Transglutaminase Activity Determines Nuclear Localization of

Subscriber access provided by UNIV OF SOUTHERN INDIANA

Article

Transglutaminase activity determines nuclear localization of serotonin immunoreactivity in the early embryos of invertebrates and vertebrates Evgeny Ivashkin, Victoria Melnikova, Anastasia Kurtova, Nadja Brun, Alexandra Obukhova, Marina Yu. Khabarova, Alexander Yakusheff, Igor Adameyko, Kristin E. Gribble, and Elena E. Voronezhskaya ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00346 • Publication Date (Web): 10 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

1 2

Transglutaminase activity determines nuclear localization of serotonin immunoreactivity in the early embryos of invertebrates and vertebrates

3 4 5

Evgeny Ivashkin1,2,3,#, Victoria Melnikova1, Anastasia Kurtova1, Nadja R. Brun4, Alexandra Obukhova1, Marina Yu. Khabarova1, Alexander Yakusheff1, Igor Adameyko2,5, Kristin E. Gribble3, Elena E. Voronezhskaya1,#

6

#-

7 8

1Department

of Developmental and Comparative Physiology, Koltsov Institute of Developmental Biology, Russian Academy of Sciences, 119334 Moscow, Russia

9

2Department

corresponding authors: [email protected], [email protected]

10 11

3Josephine

12

4Department

13 14

5Department

of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden

Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, 02543 Woods Hole, MA, USA of Biology, Woods Hole Oceanographic Institution, 02543 Woods Hole, MA, USA

of Molecular Neurosciences, Center of Brain Research, Medical University of Vienna, A-1090 Vienna, Austria

15 16

Abstract

17 18 19 20 21 22 23

Serotonin (5-HT) is a key player in many physiological processes both in the adult organism and developing embryo. One of the mechanisms for 5-HT-mediated effects is covalent binding of 5HT to the target proteins catalyzed by transglutaminases (serotonylation). Despite the implication in a variety of physiological processes, the involvement of serotonylation in embryonic development remains unclear. Here we tested the hypothesis that 5-HT serves as a substrate for transglutaminase-mediated transamidation of the nuclear proteins in the early embryos of both vertebrates and invertebrates.

24 25 26 27

For this, we demonstrated that the level of serotonin immunoreactivity (5-HT-ir) in cell nuclei increases upon the elevation of 5-HT concentration in embryos of sea urchins, mollusks, and teleost fish. Consistently, pharmacological inhibition of transglutaminase activity resulted in the reduction of both brightness and nuclear localization of anti-5-HT staining.

28 29 30 31 32

We identified specific and bright 5-HT-ir within nuclei attributed to a subset of different cell types: ectodermal and endodermal, macro- and micromeres, and blastoderm. Western blot and dot-blot confirmed the presence of 5-HT-ir epitopes in the normal embryos of all the species examined. The experimental elevation of 5-HT level led to the enhancement of 5-HT-ir-related signal on blots in a species-specific manner.

33 34 35 36

The obtained results demonstrate that 5-HT is involved in transglutaminase-dependent monoaminylation of nuclear proteins and suggest nuclear serotonylation as a possible regulatory mechanism during early embryonic development. The results reveal that this pathway is conserved in the development of both vertebrates and invertebrates.

37

Keywords: serotonylation, nucleus, early development, mollusks, sea urchins, zebrafish

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 26

38

Introduction

39 40 41 42 43 44

Serotonin (5-hydroxytryptamine, 5-HT),a neurotransmitter and neurohumoral (endo- and paracrine) substance, is involved in the regulation of a remarkable variety of biological processes throughout all animal phyla 1. During the individual development of animals, 5-HT participates in a number of neuronal and non-neuronal processes including cell proliferation and differentiation. 5-HT and 5-HT-related proteins are produced during cleavage and gastrula developmental stages of different animals ranging from a plethora of invertebrates to mammals.

45 46 47 48 49 50 51 52 53 54 55

Previously, the efforts of different laboratories revealed that changes in the level of early 5-HT or pharmacological/genetic modulations of serotonin receptors and transporters lead to various developmental malformations including disturbances of cell divisions or even a complete cell cycle arrest 2–8. However, the intracellular molecular mechanisms of the observed effects remain mostly unknown. Antidepressants, many of which are serotonin-specific reuptake inhibitors, may result in teratogenic effects in pregnant women 9,10. Additionally, the ubiquitous distribution of many industrial chemicals is associated with neurotoxic effects in the developing embryo 11,12. While numerous studies have improved our understanding of the role of 5-HT in neural stages of development, the roles and mechanisms of pre-neural serotonin remain enigmatic. All this makes the early 5-HT of special interest in reproductive technologies and developmental biology.

56 57 58 59 60 61

Growing evidence suggests that the canonical effects of monoamines via extracellular ligand-receptor interactions are accompanied by the wide involvement of monoamines in intracellular regulation via posttranslational modifications of proteins. Unlike in a classical scenario of 5-HT action, the latter process requires 5-HT to be inside of the cells and includes the transglutaminase-mediated binding of monoamines to glutamine residues in target peptides (transamidation) 1.

62 63 64 65 66 67 68 69

In some cases, protein serotonylation (or generally monoaminylation) may lead to the formation of an active or inactive molecule that remains stable until its proteolytic degradation 13. The described pathway has been confirmed to be important for regulating various physiological functions, including activation of platelet aggregation, insulin release, long term smooth muscle contraction, reorganization of dendritic spines in neurons, and many other processes 14. Recently, we have shown that monoaminylation occurs not only in an adult organism but also during embryonic development. Serotonylation of proteins in the early embryo mediates 5-HT-dependent maternal effects during the development of a freshwater snail Lymnaea stagnalis 15.

70 71 72 73 74 75 76 77 78 79 80 81 82

Consistent with the fact that nuclei contain thousands of regulatory proteins, the tissue transglutaminase (also known as transglutaminase 2, TG2) appears to be localized and active in the nuclei of different cells. Nuclear TG2 promotes post-translational modifications of transcriptional factors and other related proteins, which has a direct impact on gene expression 16. At the same time, an enigmatic presence of 5-HT within the cell nuclei has been registered in a number of early works using electron-microscopy and autoradiography techniques. Moreover, application of the radioactively marked 5-HT or its biochemical precursor 5-hydroxytryptophan (5HTP) revealed strong localization of the signal within the cell nuclei, perinuclear space or even in condensed chromosomes 17–22. Later, the immunochemical techniques also demonstrated the presence of 5-HT-immunoreactivity in adult neuronal and immune cell nuclei. These observations were confirmed by both fluorescent immunohistochemistry (IHC) and immunogold IHC combined with electron microscopy 23–25 as well as by direct identification of histone serotonylation in a recent study 26.


Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


83 84 85 86

In this study, we addressed the evolutionarily conserved role of transglutaminase activity in the nuclear localization of serotonin during early embryonic development of sea urchins, mollusks and teleost fish embryos. The regulated activity of transglutaminase might be important for specific developmental transitions and epigenetically defined adaptations to postnatal life15.

