in potential for a similar risk from high aspect ratio nanoparticles (HARN) such as carbon nanotubes. In the course of studying the potential impact of HARN on the pleura we have utilised the existing hypothesis regarding the role of the parietal pleura in the response to long fibres. This review seeks to synthesise our new data with multi-walled carbon nanotubes (CNT) with that hypothesis for the behaviour of long fibres in the lung and their retention in the parietal pleura leading to the initiation of inflammation and pleural pathology such as mesothelioma. We describe evidence that a fraction of all deposited particles reach the pleura and that a mechanism of particle clearance from the pleura exits, through stomata in the parietal pleura. We suggest that these stomata are the site of retention of long fibres which cannot negotiate them leading to inflammation and pleural pathology including mesothelioma. We cite thoracoscopic data to support the contention, as would be anticipated from the preceding, that the parietal pleura is the site of origin of pleural mesothelioma. This mechanism, if it finds support, has important implications for future research into the mesothelioma hazard from HARN and also for our current view of the origins of asbestos-initiated pleural mesothelioma and the common use of lung parenchymal asbestos fibre burden as a correlate of this tumour, which actually arises in the parietal pleura.

Background
The experience with asbestos highlighted that high aspect ratio particles (fibres) pose an additional hazard to the lung beyond that produced by conventional compact particles and gave rise to the discipline of fibre toxicology. Over several decades up to the present, fibre toxicology has evolved a structure activity paradigm that explains the pathogenicity of fibres that is the most robust in particle toxicology. Whilst this paradigm explains the relationship between fibre characteristics and their pathogenicity, the exact sequence of events, following fibre deposition leading to a fibre-type hazard to the pleura and pleural mesothelium has not been clarified. In particular we poorly understand the mechanism whereby fibres seem to selectively deliver their dose to the parietal pleura, whilst the visceral pleura is not initially affected [1]. There has been no unifying hypothesis as to exactly how, within the normal understanding of particle clearance of particles out of the lungs, sustained fibre 'dose' is delivered to the parietal pleura sufficient to produce the distinctive profile of pleural effects associated with fibre exposure in man and animals. In this paper we advance a plausible hypothetical mechanism that emphasises translocation of a fraction of all deposited particles and fibres to the pleural space but the retention of only long fibres in the parietal pleura. This retention of fibre dose at the parietal pleura then serves as the driver that initiates mesothelial injury and inflammation that over time leads to pleural pathology, including mesothelioma. This mechanism is, we contend, generalisable to carbon nanotubes and potentially to other high aspect ratio nanoparticles (HARN) that are currently a cause for concern due to their asbestos-like morphology and which represent the driving stimulus for this work.
The application of this hypothesis to nanotubes arises from our initial work regarding the similarities in length-dependent mesothelial inflammogenicity of asbestos and carbon nanotubes in the peritoneal cavity [2]. The role of failed peritoneal and pleural cavity clearance of long fibres in their pathogenicity was advanced in its essential form by Kane and co-workers [3,4]. Boutin and co-workers [5][6][7] have also made essentially the same suggestion as regards asbestos and clearance from the parietal pleura in relation to human asbestos-related mesothelioma. We therefore fully recognise these intellectual precedents in our restatement and elaboration of the hypothesis here in relation to our work with long and short nanotubes in the pleural cavity [2]. This new work, which is mentioned to a small extent in this review, is being submitted for full peer-review elsewhere (Murphy, F., Poland, C.A., Ali-Boucetta, H., Al-Jamal K. T., Duffin, R., Nunes, A., Herrero, M-A., Mather, S. J., Bianco, A., Prato, M., Kostarelos, Donaldson, K. Long but not short nanotubes are retained in the pleural space initiating sustained mesothelial inflammation. Submitted for publication)This present review sets out the anatomical and pathophysiological background concerning the behaviour of particles and fibres in the pleural space and elaborates the evolving hypothesis that might explain how long fibres and long nanotubes might deliver 'dose' to the parietal pleura.

Fibres and the pleural mesothelium
Particles tend to deliver their effects to the lung itself in the form of fibrosis or lung cancer. PM 10 particles also affect susceptible populations, exacerbating existing airways disease and cardiovascular disease, probably via pro-inflammatory effects emanating from the lungs. The additional hazard posed by fibres relates to the mesothelial lining of the pleural cavity and to some extent the peritoneal cavity. Individuals exposed to asbestos demonstrate a wide range of pleural pathologies including pleural effusion (a build up of fluid within the pleural space), pleural fibrosis and pleural mesothelioma [8]. A variable, usually small, proportion of mesotheliomas developing in individuals exposed to asbestos arise in the peritoneal cavity, likely as a result of fibre translocation from the pleural cavity to the peritoneal cavity [9]. The mechanism of production of pleural mesothelioma is not well understood although various mechanisms have been advanced [10]. However some contact between fibres and mesothelial cells is a reasonable supposition (see below) and numerous studies have demonstrated effects such as genotoxicity [11] and proinflammatory effects [10] following exposure of mesothelial cells to asbestos and other fibres in vitro.
The classical fibre pathogenicity structure:activity paradigm Several decades of fibre toxicology have lead to an overarching fibre toxicology structure:activity paradigm involving length, diameter and biopersistence (reviewed in [12] Figure 1).
The fibre paradigm identifies the geometry of fibres as their most important toxicological characteristic and not the chemical make-up, except in so far as the composition makes a contribution to biopersistence (see later). This independence from composition is evident in the fact that the paradigm embraces fibres composed of diverse materials including amphibole and serpentine asbestos minerals, vitreous and ceramic fibres and an organic fibre (reviewed in [12]). Diameter is important Figure 1 Diagram illustrating a pathogenic fibre according to the pathogenicity paradigm and the role of particle characteristics. because of the central role that fibre diameter plays in defining aerodynamic diameter (D ae ) and the dependence of pulmonary deposition on D ae [13]. Clearance from beyond the ciliated airways is dominated by slow, macrophage-mediated clearance [14] and so fibres which deposit there have the potential to contribute most to build-up of dose. Length impacts little on D ae for thin fibres [15] except when length is sufficient to cause interception, a mechanism of particle deposition that is confined to fibres, involving the centre of gravity of the fibre following the airstream at a bifurcation whilst the tip of the fibre makes contact with the wall, resulting in deposition. The penetration of long fibres (>50 μm) beyond the ciliated airways is explicable on the basis that the aerodynamic diameter of a straight fibre is around 3 times its actual diameter [15]. This results from its alignment with the airflow as the fibres move aerodynamically through these tubes, aligned along the axis of the bronchial tree.
The evidence demonstrating that length is a key factor in pathogenicity of fibres comes from a number of sources but the best data are from experimental toxicological studies where it is possible to isolate length categories and assess their effects, unlike the mixed nature of human exposures. In the seventies Stanton carried out a large number of studies aimed at understanding the role of fibre characteristics in mesothelioma using implantation of fibres in gelatin, directly onto the pleural mesothelial surface. Although this is a highly artificial exposure, in a summary of these studies [16] Stanton identified that carcinogenicity was related to 'durable' fibres longer than 10 μm. In the study by Davis et al. [17] rats were exposed in a chamber to clouds with equal airborne mass concentration of either long amosite asbestos fibre or a short fibre amosite sample obtained from it by ball-milling. After lifetime exposure there was substantial tumour and fibrosis response in those rats exposed to the long amosite and but virtually no response in rats exposed to the short amosite. Adamson et al. used long and short crocidolite and following deposition in mouse lungs reported fibrosis [18] and proliferative responses [19] at the pleura with the long, but not the short samples. The mouse peritoneal cavity has been used as a model of direct mesothelial exposure and much greater toxic [20], inflammatory [21] and granuloma-generating [4] responses were evident in mice that were exposed to high doses of long fibres than was seen with shorter fibres. In vitro systems have also demonstrated the greater potency of long compared to short fibres in assays of pro-inflammatory and genotoxic activity [22][23][24][25][26][27].
Biopersistence and length interact in determining the clearance of long fibres from the lungs since long fibres may undergo dissolution which could result in complete dissolution, or most likely weakening of the fibre such that it undergoes breakage into shorter fibres, which can be more rapidly cleared than long fibres. The retention half-time (T 1/2 ) of a compact, inert, respirable, tracer particle, or a short fibre, in the respiratory tract of a rat is commonly~60 days [28]. However long fibres (> 20 μm) are more slowly cleared as they cannot be easily enclosed by macrophages [29] leading to frustrated phagocytosis (see below). Thus long fibres are more likely to accumulate in the lungs allowing the long fibre dose to build up. Long fibres that are composed of bio-soluble (non-biopersistent) structural components can undergo weakening and breakage in the lungs [30]. The knowledge described above lead to the evolution of the fibre pathogenicity paradigm show in Figure 1 highlighting that a pathogenic fibre is one that is long, thin and biopersistent.
Carbon nanotubes and the classical fibre pathogenicity paradigm Carbon nanotubes (CNT) are one of the most important products of nanotechnology, representing significant investment and are already incorporated into a large number of products and this is likely to increase. However, the essentially fibrous structure of CNT has lead to concern that they might cause asbestos-like pathology in the lung and mesothelium [2,31]. Carbon nanotubes can exist as compact tangles of nanotubes that are essentially particles, or as longer, straighter 'fibres' and we would anticipate that the hazard from these two different forms of carbon nanotube would differ. Particle effects would be confined to the lungs as fibrosis and cancer whilst fibres, exemplified by asbestos, are known have the same types of pulmonary effect but to also affect the pleura. We previously carried out a study where we exposed the peritoneal mesothelium, as a convenient model for the pleural mesothelium, to carbon nanotubes to determine whether they showed an asbestos-like, length-dependent toxicity [2]. These studies revealed that carbon nanotubes in the form of long fibres showed a similar, or greater, propensity to produce inflammation and fibrosis in the peritoneal cavity, to that produced by long asbestos. In contrast neither short asbestos fibres nor short, tangled CNT caused any significant inflammation. One important underlying process in the toxicity of long fibres is the failure of the macrophage to completely enclose them-termed incomplete or 'frustrated' phagocytosis, which is a pro-inflammatory condition. Long carbon nanotubes very likely cause inflammation via this process when long enough, i.e. longer than about 15 μm [2]. Frustrated phagocytosis of long fibres as it likely applies to asbestos and carbon nanotubes is illustrated in Figures 2 and 3.
In terms of the fibre pathogenicity paradigm, it is possible for carbon nanotubes to be pathogenic by being thin, long and biopersistent but unlike other fibres it is also possible for CNT to exist in forms that do not comply with the paradigm for a pathogenic fibre. For example CNT can exist as short forms, and longer but tangled forms (see right side of Figure 2), neither of which would pose a problem to macrophages in terms of phagocytosis or clearance. Whilst singlet nanotubes are always thin they form tangles, ropes and wires of intertwined tubes and these can be thicker, although still likely to be thin enough to be respirable. However, in larger tangles and bundles the aerodynamic diameter may well increase beyond respirability. Graphene, the basic structural component of CNT is an exceedingly strong material [31] and so is likely to be biopersistent when the graphene is pristine, with few defects and underivatised, and that is suggested by our own data (in preparation). However CNT derivatised by some chemistries, with increased amounts of defects in the graphene structure, may be less biopersistent.
Injection of fibres into the peritoneal cavity as a surrogate for fibre effects in the pleural cavity: the role of retention of long fibres in the peritoneal cavity in long fibre-induced inflammogenicity and fibrogenicity The peritoneal cavity and its viscera are covered by a mesothelium and this was recognised as a convenient surrogate for the pleural cavity mesothelium in fibre studies over 30 years ago. Subsequently asbestos fibres were found to produce inflammation [21] and mesotheliomas [32] in the peritoneal cavity following injection. Although the peritoneal cavity would not be expected to have evolved the efficient clearance mechanisms shown by the lungs, in fact it does have a system for removing particles. Instilled particles are rapidly drawn cranially in the lymph flow through the diaphragm to the parathymic lymph nodes [33]. This involves transit through the diaphragm via stomata which are pore like structures less than 10 μm in diameter ( Figure 4) linking the peritoneal cavity to the underlying lymphatic capillaries and which were implicated in fibre effects by Kane and co-workers in 1987 [4].
Kane and co-workers [3] noted that long asbestos fibres accumulated preferentially at the peritoneal face of the diaphragm around the stomata since they could not be cleared through them due to their length. Kane et al. contended that retention of long fibres at the diaphragmatic mesothelial surface initiated inflammation, proliferation and granuloma formation. Short fibres did not cause this effect, easily exiting through the stomata. However, if short fibre were injected at such high dose that their sheer volume blocked the stomata this prevented clearance allowing retention, resulting in inflammation [3]. We confirmed that low dose exposure of the mouse peritoneal cavity to long multi walled carbon nanotubes (MWCNT) [34] resulted in accumulation of the long CNT at the diaphragm, suggesting that they are also too long or bulky to exit through the stomata. Retention of the long CNT in the peritoneal cavity Figure 2 The frustrated phagocytosis paradigm as it relates to long and short fibres of asbestos (left) and various forms of carbon nanotubes (right). When confronted by short asbestos fibres or tangled, compact carbon nanotube 'particles' the macrophage can enclose them and clear them. However the macrophage cannot extend itself sufficiently to enclose long asbestos or long nanotubes, resulting in incomplete or frustrated phagocytosis, which leads to inflammation.
initiated granulomas with classical foreign body giant cells in the peritoneal lavage ( Figure 5) and in the granulomas ( Figure 6) [2]. Short, tangled CNT were not retained and were never seen at the diaphragm or viscera and did not induce inflammation or granuloma formation, their absence from sections strongly suggesting that were cleared through the stomata.
Therefore, we would postulate that there are two important parts to the mechanism of the pro-inflammatory effects of long fibres in the peritoneal cavity, shared by both asbestos and long MWCNT i) failure of long fibres to negotiate the diaphragmatic stomata with subsequent retention of the long fibre dose at the diaphragm; this contrasts with smaller particles which easily leave the peritoneal cavity through the diaphragmatic stomata to accumulate in the parathymic nodes [33], ii) at the point where the long fibre dose accumulates at the peritoneal face of the diaphragm macrophages attempt to phagocytose the long fibres; they then undergo frustrated phagocytosis stimulating inflammation and mesothelial cell damage, leading to chronic inflammation and granuloma development [2].
The consequences of pro-inflammatory and fibrogenic effect of long fibre retention at the diaphragm were most evident in the extent of the granuloma/fibrosis response seen 6 months following instillation of 10 μg of long or short nanotubes (see [2] for a full description of the NT tang 2 and NT long 1 nanotubes used in this study). As Figure 6 shows, the presence of quite a large aggregate of short/tangled nanotubes produced very little tissue reaction ( Figure 6A and 6B). In contrast, loose aggregates and singlet fibres of long nanotubes caused a florid granuloma response ( Figure 6C and 6D). The multi-layered basketwork -like arrangement of largely acellular collagen in the granuloma is strongly reminiscent of the structure of an asbestos-induced pleural plaque (see later). Frustrated phagocytosis (arrows) and the associated acute inflammatory reaction in the bronchoalveolar lavage of mice whose lungs have been instilled with long nanotubes. Aspiration of 50 μg of long fibrous multi-walled carbon nanotubes (CNT) into the lungs of C57BL/6 mice caused an acute inflammatory reaction at 24 hrs typified by a large influx of inflammatory neutrophils (PMN) into the bronchoalveolar lavage. CNT bundles and singlet fibres were seen both within macrophages (hollow arrow) and extending outside the macrophage in the process of incomplete or frustrated phagocytosis (black arrows). All images at taken at ×1000 magnification. The issue of peritoneal mesothelioma arises and begs the question as to the extent of translocation of fibres from the pleural space to the peritoneal cavity. Little is known about this and it has never been quantified in man. The mere fact that far-and-away most mesotheliomas arise in the pleural cavity suggests that long fibres are retained there, so short fibres can reach the peritoneal cavity but, by virtue of their low pathogenicity, generally do not cause much harm there.

