M Coisy-Quivy

Alexandre Philips
email: alexandre.philips@inserm.fr

TC10 controls human myofibril organization and is activated by the sarcomeric RhoGEF obscurin

Keywords: Myofibrillogenesis, Rho GTPase, Obscurin

The contractile activity of striated muscle depends on myofibrils that are highly ordered macromolecular complexes. The protein components of myofibrils are well characterized, but it remains largely unclear how signaling at the molecular level within the sarcomere and the control of assembly are coordinated. We show that the Rho GTPase TC10 appears during differentiation of human primary skeletal myoblasts and it is active in differentiated myotubes. We identify obscurin, a sarcomere-associated protein, as a specific activator of TC10. Indeed, TC10 binds directly to obscurin via its predicted RhoGEF motif. Importantly, we demonstrate that obscurin is a specific activator of TC10 but not the Rho GTPases Rac and Cdc42.

Finally, we show that inhibition of TC10 activity by expression of a dominant-negative mutant or its knockdown by expression of specific shRNA block myofibril assembly.

Our findings reveal a novel signaling pathway in human skeletal muscle that involves obscurin and the Rho GTPase TC10 and implicate this pathway in new sarcomere formation.

Introduction

The myoplasm of adult striated muscle fibers contains highly ordered macromolecular complexes, named myofibrils, that are necessary for contraction.

The myofibril assembly takes place during the embryonic differentiation of heart and skeletal muscle, but also on a continuous basis during the physiological turnover of muscle. The thousands of protein subunits that compose sarcomeres, the smallest contractile units of myofibrils, are well characterized. On the other hand, it remains largely unclear how signaling at the molecular level within the sarcomere and the control of assembly are coordinated. Therefore, identifying and characterizing key elements of sarcomeric signal transduction and their roles in the control of myofibrillogenesis will be essential to understand the basic mechanisms of the biology of muscle under normal and pathological conditions.

The process of myofibril assembly requires both spatial and temporal coordination of protein interactions with high precision [START_REF] Gautel | Control of sarcomeric assembly: the flow of information on titin[END_REF]. In vertebrate muscle, this is mediated by three giant modular proteins: titin [START_REF] Maruyama | Connectin, an elastic protein from myofibrils[END_REF][START_REF] Wang | Titin: major myofibrillar components of striated muscle[END_REF], nebulin [START_REF] Wang | Human skeletal muscle nebulin sequence encodes a blueprint for thin filament architecture. Sequence motifs and affinity profiles of tandem repeats and terminal SH3[END_REF] and obscurin [START_REF] Young | Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly[END_REF], which act as molecular scaffolds or blueprints. Titin and nebulin regulate myofibril assembly, providing specific attachment sites for other proteins and thus specifying their sarcomeric positions [START_REF] Maruyama | Connectin, an elastic protein from myofibrils[END_REF][START_REF] Trinick | Titin as a scaffold and spring[END_REF][START_REF] Trinick | Titin: a molecular control freak[END_REF][START_REF] Wang | Titin: major myofibrillar components of striated muscle[END_REF]. In cardiac myocytes and muscle cell lines, the suppression of titin expression was associated with a significant structural disturbance of the contractile apparatus and a failure to incorporate myosin filaments in the nascent sarcomeres [START_REF] Person | Antisense oligonucleotide experiments elucidate the essential role of titin in sarcomerogenesis in adult rat cardiomyocytes in long-term culture[END_REF][START_REF] Van Der Ven | A functional knock-out of titin results in defective myofibril assembly[END_REF]. Obscurin, the third and last identified sarcomeric giant protein [START_REF] Young | Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly[END_REF], interacts with titin and myomesin [START_REF] Fukuzawa | Interactions with titin and myomesin target obscurin and obscurin-like 1 to the M-band -implications for hereditary myopathies[END_REF] and localizes mainly to the M-band in mature myofibrils [START_REF] Borisov | Dynamics of obscurin localization during differentiation and remodeling of cardiac myocytes: obscurin as an integrator of myofibrillar structure[END_REF][START_REF] Kontrogianni-Konstantopoulos | Obscurin regulates the organization of myosin into A bands[END_REF][START_REF] Young | Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly[END_REF]. Obscurin is also implicated in myofibril assembly in various organisms, but its mechanism of action remains unidentified. Defective myofibrillogenesis has been reported in cardiac myocytes following the disruption of obscurin expression by small interfering RNA-mediated gene silencing [START_REF] Borisov | Essential role of obscurin in cardiac myofibrillogenesis and hypertrophic response: evidence from small interfering RNAmediated gene silencing[END_REF][START_REF] Kontrogianni-Konstantopoulos | Obscurin modulates the assembly and organization of sarcomeres and the sarcoplasmic reticulum[END_REF]. In C. elegans, the ortholog of obscurin, Unc-89, is localized to the M-band in obliquely striated muscle and a mutation in this gene leads to loss of M-line assembly and regular A-bands [START_REF] Benian | The Caenorhabditis elegans gene unc-89, required fpr muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains[END_REF]. In Zebrafish, inhibition of obscurin expression with morpholino antisense oligos results in diminished numbers of skeletal myofibrils and impaired lateral alignment of adjacent myofibrils [START_REF] Raeker | Obscurin is required for the lateral alignment of striated myofibrils in zebrafish[END_REF].