87

Results

88 89 90 91 92 93 94 95 96 97 98 99 100

Sea Urchins. Echinoderms and particularly sea urchins are known as a classical model system widely used for investigating the early pre-neural neurotransmitter functions 27,7. We analyzed embryos of sea urchins Mesocentrotus nudus, Scaphechinus mirabilis and Paracentrotus lividus at blastula and gastrula stages to assay localization of 5-HT immunoreactivity (5-HT-ir). Sea urchin blastula represents a hollow sphere made of ciliated monolayer epithelium. M. nudus and P. lividus blastula cells exhibited positive 5-HT-ir within the nuclei of all epithelial cells (Fig. 1A1, A4; Fig 1N, O, P). Application of the 5-HT precursor 5-HTP to the embryo medium significantly enhanced the brightness of staining (Fig. 1B1, B4, D) but did not change its pattern (Fig. 1B1- B4), whereas inhibition of the transglutaminase activity by monodansylcadaverine (MDC) diminished brightness of staining (Fig. 1C1, C4, D). Colocalization analysis of 5-HT-ir and a nuclear marker confirmed that most anti-5-HT staining is associated with nuclear material (Fig. 1A2-A4). The level of colocalization was noticeably higher after 5-HTP application (Fig. 3 B2- B4) and decreased after MDC treatment (Fig. 1C2- C4).

101 102 103 104 105 106 107 108

In intact M. nudus gastrula, no bright positive 5-HT-ir was associated with nuclei of any cells (Fig. 1E, G, H), while bright 5-HT-ir spots appear at the apical region of all ectodermal cells (Fig. 1E, G). Incubation with 5-HTP resulted in an increased level of 5-HT-ir in all cells (Fig. 1F). The ectodermal cells, however, demonstrated the maximal brightness with a positive 5-HT-ir signal in nuclei (Fig. 1I), while the cells of developing archenteron (endoderm) were overall less extensively stained (Fig. 1J). Primary mesodermal cells demonstrate 5-HT-ir negative signal (Fig. 1K). A similar pattern of 5-HT-ir occurred in normal and 5-HTP-treated gastrula of S. mirabilis (Fig. 1L, M).

109 110 111 112 113 114 115

We applied a set of various commercial antibodies produced in different animals: rabbit (from two different suppliers), goat polyclonal and rat monoclonal antibodies to test the specificity of 5-HT-ir labeling. Staining of P. lividus blastula revealed a similar staining pattern for all polyclonal antibodies. In all cases, the positive signal was associated with the nuclear region (Fig. 1N, O, P). Rat monoclonal antibody revealed no bright signal in nuclei (Fig. 1Q). The omission of the primary antibody, as well as pre-adsorption with BSA-5-HT conjugate, eliminated the positive signal (Fig 1R, R’).

116 117 118 119 120

We incubated P. lividus blastula in another well-known transglutaminase activity blocker, cystamine, alone and in combination with 5-HTP. The effect was similar in all experimental series regardless of the antibody used. In all cases, incubation with 5-HTP resulted in a significant increase of 5-HT-ir brightness within nuclei. This outcome was negated by application of cystamine prior to 5-HTP (Fig. 1S, T, U).

121 122 123 124 125 126 127 128

Western blot with rabbit polyclonal anti-5-HT antibody (Immunostar) revealed several bands with the most prominent weight around ~20, ~50 and ~70 kDa in the samples of M. nudus embryos. An additional band of ~35 kDa occurred after 5-HTP treatment while bands of ~20 and ~70 kDa demonstrated a visible enhancement of signal (Fig. 2A). In the blots of P. lividus embryos ~30 kDa and ~50 kDa bands are visible with three different antibodies (Fig. 2B-D). Additional bands appear around ~15, ~30, ~32 and ~70 kDa with polyclonal rabbit anti-5-HT antibody (Fig. 2B), and additional bands with weight ~35 and ~45 kDa with polyclonal goat antibody (Fig. 2C). Despite the differences between species and antibodies applied, the selected bands ACS Paragon Plus Environment


Page 4 of 26

129 130 131 132 133 134

demonstrated consistent changes in relative density after 5-HTP and cystamine treatment. Thus, the density of ~30 kDa and ~50 kDa bands exceeded control level in 5-HTP-treated samples and were lower in cystamine-treated samples (Fig. 2E). The specificity of the visualized bands was confirmed by the decrease of the positive signal in all bands after pre-adsorption of the primary antibody with BSA-5-HT conjugate. The omission of the primary and secondary antibody also confirmed the specific character of staining (Fig. 2J).

135 136 137 138 139 140 141 142 143

To estimate the total pool of serotonylated peptides, we used dot-blot of P. lividus blastula samples. Measurements of integrated spot density revealed a reduction of the positive 5-HT-ir signal after cystamine treatment and consistent increase after 5-HTP treatment. When 5-HTP was applied after cystamine, the signal remained at the control level (Fig. 2F). All three antibodies demonstrated high specificity: they were bound to BSA-5-HT conjugate in a concentrationdependent manner but not with BSA alone (Fig. 2G), and the intensity of the dot spots diminished after pre-adsorption of the primary antibody with soluble BSA-5-HT (Fig. 2H). Representative samples demonstrated a consistent concentration-dependent increase in positive signal density after 5- HTP application in case of all three anti-5HT antibodies used in this experiment (Fig. 2I).

144 145 146 147 148 149 150

Ε-(γ-glutamyl)-lysine bond is a well-known marker for the transglutaminase activity 28. We stained blastula of M. nudus using antibody against ε-(γ-glutamyl)-lysine to estimate the subcellular pattern of transglutaminase activity. Positive staining occurred in vesicle-like granules in the cytoplasm (Fig. 2K1). Such a pattern can be associated with autophagosomes 29. We also observed a positive signal located in the nuclei and colocalized with the nuclear marker (Fig. 2K2, K3). Application of MDC considerably suppressed both brightness of the staining and colocalization of a specific signal with the nuclear marker (Fig. 2L1- L3).

151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170

Mollusks. To determine the evolutionary conservation of nuclear serotonin during embryonic development of different animals, we carried out experiments on the representatives of other invertebrate and vertebrate groups. We chose the mollusks as one of the candidates for two reasons: first, 5-HT has been found previously within early embryos of gastropod mollusks to have a somewhat nuclear pattern 6; and second, serotonylation has already been described in the early development of a snail 15. Based on this, we experimentally analyzed the distribution of 5-HT-ir in early cleaving embryos of a fresh-water snail Lymnaea stagnalis. The signal from anti5-HT staining in control embryos was either distributed equally in cytoplasm and nuclear volumes (Fig. 3A), or nuclei and mitotic spindles were had lower levels of staining as compared to the cytoplasm of dividing cells (Fig. 3B, C). Incubation with 5-HTP increased the general level of 5HT-ir within the entire embryo and changed the staining pattern so that the greatest anti-5-HT staining became associated with the nuclear region (Fig. 3A’), condensing chromosomes, and the mitotic spindles (Fig. 3B’). This pattern of 5-HT-ir was similar in both macro- and micromeres (Fig. 3D, E). Notably, in the case of elevated serotonin, the association of bright anti-5-HT staining signal with nuclear structures was observed regardless of the phase of the cell cycle. Bright positive 5-HT-ir was located either within the nuclear region in interphase stage or around the condensing chromosomes and along with the spindles at prophase, metaphase, anaphase, and telophase phases during cell division (Fig. 3F). Colocalization analysis confirmed a positive correlation of 5-HT-ir and nuclear marker (Fig. 3G1-G3) as well as enhancement of such colocalization in 5-HTP-treated embryos (Fig. 3H1-H3).