Pleural structure and function
Although the peritoneal cavity serves as a convenient model to study mesothelial impacts of fibres, the primary mesothelial target for inhaled fibres is the pleural mesothelium. The chest cavity, or pleural cavity is the cavity that surrounds the lungs and heart, comprising the ribs and associated muscles and connective tissue. This cavity is covered by the parietal pleura, which is attached to the chest wall and is covered by a continuous 'parietal' mesothelial cell layer. The lungs themselves are enclosed by the visceral pleura which is integral to the lung surface and which has a surface 'visceral' mesothelial layer. The tight fit of the lungs to the inside of the chest wall means that the two mesothelial layers are closely apposed and there is a thin space between them that contains the pleural fluid ( Figure 7) and also a population of pleural macrophages.
The visceral and parietal mesothelium are both composed of a single layer of mesothelial cells, a basal lamina of connective tissue and a loose connective tissue layer with blood and lymph vessels. Mesothelial cells have several functions in normal pleural action [35]. Pleural fluid is constantly produced by hydrostatic pressure from the sub-pleural capillaries [35], supplemented by glycoproteins secreted by the mesothelial cells [36]. The pleural fluid and its constant outflow (see below) maintains tight coupling of the lungs to the chest wall, allowing diaphragmatic muscle contraction and relaxation to expand and passively relax the lungs during breathing movements. The pleural space is a narrow, variable space, that is up to 20 μm or so in sheep rapidly fixed at death and presumed to be similar in size in humans [35]. The pleural fluid turns over rapidly [37], continuously exiting through stomata in the parietal (not the visceral) pleura via lymphatic capillaries; these stomatal openings on the parietal pleural surface are between 3 and 10 μm in diameter ( Figure 8). They are often found in association with 'milky spots', large accumulations of leukocytes present on the parietal pleura [38] and presumed to be involved in immune activity in the pleural space. The pleural fluid outflow through these stomata drains to the lung lymph nodes in the region of the mediastinum and this pathway is important in the clearance of particles and fibres that reach the pleural space (see below). The drainage of fluid from the pleural space carries particles in the lymph to the hilar lymph nodes, mediastinal lymph nodes, parasternal lymph nodes and posterior mediastinal lymphoid tissue [39]. The stomata are most densely situated in the most caudal and posterior intercostal spaces although and are more lightly scattered in more cranial and anterior intercostal regions [40].
A fraction of all deposited particles transit through the pleura, exit via the stomata and form 'black spots' around the stomata Applying the classical dose/response toxicology paradigm to the unique pleural pathology seen with asbestos and other fibre exposures, it may be assumed that since the response occurs at the pleura, the dose must be applied at the pleura. It might be argued therefore that since fibres produce pleural pathology whilst particles do not, fibres must reach the pleura and particles must not. However, a body of literature exists to the effect that in fact a proportion of all deposited particles reach the pleura, pass through the pleural space and exit through the stomata. In the process of this they elicit range of low to higher grade responses there in the form of parietal pleural 'black spots'.
Evidence that all particles pass through the pleura comes from a substantial literature concerning the almost universal existence of these "black spots", Figure 7 Diagrammatic representation of the relationship between the visceral and parietal pleurae. The visceral pleura (VP) and the parietal pleura (PP) are seen in close apposition separated by a pleural space that contains a small volume of pleural fluid (pf). Contact between the 2 pleurae is made via the mesothelial cell layers (m) on the surface of the parietal and visceral pleurae. Pleural macrophages (PM) are present in the pleural space. The rigid chest wall is tightly locked to the lungs by the adherence of the visceral pleura to the parietal pleura allowing movements of the chest wall caused by the action of the diaphragmatic muscle and intercostal muscle (IM) to expand and relax the lungs, allowing pulmonary inspiration and expiration. The pathway for particles to reach the pleural space is unknown but the path for an airborne particle (1) that deposits in the distal alveoli (2) is shown as it passes into the interstitium (3) enters the pleural space (4) and exits through a stoma in the parietal pleura (s) into a lymphatic capillary (lc, 5) to enter the lymph flow to the lymph nodes in the mediastinum and central lung. observable on the parietal pleural wall at autopsy. These mark the stomata and arise where particles must focus to exit the pleural space at the stomata and where they enter the sub-mesothelial connective tissue around the stomatal mouths. In the study by Mitchev et al. [41], 150 consecutive necropsies of urban dwellers were examined in Belgium. Of the 96 male and 54 female necropsies, whose age ranged from 22-93 years, black spots were almost invariably seen (>90% of autopsies) on the parietal pleura. The authors noted that their location appeared to be related to the structures responsible for the lymphatic drainage of the pleural cavity and they considered these to mark the points of pleural fluid resorption. Black spots were also present on the pleural face of the diaphragm, suggesting that there is pleural fluid outflow in a caudal direction. The black spots in the Mitchev study of normal individuals at autopsy clearly reflects that deposited soot particles normally pass through the pleura some of them accumulating in the parietal pleural wall forming black spots. The black spots contain particles and elicit a tissue response which is a low grade in city dwellers, where there is accumulation of dust-laden macrophages and lymphocytes. In coal miners, however, with their large exposures to particles, the mixed dust particles trigger a higher-grade inflammatory reaction of the parietal pleura with concomitant low grade fibrosis in the 'black spots' which are very pronounced [42]. Occasionally pleural inflammatory reactions to interstitialisation of the mixed dust at the black spots are more pronounced, producing more severe granulomatous structures with concentrically arranged collagen fibres [42]. In one study [42], 12 patients with black spots (8 at autopsy and 4 surgically) who were largely miners, had their black spots removed and sectioned for histological purposes. As might be expected with such high dust exposure, the black spots were extremely well-demarcated and followed the lines of lymph flow across and through the parietal pleura.
The most severe and frequently-documented example of pleural response to dust is asbestos pleural plaques. Pleural plaques are commonly seen in asbestos-exposed individuals occurring only on the parietal pleura and diaphragm as discrete, raised, irregularly shaped areas a few millimetres to 10 centimetres in size, having a greyish to ivory white colour depending on their thickness [43]. It is important to note that pleural plaques occur in greatest profusion in exactly the sites where the stomata are in greatest profusion i.e. pleural plaques are '...most commonly found on the posterior wall of the lower half of the pleural spaces, those in the intercostal space tended to have an elliptical shape and ran parallel to the ribs above and below...'. On histological section plaques can be seen to be composed of dense bands or weaves of avascular and largely acellular collagen, with only the occasional fibroblast nucleus to be seen; they are sometimes calcified [44]. These collagenous plaques, whilst commonly seen in association with asbestos exposure are not unique to it, being found following pleural infection or trauma and so can be presumed to be the way that the pleura reacts to injury [44].
Thus there is clear evidence that a proportion of all deposited particles, most commonly urban particulate matter, reach the parietal pleura where they may interstitialise around the stomata and elicit responses. The severity of the response is dependent on the intrinsic toxicity of the dust, with increasing levels of inflammatory/fibrotic response as follows:-soot < mixed mineral dust < short asbestos. The benign nature of asbestos pleural plaque-type responses is evident in the lack of reports of mesothelioma in city dwellers or coalminers despite the prevalence of black spots in these populations and the notable lack of asbestos pleural plaque progression to malignancy [45]. Since normal asbestos pleural plaques are benign and not pre-cancerous, we hypothesise that pleural plaques are a special case of a 'black' spot caused by short asbestos fibres which elicit an unusually florid collagenous response, or as a result of a very high dose of short fibres reaching the peri-stomatal wall. The emphasis on shortness here is important since the key feature of black spots, we contend, is that the particles and short fibres are small enough to negotiate the stomatal openings where they mostly clear to the lymph nodes whilst some interstitialise into the sub-mesothelial interstitium through the proximal lymphatic capillary walls. As described below, this contrasts with events that may occur with long fibres; these cannot negotiate the stomata leading to retention at the stomatal openings, initiating a very different pathobiological sequence of events culminating in a different pathological outcome.
Our knowledge regarding the pathway by which particles reach the pleura from the lung parenchyma is well summed up in a recent review '... How asbestos fibres that have impacted the airway wall migrate to the pleural surface ..... is quite obscure. ..' [1]. Lymphatic flow from the parenchyma to the pleural space is one obvious possibility [46] but such a pathway, if it exists, is not well-documented [1]. A fluid dynamics model of fibre translocation highlights two possible pathways [47], the first of these being by normal lymph flow centrally to the mediastinum and then into the blood via the thoracic duct followed by extra-vasation in the pleural capillaries during the formation of pleural fluid. This rather tortuous route disregards the filtering role of the lymph nodes and seems to us to be intuitively unlikely. The second route requires inflammation in the parenchyma, caused by the fibres, to reverse both the normal flow of lymph and the normal trans-pleural pressure, resulting in a net flow of fluid and fibres directly into the pleural space from the underlying parenchyma [47]. This latter process cannot be the explanation for normal transit of particles to the pleura that gives rise to black spots (see above) in normal people, who have no pulmonary inflammation. Therefore, even if such an inflammation-dependent pathway exists, a pathway that is independent of inflammation clearly operates for compact particles in normal people. Further research is needed to establish the mechanism of transport of fibres to the pleural space.
Long fibre retention at the stomata of the parietal pleura So from the above there is good evidence to support the contention that a fraction of all deposited particles reach the pleura by an obscure pathway and that short fibres and compact particles leave the pleura through the stomatal openings. Most of the particles are transported to lymph nodes and some enter the interstitium at the mouth of the stoma to form a 'black spot' or equivalent. Long fibres that reach the pleural space, however are an exception to this, since they have the potential to physically block the stomata due to their difficulty in negotiating the bend into the stoma which would result in interception of the ends of the fibre with the walls of the stomatal openings and with the lymph vessels walls themselves. This is likely to lead to mesothelial and endothelial cell damage at this site, inflammation and the accumulation of pleural macrophages attempting to phagocytose these retained fibres. The macrophages are likely to undergo frustrated phagocytosis in attempting to enclose the long fibres and so release cytokines and oxidants. This would lead to further inflammation, fibrosis and genotoxicity in the bystander mesothelial cells in these areas of congestion around the stomatal entrances. Direct interaction between retained long fibres and mesothelial cells around the stomata could also result in direct genotoxicity. Eventually some stomata are likely to be entirely blocked by cells and fibres. Figure 9 demonstrates diagrammatically the difference between formation of black spots and pleural plaques with particles and short fibres (A), compared to the response to long fibre retention at parietal stomata (B).
This means that that the primary lesion caused by long fibres must form at the parietal pleura, the site of retention of long fibre dose and therefore the site of response. Mesothelioma would therefore originate not at the visceral pleura but at the parietal pleura. There is good evidence to suggest that this is indeed the case, and numerous studies using thoracoscopy have confirmed that the origin of mesothelioma is the parietal pleura [6]. This is reflected in the staging of mesothelioma which recognises that early mesothelioma is confined to the parietal pleura, while more advanced mesothelioma involves the visceral pleura [48]. Indeed, the prognosis for mesothelioma when it only involves the parietal pleura is much better by around 30 months, than the prognosis arising when mesothelioma involves visceral pleura [6]. This reflects the earliness of the disease stage when it is still confined to the parietal pleura. From a toxicological viewpoint this means that the focus of attention in trying to determine whether any fibre is likely to cause mesothelioma should not be focussed on the question 'Do fibres reach the pleura?' Figure 9 A) diagram showing the events leading up to formation of black spots. A1) particles enter the pleural space; A2) in focusing to exit via the stoma (St) some particles interstitialise through the loose lymphatic capillary endothelium and macrophages begin to accumulate in response; A3) macrophages and particles form a mature 'black spot' with mesenchymal cell activation and proliferation depending on the toxicity and dose of the particle. B) In B1 a single long fibre is intercepted as it attempts to negotiate the stomatal opening and is retained; 2) other fibres are caught up and there is an accumulation of retained long fibres; 3) macrophages attempt to phagocytose the fibres and undergo frustrated phagocytosis releasing a range of pro-inflammatory, genotoxic and mitogenic mediators close to the pleural mesothelial cells. PM = pleural mesothelium; St = stoma; LC = lymph capillary. but should be focussed on the question 'Are the fibres retained in the parietal pleura?'.
Fibres found in digested human lung and visceral pleura at autopsy following death from mesothelioma are often short [49] but, as described below, these are not the site to seek the effective fibre 'dose' for mesothelioma, since the parietal pleura is the site of mesothelioma initiation. In fact the site where the effective dose for long fibres is initially applied is the parietal mesothelium, which has seldom been sampled for fibre burden or dimensions. However, in several studies, fibres recovered from the parietal pleura have also been found to be short [50,51]. This may be explained by the fact that, as described above, the location of the longer fibres is likely to be very focal, at the stomata. When this area was specifically sampled in 14 patients diagnosed with asbestos-associated diseases, including mesothelioma, much longer fibres were found concentrated there [5].
Correct sampling is key to determining the important measure of dose and since this is likely to be found only in 'hot-spots' at stomata but these are heterogeneously distributed across the parietal pleura, are very small and so difficult to sample.