These giant proteins contain potential signaling domains, suggesting that they receive and propagate signals from various pathways that remain to be characterized. Titin contains an active protein kinase domain implicated in myofibril assembly [START_REF] Mayans | Structural basis for activation of the titin kinase domain during myofibrillogenesis[END_REF], whereas nebulin and obscurin possess a src homology 3 (SH3) domain [START_REF] Labeit | The complete primary structure of human nebulin and its correlation to muscle structure[END_REF][START_REF] Young | Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly[END_REF]. In addition, obscurin contains a predicted GDP/GTP exchange factor (Rho GEF) domain [START_REF] Young | Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly[END_REF], also known as dbl-homology (DH) domain, immediately followed by a pleckstrin homology (PH) domain [START_REF] Hart | Catalysis of guanine nucleotide exchange on the CDC42Hs protein by the dbl oncogene product[END_REF]. This tandem organization is found in RhoGEF proteins, the activators of the Rho GTPases [START_REF] Rossman | GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors[END_REF]. Rho GTPases are key regulators of gene expression and actin cytosketal reorganization that control cell shape dynamics during diverse physiological processes, such as cell polarity, motility, adhesion, differentiation and apoptosis [START_REF] Etienne-Manneville | Rho GTPases in cell biology[END_REF]. Rho GTPases adopt either an active GTP-bound or an inactive GDP-bound conformation. Their activity is controlled positively by guanine nucleotide exchange factors (RhoGEF) and negatively by GTPase activating proteins (RhoGAPs). Moreover, guanine nucleotide dissociation inhibitors (GDIs) recycle Rho proteins before their reactivation [START_REF] Van Aelst | Rho GTPases and signaling networks[END_REF]. Once loaded with GTP, the GTPase binds to and activates a set of downstream effector proteins. Previous studies have implicated Rho GTPase-dependent signaling pathways in muscle differentiation. Rac1 signaling is required for myoblast fusion [START_REF] Luo | Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion[END_REF][START_REF] Nolan | Myoblast city, the Drosophila homolog of DOCK180/CED-5, is required in a Rac signaling pathway utilized for multiple developmental processes[END_REF] while RhoA positively regulates sarcomeric protein expression by modulating the transcriptional activity of tissue-specific transcription factors [START_REF] Carnac | RhoA GTPase and serum response factor control selectively the expression of MyoD without affecting Myf5 in mouse myoblasts[END_REF][START_REF] Wei | RhoA signaling via serum response factor plays an obligatory role in myogenic differentiation[END_REF].