171 172 173 174

Application of two different primary antibodies raised in rabbit and goat demonstrated a similar nuclear pattern of 5-HT-ir expression (Fig. 3I). The specificity of the staining was confirmed both by pre-adsorption with BSA-5-HT (Fig. 3J) and omission of the primary antibody (Fig. 3J’). with the antibody against the serotonin precursor 5-HTP revealed signal within the cell volume


Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


175 176

and increased intensity in all cells after 5-HTP treatment, although we observed no nucleusassociated pattern in all cases (Fig. 3K, L).

177 178 179 180 181 182 183

Inhibition of transglutaminase activity by cystamine or MDC generally diminished the 5HT-ir in all possible conditions. In contrast, application of 5-HTP resulted in general enhancement of positive staining with the bright contrasted nuclei. Combined application of cystamine and 5HTP never led to nuclear localization of 5-TH-ir (Fig. 3M). Evaluation of relative 5-HT-ir brightness within nuclei demonstrated a consistent decrease of the signal level in cystamine and MDC treated preparations and an increase of staining brightness in 5-HTP-treated preparations. Combined application of cystamine and 5-HTP resulted in negation of 5-HTP effect (Fig. 3M, N).

184 185 186 187 188 189 190 191

Western blot revealed the characteristic major band with the weight of ~90 kDa (both for rabbit and goat antibodies) as well as minor bands of ~35 and ~250 kDa (rabbit polyclonal antibody, Immunostar) and 25, 55 and 60 kDa (goat antibody, Immunostar). Quantification showed a consistent increase in relative density of ~90 kDa band in samples after 5-HTP treatment (Fig. 3Q). Dot blot analysis confirmed a reduction of the positive 5-HT-ir signal after cystamine and MDC treatment and consistent increase after 5-HTP treatment. When 5-HTP was applied after cystamine and MDC, the signal remained at a level similar to that in the control (Fig. 3R, S).

192 193 194 195 196 197 198 199 200 201 202 203 204 205

In addition to L. stagnalis, we analyzed early embryos of the marine bivalve mollusks bay mussel, Mytilus trossulus, and the Ezo giant scallop, Mizuhopecten yessoensis. In the intact embryo of both species, 5-HT-ir was either equally distributed throughout the cytoplasm and nuclear region (Fig. 4A, E) or the nuclear area was negative in contrast to the cytoplasm (Fig. 4B). Application of 5-HTP resulted in significant enhancement of staining and expression of bright 5-HT-ir in the nuclear region (Fig. 4A’, B’, F, G). In M. trossulus, this pattern was evident in an interphase nucleus while staining around the condensed chromosomes did not exceed the cytoplasm level (Fig. 4B’). In M. yessoensis, a bright signal was located around the condensing chromosomes of metaphase cells and the spindles of anaphase cells (Fig. 4G). The staining specificity was supported by omission (Fig. 4 I, J) and pre-adsorption of the primary antibody (Fig. 4I', J'). Quantification of 5-HT-ir brightness in the nuclear region confirmed the consistent increase after 5-HTP application for samples of both bivalve species (Fig. 4C, H). Western blotting of M. trossulus samples shows a characteristic band at ~40 kDa. The application of 5-HTP led to the appearance of additional bands of ~25 and ~60 kDa (Fig. 4D).

206 207 208 209 210 211 212 213 214 215

Zebrafish. Finally, we turned to teleost zebrafish Danio rerio as an essential model of vertebrate development and analyzed 64-cell and oblong blastula stage embryos. We found that 5-HT-ir is brighter in the nuclei as compared with the cytoplasm in blastoderm cells but not in the yolk syncytial layer (YSL) of control embryos at both stages (Fig. 5A, C, D, E). 5-HTP application resulted in an enhancement of 5-HT-ir within all cells. The brightest 5-HT-ir was associated with nuclei of the blastoderm cells (Fig. 5B, F), whereas bright nuclei were never observed in YSL. The positively contrasted signal appeared in the nuclear region both in the interphase nuclei and in the cell at any phase of division (Fig. 5D-F, G). It is worth noting that we have observed neither positive nor negative contrast pattern of 5-HT-ir along with the mitotic spindles both in intact and 5-HTP treated zebrafish embryos.

216 217 218 219 220

Consistently, the incubation with cystamine reduced the positive 5-HT-ir staining in the nuclei of blastoderm cells, while 5-HT application increased signal intensity as expected. Combined application of cystamine and 5-HT resulted in the diminishing of the nuclear staining pattern (Fig. 5H, I). No positive signal occurred in control preparations after omission (Fig. 5J2) or pre-adsorption of the primary antibody (Fig. 5J3). ACS Paragon Plus Environment


Page 6 of 26

221 222 223

Discussion

224 225 226 227 228 229 230 231 232 233

The role of serotonin during early embryogenesis remains largely unexplored yet is of a broad interest in general developmental biology and toxicology. To summarize, our results demonstrate the presence of 5-HT-ir within the nuclear region of early embryonic cells of both invertebrate and vertebrate animals. According to our observations, the level of 5-HT-ir colocalization with nuclear material increases after elevation of 5-HT within the cytoplasm, while inhibition of transglutaminase activity reduces this nuclear-specific pattern. The nuclear localization of 5-HT-ir is prominent in all examined animals: sea urchin, mollusk and teleost fish. Distribution of nuclear 5-HT-ir is specific for different cells and tissues. In some cases, it is attributed to certain nucleus-related compartments (condensed chromosomes, perinuclear area, mitotic spindles).

234 235 236 237 238 239 240 241 242 243 244 245 246 247

Commercially available anti-5-HT antibodies were shown to recognize other small molecules similar to 5-HT 30 and serotonylated proteins in parallel with their primary target – 5-HT itself. Such anti-5-HT antibodies are typically produced using 5-HT linked to BSA by formaldehyde fixation as an antigen. The NH2- group in the side chain of glutamine residues and primarily amine group in the molecule of 5-HT represent sites for the crosslinking during formaldehyde fixation. The resulting structure is similar to 5-HT bound to the glutamine, with transamidation in the serotonylated peptide chains. The specificity of such epitope recognition by polyclonal anti-5-HT antibodies was previously confirmed by both inhibiting and knocking out the catalyzing enzyme TG2 31,32. However, in previous studies, anti-5-HT antibody was used to assess serotonylation in blots only. In our study, we applied in vivo pharmacological inhibition of TGases activity in a combination with both IHC and blotting. The similarity of the results obtained via different methods suggests that serotonylation of proteins may have a significant impact on the brightness and pattern of anti-5-HT staining in tissues. This fact is not usually considered by the researchers who apply anti-5-HT (and highly likely other monoamines) IHC staining.