Fibre biopersistence and the hypothetical model
In the case of non-biopersistent fibres, the degree of their biopersistence, specified by the retention half-time, will dictate the likelihood that they will reach the pleura and the impact that they will have there. For fibres of very low biopersistence such as the chrysotile fibres with half-lives of around 1 day [52] it seems likely such chrysotile fibres undergo dissolution and breakage in the lung parenchyma in the hours following deposition, such that no long fibres are likely to reach the pleura; only short fibres, if any fibre-like structures remain at all, are likely to transit to the pleural space. In the case of fibres that are moderately biopersistent, the long fibres may retain their structure en route to the pleural space, all the while undergoing dissolution/breakage. If long fibres are sufficiently biopersistent to retain their fibrous structure long enough to enter the pleural space they may be retained at the parietal stomata, initiating frustrated phagocytosis and granuloma formation. Depending on the extent of their biopersistence, however, fibres could still dissolve and break within the macrophages as a result of the high pH within the phagolysosomes, allowing the granuloma to resolve. The exact tempo of translocation of particles and fibres to the pleural space is unknown, but in less than 1 day following inhalation of short, essentially particulate, CNT in mice, the CNT were evident in the sub-pleural extracellular matrix [53]. This suggests that compact particles or very short HARN may reach the pleural space rapidly following deposition; however such short HARN and compact particles are not likely to be retained at the stomata. No inhalation study with long CNT has yet been carried out but fibres long enough to be retained at the parietal stomata may move more slowly through the parenchyma, to the pleura, because of their greater dimensions causing 'drag' to their movement through fluid. Research is needed to elucidate the relationship between fibre length and biopersistence in leading to pleural transport, mesothelial injury, inflammation and mesothelioma.