Recently, we have established a role of TC10 in myofibrillogenesis in an ascidian model [START_REF] Coisy-Quivy | Identification of Rho GTPases implicated in terminal differentiation of muscle cells in ascidia[END_REF]. Inhibition of TC10 activity by expression of a dominant negative mutant in tail muscle cells of Ciona intestinalis larva impairs myofibril assembly. Consistent with our results in ascidia, TC10 mRNA is easily detected in striated muscle tissues from mouse and human [START_REF] Abe | Small GTPase Tc10 and its homologue RhoT induce N-WASP-mediated long process formation and neurite outgrowth[END_REF][START_REF] Neudauer | Distinct cellular effects and interactions of the Rho-family GTPase TC10[END_REF]. In the mouse C2C12 cell line, TC10 mRNA is detected at a moderate level in undifferentiated myoblasts. It is then induced in myotubes and reaches high levels in terminally differentiated myotubes [START_REF] Abe | Small GTPase Tc10 and its homologue RhoT induce N-WASP-mediated long process formation and neurite outgrowth[END_REF]. Although Jebailey et al. observed TC10 transcripts in differentiated rat L6 skeletal muscle cells and 3T3-L1 adipocytes, the protein was only detected in adipocytes [START_REF] Jebailey | Skeletal muscle cells and adipocytes differ in their reliance on TC10 and Rac for insulin-induced actin remodeling[END_REF]). In the latter, TC10 regulated insulin-induced translocation of the GLUT4 glucose transporter protein from intracellular storage sites to the plasma membrane.

Since this function appears to be performed by Rac in rat L6 skeletal muscle cells, the functional role of TC10 remains unknown in vertebrate striated muscles [START_REF] Jebailey | Skeletal muscle cells and adipocytes differ in their reliance on TC10 and Rac for insulin-induced actin remodeling[END_REF][START_REF] Khayat | Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes[END_REF].

In this study, we show that TC10 is present and active in human differentiated skeletal muscle cells. Moreover, we find that obscurin binds directly to TC10 and promotes its activation in human primary myotubes. Our data provide the first evidence that obscurin is a functional RhoGEF. Finally, we demonstrate that TC10 expression and activity are essential for human myofibrillogenesis, indicating that this function of TC10 is conserved from ascidia to humans.

Results

TC10 is expressed and active in human differentiated myotubes

We recently demonstrated the role of TC10 in myofibrillogenesis in an ascidian model [START_REF] Coisy-Quivy | Identification of Rho GTPases implicated in terminal differentiation of muscle cells in ascidia[END_REF]. To explore its role in human myofibrillogenesis, we first examined TC10 expression during differentiation of cultured human primary myoblasts. Immunoblot analysis showed that TC10 is not expressed, or at very low levels, in proliferating undifferentiated myoblasts (Fig. 1A). TC10 appeared after myoblast fusion, which is marked by a strong expression of the myogenic marker myogenin and by the appearance of differentiation-specific proteins such as obscurin, myosin heavy chain (MHC) and sarcomeric α-actinin (Fig. 1A). During differentiation, TC10 expression was maintained (Fig. 1A). This suggests that TC10 is probably not necessary for myoblast fusion, but rather for later processes of differentiation. We next examined whether TC10 was active in differentiated myotubes, incubating lysates from proliferating myoblasts and differentiated myotubes with the p21-binding domain (PBD) of PAK1 fused to glutathione S-transferase (GST) (Fig. 1B). The PBD of PAK1 binds specifically to GTP-bound active forms of TC10, Cdc42 and Rac [START_REF] Benard | Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases[END_REF][START_REF] Neudauer | Distinct cellular effects and interactions of the Rho-family GTPase TC10[END_REF]. Endogenous TC10 was detected neither in lysates nor in GST-PBD precipitates from undifferentiated proliferating myoblasts (Fig. 1B). In contrast, endogenous TC10 associated with GST-PBD and was therefore active in extracts from differentiated myotubes (Fig. 1B). These data present the first evidence that TC10 is present and active in differentiated myotubes.

TC10 binds to the DH domain of obscurin in cells and in vitro

The activation of Rho GTPase results from an exchange of a GDP by a GTP that is mediated by a dbl homology (DH) domain, immediately followed by a pleckstrin homology (PH) domain in Rho guanine nucleotide exchange factor (Rho GEF). A similar DH-PH module in obscurin [START_REF] Young | Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly[END_REF] interested us because this sarcomeric protein is essential for de novo myofibril assembly [START_REF] Borisov | Essential role of obscurin in cardiac myofibrillogenesis and hypertrophic response: evidence from small interfering RNAmediated gene silencing[END_REF].