248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267

The increased level of 5-HT in nuclei was described in different 5-HT-expressing cells in the 1970s. In hagfish and rat, 5-HT is localized in fibroblast nuclei after incubation with tritiated 5HT 18. Later, the presence of serotonin and other monoamines was shown in the nuclei of various cells such as immune cells, peritoneal mast cells, and adrenal medulla cells of mammals 22–24. These studies have also highlighted the role of serotonin synthesis, transport and catabolism on the nuclear pattern of 5-HT subcellular localization. In addition to differentiated tissues, 5-HT-ir expression was noted in the nuclei of early embryos as well. For example, the electronmicroscope autoradiographs have shown nuclear localization of serotonin in the 2-4-cell embryos of a polychaete supplied with 3H-5-HTP 17. However, despite the descriptive evidence of the nuclear localization of serotonin and its association with intranuclear structures in early embryos, the possible mechanisms of such patterns have not been previously extensively investigated. Our results clearly indicate that TGase activity has a large impact on the nuclear localization of 5HT-ir. Nevertheless, despite presence of 5-HT in early embryos of a wide range of animal taxa, the nuclear localization pattern of 5-HT has some exceptions. In normal mouse cleaving embryos, 5-HT preferentially occupies cytoplasm. A ring-like 5-HT positive staining was observed around the nucleus in some embryos only 33, while after the elevation of intracellular 5-HT, anti-5-HT immunostaining was distributed uniformly both within cytoplasm and nucleus 34. We also could see obvious diversity in the level and pattern of nuclear transglutaminase-dependent 5-HT-ir both among our objects and between different cells of the same embryo. The meaning and origination of these differences require further study.


Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293

Nuclear localization of TG2 itself was reported for a number of cell types in mammals. Soon after the first description, the capability of transglutaminase activity to mediate nuclear conjugation of polyamines was found in the cells of regenerating liver 35. The authors performed isolation of nuclei from rat livers and described increased incorporation of polyamines after partial hepatectomy. In later works, nuclear localization of transamidation activity was directly demonstrated by using in situ technique 36. In the following years, numerous works confirmed that up to 10% of TG2 activity is localized within nucleus volume in the TG2 expressing cells 16. For the case of invertebrates, it is known that immunohistochemistry reveals a strong nuclear pattern of TG2 localization in sea urchin embryonic cells. A variety of nuclear proteins such as E2F1, hypoxia-inducible factor 1 and Sp1 are the targets for transglutaminases 16. Other important nuclear substrates for transglutaminases are histones. TG2 is involved in the crosslinking of the H2A, H2B, H3 and H4 histones in vertebrates 37 and invertebrates 38,39. Histone crosslinking affects chromatin condensation in vitro and indicates the onset of apoptosis 40. Our experiments revealed a strong colocalization of 5-HT-ir with condensed chromosomes, suggesting histones are probable candidates for serotonylation in early embryos. Such a mechanism may directly contribute to pro- and antiapoptotic action of 5-HT in addition to the earlier described action via activating specific 5-HT receptors 41. Finally, in very recent work published during the revision of the current paper, serotonylation of histone H3 was described as a modification that can modulate transcription factor II D binding to H3K4me3 thus affecting permissive gene expression in serotonergic neurons 26. In our experiments, the increase of intracellular 5-HT level led to a very high brightness of anti-5-HT staining in nuclei. While the application of TGase blockers, almost completely vanished this pattern. On the other hand, in some cases, we observed transglutaminase-dependent 5-HT-ir both in the nuclei and along with the mitotic spindles during cell division. Such a high amplitude of brightness variation and the apparent variety of affected nuclear structures, suggests considerable diversity and abundance of proteins in nuclei which may be serotonylated.

294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314

According to the existing data, TG2 has low specificity requirements to the amino acid components adjusting to glutamine in the transamidation site 42. Thus, nearly any glutamine residues can be either be (i) bound to the lysine residues, (ii) transamidated utilizing any of monoamines available, or (iii) deamidated and turned into glutamate (in cases where other substrates are lacking) if it is available for the enzyme, according to the predicted protein structure. That means the substrates for transamidation may serve as concurrent inhibitors for each other. An example of such inhibition can be represented by antagonizing action of 5-HT to Factor XIII-A-mediated plasma fibronectin matrix assembly and crosslinking in osteoblast cultures via direct competition to glutamine-lysine transamidation 43. It is also worth mentioning that enzymes with opposite activity to transamidation have not been found in eukaryotes to date. Moreover, there is abundant evidence that protein complexes generated by transamidation are highly stable. Thrombus formation during blood coagulation 44 or egg envelope formation soon after the fertilization in echinoderm, teleost and amphibian 45–47 are characteristic examples of such highly stable complexes based on the TGases’ activity. In the cases of thrombus and egg envelope, the peptides undergo degradation via proteolysis without previous glutamine-lysine unlinking 48. This is consistent with the fact that monoaminylated proteins remain stable upon ubiquitin-dependent degradation 13. Thus, the target glutamine residue in the monoaminylated protein appears locked for transamidation with other protein’s lysine. And, vice versa, the proteins crosslinked through transamidation cannot be exposed to the monoaminylation at the target site. This alternative mode of transamidation renders the monoamines as an important regulatory component of the transglutaminase-dependent molecular processes in the nucleus.



Page 8 of 26

315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344

TG2 is known to take part in a variety of processes during embryonic development. For instance, it contributes to the regulation of germinal vesicle breakdown in mouse and amphibian oocytes 49,50. Moreover, TG2 and its activity can be targeted to the germinal vesicle nucleoplasm but not to the nucleolus. This feature closely matches our observation of 5-HT-ir intranuclear distribution, especially when the 5-HT positive signal is attributed to the nuclei but is absent in nucleolus-like structures of the interphase cell (Fig.3 F, H1). Little is known about TGases functions in developmental processes during the period between oocyte maturation and later cell differentiation/organogenesis. In our previous work, we hypothesized that TGases in the zygote and pre-neural stages take part in the serotonin-mediated maternal effects 15. In mammals, TG2 is involved in the regulation of adhesion and cell migration, which play an important role in establishing the embryo-maternal interface in the trophoblasts of implanting blastocyst 51. In our previous work on L. stagnalis, we have found that increasing but not decreasing of early preneural serotonin led to delayed developmental effects. Moreover, the exogenous elevation of the 5-HT level at a certain developmental stage resulted in gastrulation abnormalities prevented by the application of transglutaminase inhibitors 15. 5-HT was found to have an important role in the establishment of left-right polarity in the early embryo of chicken and frog 52,53. This mechanism includes gradient distribution of 5-HT between blastomeres and involves 5-HT binding to histone deacetylase partner protein Mad3 54. Serotonylation of nuclear proteins also depends on the intracellular level of 5-HT within cells and can act in conjunction with this process. However, neither in mollusks 55,15 nor in echinoderms or teleost fish (unpublished data of the authors), did the treatment of zygotes, cleaving embryos or gastrulae with inhibitors of TGases demonstrate any significant disturbances of development (including violation of left-right polarity). These results reveal an overall moderating rather than a master-regulating role of serotonylation and TGases activity during early development. In line with that, serotonylation of nuclear proteins might serve as a precise and accurate regulatory mechanism interlinking any of the serotonylation-related processes in organogenesis and tissue differentiation 56–58 with early events in the development and external factors such as mother’s physiology or embryonic environment. The fact that we found serotonylation in such diverse taxa suggests this process is highly conserved among animals and underlines the important role of serotonylation in the regulation of development.