Carbon nanotubes in the pleura
We approached the issue of the potential mesothelial toxicity and pleural toxicity of carbon nanotubes by first attempting to determine whether, similar to asbestos, carbon nanotubes showed length-dependent toxicity to the mesothelium. Based on the above argument we also hypothesise that the long CNT would be retained at the parietal pleural around the stomata. In early studies we used the peritoneal mesothelium lining the peritoneal cavity as the target mesothelium. These studies demonstrated that there was indeed length-dependent inflammogenicity and fibrogenicity of carbon nanotubes in the peritoneal cavity, mimicking asbestos [2]. As predicted from the fibre pathogenicity paradigm, the key length appeared to be between 15-20 μm, the length beyond which macrophages cannot stretch and enclose fibres, thus eliciting frustrated phagocytosis. In the follow up to the studies we utilised the pleural mesothelium and developed a model of injection of nanotubes and asbestos into the pleural space of mice. This can be affected quickly in non-anaesthetised mice using a very fine needle with a collar at the level of the bevel in the needle to restrict penetration through the chest wall allowing injection only into the pleural space. Following injection, the injectate distributes through the pleural space as was evident by inspection of lungs immediately following injection. In these studies we injected the same panel of fibres used in the peritoneal cavity in the Poland studies, i.e. long and short amosite asbestos samples, two long nanotubes samples and two short/tangled nanotubes samples and nanoparticulate carbon black as a graphene control. Following injection the pleural cavity was lavaged to determine the inflammatory response. We found clear evidence of length-related inflammation in the pleural space with both the long amosite and the two long nanotubes samples causing inflammation while all the other short samples failed to elicit significant inflammation (Figure 10).
In a time course, the long nanotubes caused a sustained high level of inflammation at 7 days, which was the same as was present at 1 day. This is in contrast to the events in the peritoneal space where there is a decline in inflammatory response over 7 days to levels of about one-fifth present on day 1 by day 7. Based on our hypothesis that long fibres were retained at the parietal pleural and that short fibres were not, we used paraffin wax histology to examine the parietal pleural surfaces. In keeping with the hypothesis, long fibres which were visible on day 1 following injection were still visible in granulomas at the surface of the parietal pleura on day 7 ( Figure 11). No short fibres were visible in sections of parietal pleura at 1 or 7 days, however the activated, thickened mesothelium seen at day 1 had returned to normal by day 7 suggesting that the short fibres had been cleared (Murphy, F., Poland, C. A., Ali-Boucetta, H., Al-Jamal K.T., Duffin, R., Nunes, A., Herrero, M-A., Mather, S. J., Bianco, A., Prato, M., Kostarelos, Donaldson, K. Long but not short nanotubes are retained in the pleural space initiating sustained mesothelial inflammation. Submitted for publication).
We therefore hypothesise that the retention of long fires at the stomatal openings on the parietal pleura, coupled with frustrated phagocytosis of pleural leukocytes that attempt to ingest them, produce a chronic pleural mesothelial inflammatory response. Chronic inflammation is known to be a driver for proliferation, genotoxicity, growth factor synthesis and release that are likely to culminate in pathology such as fibrosis, pleural effusion and mesothelioma ( Figure 12).

Implications for testing of high aspect ratio nanoparticles (HARN)
The foregoing discussion has highlighted the importance of the parietal pleura as the target for the long fibre hazard following pulmonary deposition and the site of initiation of mesothelioma. In addition to carbon nanotubes, there are a whole new generation of high aspect ratio nanoparticles (HARN), such as nanorods and nanowires. These are made of a wide range of materials, including silica, silver, nickel and various forms of carbon. There is a need to test these materials and understand their potential for causing mesothelioma. Mesothelioma has a very long latent period and in rats, following inhalation of asbestos, mesothelioma commonly does not develop until near the end of life in a small proportion of animals. The peritoneal cavity has been used as a more efficient model for mesothelioma induction but has been criticised because of its non-physiological nature and irrelevance for risk assessment. However, the peritoneal cavity does show size-restricted clearance and subsequent sensitivity to retained long fibres. An appreciation of the role of the parietal pleura as the site where fibres are retained leading to pleural pathology of various sorts which accompany exposure to fibres, means that a rational testing strategy could attempt to identify early changes in this tissue following fibre exposure. New techniques that allow investigators to home in on specific areas, eg laser capture, would enable the areas of the parietal pleura where the stomatal openings occur to be identified and studied in detail for the presence of fibres and their molecular consequences. Thus there is the prospect of studying oxidative stress, inflammation and genotoxicity at an early stage in the very target tissue where mesothelioma is likely to arise. This should revolutionise the ability to screen for pathogenic fibres amongst emerging HARN and allow us to look anew at the effects of asbestos and more conventional fibres that affect the pleural mesothelium and we look forward to future studies that utilise this knowledge. Figure 10 Cytospin preparations of pleural lavage cells from mice treated with short/tangled CNT (left) and long CNT (right). Arrows indicate granulocytes. Note PMN and eosinophils (arrows) indicative of inflammation in pleural leukocytes from rats exposed to the long NT only.
Donaldson et al. Particle and Fibre Toxicology 2010, 7:5 http://www.particleandfibretoxicology.com/content/7/1/5 Figure 11 Histological sections through the chest wall of mice treated with long straight nanotubes (A, B) or short, tangled nanotubes (C, D) for 1 or 7 days. Note the progressive thickening of the mesothelium in A and B in response to long straight nanotubes; this contrasts with the acute thickening of the pleural layer with short/tangled CNT which shows resolution to normal mesothelium by 7 days (D). Scale bars represent 20 μm. Figure 12 Hypothesised sequence of events leading to pleural responses as a consequence of long fibre retention at the parietal pleural stomatal openings.

Implications for asbestos and mesothelioma
The emphasis on the parietal pleura as the site of retention and focus for the long fibre dose has important implications for our understanding of the origins of mesothelioma, a subject of considerable scientific and, arguably, even greater medico-legal significance. Mesothelioma continues to be a global problem due to ongoing exposure to fibres and as a legacy from past exposure to asbestos, even in countries where asbestos is currently banned or has been regulated out of use for decades [54]. Lung tissue burdens of asbestos fibres have long been used as an index of exposure, but the above discussion highlighting the parietal pleura, not the lung tissue, as the site of origin of mesothelioma calls into question the relevance of parenchymal lung fibre burdens as a correlate of mesothelioma. The lung diseases caused by asbestos i.e. lung cancer and asbestosis -may well be related to the lung parenchyma tissue burdens, since one would reasonably look in the target tissue for the effective dose. However the same logic would dictate that the effective dose for mesothelioma, which arises in the parietal pleura, should be sought in that tissue. In fact the parietal pleura fibre burden has been studied, but only very occasionally; for example Dodson and Atkinson [55] cite Sebastien [56] as stating that, for asbestos fibre burden "lung parenchymal retention is not a good indicator of pleural retention: indeed, there was no relationship between parenchymal and pleural concentrations". This would be predicted from the arguments presented in this paper. Therefore, whilst the lung parenchyma is a site of fibre accumulation that is likely related to exposure, the lung parenchyma is not expected to focus the effective dose for mesothelioma in the way that the parietal pleura does [5] through its action as a kind of 'sieve' that selectively retains long fibres.
Even supposing the parietal pleura were to be chosen as the tissue of choice for assessing effective dose of long fibre for the mesothelioma hazard, a considerable problem is posed in sampling it for fibre-burden analysis because of their small size and the heterogeneous distribution of the stomata over the parietal pleura. Yet these are exactly the sites that should be sampled in order to find the dose responsible for the mesothelioma response or to sample the site of developing mesothelioma in order to determine its molecular ontogeny. These 'hotspots' of dose could not be easily selected at autopsy by a pathologist unless they knew specifically where to look and even then the diluting effect of non-stomatal tissue in the immediate vicinity could easily confound any attempt to determine the specific long fibre dose at the stomata.

Conclusion
We have reviewed the evidence for the hypothesis for the behaviour of long fibres in the parietal pleura, focusing on nanotubes as a new potential pleural hazard, although the discussion is relevant to all fibres including asbestos. We argue from existing evidence that a fraction of all deposited particles reach the pleura and from evidence on the mechanism of particle clearance from the pleura, to argue that the parietal pleura is the site of retention of long fibres ( Figure 10). We suggest that their retention there, a consequence of length-restricted clearance through the normal stomatal clearance system, initiates inflammation and pleural pathology including mesothelioma. We cite data from thoracoscopy to support the contention that, as would be anticipated from the foregoing, the parietal pleura is the site of origin of pleural mesothelioma. This general hypothesis on the key role of fibre length-restricted clearance from the pleural space as a mechanism for delivering a high, focussed, effective dose of long fibres to the mesothelial cells around the parietal pleural stomata, has important implications. These lie in future research into the mesothelioma hazard from HARN but also for our current view of the origins of asbestos-initiated pleural mesothelioma and the use of lung parenchymal fibre burden as a correlate of this tumour, which arises in the parietal pleura, not the lung parenchyma or visceral pleura.


extfieldsofstudy: Biology Medicine

text:
Mechanism of Processive Movement of Monomeric and Dimeric Kinesin Molecules

Kinesin molecules are motor proteins capable of moving along microtubule by hydrolyzing ATP. They generally have several forms of construct. This review focuses on two of the most studied forms: monomers such as KIF1A (kinesin-3 family) and dimers such as conventional kinesin (kinesin-1 family), both of which can move processively towards the microtubule plus end. There now exist numerous models that try to explain how the kinesin molecules convert the chemical energy of ATP hydrolysis into the mechanical energy to “power” their proceesive movement along microtubule. Here, we attempt to present a comprehensive review of these models. We further propose a new hybrid model for the dimeric kinesin by combining the existing models and provide a framework for future studies in this subject.

