The journey of tetanus and botulinum neurotoxins in neurons

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The journey of tetanus and botulinum neurotoxins in neuronsq Giovanna Lalli1, Stephanie Bohnert2, Katrin Deinhardt2, Carole Verastegui2 and Giampietro Schiavo2 1

MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, UK Molecular NeuroPathobiology Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK 2

Anaerobic bacteria of the genus Clostridia are a major threat to human and animal health, being responsible for pathologies ranging from food poisoning to gas gangrene. In each of these, the production of sophisticated exotoxins is the main cause of disease. The most powerful clostridial toxins are tetanus and botulinum neurotoxins, the causative agents of tetanus and botulism. They are structurally organized into three domains endowed with distinct functions: high affinity binding to neurons, membrane translocation and specific cleavage of proteins controlling neuroexocytosis. Recent discoveries regarding the mechanism of membrane recruitment and sorting of these neurotoxins within neurons make them ideal tools to uncover essential aspects of neuronal physiology in health and disease. During the last century, the study of host –pathogen interactions has offered scientists a glimpse of the different strategies that have resulted from the evolutionary race between eukaryotic cells and competing microorganisms. One outcome of this evolutionary pressure was the refinement of specific virulence factors that interfere with fundamental cellular processes, which are hijacked to the advantage of pathogenic organisms [1,2]. The biochemical analyses of these molecules and the characterization of their cellular mechanisms of action have yielded a repertoire of targets for vaccine development and therapeutic intervention. Furthermore, these findings have armed biologists with a toolkit to dissect cellular functions of eukaryotic cells in health and disease [1]. Among bacterial virulence factors, tetanus (TeNT) and botulinum neurotoxins (BoNTs) emerge as the most powerful with respect to specificity, overall toxicity and importance for human and animal health [3,4]. TeNT and BoNTs (seven different serotypes named from A to G) form the clostridial neurotoxin family and are the causative agents of tetanus and botulism, respectively. They are structurally organized into heavy and light chains linked by a disulphide bond [3,5] (Fig. 1a). The light chains are zinc-endopeptidases specific for core components of the neurotransmitter release apparatus [3,5]. q Supplementary data associated with this article can be found at doi: 10.1016/ S0966-842X(03)00210-5 Corresponding authors: Giovanna Lalli ([email protected]), Giampietro Schiavo ([email protected]).

These proteins (VAMP/synaptobrevin, SNAP-25 and syntaxin 1) are necessary for the fusion of synaptic vesicles (SV) at the nerve terminal and constitute the synaptic members of the SNARE family [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor]. SNAREs play a central role in all membrane fusion events occurring in eukaryotic cells [6]. The selective proteolysis of synaptic SNAREs accounts for the total block of neurotransmitter release caused by clostridial neurotoxins in vivo [5,7]. Following the identification of their intracellular targets, clostridial neurotoxins became the tool of choice to study synaptic SNARE function in vitro and in vivo [5,7]. However, other aspects of TeNT and BoNT pathogenesis remain poorly understood. In particular, little is known about the mechanisms used by these toxins to interact with the neuronal membrane and the sorting events responsible for their intracellular trafficking. Remarkably, both BoNTs and TeNT bind and are internalized at the neuromuscular junction (NMJ). However, at physiologically relevant concentrations, TeNT enters the axonal retrograde transport pathway and reaches the soma of motor neurons located in the spinal cord (Fig. 2). TeNT is then transcytosed to inhibitory interneurons where it blocks neuroexocytosis [5,7]. By contrast, BoNTs remain in the periphery, blocking acetylcholine release at the NMJ (Fig. 2). At high concentrations, BoNT-A is also retrogradely transported to the spinal cord [8– 10], a phenomenon which might explain the effects on the central nervous system observed in vivo with this serotype [11]. This review highlights recent findings on the interaction of clostridial neurotoxins with the neuronal surface and the use of TeNT to monitor axonal retrograde transport in neurons. The HC fragment and its binding to neurons The absolute neurospecificity of clostridial neurotoxins is a major determinant of their outstanding toxicity [3]. The C-terminal portion of the heavy chain (HC) is responsible for neurospecific binding and, in the case of TeNT, retrograde transport [7] (Fig. 1a). Recombinant HC fragments bind to the functional receptors of TeNT and BoNTs, because they counteract the paralysis induced by parental neurotoxins in cultured neurons and isolated NMJ [12]. The crystal structures of the HC fragment of

http://www.trends.com 0966-842X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0966-842X(03)00210-5