345 346 347 348 349 350 351

Serotonylation-related processes largely depend on intracellular 5-HT and are known to be sensitive to the blockade of 5-HT transport activity13. In humans, treatment with serotoninspecific reuptake inhibitor antidepressants during the earliest period of pregnancy affects the expression of proteins related to cell growth, survival, proliferation, and inflammatory response 9, and additionally, may show teratogenic effects 10. Our results suggest that serotonylation of proteins in early embryos must be carefully studied in order to expand our knowledge about the action of antidepressants during early development.


Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


352

Materials & Methods

353 354 355 356 357 358 359 360 361 362 363

Animals maintenance and embryos production. Adult individuals of sea urchins (Mesocentrotus nudus and Scaphechinus mirabilis) and bivalve mollusks (bay mussel Mytilus trossulus and the giant Ezo scallop Mizuhopecten yessoensis) were collected in Vostok Bay, the Sea of Japan and maintained in the laboratory conditions. Embryos of Paracentrotus lividus were obtained from the adult specimens maintained in the Resource Center for Ecological Safety Observatory, St. Petersburg University. Gastropod mollusk (Lymnaea stagnalis) and teleost fish (Danio rerio) were from laboratory culture. All animals were maintained in conditions optimal for reproduction and eggs were obtained according to standard protocols described elsewhere 59– 61,15. All the animals’ maintenance and manipulation procedures were done according to the local rules and regulations acting at the territory of the Russian Federation (for mollusks and sea urchins) or Sweden (for zebrafish).

364 365 366 367 368 369 370

Pharmacological treatments. Serotonin hydrochloride (5-HT), 5-HT precursor 5-hydroxy-Ltryptophan (5-HTP) and the inhibitors of transglutaminases monodansylcadaverine (MDC) and cystamine hydrochloride (cystamine) were obtained from Sigma-Aldrich. Final concentrations were 0.25 mM for MDC, 2.5 mM for cystamine, 0.01 mM for 5-HT and 0.1 mM for 5-HTP. Incubation solutions were made from 10 mM stock solutions freshly prepared in distilled water (5HT, 5-HTP, cystamine) or DMSO (MDC). Respective DMSO concentration was used as a control and was found not to affect normal development.

371 372 373 374 375 376 377

For pharmacological treatments, eggs or embryos at the respective developmental stage were transferred into 2 ml of medium and held in a dark humid chamber for 1 hour at 18±0,2 °C for bivalve mollusks, 20±0,5 °C for sea urchins, 25±0,5 °C for gastropod mollusk and 27±0,5 °C for zebrafish. Embryos from the same egg mass or fertilization event were used for every experimental trial. At least three replicates were done for each species. Embryos were washed from the incubation solutions with respective fresh medium prior fixation and proceeded to the IHC and WB protocol.

378 379 380 381 382 383 384 385 386 387 388 389

Immunohistochemistry. IHC staining was performed according to the standard protocol for whole mount staining described earlier15. 5-HT-ir was visualized using polyclonal rabbit antibody against 5-HT (Immunostar, Hudson, USA, #20080, dilution 1:1000), polyclonal goat antibody against 5-HT (Immunostar, Hudson, USA, #20079, dilution 1:500, 1:300), polyclonal rabbit antibody against 5-HT (Sigma, USA, # S 5545, dilution 1:500), monoclonal rat antibody against 5-HT (Chemicon, USA, # MAB 352, dilution 1:300), polyclonal rabbit antibody against 5-HTP (Immunostar, Hudson, USA, # 24446, dilution 1:1000). The activity of TGases in sea urchin embryos was identified with primary antibodies against ε-(γ-glutamyl)-lysine (Abcam, # ab424, dilution 1:1000). Primary antibodies were detected with goat-anti-rabbit, donkey-anti-goat or goatanti-rat Alexa 488 or Alexa 555 conjugated IgG (Molecular Probes, USA, diluted 1:800 in PBS). Cell nuclei were stained with DAPI, Hoechst 33342 or TOTO-3 Iodide (ToTo, Thermofisher, # T3604) dye.

390 391 392

The omission of primary antibodies and, alternatively, pre-adsorption of primary antibodies with BSA-5-HT conjugate (Immunostar # 20081) were used as controls. We detected no positive signals in the first case and a significant reduction of brightness in the second one.

393 394 395 396

Imaging and analysis. For imaging, embryos were cleared in 2,2’-thiodiethanol. Imaging was performed using Zeiss LSM 510 Meta, LSM 700, LSM 780 and Leica TCS SP5 confocal systems. Z-stack projections, optical sections, fluorograms, and colocalization channel images were built using Imaris software (Bitplane).



Page 10 of 26

397 398 399 400 401 402 403 404

Western and dot-blot analysis. Western blotting was performed according to the protocol described earlier 15. In brief, the same anti-5-HT antibodies as used for IHC were applied (Immunostar, Hudson, USA, #20080, dilution 1:2500; #20079, dilution 1:1000; Sigma, USA, # S 5545, dilution 1:2000). Staining of blots with antibodies against acetylated α-tubulin (SigmaAldrich, T-6793, dilution 1:4000) was used for normalization. The secondary antibody conjugated with horseradish peroxidase to mouse or rabbit IgG (1:10000; Jackson Immunoresearch, USA) followed by detection using an ECL detection kit (Amersham Biosciences, UK) were applied for signal visualization.

405 406 407 408 409 410 411

Dot-blots were made using standard protocol 62. Pieces of properly sized nitrocellulose membrane were positioned on top of a 96-well microtiter plate and placed on a light box. The round well boundaries were easily discernible on the membrane and provided a guide when applying the samples at regular distances. Two-microliter aliquots of homogenates were spotted into the centers of individual circles, membranes were dried for 10–15 min. Homogenates were diluted to the final solutions with Tris buffer. Samples were loaded in triplicate. Immunodetection was performed the same way as in Western blot analysis (described above).