Introduction
Kinesin is a motor protein that transports cargo across cells by moving along microtubule (MT) filaments by hydrolysis of ATP [1][2][3][4][5]. Native kinesins generally have several forms of construct. This review focuses on two forms of them which have been studied extensively. One is the monomer such as KIF1A (kinesin-3 family [6]) that has one N-terminal motor domain or head, whereas the other one is the dimer such as conventional kinesin (kinesin-1 family [6]) that consists of two identical N-terminal motor domains or heads that are connected together by a rod-shaped, coiled-coil stalk through their neck linkers (NLs). Both forms can move towards the MT plus end.
For the two-headed kinesin-1, it has been determined that the dimer advances stepwise over the MT surface lattice in about 8 nm increments, the tubulin dimer repeat distance. A single molecule can generate hundreds of steps during a single encounter with a MT [7,8] and can exert a maximal force of about 5-7.5 pN [9][10][11][12]. During the processive movement, the dimer only occasionally steps backwards under low backward loads [13][14][15]. It is now well known that the dimer moves along MT in a hand-over-hand manner [16,17]: a given head is displaced in discrete steps with a size of about 16 nm [17], resulting in the step size of the dimer of about 8 nm.
Among single-headed kinesins, it has been experimentally demonstrated that a single KIF1A molecule, like the dimeric kinesin-1 molecule, can also move processively along MT [18,19]. However, in contrast to the dimer, the step size of the monomer is distributed stochastically around multiples of 8 nm with a Gaussian-like envelope [20]. A single KIF1A molecule can only exert a maximal force of about 0.15 pN [20]. Interestingly, among truncated single-headed conventional kinesins, one has also been observed to move processively along MT [21,22], sim-ilar to KIF1A, while others have been shown to have very low or no processivity [2326].
To understand the mechanism on how kinesin molecules convert chemical energy of ATP hydrolysis into mechanical energy to "power" their proceesive movement along MT, numerous models have been proposed for both monomeric and dimeric kinesin molecules [4,5,1520,2747]. In this review, we provide a broad classification to these models. We also present an intuitive description of each class of the models. The consistency and/or inconsistency between each class of the models and the available experimental data are discussed briefly. Based on these previous models, we propose a hybrid model for the dimeric kinesin and a framework for future studies.

Models for Processive Movement of Monomeric Kinesin
Broadly speaking, the models for the unidirectional motion of monomeric kinesin can be divided into two classes, which are described in the following two sections.

Brownian ratchet model with fluctuation of global potential
The prevailing model for the unidirectional motion of the monomeric kinesin along MT is described by the potentials as shown in Fig. 1 [27,28] or those with minor variants [1820]. Here, we take Fig. 1 as an example to discuss this class of models. For convenience, the model is termed as Brownian ratchet model with fluctuation of global potential (FGP model). In the model, the monomer is viewed as a Brownian particle moving in a periodic, piecewise but spatially asymmetric potential. The change in nucleotide states of the motor induces its conformational change [48,49], which in turn induces the potential switching between "on" (corresponding to the kinesin in nucleotide-free, ATP or ADP.Pi state) and "off" states (corresponding to the kinesin in ADP state). When the potential is on, the particle is pinned near the bottom of one potential well (for example, at x = 0) (Fig. 1A). When the potential is off, the particle diffuses freely due to the thermal noise. After a time, t off , the probability distribution of the particle's position has the form Where D=k B T/Γ, Γ=6πηr, is the drag coefficient, where η=0.01g cm -1 s -1 is the viscosity of the aqueous medium. The motor is approximated as a sphere of radius r and F is the external force acting on the motor and pointing towards the MT minus end. Although the FGP model is very simple and elegant, it has the following problems when it is applied to monomeric KIF1A. First, as noted from Fig. 1, even under F = 0, the motor shows a much larger probability to stay in the original position than to make a forward step during one ATPase cycle. This gives a very small mechanochemical coupling efficiency. Secondly, using Eq. (1) one can easily obtain the stall force to be F stall = Γ L(1/2-α)/ t off . From the available measured ADP-release rate of about 250 s -1 [4,50], we have t off ≈ 4ms. Taking r = 3 nm and L = 8 nm, we obtain F stall ≈ 5.65×10 -5 pN even for α→0. This value is much smaller than the measured stall force of about 0.15 pN for monomeric KIF1A [20]. Finally, during the off state ( Fig. 1B) with t off ≈ 4 ms, since the kinesin has a very weak affinity for MT, the motor has 100% probability to detach from MT. Thus, the motor shows no processivity, which is inconsistent with the experiments, which showed KIF1A to be of high processivity [1820].

Brownian ratchet model with fluctuation of both global and local potential
More and more structural studies indicate that the kinesin conformation should also be investigated with MT to understand the moving mechanism of kinesin along MT [51]. For example, in the absence of MT, the conformation of kinesin in ATP state [48] has been observed in the presence of ADP, while the conformation in ADP state [48] has been observed in the presence of AMP-PNP (an ATP analog) [52]. The two conformations are independent of the bound nucleotide [53,54]. However, in the presence of MT, the conformation of the plus-end directed kinesin seems to be tightly coupled with the bound nucleotide, as reviewed by Kikkawa [51]. Thus, the interaction of MT with kinesin seems to affect the conformation of the kinesin. Conversely, it has been reported that a significant structural change in MT is also induced by the strong binding of kinesin [5558]. It is thus expected that both the conformational change of kinesin and that of MT induced by the strong binding of the kinesin should be taken into account in eliciting the mechanism of kinesin moving along MT. In light of the above results, the binding affinity of MT for kinesin due to nucleotide transitions is hypothesized to have the form shown in Fig. 2 [29]. In nucleotide-free, ATP or ADP.Pi state, the strong interaction potential of kinesin with MT has a periodic, piecewise but spatially asymmetric form ( Fig. 2A), same as Fig. 1A. As just mentioned above, the strong interaction of kinesin with MT induces a significant structural change in the local binding site of MT where the kinesin is binding, making it different from other unaffected binding sites of MT. Thus the affinity of the local binding site on MT should be different from that of the other unaffected binding sites for the ADP-kinesin that has a different conformation near its MT-binding site from that in ATP or ADP.Pi state. It is reasonable to assume that the ADP-kinesin has a further weaker interaction with the local binding site on MT (Fig. 2B). In a time of t r after Pi release from the kinesin, the local binding site of MT relaxes to its normal conformation and, thus, the interaction potential of ADP-kinesin with MT changes from that schematically shown in Fig As demonstrated in Ref. [29], the numerical results based on the FGLP model have shown good quantitative agreement with the available experimental data for monomeric kinesin molecules. Some typical characteristics are stated as follows. (1) As noted evidently from Fig. 2B, the motor shows a very small probability to stay in the original position during one ATPase cycle. Moreover, the asymmetric potential provides a larger probability for forward stepping than backward stepping. This thus gives a large mechanochemical coupling efficiency, in contrast to the FGP model. (2) In the FGLP model, the stall force corresponds to the backward force under which the particle has equal probability to diffuse to x = αL and to x = (α-1)L from position x = 0 with the potential shown in Fig. 2B, which is very different from the case of FGP model (Fig. 1B). This thus gives a stall force much larger than that for the FGP model. (3) Another important conclusion deduced from the FGLP model is that the processivity of a monomeric kinesin is determined by its binding affinity for MT in ADP state: a weak affinity giving a low processivity while a high affinity giving a high affinity. Due to the presence of K loop, KIF1A shows a high affinity for MT, thus giving a high processivity. In particular, with the FGLP model the predicted binding affinity (~ 13 k B T) of ADP-kinesin for MT which is obtained using the measured processivity [29] is consistent with that (13±2 k B T) which is obtained using the frictional drag coefficient measured recently by Bormuth et al. [59].

Models for Processive Movement of Dimeric Kinesin
Since it has been determined that the dimeric kinesin moves hand-over-hand along MT [16,17], we exclude here the discussion of models in which the dimer moves in other manners such as inchworm manner [60] other than in the hand-over-hand manner. A lot of models have been proposed for the hand-over-hand motion of the dimer [4,5,16,17,3047]. Broadly speaking, the models can be divided into two classes. One prevailing class, although each model in this class has minor variants, is that the forward movement of the trailing ADP-head to the leading position results mainly from the conformational change of the NL, with all binding sites on MT having the same affinity for the ADP-head. The other one is that the forward movement of the trailing ADP-head to the leading position is due mainly to the difference in the binding affinity for ADP-head between the next forward binding site on MT and the original one and to the affinity between the two heads.

Neck-linker-docking model
A pronounced conformational change in kinesin's NL has been observed [61,62]: the NL is mobile when the MT-bound kinesin is nucleotide free, but the NL becomes immobilized and extended towards the MT plus end after the binding of AMP-PNP. Based on this evidence, the neck-linker-docking (NLD) model has been proposed (Fig. 3) [5,16,30,3440,46,47]. Recent computational modeling of MT-unbound kinesin suggested that the docking of the NL to the motor domain may be realized through the interaction between the N-terminal peptide and the NL in the ATP state [63]. More recently, based on their observations by using cryoelectron microscopy, Sindelar and Downing [64] proposed a mechanism in which the switch loops of the MT-bound kinesin, triggering by ATP binding, propel their side of the motor domain down and thereby elicit docking of the NL on the opposite side of the seesaw. In the NLD model, the docking of the NL of the leading head upon ATP binding delivers the trailing ADP-head that is weakly bound to MT to the leading position (Fig. 3B). Then, due to the thermal noise, the tethered ADP-head diffuses to the nearest binding site (Fig. 3C). The strain between the two NLs induced by strong bindings of both heads to MT accelerates the hydrolysis of ATP to ADP.Pi in the trailing head (blue) but reduces the rate of ATP binding to the leading head (red). (E) After Pi is released from the trailing head (blue), the state of dimeric kinesin becomes that of (A) except that the dimer has moved forwards by one step.
The model requires that the tethered ADP-head can distinguish between forward-and backward-binding sites. The possible mechanism for this biased binding is supposed as follows. In the dimer, the kinesin heads are connected by the NLs and the binding of the tethered ADP-head to the forward-binding site induces a backward force on the ADP-head. If the backward force increases ADP-release rate, the ADP release and subsequent strong binding to the MT most probably happens at the forward-binding site. This is consistent with the experimental data on distributions of unbinding force for dimeric kinesin molecules, which showed that, at intermediate ADP concentrations where populations of strong and weak MT-binding kinesins coexist, backward loading increases the proportion of strong binding molecules, while forward loading increases the proportion of weak binding molecules [65].
As mentioned elsewhere (see, Refs. [5,51,66,67]), a persistent problem for the NLD model is that the free energy change associated with the NL docking is only about 1.5 k B T [68]. This implies that a small load of only 1 pN, which is much smaller than the stall force of about 7 pN, can prevent the NL docking which, based on the NLD model, should drive the trailing ADP head to move forwards by more than 8 nm. Indeed, it has been shown recently that, with the aid of only a 1.5-pN forward load, the walking behavior of the mutant kinesin with deletion of the N-terminal peptide, potentially defecting the docking of the NL, restored much of the character of the wild-type kinesin [69]. In addition, as mentioned above, using the measured frictional drag coefficient, Bormuth et al. [59] have determined that the well depth of the interacting potential between the ADP-head and a binding site on MT is 13±2 k B T. Consequently, the small free energy change of only about 1.5 k B T associated with NL docking is far from being sufficient to drive the ADP-head to escape from the potential well of the ADP-head interacting with binding site (I) (see Fig. 3). Here it is interesting to note that recent molecular dynamics simulations showed a larger free energy change associated with NL docking upon ATP binding [63]. However, the molecular dynamics simulations were performed on kinesin alone. As mentioned above, both the experimental data [61,62] and structural studies [5154] have showed that the docking or undocking of the NL seems to be independent of the nucleotide state of kinesin without bound to MT. Thus, the molecular dynamics simulations on the nucleotide-dependent conformational change of kinesin's NL in the absence of MT seem not to be credible.
Therefore, the docking of the NL upon ATP binding seems not to be the only mechanism for the forward stepping of the dimeric kinesin, and other mechanisms must be involved.

