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Molecular & Biochemical Parasitology 183 (2012) 63– 69

Contents lists available at SciVerse ScienceDirect

Molecular & Biochemical Parasitology

mmunofluorescent localization of thymidylate synthase in the development ofrichinella spiralis and Caenorhabditis elegans

arbara Gołosa, Magdalena Dabrowskaa, Elzbieta Wałajtys-Rodeb, Zbigniew Zielinski a,atrycja Winskaa, Joanna Cieslaa, Elzbieta Jagielskaa, Tadeusz Moczonc, Wojciech Rodea,∗

Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warszawa, PolandUniversity of Information Technology and Management in Rzeszów, Chair of Cosmetology, 2 Sucharskiego Street, 35-225 Rzeszów, PolandInstitute of Parasitology, Polish Academy of Sciences, 51/55 Twarda Street, 00-818 Warszawa, Poland

r t i c l e i n f o

rticle history:eceived 23 December 2011eceived in revised form 2 February 2012ccepted 5 February 2012vailable online 13 February 2012

eywords:

a b s t r a c t

Localization of thymidylate synthase protein in Trichinella spiralis and Caenorhabditis elegans developmentwas followed with the use of confocal microscopy, revealing similar expression patterns in both nematodespecies. In T. spiralis premature muscle larvae and C. elegans dauer, L3 and L4 larvae, thymidylate synthasewas detected in the nerve ring and gonad primordia, as well as T. spiralis stichosome and C. eleganspharyngeal glandular cells. In developmentally arrested T. spiralis muscle larvae, the enzyme was foundlocalized to the gonad primordia and stichosome. High enzyme level was also observed in the embryos

hymidylate synthaseonfocal microscopyrichinella spiraliseanorhabditis elegans

developing in uteri of T. spiralis female adult and C. elegans hermaphrodite forms. In the case of T. spiralisadult forms, thymidylate synthase was detected in stichosome, along esophagus wall, as well as in eggand sperm cells. While the enzyme protein present in the embryos remains in accord with its knownassociation with proliferating systems, thymidylate synthase presence in the nerve ring, and reproductive

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. Introduction

Thymidylate synthase (EC 2.1.1.45), catalyzing the N5,10-ethylenetetrahydrofolate-dependent C(5) methylation of 2′-

eoxyuridylate (dUMP), is the sole cellular source of de novoynthesis of dTMP, and thus a target in chemotheraphy [1].lthough thymidylate synthase specific activity is impossible toetect in quiescent cells [2], it has been found at high level in cer-ain nonproliferating systems, including unfertilized eggs of severalnvertebrates [3–5], the latter in accord with oocytes being arrestedn the cell cycle [6], hence not quiescent or out of the cell cycle G0hase [cf. ref. 7]. Therefore having found in non-growing Trichinellapiralis muscle larvae a surprisingly high thymidylate synthase spe-ific activity [7,8], persisting for at least two years after infectionnd accompanied by a high mRNA level [9], we hypothesized it to be

consequence of a global cell cycle arrest [7,9]. In accord with thisypothesis, high thymidylate synthase expression, demonstrated

t the mRNA, protein and specific activity levels, was found alson Caenorhabditis elegans developmentally arrested dauer larvae10], a life cycle stage corresponding to T. spiralis muscle larva [11],

∗ Corresponding author at: Nencki Institute of Experimental Biology, Polishcademy of Sciences, 3 Pasteur St., 02-093 Warszawa, Poland. Tel.: +48 22 5892477;

ax: +48 22 8225342.E-mail address: w.rode@nencki.gov.pl (W. Rode).

166-6851/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.molbiopara.2012.02.002

somal and C. elegans pharyngeal glandular cells) systems, points to a state to preserve the enzyme protein.

© 2012 Elsevier B.V. All rights reserved.

where a state of global cell cycle arrest has been considered alsobased on other observations [12]. Furthermore, the phenomenonmay be characteristic for developmentally arrested larvae of dif-ferent nematodes.