412 413 414 415 416 417

Measurements and statistical analysis. Measurement of nuclei brightness was made with Bitplane Imaris software. We used the signal of nuclear marker staining as a mask to build isosurface volumes and measured the mean brightness of 5-HT-ir signal inside of them. FIJI (ImageJ) software was used to calculate relative density of Western blot bands and the integrated density of dot-blots. Statistical analysis was performed with GraphPad Prism software. See Figure legends for the details.

418 419 420

Acknowledgements

421 422 423 424 425 426

The authors are grateful to the staff of the Vostok Biological Station of NSCMB FEB RAS, Resource Center for ESO (St. Petersburg University) and Zebrafish Core Facility of Karolinska Institutet (Stockholm, Sweden). We also thank Dr. Vyacheslav Dyachuk for the help with bivalve Western blotting. The research was done using the equipment of the Core Centrum of IDB RAS and Far East Center of Electron Microscopy of NSCMB FEB RAS. The work was supported by the Russian Science Foundation grant No. 17-14-01353.

427 428

Author Contributions

429 430 431 432

EI and EEV conceived and planned the experiments. EI, VM, AK, NB, and EEV carried out the experiments. AO, MYK and AY contributed to sample preparation. EI analyzed data. EI and EEV wrote the manuscript in consultation with KEG, IA and NB. EEV, IA and KEG obtained the funding. All authors provided critical feedback.

433


Page 11 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


434

Bibliography

435 436

(1) Berger, M., Gray, J. A., and Roth, B. L. (2009) The expanded biology of serotonin. Annu. Rev. Med. 60, 355–366.

437 438

(2) Hämäläinen, M., and Kohonen, J. (1989) Studies on the effect of monoamine antagonists on the morphogenesis of the newt. Int. J. Dev. Biol. 33, 157–163.

439 440 441

(3) Markova, L. N., Sadykova, K. A., and Sakharova, N. I. (1990) [The effect of biogenic monoamine antagonists on the development of preimplantation mouse embryos cultured in vitro]. Zh Evol Biokhim Fiziol 26, 726–732.

442 443 444

(4) Emanuelsson, H. (1992) Autoradiographic localization in polychaete embryos of tritiated mesulergine, a selective antagonist of serotonin receptors that inhibits early polychaete development. Int. J. Dev. Biol. 36, 293–302.

445 446

(5) Khozhaĭ, L. I., Puchkov, V. F., and Otellin, V. A. (1995) [The effect of a serotonin deficiency on mammalian embryonic development]. Ontogenez 26, 350–355.

447 448 449

(6) Buznikov, G. A., Nikitina, L. A., Voronezhskaya, E. E., Bezuglov, V. V., Dennis Willows, A. O., and Nezlin, L. P. (2003) Localization of serotonin and its possible role in early embryos of Tritonia diomedea(Mollusca: Nudibranchia). Cell Tissue Res. 311, 259–266.

450 451 452

(7) Buznikov, G. A., Peterson, R. E., Nikitina, L. A., Bezuglov, V. V., and Lauder, J. M. (2005) The pre-nervous serotonergic system of developing sea urchin embryos and larvae: pharmacologic and immunocytochemical evidence. Neurochem. Res. 30, 825–837.

453 454 455

(8) Glebov, K., Voronezhskaya, E. E., Khabarova, M. Y., Ivashkin, E., Nezlin, L. P., and Ponimaskin, E. G. (2014) Mechanisms underlying dual effects of serotonin during development of Helisoma trivolvis (Mollusca). BMC Dev. Biol. 14, 14.

456 457 458

(9) Kaihola, H., Yaldir, F. G., Hreinsson, J., Hörnaeus, K., Bergquist, J., Olivier, J. D. A., Åkerud, H., and Sundström-Poromaa, I. (2016) Effects of fluoxetine on human embryo development. Front. Cell Neurosci. 10, 160.

459 460 461

(10) Warkus, E. L. L., and Marikawa, Y. (2018) Fluoxetine inhibits canonical wnt signaling to impair embryoid body morphogenesis: potential teratogenic mechanisms of a commonly used antidepressant. Toxicol. Sci. 165, 372–388.

462 463

(11) Grandjean, P., and Landrigan, P. J. (2014) Neurobehavioural effects of developmental toxicity. Lancet Neurol. 13, 330–338.

464 465 466 467 468 469 470 471

(12) Landrigan, P. J., Fuller, R., Acosta, N. J. R., Adeyi, O., Arnold, R., Basu, N. N., Baldé, A. B., Bertollini, R., Bose-O’Reilly, S., Boufford, J. I., Breysse, P. N., Chiles, T., Mahidol, C., CollSeck, A. M., Cropper, M. L., Fobil, J., Fuster, V., Greenstone, M., Haines, A., Hanrahan, D., Hunter, D., Khare, M., Krupnick, A., Lanphear, B., Lohani, B., Martin, K., Mathiasen, K. V., McTeer, M. A., Murray, C. J. L., Ndahimananjara, J. D., Perera, F., Potočnik, J., Preker, A. S., Ramesh, J., Rockström, J., Salinas, C., Samson, L. D., Sandilya, K., Sly, P. D., Smith, K. R., Steiner, A., Stewart, R. B., Suk, W. A., van Schayck, O. C. P., Yadama, G. N., Yumkella, K., and Zhong, M. (2018) The Lancet Commission on pollution and health. Lancet 391, 462–512.

472 473 474

(13) Guilluy, C., Rolli-Derkinderen, M., Tharaux, P.-L., Melino, G., Pacaud, P., and Loirand, G. (2007) Transglutaminase-dependent RhoA activation and depletion by serotonin in vascular smooth muscle cells. J. Biol. Chem. 282, 2918–2928. ACS Paragon Plus Environment


Page 12 of 26

475 476

(14) Muma, N. A., and Mi, Z. (2015) Serotonylation and transamidation of other monoamines. ACS Chem. Neurosci. 6, 961–969.

477 478 479 480

(15) Ivashkin, E., Khabarova, M. Y., Melnikova, V., Nezlin, L. P., Kharchenko, O., Voronezhskaya, E. E., and Adameyko, I. (2015) Serotonin mediates maternal effects and directs developmental and behavioral changes in the progeny of snails. Cell Rep. 12, 1144– 1158.

481 482

(16) Kuo, T.-F., Tatsukawa, H., and Kojima, S. (2011) New insights into the functions and localization of nuclear transglutaminase 2. FEBS J. 278, 4756–4767.

483 484

(17) Emanuelsson, H. (1974) Localization of serotonin in cleavage embryos of Ophryotrocha labronica La Greca and Bacci. Wilhelm Roux. Arch. Entwickl. Mech. Org. 175, 253–271.

485 486

(18) Korneliussen, H. (1976) 5-Hydroxytryptamine: Autoradiographic evidence for uptake into fibroblast cell nuclei. Experientia 32, 443–445.