Two-heads-interacting model
An alternative model for the hand-over-hand walking of the dimer has been constructed based both on the difference in the binding affinity between the next binding site on MT and the original one (see Fig.  2), as argued above, and on the existence of the interaction between the two heads of the dimer [4245]. This model is called two-heads-interacting (THI) model here.
As noted from the crystal structure of dimeric kinesin determined by Kozielski et al. [70], there exists a local interaction between loop L8b from one head and loop L10 from another head (Fig. 4). This local interaction maintains the dimer in the equilibrium conformation as shown in Fig. 4. Marx et al. [71] further revealed that the dimer has this equilibrium conformation in solution. Based on this evidence and the interaction potential of the kinesin head with MT, as argued above (Fig. 2), the THI model has been proposed (Fig. 5) [4245]. In contrast to the NLD model, where the movement of the trailing ADP-head to the leading position follows ATP binding to the leading head (the leading head is gated) (Fig. 3B), in the THI model the movement of the trailing head to the leading position follows Pi release from the trailing head (the trailing head is gated) (Fig. 5B). This is similar to the kinetic model proposed by Cross and his coworkers [4,15,72]. To explain their experimental data on kinesin backsteps induced by nucleotide, Guydosh and Block [73] have proposed that the leading head is gated. However, as shown in the Supplementary Material of Xie et al. [45], the experimental data can also be well explained by using the THI model. In the THI model, the interaction between the two heads makes a contribution to the movement of the trailing ADP-head to the leading position (Fig.  5B). Then, due to the thermal noise, the tethered ADP-head diffuses to the nearest binding site (III) by overcoming its interaction with the other head. Note that, in Fig. 5B, since the ADP-head is nearer to binding site (III) than to binding site (I), the ADP-head would have larger probability to diffuse to site (III) than to site (I) even if the two sites had the same affinity for the ADP-head. Thus, the interaction between the two heads in the model plays the similar role to the NL docking in the NLD model in the forward movement of the trailing ADP-head.
As in the NLD model, the THI model also requires that the tethered ADP-head can distinguish between forward-and backward-binding sites. Besides the possible biased-binding mechanism, as discussed in the NLD model, another more important mechanism is present in the THI model. Immediately after Pi release, the binding affinity of binding site (III) for ADP-head is higher than that of site (I) (see Figs. 2 and 5). This results in that the ADP release nearly always happens at the binding site (III), i.e., the forward-binding site. The two biased-binding mechanisms give a very high mechanochmeical coupling efficiency in the THI model.

Two-heads-interacting plus neck-linker-docking model
As discussed above, both the NL docking and the interaction between the two heads are determined characters for dimeric kinesin [61,62,70,71]. Thus, both characters should be included in the moving mechanism of the dimer along MT. Moreover, both the NL docking in NLD model and the interaction between the two heads in THI model play the same role in reducing the probability for the ADP-head in the leading position from moving backwards to the trailing position (see Figs. 3B and 5B). Based on these considerations, a hybrid model that is termed as "two-heads-interacting plus neck-linker-docking" (THI-NLD) model is proposed here (see also Fig. 5). As in the THI model, in the THI-NLD model the trailing ADP-head (red) also moves to the leading position after Pi release (Fig. 5B). Then the docking of the NL to the blue head after ATP binding further increases the energy barrier by ~ 1.5 k B T [68] to prevent the ADP-head (red) from moving backwards to the trailing position (Fig. 5D), but the docking has no effect on the energy barrier for the ADP-head to move from the equilibrium position (dotted red) to the MT-bound position (solid red).  The cycle begins with both heads binding to MT with the trailing head (red) in ATP state and the leading head (blue) in nucleotide-free state. The strain between the two NLs induced by strong bindings of both heads to MT accelerates the ATP hydrolysis to ADP.Pi and Pi release in the trailing head (red) but reduces the rate of ATP binding to the leading head (blue). (B) After Pi is released, due to the very weak affinity between the ADP-head and the local binding site (I) (see Fig. 2B), the trailing ADP-head (red) is moved to the equilibrium position relative to the other head (blue) due to the affinity between two heads. (C) The new leading head (red) diffuses to the nearest binding site (III). Because the NL of the nucleotide-free new trailing head (blue) cannot be docked, the length of the NLs along MT is not long enough to allow the leading ADP head (red) binding appropriately to the binding site (III). Thus, the release of ADP from the leading head (red) is inhibited without the MT activation. Moreover, due to the weak interaction between ADP-head (red) and the binding site (III), the ADP-head (red) often diffuses backwards to the equilibrium position relative to the other head (blue), which is indicated by the arrow pointing from (C) to (B). (D) After an ATP binding to the trailing head (blue), its NL is docked, allowing the leading head (red) to bind appropriately to MT. Note that, before ADP release, the ADP-head (red) may also occasionally diffuse backwards to the equilibrium position (red head with dotted outline) relative to the other head (blue). (E) When ADP-head (red) binds appropriately to MT, activated by MT, ADP is released. Then, the state of dimeric kinesin becomes that of (A) except that the dimer has moved forwards by one step. Futile mechanochemical cycle: (A') Kinesin is in the same state as that in (A). (B') Consider an ATP binding to the leading head (blue) before ATP hydrolysis occurs in the trailing head (read). Note that, because the strain between the two NLs accelerates the hydrolysis of ATP in the trailing head (red) but reduces the rate of ATP binding to the leading head (blue), this case rarely occurs. (C') After ATP is hydrolyzed and Pi is released, the leading head (blue) diffuses to the equilibrium position. After the conformation of the binding site (II) relaxes to its normal form (see Fig. 2), the leading head (blue) rebinds to it and releases ADP. Then, the state of dimeric kinesin becomes that of (A). In this case, although an ATP is consumed, no mechanical step is made.

Concluding Remarks and Perspectives
In this review we have focused on the models for unidirectional motion of both monomeric and dimeric kinesin molecules and models for other behaviors such as limping of the dimer [16,74] have not been discussed. For either monomers or dimers, two classes of models for the unidirectional motion have been discussed. Moreover, a hybrid model has also been proposed based on the two classes of the model for the dimers.
For the monomer, while the FGP model is simpler than the FGLP model, it cannot explain several aspects of the dynamics of the monomeric kinesin. On the contrary, the latter model agrees with the available experimental data. For the dimer, though very differences between the NLD and THI models, most of the experimental observations for wild-type dimers can be well reproduced by using either of the two models [4146,7577]. However, the puzzling dynamic behaviors for a number of mutant homodimeric and heterodimeric kinesin molecules observed by Kaseda et al. [78,79] could not be described by the NLD model [80], but can be easily explained by the THI model [43]. Similarly, the experimental data for the wild-type dimers can also be well reproduced and the puzzling results for the mutant dimers can also be understandable by using the THI-NLD model. Moreover, based on the FGLP model for the monomer and the THI-NLD model for the dimer, it is evident that, whether the monomer is nonprocessive (corresponding to a weak binding affinity for MT in ADP state) or processive (corresponding to a high binding affinity), the dimerization can convert the diffusion motion along MT (monomeric mechanism) into the hand-over-hand motion (dimeric mechanism) [8183]. If the neck linkers of the dimerized molecule are longer than 8 nm, no internal strain between the two heads would exist. As a result, the ATPase rates of the two heads are comparable, resulting in that the two heads are sometimes in ADP state simultaneously. Thus, the dimer would display motility properties of both diffusion and hand-over-hand walking along MT, consistent with the recent experimental data of Hammond et al. [82].
As discussed above, the FGLP and THI-NLD models are likely the best candidates to describe the mechanochemistry of monomeric and dimeric kinesin molecules, respectively. The basis for the two models is that the conformational change of MT induced by the strong interaction with kinesin plays a critical role in the kinesin motility, which has not been paid much attention up to now. Thus, in order to elucidate the moving mechanism of kinesin along MT, the future studies, besides on the more detailed and subtle kinetics and dynamics of wild-type and mutant kinesins, should also be on the effect of MT such as the effect of its conformational change on the affinity for kinesin in different nucleotide states.
For this purpose, we can resort to the structural studies, for example, using high-resolution cryoelectron microscopy to study the structural difference between MT decorated with AMP-PNP-kinesin and that with ADP-kinesin, and, more interestingly, using X-ray crystallography to study the difference between structure of α-and β-tubulin molecules complexed with AMP-PNP-kinesin and that with ADP-kinesin. We can also resort to biochemical assays. For example, in one assay, MT is preincubated with nucleotide-free KIF1A of very high density so that nearly all α-and β-tubulin molecules are bound strongly by KIF1A. After adding ATP into the solution, it would be expected that the kinesin molecules detach from MT after ATP hydrolysis and Pi release, because, based on the FGLP model, the affinity for ADP-kinesin of nearly all bare binding sites on MT surface now becomes very weak for a short time. In another assay, when mixing MT with preincubated ADP-KIF1A, one would expect to observe the cosedimentation between the ADP-kinesin and MT, because the interaction of the ADP-kinesin with the unaffected MT is not weak.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy:
Biology
Computer Science

text:
Mouse Tissues Express Multiple Splice Variants of Prominin-1

Prominin-1, a heavily glycosylated pentaspan membrane protein, is mainly known for its function as a marker for (cancer) stem cells, although it can also be detected on differentiated cells. Mouse prominin-1 expression is heavily regulated by splicing in eight different variants. The function or the expression pattern of prominin-1 and its splice variants (SVs) is thus far unknown. In this study, we analyzed the expression of the prominin-1 splice variants on mRNA level in several mouse tissues and found a broad tissue expression of the majority of SVs, but a specific set of SVs had a much more restricted expression profile. For instance, the testis expressed only SV3 and SV7. Moreover, SV8 was solely detected in the eye. Intriguingly, prominin-1 knockout mice do not suffer from gross abnormalities, but do show signs of blindness, which suggest that SV8 has a specific function in this tissue. In addition, databases searches for putative promoter regions in the mouse prominin-1 gene revealed three potential promoter regions that could be linked to specific SVs. Interestingly, for both SV7 and SV8, a specific potential promoter region could be identified. To conclude, the majority of mouse prominin-1 splice variants are widely expressed in mouse tissues. However, specific expression of a few variants, likely driven by specific promoters, suggests distinct regulation and a potential important function for these variants in certain tissues.