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Fig. 1. Scheme of clostridial neurotoxins and their domains. (a) Tetanus (TeNT) and Botulinum neurotoxins (BoNTs) are composed of two chains, heavy (H) and light (L), held together by a disulphide bridge and non-covalent interactions. The L chain (blue) is a zinc-endopeptidase specific for SNARE proteins, which are core components of the neurotransmitter release apparatus. Several residues paving the active cleft coordinate the zinc ion and play an essential role in catalysis. Among these, two histidines belonging to catalytic motif of zinc-endopeptidases (HExxH) and a glutamic acid form the coordinating inner shell. In addition, a tyrosine and an arginine are involved in transition state stabilization and are crucial for the endopeptidase activity [62–64]. The H chain is composed of a 50 kDa N-terminal domain (HN; green) involved in membrane translocation [5,45] and a C-terminal portion (HC) responsible for neurospecific binding and retrograde transport. This domain is formed by two independently folded sub-domains: HCN (orange) and HCC (red). BoNT and TeNT HC fragments can be expressed as recombinant proteins with a cysteine-rich tag at the N-terminus, which binds the fluorescein arsenical helix binder, bis-1,2 ethanedithiol adduct (FLASH) or Alexa maleimides. Recently, these fragments have been shown to be very useful tools to monitor membrane dynamics and retrograde axonal transport in neurons [43,44,52]. (b) Structure of the TeNT HC fragment with a soluble analogue of ganglioside GT1b (in space-filling mode) bound to one of its four carbohydrate-binding sites [15] (PDB accession number 1AFV2).

TeNT [13 – 15] and of BoNT-A and -B [16– 18] clearly show that HC is composed of two distinct sub-domains of almost identical size (Fig. 1b). The structure of the N-terminal domain (HCN) resembles that of the carbohydrate-binding moiety found in lectins and is well conserved throughout the clostridial neurotoxin family [19]. The highest sequence divergence between BoNTs and TeNT is present in the C-terminal domain (HCC) and probably accounts for the distinct binding properties and sorting of TeNT and BoNTs. HCC adopts a modified b-trefoil fold, which presents four distinct carbohydrate-binding regions [14,15]. The most structurally conserved oligosaccharidebinding pocket, initially crystallized with bound lactose [14], plays a major role in the interaction with surface receptors containing sialic acid, such as polysialogangliosides and, possibly, neuronal glycoproteins (see below). Mutagenic analysis of this region has shown that it is required for high-affinity interaction with neuronal membranes [20,21]. This carbohydrate-binding domain also interacts with a soluble derivative of ganglioside GT1b [15] (Fig. 1b) and has been predicted to be the gangliosidebinding site in BoNT-A. However, this is not the only determinant for HC binding: another carbohydrate-binding pocket, which has been shown to dock sialic acid [14], is also required for high-affinity binding to neuronal membranes and TeNT toxicity [21]. Accordingly, the deletion of two of the three loops underlying this pocket causes loss of binding to motor neurons and inhibition in the ability of TeNT to undergo retrograde transport in vivo [22]. Thus, HC is a multivalent oligosaccharide-binding protein and its multiple sugar-binding sites mediate the recruitment of TeNT and BoNTs to specific carbohydrate-containing receptors on the neuronal surface. http://www.trends.com