Since catalytic activity of thymidylate synthase in non-growinglarvae appears irrelevant [7; cf. ref. 13], a question of whetherthymidylate synthase protein may play a regulatory role in cellcycle/metabolism must be considered. It should be mentionedthat a potential regulatory role of thymidylate synthase protein issuggested also by its described non-catalytic activities, such as acapacity to bind and repress translation of its own and several othermRNAs [14] or oncogene-like activity [15]. T. spiralis is known toproduce within its excretory/secretory organ, stichosome, proteinsdetected in the cytosol and nuclei of infected muscle cells and foundto be secreted into epithelial cells [reviewed in ref. 16]. However,although larval excretory–secretory products are implicated in theformation/maintenance of the parasite intracellular habitat, called“nurse cell” [17], and their contents is being intensively studied[16,18,19], little is known of the inductive and regulatory mecha-nisms involved in reorganization of host muscles leading to nursecell formation or their meaning for infective larva penetration intoand adaptation in epithelial cells.

In order to learn localization of thymidylate synthase protein inT. spiralis and C. elegans development, immunofluorescence meth-ods were used to study larval forms, including developmentallyarrested T. spiralis muscle larvae and C. elegans dauer larvae, and

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dult worms of both species. In case of both species, apart fromeveloping embryos, high thymidylate synthase level was alsoound in gonad primordia and nerve ring of larval forms, as wells in excretory–secretory cells of T. spiralis larval and adult forms’tichosome and secretory cells of C. elegans L3 and L4 larvae phar-nx. These results point to gonadal, nerve ring and secretory cellspening to the esophagus in the case of T. spiralis, and to the phar-nx in the case of C. elegans, as potentially cell cycle-arrested, thehenomenon appearently characteristic for nematods rather than

ust T. spiralis.

. Material and methods

.1. Oligonucleotides

Digoxin-labeled oligonucleotide probes (Green StarTM DIG10),ere designed and synthesized by GeneDetect. Each 48-nt longrobe has a sequence complementary to the following regions ofhymidylate synthase coding sequence: (i) 56–103: 5′ GTGAAC-TGCTGTTGAACGGTTTTTACGACATCAGAAGGCGCATCGGC 3′,72–619: 5′ GTGACACGGTGGCAGAACCATTTGTCAAAGATCCGACG-ATTCCACGC 3′ and 832–879: 5′ CGCGTGAATCGAATTTTGGGGAA-GCGTATGGCTCACGGTCCAGTTGA 3′. DIG-labeled poly(dT) controlrobe was also 48-nt long and synthesized by GeneDetect.

.2. Nematode culture

T. spiralis human isolate H2 from the Instituto Superiore dianita, Rome, Italy, ref. no. MHOM/PO/88/ISS67 (T1) was used. Thearasite maintenance, mature muscle larvae (at least 30 days postral infection) and adult worm isolation were performed as previ-usly reported [9]. Premature muscle larvae were isolated 20 daysost oral infection, by incubation of minced mouse carcasses inhosphate buffered saline (PBS) at 37 ◦C for 2 h, followed by exten-ive washing with PBS. Intact nurse cells were isolated from theiaphragms of infected mice, at day 30 post infection, by restrictiveigestion with 0.25% trypsin/EDTA in PBS.

C. elegans wild type strain N2, variety Bristol, was maintainednd worms at specific developmental stages were obtained as pre-iously described [10].

.3. Anti-thymidylate synthase antibodies

The monoclonal antibodies were generated with recombinantat hepatoma thymidylate synthase, used as an antigen, andurified by thiophilic adsorption chromatography, as earlier pre-ented [24]. Immunoreactivity of monoclonal antibodies with T.piralis enzyme and lack of specificity towards mouse enzyme,ere demonstrated by immunoblot analysis presented as Sup-lementary Fig. I. The polyclonal antibodies were generated withecombinant C. elegans thymidylate synthase used as an antigen,s previously described [10]. Reactivity of thus obtained antibodiesas confirmed by immunoblot analysis presented as Supplemen-

ary Fig. II.Supplementary Figures I and II related to this article found, in

he online version, at doi:10.1016/j.molbiopara.2012.02.002.