Introduction
The pentaspan membrane glycoprotein prominin-1 is widely studied as a stem cell surface marker, both in human [1,2,3,4,5,6,7,8] and mouse [9,10]. The human orthologue of prominin-1, called CD133 has been used as a marker in a variety of cancers to isolate cancer stem cells (CSCs) [1,2,3,4,5,6] as well as hematopoietic stem cells [7,8]. Even though CD133 is broadly used as a marker for (cancer) stem cells, the protein is also detected in more differentiated cell types [11,12,13,14] and its exact function of CD133 on (cancer) stem cells remains enigmatic. However, it is quite evident that the expression of CD133 is heavily regulated. Human CD133 is reported to have seven splice variants (SVs) [15], which are under the control of five different promoters [16]. In addition, the CD133 protein is glycosylated, which, as we have shown before, is dependent on differentiation status of the (cancer) cell [15].
As in human, the murine prominin-1 is also expressed in differentiated cell types, shown by immunostainings for prominin-1 on mouse tissues [9,14,17] as well as by a prominin-1 LacZ/+ mice, that express LacZ in all prominin-1 expressing cells [9,10,14]. In addition, colon tumors displayed an overall expression of prominin-1 [14], suggesting that also in mouse, prominin-1 expression is not restricted to a (cancer) stem cell state. Interestingly, prominin-1 did only mark the stem cell fraction in the small intestine [9,10], indicating that the regulation of this protein might be different in this tissue.
Like the human orthologue, the prominin-1 protein can undergo heavy modification by glycosylation of its eight different N-linked glycosylation sites. In addition, the existence of a minimum of the eight SVs [10,17] point to the possibility that mouse prominin-1 is highly regulated, although its promoters have not been identified yet.
The alternative splicing mostly affects the cytoplasmic Cterminus, resulting in four different C-terminal tails [17]. Differentially splicing of C-terminal tails suggest that prominin-1 SVs might interact with distinct cytoplasmic binding partners, potentially inducing specific signaling pathways and thereby exerting separate functions. Although cytoplasmic binding partners have not been identified for prominin-1, the C-terminal tail of SV3-5 resembles a class II PDZ-binding domain, while the C-terminal tail of SV1-2 and SV7-8 harbors characteristics of a class I PDZ-binding domain [18]. PDZbinding domains are are thought to organize and regulate signaling complexes via protein-protein interactions. In agreement, a yeast two-hybrid screen showed that SV2 binds to a PDZ-domain containing novel splice variant of the glutamate receptor-interacting protein [18]. In human, Src and Fyn can phosphorylate two tyrosine residues on the C-terminal part of prominin-1 [19]. Altogether, this suggests that regulation of SV expression might influence the (signaling) function of the prominin-1 protein.
To gain more insight in the regulation of prominin-1 in mice, we decided to study the prominin-1 SVs by analyzing their expression pattern on protein and mRNA in several mouse tissues. We found that most SVs were expressed in all tissues. However, SV8 was specifically expressed in the eyes, whereas SV3 was only found in the eyes, testis and colorectal (CRC) cell line CMT93. In addition, SV7 was highly expressed in the testis. Interestingly, via database searches, we were able to identify a specific potential promoter region for both SV7 and SV8, suggesting that these two SVs have a more directed regulation and could therefore have a specific function.

Ethics Statement
Mice were maintained and experimented on in accordance with the guidelines of and after approval by the Dier Experimenten Commissie (DEC) of the Academic Medical Institute under permit number DIX100578.

Mice tissues
Mouse tissues were obtained from C57BL/6J (WT) mice. The APC Min colon and polyp were obtained from C57BL/6J-ApcMin/J mice. After the animals were sacrificed, the tissues were retrieved and snap-frozen in liquid nitrogen.

PCR
Total RNA was isolated from homogenized tissues by TRizol extraction (Invitrogen) according to manufacturers' protocol. Quality and quantity of the RNA was measured on the Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies). With equal input of total RNA, cDNA was prepared with reverse transcriptase III (Invitrogen) using random primers. The PCR primers used for the splice variants are listed in Table 1. For GAPDH, the following promers were used: forward 59-AT-GTGTCCGTCGTGGATCTGA-39and reverse 59-ATGCCT-GCTTCACCACCTTCT-39. The PCRs were run at either 53uC or 54uC according to the following PCR program: 95uC for 4 min, then cycling for 26-34 cycles with 95uC for 30 s, 53/54uC for 30 s, 54uC for 30 s 72uC for 30 s followed by a final elongation step at 72uC for 10 min. PCRs were run on a 2% agarose gel.

Protein isolation, immunoblotting and treatment of lysates with PNGaseF
For protein extraction, tissues were homogenized in lysis buffer (20 mM Tris HCl, pH 7.4, 137 mM NaCl, 10% Glycerin, 1% Triton-X-100, 2 mM EDTA, SPI and 1 mM PMSF). Protein amounts were measured by BCA protein assay according to manufacturer's protocol. For deglycosylation experiments, protein lysates (40 mg of protein) were incubated with glycoprotein denaturing buffer (5% SDS, 0.4 M DTT) (New England Biolabs #B1704S) for 10 min at 95uC. After denaturation, G7 buffer (#B3704S) and 10% NP-40 (#B2704S) were added to the samples. Subsequently, the samples were split in two. One aliquot treated with 500 units/ml PNGase F whereas the other served as an untreated control. Both aliquots were incubated overnight at 37uC. Per lane, 20 mg protein was loaded on 8% acryl amide gels and consecutively blotted onto PDVF membrane. Unspecific binding was blocked by 5% milk in PBS-0.1% Tween for 2 hours. Blots were incubated with either rat-anti-mouse prominin-1 (13A4, eBioscience) or rabbit-anti-actin (I-19, sc-1616, Santa Cruz) overnight in blocking buffer, subsequently washed in PBS containing 0.2% Tween and incubated with HRP-labeled goatanti-rat IgG (I1306, Santa Cruz Biotechnology) or goat anti-rabbit IgG (7074, Cell signaling) for 1 hour. For chemoluminescent visualization, ECL from Amersham Biosciences was used. The 13A4 blots were developed by the use of films, whereas the actin blots were developed by use of the FujiFilm LAS-3000.

mRNA and putative promoter database and computer analyses
Analysis of mRNA and EST sequences was performed used the National Center for Biotechnology Information database (http:// www.ncbi.nlm.nih.gov). Sequences were aligned in Vector NTI. Putative promoter regions were analyzed by comparing them to the genomice sequence (http://genome.ucsc.edu/). Potential transcription factor binding sites on the putative promoter regions were studied by using TFSearch (http://molsun1.cbrc.aist.go. jp/research/db/TFSEARCH.html) and Alibaba (http://www. gene-regulation.com/pub/programs/alibaba2/index.html). The putative promoter region was analyzed for a potential CpG island in CpG Island Searcher (http://cpgislands.usc.edu/cpg.aspx).