Because HCC is both necessary and sufficient for binding to the neuronal surface and internalization [23], what is the cellular function of the HCN domain? Its structural homology with lectins suggests a role in the recognition of oligosaccharide-containing molecules, possibly during a late step in the intoxication process. HCN might play a role in the intracellular sorting of TeNT, which is similar to the mechanism proposed for ERGIC-53 and VIP-36 [24]. These animal lectins modulate the trafficking of glycoproteins and glycolipids in the secretory pathway, and their carbohydrate-binding activity is essential for their function. Thus, HCN might direct TeNT to a specific trafficking route or sort it to the transcytotic pathway, leading to its delivery at adjacent inhibitory interneurons. TeNT and BoNTs receptors TeNT and BoNTs bind to polysialogangliosides [7] and show a reduced activity in neurons in which ganglioside synthesis has been inhibited [25] or ablated [26]. However, their absolute neurospecificity and the overall lack of binding competition between TeNT and BoNTs [5] make it unlikely that polysialogangliosides represent the sole determinants for their binding to the neuronal surface. In agreement with the double lipid and protein receptor model [27], specific protein co-receptors for clostridial neurotoxins have been characterized. BoNT-B interacts with members of the synaptotagmin family [28– 30], which constitute the main calcium sensors located at the synapse [31]. Recently, a 15 kDa glycosylphosphatidylinositol (GPI)-anchored glycoprotein that interacts with TeNT and its HCC fragment, but not with BoNTs, was isolated [23,32] (Fig. 3; inset). This TeNT-interacting protein has been identified as Thy-1 in differentiated PC12 cells [33].

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Fig. 2. Schematic view of a mammalian motor neuron and an interacting spinal inhibitory interneuron. The sites of action of tetanus (TeNT; green) and botulinum neurotoxins (BoNTs; blue) are shown, together with their specific intracellular trafficking route. At the neuromuscular junction (NMJ), BoNTs are internalized in synaptic endosomal compartments, one of which might coincide with synaptic vesicles. By contrast, TeNT is sorted to the retrograde transport pathway. Microtubule tracks are shown in dark brown, whereas actin microfilaments are in red. Both cytoskeletal elements are required for fast retrograde transport of TeNT in motor neurons. Red crosses indicate the preferential sites of neurotransmitter release inhibition caused by BoNTs (NMJ) and TeNT (inhibitory interneuron synapse of the spinal cord).

Thy-1 is a GPI-anchored glycoprotein present in the brain, and has been implicated in several cellular functions, including signaling and neurite outgrowth [34,35]. Despite this evidence, it is improbable that Thy-1 is the unique protein receptor for TeNT as Thy-1 knockout mice have been shown to retain sensitivity to TeNT [33] and spinal cord cells isolated from these animals were able to bind and internalize TeNT HC (J. Herreros et al., unpublished). However, treatment of neurons with a phospholipase that http://www.trends.com

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cleaves the lipid anchor of GPI-anchored proteins protects neurons from TeNT intoxication [36], which suggests that one or more unidentified GPI-anchored glycoproteins act as a physiological TeNT receptor. Lipid rafts and clostridial neurotoxin binding GPI-anchored proteins, together with cholesterol, gangliosides, and other sphingolipids, are enriched in microdomains of the plasma membrane termed lipid rafts, which act as functional platforms for signaling, ligand recognition and sorting [37,38]. The interaction of TeNT HC with two classes of raft-associated components, polysialogangliosides and GPI-anchored proteins, suggests that the binding of TeNT to neurons is mediated by lipid microdomains (Fig. 3). Accordingly, TeNT HC, as well as BoNT HC, displays a punctate distribution on the surface of neurons, which closely resembles that of lipid raft markers [12,33]. In neuronal cells, HC fragments of TeNT and BoNTs, as well as native neurotoxins, associate with detergent-insoluble glycolipid-enriched membranes (DIGs) [33,39]. Disruption of lipid microdomains with cholesterol-sequestering drugs, such as methyl-b-cyclodextrin, inhibits the association of TeNT HC with DIGs in spinal neurons [33]. In addition, treatment with methyl-bcyclodextrin abolishes TeNT HC internalization [33] and protects neurons from the endopeptidase activity of TeNT on VAMP/synaptobrevin [33,36], implicating lipid rafts in TeNT trafficking. Therefore, lipid rafts represent a common requisite for the recruitment of BoNTs and TeNT to the neuronal plasma membrane. However, the characteristic trafficking of BoNTs and TeNT predicts that these neurotoxins bind to distinct lipid rafts. The different type and composition of these lipid microdomains is expected to determine the sorting events responsible for the distinctive intraneuronal targeting of BoNTs and TeNT. Evidence that supports the presence of differently-regulated membrane microdomains is rapidly accumulating, together with the notion that the organization and distribution of rafts within the plasma membrane is likely to determine their distinct signal outputs and, ultimately, their cellular fate [37]. Lipid rafts are widely exploited by pathogens and virulence factors for their entry into cells [2,40,41]. This common strategy for cellular invasion could be due to the fact that lipid microdomains provide efficient and dynamic means for receptor clustering. Moreover, lipid rafts allow the oligomerization of virulence factors when locally concentrated on restricted domains of the plasma membrane [2,41]. Multivalent binding of the HC domain to polysialogangliosides [15] and proteins within lipid rafts might trap clostridial neurotoxins on the neuronal surface, endowing them with the high affinity observed in vivo and in vitro [5,7]. Interaction with lipid rafts could also result in clostridial neurotoxins being targeted to specialized sites for endocytosis and sorting, where the neurotoxins could take advantage of a pre-assembled signaling machinery for their internalization. In this regard, the binding of TeNT HC to brain synaptosomes and cortical neurons activates neurotrophin tyrosine receptor kinase (Trk) receptor-dependent signaling [42]. This mimics the signaling cascade triggered by neurotrophin binding,