To explore whether obscurin could activate TC10 during myofibrillogenesis, we first tested the interaction between these two proteins in the human cell line 293T. Two myc-tagged expression vectors for obscurin were constructed. The first encodes the C-terminal sequences in obscurin (ObsCT) that contain the DH-PH domains and two immunoglobulin-like repeats (Fig. 2A). The second uniquely contains the DH domain (ObsDH) (Fig. 2A), which is usually sufficient for interaction between Rho-GEF and Rho GTPase family proteins. Myc-ObsCT or myc-ObsDH were co-expressed in 293T cells with either HA-tagged TC10 or HA-tagged control protein (HA-Elmo). Cell lysates were incubated with anti-HA antibody and the immunoprecipitated proteins were detected by western blot analysis. Myc-ObsCT and myc-ObsDH coimmunoprecipitated with HA-TC10 but not with the control HA-Elmo (Fig. 2B). To confirm the interaction between obscurin and TC10, lysates from 293T cells overexpressing HA-TC10 and myc-ObsCT were incubated with either control rabbit immunoglobulin (IgG) or an anti-obscurin antibody that recognizes the C-terminus region of obscurin. HA-TC10 specifically coimmunoprecipitated with myc-ObsCT (Fig. 2C, lane 6). As obscurin is not expressed in 293T cells, no HA-TC10 was immunoprecipitated by the obscurin-specific antibody without myc-ObsCT expression (Fig. 2C, lane 4). Next, we analyzed the interaction of obscurin with constitutively activated mutant (HA-TC10CA) and the dominant negative form of TC10 (HA-TC10DN). The constitutively-activated mutants of Rho GTPase, which cannot hydrolyse GTP, bind to and activate their downstream effector. In contrast, dominantnegative mutants of Rho GTPases are unable to adopt the GTP-bound conformation and titrate out GEFs, leading to the inhibition of a Rho GTPase-dependent pathway.

Lysates from 293T cells overexpressing either HA-TC10CA or HA-TC10DN and either myc-ObsCT or its DH-PH deleted version (Myc-ObsCT(ΔDH-PH)) (Fig. 2A) were incubated with the anti-obscurin antibody. As expected, HA-TC10DN coimmunoprecipitated with myc-ObsCT and very slightly with Myc-ObsCT(ΔDH-PH) (Fig. 2D). This result confirms that TC10 interacts with the DH-PH domain of obscurin, although we cannot exclude that an additional domain of obscurin contributes to this interaction. In contrast, HA-TC10CA did not coimmunoprecipitate with myc-ObsCT or Myc-ObsCT(ΔDH-PH) (Fig. 2D), indicating clearly that obscurin is not an effector of TC10.

To determine whether the endogenous obscurin also interacts with TC10, lysates from human primary myotubes overexpressing HA-TC10 were incubated with the anti-obscurin antibody. We found that HA-TC10 was specifically coimmunoprecipitated with endogenous obscurin (Fig. 3A). To establish whether obscurin and TC10 interacted directly, we tested their association in vitro. Full-length TC10 was produced in bacteria as a MBP fusion protein and bound to amylose resin.

The DH domain of obscurin was produced in bacteria as a GST fusion protein, purified on glutathione-agarose beads and eluted with glutathione. GST protein alone or GST-ObsDH were incubated with immobilized MBP-TC10. GST-ObsDH, but not GST, interacted with MBP-TC10, indicating that obscurin binds directly to TC10 via its DH domain (Fig. 3B).