.4. Thymidylate synthase immuno-localization studies

The procedure of specimen preparation was adapted from Millernd Shakes [20].

Fixation. Fixation was done with Ruvkun buffer containing 2%ormaldehyde in the case of T. spiralis or 1% formaldehyde and% glutaraldehyde in the case of C. elegans forms. Fixed samplesere stored at −80 ◦C. After thawing, glutaraldehyde was added to

l Parasitology 183 (2012) 63– 69

T. spiralis samples to a final concentration of 1%, samples were incu-bated for 1 h and washed three times in Tris buffered saline Tween20 (TBST). Delipidation of C. elegans forms was performed afterthawing, by five washes with solutions of increasing ethyl alcoholconcentrations (10%, 30%, 50%, 75%, 95%), acetone and subsequentlyethanol solutions used in the reversed order. Final wash steps wereperformed using TBST.

Permeabilization. This was done by incubating samplesovernight at 37 ◦C in TBST containing 1% �-mercaptoethanol, fol-lowed by two washes in borate buffer containing 0.01% Triton X100(or 1% Tween20 with C. elegans samples), incubating 15 min (30 minwith C. elegans) in the same buffer containing 10 mM dithiothreitol,washing again in the buffer alone and incubating for 15 min (30 minfor C. elegans samples) in the same buffer containing 0.3% H2O2.After final washes with TBST the samples were suspended in TBSTcontaining 3% BSA (1% for C. elegans forms) and 0.005% sodium azideand incubated overnight at 4 ◦C. Photobleaching of T. spiralis wormsas well as cytoplasm of nurse cells, was performed by exposure tosolar radiation for over two weeks, maintaining anti-UV protection.

Immunohistology. For T. spiralis thymidylate synthase proteinlocalization mouse monoclonal IgG 2a type antibodies were usedas a primary antibody, and goat, anti-mouse IgG-TRITC conjugate(Sigma–Aldrich) was applied as a secondary antibody. For local-ization of thymidylate synthase protein in C. elegans forms rabbitpolyclonal IgG was used as a primary antibody and goat, anti-rabbitIgG-TRITC conjugate (Sigma–Aldrich) was used as a secondary anti-body. Specificity of monoclonal antibodies binding was assessedby the following control reactions: (i) incubation of the primaryantibody with an excess of purified recombinant rat hepatomathymidylate synthase preceding incubation of worms with thisantibody, (ii) omission of the primary antibody treatment of worms(to test possible non-specific binding of the secondary antibody),(iii) treatment of worms with serum of T. spiralis-infected mice(considered to contain antibodies specific to cuticular antigens), inplace of the primary antibody (a positive activity control of the sec-ondary antibody). Immunofluorescence signal was detected only inthe third control reaction, confirming the applied antibodies com-bination to reflect thymidylate synthase-specific binding. In thecase of polyclonal antibodies, only lack of cross-reactivity of thesecondary antibody with the worm-specific epitopes was demon-strated not to occur.

2.5. Thymidylate synthase in situ hybridization studies

Preliminary permeabilization and prefixation of worms wereperformed as described by Miller and Shakes [20], with freeze-cracking repeated 3 times in 2-ml Eppendorf caps instead of onslides. After incubation in 2% formaldehyde for 30 min at 4 ◦C, prote-olysis, fixation and permeabilization were carried out as elsewheredescribed [21], with H3BO4 used at concentration 0.2 M, insteadof 1 M, and proteolysis ran overnight at 37 ◦C. Hybridization andprobe detection were performed according to In situ hybridizationusing GeneDetect oligonucleotides probes protocol, Version 1.4,provided by oligonucleotide manufacturer. Following hybridiza-tion, digoxin-labeled antisense thymidylate synthase and poly(dT)probes, used at final concentration of 1–2 �g/ml and 0.2 �g/ml,respectively, were detected using rhodamine-coupled anti-DIGantibodies (Roche), applied at 1:4–1:20 dilution range. Specificityof anti-thymidylate synthase probe hybridization was assessed byreferral to hybridization of poly(dT) probe, the latter resulting instaining of the whole worm specimen.