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Fig. 3. The four-step cellular mechanism of action of clostridial neurotoxins. Similar to other bacterial protein toxins of the A-B family [65], tetanus (TeNT) and botulinum neurotoxins (BoNTs) follow a four-step mechanism to enter and inhibit neurons: membrane binding (1), internalization (2), translocation (3) and intracellular action (4). (1) Clostridial neurotoxins bind to polysialogangliosides, including GT1b, and specific proteins on the surface of neuronal cells. These lipid and protein receptors cluster in lipid microdomains (yellow segments), which are enriched in cholesterol (in green; inset) and sphingolipids (in yellow). In neuronal cells, TeNT binds polysialogangliosides (in magenta) and GPI-anchored proteins (in blue), including Thy-1, within lipid rafts. (2) Neurospecific binding is followed by internalization and sorting to specific intracellular routes which differ for BoNTs and TeNT. TeNT enters non-acidified carriers that are recruited to the fast retrograde transport pathway and then reaches adjacent inhibitory interneurons via transcytosis. BoNT-containing endocytic structures instead remain at the neuromuscular junction. (3) Upon arrival at their final destination, the light (L) chain has to cross the endocytic membrane to reach the cytoplasm. This translocation process is assisted by the N-terminal portion of the heavy chain (HN) [5,45] and is triggered by acidification of the endosomal lumen. Acidic pH triggers a conformational change in the HN domain enabling its insertion into the lipid bilayer and the formation of a trans-membrane channel large enough to accommodate the unfolded L chain [65]. (4) Different L chains specifically cleave distinct members of the SNARE family. TeNT (T) and BoNT serotype B, D, F and G act on VAMP/synaptobrevin (in green) localized on SV. BoNT-A and E cleave SNAP-25 (in pink), whereas BoNT-C cleaves both syntaxin 1 (in cyan) and SNAP-25, two proteins of the pre-synaptic plasma membrane.

which is regulated by lipid rafts [38]. These events might functionally couple TeNT endocytosis with the transport machinery responsible for the axonal retrograde traffic of neurotrophins and other endogenous ligands in spinal cord motor neurons [43,44]. Intracellular sorting and axonal retrograde transport After binding to the neuronal plasma membrane, BoNTs and TeNT are targeted towards distinct regions of motor neurons (MNs) (Fig. 2). The action of BoNTs is mainly restricted to the NMJ, where they cause a long-lasting blockade of acetylcholine release [4,7]. The peripheral targeting of BoNTs is probably determined by the cellular properties of their protein receptors. BoNT-B binds to the intraluminal portion of synaptotagmin I and II [28], which is glycosylated, in a GT1b-dependent manner. This region is exposed to the extracellular milieu during neurotransmitter release, becoming accessible to large extracellular ligands. BoNTs might therefore exploit SV endocytosis and recycling for their entry into MNs. Moreover, the acidification of SV, which is necessary for their reloading with neurotransmitters, might mediate the acid-driven insertion of the H chain [5,45] into the SV membrane and the translocation of the L chain into the NMJ cytoplasm (Fig. 3). As shown recently for diphtheria toxin, translocation http://www.trends.com