Protein expression of prominin-1 splice variants in mouse tissues
Prominin-1 is a five transmembrane protein consisting of an extracellular N-terminal part, two large extracellular loops and an Table 1. PCR primers used for discriminating prominin-1 splice variants. intracellular C-terminal tail. Extensive splicing of prominin-1 has been reported and this mostly affects the composition of intracellular C-terminal tail [17,21]. In addition, some differentially spliced exons are localized in regions that are transcribed as part of the extracellular loops of the prominin-1 protein (Fig. 1A). Altogether, differential splicing results in distinct protein sizes, with different molecular weights and native iso-electric points ( Table 2).
To gain more insight in the prominin-1 expression on protein, several organs from wild type C57Bl\6 mice, like brain, eyes, kidney, pancreas and testis and CRC cell lines like MC38, CC26, CC36 and CMT93 were lysed and protein samples were loaded on an immunoblot. Blotting with the 13A4 antibody detected the prominin-1 protein as one or multiple bands in these organs (Fig. 1B), ranging from ,90 to 116 kD. The protein's apparent molecular weight was much higher than predicted as in Table 2. As prominin-1 is a highly glycosylated protein [17,22] with eight potential N-linked glycosylation sites on two extracellular loops, the difference between the detected molecular weight and the predicted molecular weight was likely caused by glycosylation of the prominin-1 protein. Although differential splicing does not remove any N-linked glycosylation sites on the protein (Table 3), the glycosylation of the prominin-1 protein might be dependent on splicing and on the tissue in which it is expressed.
We therefore treated the lysates with PNGaseF to remove Nlinked glycans and analyzed the prominin-1 detection on an immunoblot (Fig. 1C). Deglycosylation of the protein reduced, as expected, the molecular weight to 90-98 kD. In addition, multiple sizes of prominin-1 protein were visible on the blot, suggesting that different tissues express distinct SVs. In principle, it should be possible to discriminate between SV4/5 (,90 kD), SV6/7 (,92 kD), SV 3/8 (,94 kD) and SV1/2 (,97 kD) on a immunoblot, but in practice, this was difficult to achieve, since the difference in size were too small and different bands could be linked to several SVs.
Altogether, these data indicate that protein detection of prominin-1 SVs in different mouse tissue reveals the expression of different SVs. However, it is not a sufficiently discriminative approach to analyze differential usage of SVs. This is in part due to differential glycosylation, but also due to relatively similar sizes of some SVs after deglycosylation.
Multiple prominin-1 splice variants are expressed in mouse organs To obtain more insight into the prominin-1 SV expression in mouse tissues, we decided to study their expression on mRNA level. Aligning all mRNA and expressed sequence tags (ESTs) sequences of prominin-1 from the NCBI database, we confirmed the eight known SVs and their exon organization, as has been published before ( Fig. 2A) [18]. To evaluate the expression of the different prominin-1 SVs in several tissues on mRNA level, we designed several distinct primer sets to specifically amplify the alternatively spliced regions (Table 3, Fig. 2C-H). We isolated mRNA from several mouse tissues like eye, heart, pancreas, testis and different parts of the gastrointestinal tract as confirmed by a GAPDH PCR (Fig. 2B).
SV1 and SV2 were discriminated from the other SVs by using primers that amplify the region from exon 2 to exon 25, which is specifically expressed in SV1/2 (Fig. 2C). To discriminate between SV1 and SV2, a nested PCR on this exon 2-25 PCR product was performed, amplifying exon 3a to 4b (Fig. 2C), because SV1 splices out exon 3b, whereas SV2 retains this exon. The data from this PCR showed that all tissues expressed SV1 as well as SV2, but the eyes showed the highest SV2 expression. Although this nested PCR is able to indicate if SV1 and SV2 are expressed in these tissues, the data can not be used to interpret differences in expression. This is illustrated by figure 2G, in which SV1 and SV2 are analyzed together in a direct PCR, which shows that there are quite some expression differences between tissues, e.g. there is very low expression of SV1/2 in the heart, liver and pancreas, whereas the kidney and all parts of the gastrointestinal tract highly express these SVs (Fig.2G).
The expression pattern of SV3 in mouse tissue was detected by amplifying exon 9 to exon 24a, which is specifically expressed in SV3 and the longer SV6 (Fig. 2D). Interestingly, the PCR data showed that SV3 is exclusively expressed by the eyes, the testis and CRC cell line CMT93 (Fig. 2D), suggesting that this SV has restricted expression in mouse tissues.
SV4 and SV5 were analyzed together, due to only a minor difference (15 bp) between the two variants. Also, these SVs have been shown earlier to be unable to reach the cell surface and therefore they are unlikely to have a prominent function. Primers amplifying the region from exon 8 to exon 11 only were designed to discriminate SV4/5 (which do not express exon 9) from the other SVs and the PCR data showed that SV4/5 were expressed to a low extend in all tissues (Fig. 2E). In addition, by comparing Figure 2F, in which we analyze SV3-5 together, with Figure 2D, in which only SV3 and SV6 were analyzed, we can conclude that the expression level of SV4/5 is very low, consistent with the idea that these would encode non-functional, potentially aberrant splice variants.
The SV6 expression pattern was analyzed by making use of the fact that this splice variant retains intron 23. The retention will induce an alternative stop codon, thereby inducing a shorter protein, although the mRNA sequence will be longer due to this retention. A PCR performed with primers amplifying exon 18 to exon 24a can therefore discriminate between SV6 and SV3-5 and showed that SV6 is expressed in a range of mouse tissues, but is virtually absent in the heart, liver, stomach and testis (Fig. 2F). These data are confirmed by the PCR performed in Figure 2D.
SV7 and 8 can be distinguished from the other SVs by using primers that amplify from exon 18 to exon 26, because only SV7 and SV8 express exon 26 without exon 24b and 25. To discriminate between SV7 and SV8, the PCR product includes the differentially spliced exon 19a. SV7 was found to be expressed by all tissues except for brain, heart, liver and pancreas (Fig. 2G). SV8 was only found to be expressed in the eye. A separate PCR was performed to confirm the SV8 expression profile, using primers to amplify exon 19 (specifically expressed by SV8) to exon 23. This also showed that SV8 was only expressed in the eyes (Fig. 2H).
Altogether, we can conclude from the prominin-1 mRNA SV expression pattern (Fig. 2, Table 4) that 1) the heart, liver and pancreas hardly express prominin-1; 2) SV1/2 are expressed in all other tissues, although at varying levels; 3) SV4/5 are hardly expressed in any of the tissues; 4) SV3 is only expressed in the eyes, testis and CRC cell line CMT93; 5) SV8 is uniquely expressed in the eyes; 6) the testis only expressed SV3 and SV7 and 7) the eye expressed SV2, SV3, SV6 and SV8. Altogether,this suggests that only SV3 and SV8 might have a more specific function in these tissues dependent on their splicing. The other SVs probably have either identical and/or redundant functions.

Putative promoter region of prominin-1
For human, it has been shown that multiple promoters exist that can drive the expression of prominin-1 [16], although no correlation was shown so far between specific promoter activity and distinct SV expression. We hypothesized that mouse prominin-1 is also regulated by several promoters. To this end, all 59UTR of the different splice variants and the expressed sequence tags (ESTs) as described in the NCBI database were analyzed ( Table 5). The alignment of the 59UTRs to the genomic DNA revealed two extra exons in the 59UTR region of the prominin-1 gene in addition to the previous identified exon (Fig. 3A). One exon (exon A in Fig. 3A) was found approximately 7.2 kb upstream of exon 2. This exon was specifically found in the 59UTR of SV8. Exon 1, referred to as exon B in Fig. 3A, is located 6.9 kb upstream of exon 2, and seems to be mainly expressed in the 59UTR of SV1 and SV2. A third exon (exon C), located 4.3 kb upstream of exon 2, was only found in the 59UTR of SV7. SV3-6 did not contain a 59UTR as described on the NCBI database and therefore, no additional 59UTR exons and putative promoter  regions could be identified for these SVs. Interestingly, two of the three found 59UTRs were linked to SVs that show specific expression. Exon A was found in SV8, which is specifically expressed in the eye. Exon B was found in SV7, which is expressed in multiple tissues, but highly in the testis. Next, we analyzed if these putative promoter regions contain putative transcription factor binding sites, CpG islands or TATA box regions. No TATA boxes were found, but a potential CpG island was identified starting in putative promoter 2, spanning approximately 1500 bp (data not shown). In addition, many putative transcription factor binding sites were found in each of the putative promoter regions ( Fig. 3B and C).
To conclude, specific 59UTRs for SV1/2, SV7 and SV8 were found, suggesting that these SVs can be alternatively regulated by putative promoters. Different potential transcription factor binding sites were found in each of the potential promoter regions, indicating that the expression of these prominin-1 SVs might be differentially regulated.

Discussion
In this study, we showed that several mouse organs express multiple prominin-1 splice variants on mRNA, indicating that these different variants probably have partially overlapping functions. The distinct expression of SV3 and SV8 compared to the other SVs hint towards a specific regulation of these SVs, which suggests that these SVS have a unique role compared to the other SVs. In literature, there are some indications that prominin-1 SV expression is regulated. For example, SV1 is downregulated in the brain during development, whereas SV3 is upregulated [21], hinting towards specific regulation of SV expression. In contrast, we did not detect SV3 expression in the adult brain. Although this SV is almost identical to SV6 on both mRNA and protein and could therefore easily been mistaken for SV6, our PCR strategy was designed as such that the 39UTR of SV6, which is not present in SV3, could be indentified by PCR. Therefore we can conclude that SV6 is present in the brain and not SV3.
Although the exact function of prominin-1 and its SVs are thus far unknown, it has been shown to play an important role in morphogenesis of photoreceptor cells. For instance, patients that harbor mutations in the prominin-1 gene suffer from retinal degeneration [23,24,25]. In addition, prominin-1 deficient mice, which are viable and fertile, display blindness due to complete degeneration of mature photoreceptors [26]. Human CD133 was found to be important for morphogenesis of new disk membranes by interacting with actin (decreased by R373C mutation) and protocadherin 21 (PCDH21). One of the missense mutations found in patients with macular degeneration (R373C mutation) decreases this interaction between actin and CD133 and causes mislocalization of both CD133 and PCDH21 [25]. Interestingly, SV8 was only found to be expressed in the eye and in none of the other organs, which could potentially indicate that this SV may be of importance to the development of the eye. SV8 is the only SV that retains exon 19a, which is located in the second extracellular loop. In human, a similar CD133 SV has not been identified yet.
As been shown for human [16], we found several distinct putative promoter regions for the mouse prominin-1 gene. Table 4. Expression of prominin-1 splice variants in mouse tissues.
Organs SV 1 SV 2 SV 3 SV 4&5 SV 6 SV 7 SV 8 Semi-quantitative analysis of prominin-1 splice variant PCR products. V means that the SV is expressed in the tissue, X means that no PCR product is observed. doi:10.1371/journal.pone.0012325.t004 Figure 2. Expression of prominin-1 splice variants in mouse organs. A. Schematic representation of prominin-1 splicing. Differentially spliced exons are indicated by distinct colors. B. PCR performed on mouse tissues with primers amplifying GAPDH to confirm mRNA expression C-H PCR data representing prominin-1 SV expression in mouse tissues. Each subpanel contains the analysed SVs, the location were the primers bind (arrows) and the acquired products after performing the PCR. Primers sequences can be found in Table 3. C. PCR to specifically detect SV1 and SV2 with primers amplifying exon 2 to 25, only SV1 and SV2 products were obtained. Additionally, a nested PCR amplifying regions specific for SV1 and SV2 was performed. N1 and N2 stand for nested primer sets. D. SV3 was discriminated from the other SVs by amplifying the region from exon 9 to exon 24a. E. SV4 and SV5 were analysed together due to only a minor difference between these SVs (15 bp Although the functionality of these promoters remains to be defined, the specific region found in the 59UTR of SV8, the SV that is only expressed in the eye, hints towards a selective regulation of this prominin-1 SV expression by an eye-selective promoter. In addition, as shown for human CD133 promoters [27,28] , regulation by methylation could explain for example the selective expression of SV8 in the eye. Also, the many putative binding sites for the transcription factor 'heat shock factor' (HSF) in the putative promoter region 3, which appears to be linked to the on testis highly expressed SV7, are striking. Especially as HSF is known to be involved in chromatin reorganization in sperm [29,30], suggesting that this transcription factor might regulate prominin-1 SV7 expression in the testis. We have shown that most of prominin-1 SVs are differentially expressed in the majority of mouse tissues. Especially, SV3 and SV8 expression is retained to specific tissues. Further studies will have to reveal if these SVs have the capacity to interact with specific binding partners or induce specific signaling. Specific prominin-1 SV expression combined with selective promoter usage hints towards a complex regulation of prominin-1 in mice, as was found for humans. Additional studies, revealing the function, ligand and/or binding partners of prominin-1 will shed more light on the possible function of this heavy regulation.