2.6. Confocal microscopy

For enzyme protein immunolocalization and in situ hybridiza-tion studies the specimens were mounted in 50–90% glycerol in PBS

B. Gołos et al. / Molecular & Biochemical Parasitology 183 (2012) 63– 69 65

Fig. 1. Thymidylate synthase immunolocalization during T. spiralis development. (1): A 300 �m-long premature muscle larva showing high fluorescence signal in the nervering (arrowhead), developing stichosome (arrow), its magnified image shown in (2), and gonad primordium (empty arrowhead); m – midgut. (3): An 800 �m-long prematuremuscle larva showing fluorescence signal distribution similar to that in 1. (4): Anterior part of an 800 �m-long premature muscle larva showing high fluorescence signallevel in the nerve ring (arrow) and the first anterior pair of stichocytes (arrowhead). (5): Magnification of the nerve ring of 800 �m-long premature muscle larva shown in(4) (the structure indicated by both arrows) and individual neurons (arrowheads). (6): A female muscle larva showing high signal in or around the stichosome nuclei. (7): Afemale muscle larva showing high fluorescence signal in the uterus primordium (arrowhead), its in silico magnified image shown in (8), and ovary primordium (arrow), itsmagnified image shown in (9); in (8) and (9) note the dark spots, corresponding to the nuclei positions, and cytoplasmic localization of the signal. (10): A male muscle larvawith high fluorescence signal in or around the stichosome (arrowhead) nuclei and in gonad primordium (arrow). (11): An adult female stichosome with high fluorescencesignal in the nuclear region (the arrows indicate positions of nuclei laying out of focus). (12): An adult female stichosome with high signal in the canalicular tree (arrowheads)and esophagus wall (arrows); panels (11) and (12) present different cross-sections of the same segment of an isolated stichosome.

66 B. Gołos et al. / Molecular & Biochemical Parasitology 183 (2012) 63– 69

Fig. 2. Thymidylate synthase immunolocalization during T. spiralis development (continued). (1): An adult female ovary, its in silico magnified image shown in (2), with highfluorescence signal in egg cells. (3): High signal in embryos developing in uterus of an adult female, a magnified image of the structure shown in (4). (5) and (6): High signalsdemonstrated by larvae before birth (5) and a larva isolated from the uterus (6), with arrows indicating dark spots corresponding to the nuclei positions. (7): Posterior partof a male adult showing fluorescence signal accumulation in the testis (t), seminal vesicle (sv), and secondary spermatocytes (arrow); a magnified cluster of spermatocytes isshown in the insertion box. (8): An in silico magnified image of the seminal vesicle shown in (7) with high fluorescence signal from secondary spermatocytes (arrowheads),t hin nup

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wo of which are magnified in the insertion box. (9): A muscle larva encysted witerinuclear localization of the signal in the stichosome).

nd examined by scanning confocal laser system, LSM 510-Zeiss,aser HeNe1 543 nm, LP 560 nm in the case of immunolocaliza-ion detection and Leica TCS SP2 Spectral Confocal, Laser GeNe43 nm, LP 576 nm, in the case of in situ hybridization visualiza-ion. In the case of immunohistological studies fluorescent imagesre presented, each reflecting signals collected from a cross section,nd for certain images also transformation into black and whiteounterparts is shown, devoid of excess glow.