Obscurin specifically activates TC10 in myotubes

We next tested whether obscurin could activate TC10 in cells. The GTP-bound, active form of TC10 was assayed via its interaction with the PBD of PAK1, as described in Figure1. We cotransfected 293T cells with expression vectors for obscurin (Myc-ObsCT), wild-type TC10 (HA-TC10wt) or its constitutively active mutant (HA-TC10CA), and the PBD of PAK1 linked to GFP (GFP-PBD). As expected, the constitutively active mutant of TC10 (HA-TC10 CA) was strongly immunoprecipitated by the PBD (Fig. 4A, lane 3). While a low level of active HA-TC10wt bound PBD in the absence of obscurin (Fig. 4A, lane 1), this was strongly increased when obscurin (Myc-ObsCT) was coexpressed (Fig. 4A, lane 2).

Quantification revealed a four-fold increase in active TC10 in the presence of obscurin, and a six-fold increase with overexpressed HA-TC10CA (Fig. 4A). These results suggest that obscurin can mediate GTP exchange on TC10 in cells. To verify whether obscurin can activate endogenous TC10 in human primary myotubes and to evaluate its specificity, we performed the same assays for endogenous TC10, Cdc42

and Rac. All are expressed in myotubes and interact with PBD of PAK1 when activated. Overexpression of myc-ObsCT in differentiated myotubes induced an increase of endogenous active TC10 (Fig. 4B), but did not significantly alter the level of active Rac and Cdc42 (Fig. 4B). As expected, the TC10 activation is dependent on the DH-PH domain, since overexpression of Myc-ObsCT(ΔDH-PH) failed to activate TC10 (Fig. 4B). Thus, obscurin specifically activates TC10 in human primary myotubes. To further confirm this observation, we directly analyzed PAK activation induced by TC10 in human primary myotubes. The activation of PAK1 by Rho GTPases leads to its phosphorylation at Ser199 and/or Ser204 which can be detected with an antibody specific for this modification [START_REF] Chong | The mechanism of PAK activation. Autophosphorylation events in both regulatory and kinase domains control activity[END_REF].

Phosphorylated PAK was detected in myoblasts; however, its level increased after fusion and during differentiation of myotubes (Fig. 5A). We next verified that the phosphorylation level of PAK in human skeletal muscle cells depended on TC10 activity. For this, we transduced human primary myoblasts with lentivirus vectors expressing either a short hairpin RNA (shRNA) directed against human TC10 (TC10 shRNA) or lacZ control gene (C shRNA). Five days post-fusion, myotubes were lysed and proteins were evaluated by immunoblotting. TC10 expression was reduced by the shRNA (Fig. 5B). This did not affect the expression of PAK but led to a 50% reduction of its phosphorylation on Ser199 and 204. This result suggests that PAK is an effector of TC10 in human skeletal cells, and thus can serve as a measure of the level of active TC10 in cells. To test this, we overexpressed myc-ObsCT in human primary myotubes and examined the level of phosphorylated PAK. Myc-ObsCT or HA-TC10wt expression slightly increased the phosphorylation of endogenous PAK (Fig. 5C, lanes 2 and 3 respectively). Interestingly, myotubes ectopically expressing both proteins showed a higher level of phosphorylated PAK (Fig. 5C, lane 4), matching that induced by the constitutively active mutant of TC10 (Fig. 5C, lane 5).

Altogether, these data provide the first demonstration that obscurin is a functional RhoGEF and it displays specific activity toward TC10 in human differentiated myotubes.

TC10 activity is required for myofibrillogenesis

As obscurin is essential for de novo myofibril assembly [START_REF] Borisov | Essential role of obscurin in cardiac myofibrillogenesis and hypertrophic response: evidence from small interfering RNAmediated gene silencing[END_REF][START_REF] Kontrogianni-Konstantopoulos | Obscurin modulates the assembly and organization of sarcomeres and the sarcoplasmic reticulum[END_REF] and for TC10 activation, we explored whether TC10-dependent signaling was implicated in myofibrillogenesis of human skeletal cells. Previous studies have shown that the Rho-family GTPase RhoA can regulate sarcomeric protein expression by modulating the activity of the myogenic transcription factor SRF [START_REF] Carnac | RhoA GTPase and serum response factor control selectively the expression of MyoD without affecting Myf5 in mouse myoblasts[END_REF][START_REF] Wei | RhoA signaling via serum response factor plays an obligatory role in myogenic differentiation[END_REF]. We investigated whether the deregulation of TC10 activity affected sarcomeric protein expression, using two types of lentiviral vectors. One reduced TC10 levels and thereby TC10 activity via a shRNA whereas the second increased TC10 activity by expressing either HA-tagged wild-type or constitutively active TC10. Since sarcomeric proteins are induced and begin to assemble rapidly during differentiation, we infected undifferentiated human skeletal cells with lentivirus at the myoblast stage. Five days post-fusion, infected myotubes were lysed and protein expression was analyzed by immunoblot. The level of TC10 was dramatically downregulated in myotubes expressing the TC10 shRNA compared to the control lacZ shRNA (Fig. 6A and6C).