487 488

(19) Csaba, G., and Sudár, F. (1978) Localization of radioactively labelled serotonin in the nucleus of adrenal medulla cells. Acta Anat. (Basel) 100, 237–240.

489 490 491

(20) Calas, A., Dupuy, J. J., Gamrani, H., Gonella, J., Mourre, C., Condamin, M., Pellissier, J. F., and Van den Bosch, P. (1981) Radioautographic investigation of serotonin cells. Adv. Exp. Med. Biol. 133, 51–66.

492 493

(21) Bosler, O., and Calas, A. (1982) Radioautographic investigation of monoaminergic neurons: An evaluation. Brain Res. Bull. 9, 151–169.

494 495 496

(22) Csaba, G., Sudár, F., and Ubornyák, L. (1983) Comparative study of the internalization and nuclear localization of amino acid type hormones in Tetrahymena and rat lymphocytes. Exp Clin Endocrinol 82, 61–67.

497 498

(23) Csaba, G., and Kovács, P. (2006) Perinuclear localization of biogenic amines (serotonin and histamine) in rat immune cells. Cell Biol. Int. 30, 861–865.

499 500 501

(24) Csaba, G., Kovács, P., and Pállinger, E. (2006) Hormones in the nucleus. Immunologically demonstrable biogenic amines (serotonin, histamine) in the nucleus of rat peritoneal mast cells. Life Sci. 78, 1871–1877.

502 503

(25) Czaker, R. (2006) Serotonin immunoreactivity in a highly enigmatic metazoan phylum, the pre-nervous Dicyemida. Cell Tissue Res. 326, 843–850.

504 505 506 507 508 509

(26) Farrelly, L. A., Thompson, R. E., Zhao, S., Lepack, A. E., Lyu, Y., Bhanu, N. V., Zhang, B., Loh, Y.-H. E., Ramakrishnan, A., Vadodaria, K. C., Heard, K. J., Erikson, G., Nakadai, T., Bastle, R. M., Lukasak, B. J., Zebroski, H., Alenina, N., Bader, M., Berton, O., Roeder, R. G., Molina, H., Gage, F. H., Shen, L., Garcia, B. A., Li, H., Muir, T. W., and Maze, I. (2019) Histone serotonylation is a permissive modification that enhances TFIID binding to H3K4me3. Nature 567, 535–539.

510 511 512

(27) Buznikov, G. A., Chudakova, I. V., and Zvezdina, N. D. (1964) The r ole of neurohumours in early embryogenesis. i. serotonin content of developing embryos of sea urchin and loach. J Embryol Exp Morphol 12, 563–573.

513 514

(28) Watts, S. W., Priestley, J. R. C., and Thompson, J. M. (2009) Serotonylation of vascular proteins important to contraction. PLoS One 4, e5682. ACS Paragon Plus Environment

Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


515 516 517 518

(29) Altuntas, S., Rossin, F., Marsella, C., D’Eletto, M., Diaz-Hidalgo, L., Farrace, M. G., Campanella, M., Antonioli, M., Fimia, G. M., and Piacentini, M. (2015) The transglutaminase type 2 and pyruvate kinase isoenzyme M2 interplay in autophagy regulation. Oncotarget 6, 44941–44954.

519 520

(30) Levin, M. (2004) A novel immunohistochemical method for evaluation of antibody specificity and detection of labile targets in biological tissue. J Biochem Biophys Methods 58, 85–96.

521 522 523

(31) Liu, Y., Wei, L., Laskin, D. L., and Fanburg, B. L. (2011) Role of protein transamidation in serotonin-induced proliferation and migration of pulmonary artery smooth muscle cells. Am. J. Respir. Cell Mol. Biol. 44, 548–555.

524 525 526

(32) Salter, N. W., Ande, S. R., Nguyen, H. K., Nyomba, B. L. G., and Mishra, S. (2012) Functional characterization of naturally occurring transglutaminase 2 mutants implicated in early-onset type 2 diabetes. J. Mol. Endocrinol. 48, 203–216.

527 528 529

(33) Basu, B., Desai, R., Balaji, J., Chaerkady, R., Sriram, V., Maiti, S., and Panicker, M. M. (2008) Serotonin in pre-implantation mouse embryos is localized to the mitochondria and can modulate mitochondrial potential. Reproduction 135, 657–669.

530 531

(34) Amireault, P., and Dubé, F. (2005) Serotonin and its antidepressant-sensitive transport in mouse cumulus-oocyte complexes and early embryos. Biol. Reprod. 73, 358–365.

532 533

(35) Haddox, M. K., and Russell, D. H. (1981) Increased nuclear conjugated polyamines and transglutaminase during liver regeneration. Proc. Natl. Acad. Sci. USA 78, 1712–1716.

534 535

(36) Lesort, M., Attanavanich, K., Zhang, J., and Johnson, G. V. (1998) Distinct nuclear localization and activity of tissue transglutaminase. J. Biol. Chem. 273, 11991–11994.

536 537

(37) Ballestar, E., Abad, C., and Franco, L. (1996) Core histones are glutaminyl substrates for tissue transglutaminase. J. Biol. Chem. 271, 18817–18824.

538 539

(38) Shimizu, T., Hozumi, K., Horiike, S., Nunomura, K., Ikegami, S., Takao, T., and Shimonishi, Y. (1996) A covalently crosslinked histone. Nature 380, 32.

540 541 542

(39) Shimizu, T., Takao, T., Hozumi, K., Nunomura, K., Ohta, S., Shimonishi, Y., and Ikegami, S. (1997) Structure of a covalently cross-linked form of core histones present in the starfish sperm. Biochemistry 36, 12071–12079.

543 544 545

(40) Milakovic, T., Tucholski, J., McCoy, E., and Johnson, G. V. W. (2004) Intracellular localization and activity state of tissue transglutaminase differentially impacts cell death. J. Biol. Chem. 279, 8715–8722.

546 547 548

(41) Serafeim, A., Grafton, G., Chamba, A., Gregory, C. D., Blakely, R. D., Bowery, N. G., Barnes, N. M., and Gordon, J. (2002) 5-Hydroxytryptamine drives apoptosis in biopsylike Burkitt lymphoma cells: reversal by selective serotonin reuptake inhibitors. Blood 99, 2545–2553.

549 550 551

(42) Hitomi, K., Kitamura, M., and Sugimura, Y. (2009) Preferred substrate sequences for transglutaminase 2: screening using a phage-displayed peptide library. Amino Acids 36, 619– 624.

552 553 554

(43) Cui, C., and Kaartinen, M. T. (2015) Serotonin (5-HT) inhibits Factor XIII-A-mediated plasma fibronectin matrix assembly and crosslinking in osteoblast cultures via direct competition with transamidation. Bone 72, 43–52. ACS Paragon Plus Environment


Page 14 of 26

555 556

(44) Stump, D. C., and Mann, K. G. (1988) Mechanisms of thrombus formation and lysis. Ann. Emerg. Med. 17, 1138–1147.