. Results

.1. Thymidylate synthase protein localization during T. spiralis

evelopment

High thymidylate synthase protein level was found in the gonadrimordia and stichosome of T. spiralis premature and infective

rse cell, with high signal displayed by larva but not nurse cell; note an apparent

muscle larvae of both sexes (Fig. 1, panels 1–3 and 6–10). Whilein ovary and uterus primordia of female muscle larvae the enzymeshowed cytoplasmic localization (Fig. 1, panels 7–9), in stichocytesof both sexes, it was present in cytoplasm and around nuclei (Fig. 1,panels 6 and 10). In premature muscle larvae high enzyme level wasalso observed in the nerve ring structure (Fig. 1, panels 4 and 5). Inovary tissue of T. spiralis adult female forms, thymidylate synthaseprotein was found in both cytoplasmic and in nuclear regions ofegg cells (Fig. 2, panels 1 and 2), and in embryos developing withinuterus (Fig. 2, panels 3–6). It was also seen in male seminal vesicle,located mainly in sperm cell nuclear region (Fig. 2, panels 7 and 8). T.spiralis adult forms, similar to muscle larvae, also showed thymidy-

late synthase protein in stichocytes, localized around nuclei (Fig. 1,panel 11), as well as in excretory–secretory canalicular tree andalong esophagus wall (Fig. 1, panel 12). Although the latter indi-cated that the enzyme protein might belong to secretory products

B. Gołos et al. / Molecular & Biochemical Parasitology 183 (2012) 63– 69 67

Fig. 3. Localization of thymidylate synthase protein (immunospecific) and mRNA (nucleotide sequence-specific) during C. elegans development. (1): Dauer (a) and 465 �m-long L3 (b) larvae showing high fluorescence signal in the nerve ring (arrowhead) and gonad primordia (along the body wall). Of note is also the signal from pharyngealgland cells, placed in the L3 larva similarly as in L4 larva (cf. panel 2a, empty arrowhead). (2): A 645 �m-long L4 larva (a) showing high fluorescence signal in the nerve ring(arrowhead), glandular cells occupying distal pharyngeal bulb (empty arrowhead) and gonad primordia (along the body wall), and a magnified image (b), with inverted lightand shade, showing location of the pharynx anterior bulb (drumstick) and nerve ring (arrowhead) of the L4 larva shown in (a). (3) and (4): Genital primordium (gp) of L3larvae, 474 �m- and 491 �m-long, respectively, showing high in situ hybridization-reflecting signal, corresponding to the enzyme mRNA level. (5): An adult hermaphroditedisplaying high fluorescence signal in developing embryos, with an enlarged image showing dark spots corresponding to the nuclei positions. (6), (7) and (8): Embryosextracted from hermaphrodite uterus at gastrulation (6), morphogenesis (7) and 3-fold (8) stages, showing overall high fluorescence signal; note in (6) dark spots, markedby arrowheads, indicating the nuclei positions. (9): High in situ hybridization-reflecting signal corresponding to the enzyme mRNA level, in the glandular cells (arrowheads)of posterior pharyngeal bulb (asterisk); m – midgut.

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f the parasite, it was not found within nurse cell, even if it was stillositively visualized in stichosome and gonad promordia of larvaeresent within that cell (Fig. 2, panel 9).

.2. Thymidylate synthase protein localization during C. elegansevelopment

Developmental pattern of thymidylate synthase protein local-zation in C. elegans resembled that of T. spiralis. In the case ofevelopmentally arrested dauer larvae, the enzyme was found inhe nerve ring and along the body wall with the apparent lack ofuorescence signal in the gut (Fig. 3, panel 1a). The latter loca-ion (along the body wall) reflects presumably the enzyme proteinresent in the gonad primordium. High enzyme level was alsoound in the nerve ring and gonad primordia of L3 and L4 larvaeFig. 3, panels 1b and 2). Additionally, in L3 larvae, high level of thenzyme mRNA was also demonstrated by in situ hybridization inonad primordium (Fig. 3, panels 3 and 4). Similar to T. spiralis sti-hosome, constituting excretory–secretory system opening to thesophagus, also C. elegans L3 and L4 larva pharyngeal glandularells, extending ducts to both anterior and posterior bulbs of theharynx, revealed the presence of high thymidylate synthase bothrotein and mRNA levels (Fig. 3, panels 1, 2 and 9). In C. elegansermaphrodite forms, high enzyme level was found in embryoseveloping in the uterus during gastrulation and morphogenesistages, as well as in fully developed embryos (Fig. 3, panels 5–8).