and cytoplasmic refolding of the L chain might be assisted by a host cell translocation complex, which comprises heat shock proteins and thioredoxin reductase [46]. The latter enzyme might also play a role in the reduction of the disulfide bond joining H and L chains [5], thus allowing the release of the catalytically-active subunit in the cytoplasm. However, the role of synaptotagmins as physiological BoNT receptors remains controversial because antibodies against this synaptotagmin domain fail to antagonize the binding and activity of BoNT-B at the NMJ [47]. By contrast, TeNT is sorted to the fast axonal retrograde transport route, which is responsible for its delivery to the MN soma located in the spinal cord (Fig. 2). Axonal retrograde transport is required for neuronal growth and survival and allows communication over long distances [48]. To study this essential neuronal pathway, we established a real-time assay for the visualization and quantitative analysis of the retrograde transport of fluorescent TeNT HC [43]. In spinal cord MNs, two main groups of organelles are responsible for the retrograde transport of TeNT HC: round vesicles and tubules. Both types of carriers are characterized by distinct kinetic behaviour; vesicles move discontinuously, alternating frequent pauses to phases of movement, whereas tubules are faster and display a more continuous transport [43].

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Conflicting results on the retrograde transport efficiency of TeNT HC compared with full-length TeNT have emerged from previous studies [49,50]. However, MNs internalize fluorescently-labeled full-length TeNT in endocytic structures morphologically identical to those carrying TeNT HC [43] (Fig. 4a). Moreover, kinetic analysis of TeNT carriers shows a speed distribution that is strikingly similar to that observed for the TeNT HC-positive compartment (Fig. 4b), and overlapping with the rates calculated for TeNT retrograde transport in vivo (0.8– 3.6 mm/s) [5]. TeNT and TeNT HC display an extensive co-localization both in axons and somas of living MNs, although cells incubated with TeNT show less plasma membrane staining than with TeNT HC. This probably occurs as a result of a

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Fig. 4. TeNT and its heavy chain C-terminal fragment (HC) are transported along motor neuron (MN) axons in retrograde carriers with identical kinetic properties. (a) Cells were incubated with tetanus neurotoxin (TeNT) labeled with Texas Red and imaged by low-light time-lapse microscopy. Time series imaging of an MN axon (from left to right) shows TeNT-positive retrograde carriers (arrowhead, arrow, asterisk). The MN soma is located out of view at the bottom of the image. Intervals between frames are 5 s. (b) TeNT and TeNT HC carriers show overlapping kinetic behaviour. Relative frequencies of speed values observed between two consecutive frames for TeNT HC-Alexa488 (364 carriers, orange bars) and TeNTTexas Red (88 carriers, blue bars). Retrograde movement is conventionally shown as positive. http://www.trends.com

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faster TeNT internalization kinetic. Therefore, TeNT HC retains the ability to enter into retrograde compartments with the same transport features as those containing fulllength TeNT. However, additional TeNT domains, other than the HC portion, might be necessary to ensure fast internalization and optimal sorting to this trafficking route [50]. Fast retrograde transport of TeNT HC in MNs requires both F-actin and microtubules [44] and relies on dynein as well as kinesins and myosins [51]. Dynein acts as the main motor of these carriers. Mutations in the dynein heavy chain specifically impair fast retrograde transport of TeNT HC in isolated MNs and generate pathological conditions closely resembling amyotrophic lateral sclerosis [52]. Dynein could also act as a regulator of other molecular motors because its pharmacological inhibition affects the contribution of kinesins and myosins to retrograde transport [44]. As predicted by the need for F-actin, TeNT transport requires members of the myosin superfamily and, in particular, myosin Va. MNs from myosin Va-null embryos had slower retrograde transport than wild-type cells [44]. Therefore, the coordination of myosin Va and microtubule-dependent motors is required for fast axonal retrograde transport of TeNT HC in MNs. TeNT HC carriers lack markers of the classical endocytic pathway and SV. Notably, these retrograde structures are not acidified during transport [43], a condition necessary to retain TeNT within their lumen and to ensure its delivery to spinal inhibitory neurons. SV40 virus and Escherichia coli are internalized into intracellular compartments that do not belong to the endosomal system, involving cholesterol and glycosphingolipid-rich caveolae [40]. TeNT might therefore use a distinct endocytic pathway to bypass the endosomal – lysosomal system and escape degradation, as suggested by the long half-life of TeNT in spinal neurons [5]. Studies have also shown that TeNT, but not BoNTs, enter hippocampal neurons through SV recycling [53,54]. In MNs, TeNT HC is internalized in the absence of depolarization and displays very limited co-localization with the SV protein VAMP/synaptobrevin. This is in agreement with studies demonstrating that both TeNT uptake and retrograde transport are unaffected at NMJ in which neurotransmitter release has been inhibited [8]. The endocytic mechanism of TeNT in MNs could therefore be different from that occurring in other neurons, which suggests that TeNT might follow a pathway linked to SV recycling [51] to enter inhibitory interneurons within the spinal cord (Fig. 2). Which endogenous cargoes are transported on the intracellular route used by TeNT in MN axons? In a similar manner to TeNT, endogenous ligands might use the same pathway to escape degradation, reaching the neuronal cell body in a biologically active form. Recently, TeNT HC and nerve growth factor (NGF) have been shown to share the same retrograde organelles in MNs, a subpopulation of which contains the neurotrophin receptor p75NTR [43]. Similar retrograde transport rates for TeNT and NGF have been reported in adrenergic neurons in vivo, consistent with the speed calculated from time-lapse experiments in MNs [43]. Moreover, NGF is retrogradely