557 558 559

(45) Larabell, C., and Chandler, D. E. (1991) Fertilization-induced changes in the vitelline envelope of echinoderm and amphibian eggs: self-assembly of an extracellular matrix. J. Electron Microsc. Tech. 17, 294–318.

560 561

(46) Cariello, L., Zanetti, L., and Lorand, L. (1994) Effects of inhibiting transglutaminase during egg fertilization and development. Biochem. Biophys. Res. Commun. 205, 565–569.

562 563 564

(47) Ha, C. R., and Iuchi, I. (1997) Purification and partial characterization of 76 kDa transglutaminase in the egg envelope (chorion) of rainbow trout, Oncorhynchus mykiss. J. Biochem. 122, 947–954.

565 566 567

(48) Sano, K., Inohaya, K., Kawaguchi, M., Yoshizaki, N., Iuchi, I., and Yasumasu, S. (2008) Purification and characterization of zebrafish hatching enzyme - an evolutionary aspect of the mechanism of egg envelope digestion. FEBS J. 275, 5934–5946.

568 569

(49) Zhang, J., and Masui, Y. (1997) Role of amphibian egg transglutaminase in the development of secondary cytostatic factor in vitro. Mol. Reprod. Dev.

570 571 572

(50) Kim, S. W., Lee, Z. W., Lee, C., Im, K. S., and Ha, K. S. (2001) The role of tissue transglutaminase in the germinal vesicle breakdown of mouse oocytes. Biochem. Biophys. Res. Commun. 286, 229–234.

573 574 575

(51) Kabir-Salmani, M., Shiokawa, S., Akimoto, Y., Sakai, K., Sakai, K., and Iwashita, M. (2005) Tissue transglutaminase at embryo-maternal interface. J. Clin. Endocrinol. Metab. 90, 4694– 4702.

576 577

(52) Fukumoto, T., Blakely, R., and Levin, M. (2005) Serotonin transporter function is an early step in left-right patterning in chick and frog embryos. Dev Neurosci 27, 349–363.

578 579

(53) Fukumoto, T., Kema, I. P., and Levin, M. (2005) Serotonin signaling is a very early step in patterning of the left-right axis in chick and frog embryos. Curr. Biol. 15, 794–803.

580 581 582

(54) Carneiro, K., Donnet, C., Rejtar, T., Karger, B. L., Barisone, G. A., Díaz, E., Kortagere, S., Lemire, J. M., and Levin, M. (2011) Histone deacetylase activity is necessary for left-right patterning during vertebrate development. BMC Dev. Biol. 11, 29.

583 584 585

(55) Ivashkin, E., Khabarova, M., and Voronezhskaya, E. (2012) Serotonin transport and synthesis systems during early development of invertebrates: Functional analysis on a bivalve model. Acta Biologica Hungarica 63, 217–220.

586 587 588

(56) Thomázy, V. A., and Davies, P. J. (1999) Expression of tissue transglutaminase in the developing chicken limb is associated both with apoptosis and endochondral ossification. Cell Death Differ. 6, 146–154.

589

(57) Ahern, G. P. (2011) 5-HT and the immune system. Curr. Opin. Pharmacol. 11, 29–33.

590 591 592

(58) Chabbi-Achengli, Y., Coudert, A. E., Callebert, J., Geoffroy, V., Côté, F., Collet, C., and de Vernejoul, M.-C. (2012) Decreased osteoclastogenesis in serotonin-deficient mice. Proc. Natl. Acad. Sci. USA 109, 2567–2572.


Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


593 594

(59) Croll, R. P., Jackson, D. L., and Voronezhskaya, E. E. (1997) Catecholamine-Containing Cells in Larval and Postlarval Bivalve Molluscs. Biol Bull 193, 116–124.

595 596

(60) Westerfield, M. (2007) The zebrafish book: a guide for the laboratory use of zebrafish (Danio rerio).

597 598

(61) Voronezhskaya, E. E., Khabarova, M. Y., Nezlin, L. P., and Ivashkin, E. G. (2012) Delayed action of serotonin in molluscan development. Acta Biol. Hung. 63 Suppl 2, 210–216.

599 600

(62) Corthell, J. T. (2014) Immunoblotting (Western Blot), in Basic molecular protocols in neuroscience: tips, tricks, and pitfalls, pp 55–75. Elsevier.

601



Page 16 of 26

602 603 604 605 606

Figure legends

607 608

A, E, G, H, N-Q, L – intact embryo; B, F, I-K, M – 5-HTP-treated embryo, C – embryo after application of monodansylcadaverine (MDC).

609 610 611 612 613

A-C– Representative image of normal (A1, A4), 5-HTP-treated (B1, B4) and MDC-treated (C1, C4) M. nudus blastula. A2, B2, C2 –Fluorogram showing brightness of green (5-HT-ir) and blue (DAPI) channels. Dots within rectangles were selected to build colocalization images shown in A3, B3. C3. Note a higher level of colocalization within the nuclei after 5-HTP-treatment and negation of colocalization after MDC-treatment.

614

D – Relative estimation of nuclear 5-HT-ir level after incubation in 5-HTP and MDC.

615 616 617 618 619 620

E-K – Sagittal optical section of normal and 5-HTP-treated M. nudus early gastrula. Ectodermal (arrows), endodermal (double arrows) and primary mesodermal (arrowhead) cells are marked. In intact gastrula, 5-HT-ir both in ectodermal and endodermal cell nuclei does not prevail over cytoplasmic level. Note strong positive 5-HT-ir signal within ectodermal cell nuclei, unlike in endodermal and mesodermal cells of 5-HTP-treated embryos. Bright 5-HT-ir spots occur in E, F, G and I located apically to the nuclei of ectoderm cells.

621 622 623 624 625

L-M – Sagittal optical section of intact and 5-HTP-treated S. mirabilis gastrula. Bright 5-HT-ir occurs in ectoderm (arrow) and endoderm (double arrows) as well as in primary mesenchyme (arrowhead) nuclei. After 5-HTP application, brightness of 5-HT-ir is more pronounced in ectodermal cell nuclei (arrow) than in endodermal (double arrows) and primary mesenchyme cells (arrowhead) nuclei.

626 627

N-Q – Comparison of 5-HT-ir pattern in P. lividus blastula revealed by different antibody staining. All polyclonal antibodies (N-P) demonstrate positive signal in nuclei unlike monoclonal one (Q).

628 629

R, R’ – Controls of staining specificity (rabbit polyclonal antibody, Immunostar); primary antibody omission (R) and pre-adsorption with BSA-5HT (R’).

630 631

S-U – Relative estimation of nuclear 5-HT-ir level visualized with various antibodies after incubation in 5-HTP and cystamine.

632

an – animal pole, ae – archenteron.

633 634

D, S-U – Mean is given, box represents 25th and 75th quartile, whiskers show min and max; n=9001600 nuclei; Mann-Whitney U test; *** - p

Transglutaminase Activity Determines Nuclear Localization of

Recommend Documents