. Discussion

In nematode life cycle, cell proliferation is restricted to a largextent, but not absolutely, to the embryonic development period22], hence high thymidylate synthase levels present in embryoseveloping in uteri of the two species investigated, T. spiralis and. elegans, remains in accord with the enzyme expression being aarker of proliferating tissues.Although thymidylate synthase has been demonstrated to be

resent in cycling cells [3], localization of the enzyme in egg cells of. spiralis is not unexpected, as unfertilized animal eggs are knowno be cell cycle-arrested [6; discussed in ref. 7]. Moreover, thenzyme found in gonad primordia of both species may point to aather general property of cells of reproductive system of nema-ode larvae to be cell cycle-arrested. However, in view of rarityf cell divisions in the postembryonic development of nematodes22], explanation of high levels of the enzyme protein localized ineproductive and nervous system cells of both species, and T. spiralisxcretory–secretory system and C. elegans pharyngeal secretoryells, of postembryonic developmental forms should probably alsossume cell cycle arrest.

It was unexpected to find a high thymidylate synthase level inhe nerve ring of developing T. spiralis larvae, as well as in dauer,3 and L4 C. elegans larvae. This phenomenon, not apparent in fullyeveloped muscle larvae, may reflect some equilibrium betweenifferentiation and accompanying cell cycle arrest [23].

Of interest is that the state of putative cell cycle arrest is foundn the two developmentally arrested live cycle forms [11], i.e. T.piralis muscle, and C. elegans dauer, larvae. In support of the cellycle arrest hypothesis, T. spiralis muscle larvae contained high lev-ls [24], comparable with those found in mouse leukemia L1210ells, of activities corresponding to DNA polymerase � and ribonu-leotide reductase, known to be regulated in association withell proliferation and cell cycle, with the two genes apparently

xpressed in nematodes [discussed in ref. 24].

Notably, the possibility of global cell cycle arrest in certain tis-ues of non-developing T. spiralis muscle larvae poses a question ofhe relation of this unusual cell cycle regulation to changes induced

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l Parasitology 183 (2012) 63– 69

by the larvae in muscle cells. Those changes, involving cell cycle re-entry and induction of DNA synthesis, followed by cell cycle arrest,believed to be G2/M arrest [25] but recently suggested to be ofsenescence-associated G1-like type [26], point to muscle larvae asa source of cell cycle-specific signals. In this respect, of interestis immunolocalization of thymidylate synthase around stichocytenuclei, as well as in extracellular region of stichocyte, in canalicularducts and lumen of adult female esophagus, pointing to a possibilitythat the enzyme might be secreted. However, no further evidenceof such secretion is available so far, as the enzyme was detectedneither within the nurse cell with the use of confocal microscopynor in the products secreted by T. spiralis muscle larvae with theuse of immunoblotting (data not shown).

A possibility of the enzyme playing a non-catalytic role in T.spiralis and/or C. elegans (see Introduction), appears attractive fromthe point of view its potential chemotherapeutic exploitation.

Of note is the potential presence of thymidylate synthase innuclei that, based on those images showing perinuclear localiza-tion, may not be excluded. So far only a few reports demonstratedthe enzyme to be present in nuclei [27,28; cf. ref. 29], with nuclearimport of the enzyme suggested to be SUMOylation-dependentand occur during S phase of the cell cycle [28]. Searching T. spi-ralis and C. elegans thymidylate synthase sequences for SUMOmodification consensus, using the SUMOplotTM Analysis Program(http://www.abgent.com) that predicts and scores sumoylationsites, we found one high probability motif in each sequence, i.e.LK238PG in T. spiralis and LK246PG in C. elegans [cf. ref.30], with 0.73score in both cases.

Acknowledgements

Supported by the Ministry of Science and Higher EducationGrants Nos. 6 P04C 021 18, 6 P04C 003 21 and the National ScienceCentre Grant No. 2011/01/B/NZ6/01781.

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