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transported via the neurotrophin receptor p75NTR in spinal cord MNs and accumulates in their somas without being degraded [55,56]. Although the endocytic compartment responsible for p75NTR trafficking has not been completely characterized, recent studies have highlighted the association of p75NTR with caveolae [38]. On this basis, lipid domains that are involved in the retrograde transport of p75NTR might play an important role in the trafficking of TeNT and other factors through a specialized endocytic route avoiding lysosomal targeting.

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picture. Frames were taken every 5 s. Shown here is a movie consisting of 45 frames played at 5 frames/s. The image is 7 £ 37 mm. Acknowledgements We thank the many colleagues whose work could not be cited here owing to space limitations. We are indebted to O. Rossetto, J. Herreros and N. Fairweather for helpful comments during the preparation of this manuscript. Laboratory work by the authors is supported by Cancer Research UK.

References

Conclusions Several studies on clostridial neurotoxins’ mechanism of action have advanced our understanding of the process of neuroexocytosis, promoting the functional characterization of SNARE proteins and their role in membrane fusion. However, novel applications based on the unique properties of BoNTs and TeNT still await full exploitation. One of the most attractive of these is the use of neurotoxins to dissect the neuronal basis of behaviour. The targeted expression of the L chain of TeNT in different model systems, such as Caenorhabditis elegans and Drosophila, represents a very powerful method to block neurotransmission in a restricted panel of neurons, selectively impairing their functions [57]. This non-invasive approach allows behavioural observations in freely moving animals and promises to provide, coupled with novel inducible expression systems, a very helpful strategy for the mapping of neuronal circuitry underlying specific functions. The absolute neurospecificity of clostridial neurotoxins, and the ability of TeNT to undergo axonal retrograde transport, make them ideal tools to study endocytosis and sorting at the synapse, both in vitro [43,44] and in vivo [58,59]. These processes, which are still poorly understood at the molecular level, represent an exciting area of application for clostridial neurotoxins and their binding fragments. This is not the only aspect of membrane dynamics at the nerve terminal that could take advantage of the unique properties of these toxins: BoNT-A has been found to block neurotransmitter release without arresting SV endocytosis, thus uncoupling the exocytic step from synaptic membrane recycling [60]. Finally, the regulation of neurotoxin synthesis in toxigenic bacteria represents an area of new investigation, which could have a profound impact on understanding the evolutionary steps that allowed the selection of such molecules. In this regard, the recent release of the complete genome sequence of Clostridium tetani [61] and Clostridium botulinum Hall strain A (http://www.sanger. ac.uk/Projects/C_botulinum/) provides the necessary resources for comparative studies between toxigenic Clostridia and their long-lasting hosts. Supplementary material See the supplementary video supplied with this article. Fluorescent tetanus neurotoxin (TeNT) is retrogradely transported in living rat motor neurons. Cells were incubated with 40 nM TeNT-Texas Red for 15 min at 378C, washed and imaged by time-lapse, low-light microscopy. The cell body is located at the bottom of the http://www.trends.com

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