TC10 downregulation did not significantly affect the expression of obscurin, α-actinin, myogenin and α-tubulin (Fig. 6A and6C), whereas it led to reduced levels of myosin heavy chain (34%) and troponin T (33%) (Fig. 6A and6C). In contrast, the level of these proteins was not significantly modified in myotubes infected with lentiviral vectors expressing HA-tagged wild-type (HA-TC10wt) or constitutively active TC10

(HA-TC10CA) (Fig. 6B, lanes 2 and 3 and 6C). This result suggests that the reduction obtained in myotubes containing TC10 shRNA is primarily due to inhibition of myofibril assembly. Indeed, the stability of myosin and myomesin is reduced when they are not assembled in myofibrils [START_REF] Kontrogianni-Konstantopoulos | Obscurin modulates the assembly and organization of sarcomeres and the sarcoplasmic reticulum[END_REF].

These results suggest that TC10 activity is crucial for myofibril assembly.

To confirm this idea, we analyzed myofibril organization in human primary myotubes expressing HA-tagged wild-type TC10, a dominant negative mutant or GFP as a control. At 5 days post-fusion, cultures were immunostained for α-actinin and HAtagged protein. Mature myofibrils, revealed by the cross striated organization of αactinin, were observed in the majority of myotubes expressing GFP or wtTC10 (Fig. 7A). In contrast, most myotubes expressing the dominant negative form of TC10 (HA-TC10 DN) contained no mature myofibrils but only nascent myofibrils (Fig. 7A).

These results show that TC10 signaling is critical for normal myofibril assembly during human skeletal differentiation.

Inhibition of TC10 expression by shRNA results in impaired myofibril maturation

To further confirm the importance of TC10 in myofibrillogenesis, we used a RNA interference to reduce TC10 levels and activity. Myoblasts were infected with lentiviral vectors containing TC10 or control shRNAs (shTC10 and shCtl). Five days post-fusion, myotubes were fixed and immunostained for α-actinin to evaluate myofibril organization. The majority of myotubes expressing the control shRNA showed mature myofibrils (Fig. 7B), while those expressing the shRNA directed against TC10 contained immature myofibrils (Fig. 7B). Quantitative analysis showed more than 72% of control myotubes contained mature myofibrils, compared to only 9% of myotubes where TC10 expression is disrupted (Fig. 7C). The latter did not change even after seven days in differentiating conditions (data not shown),

indicating that myofibrillogenesis is efficiently blocked and not simply delayed. Taken together, our results demonstrate that TC10 signaling is required for normal myofibril organization in human skeletal muscle cells.

Discussion

Our study presents the first evidence for the function of TC10 in vertebrate skeletal muscle and identifies obscurin as a specific activator for this Rho GTPase. First, we

show that TC10 expression and activity are required for myofibril assembly. Second, we provide evidence that TC10 binds directly to a predicted RhoGEF motif of the sarcomeric protein obscurin. Finally, we demonstrate that this RhoGEF domain is functional and specifically activates TC10 in human primary myotubes.

The implication of TC10 in myofibrillogenesis

TC10 exhibits a tissue-specific expression pattern with the highest level of TC10 mRNA found in the heart and skeletal muscle [START_REF] Abe | Small GTPase Tc10 and its homologue RhoT induce N-WASP-mediated long process formation and neurite outgrowth[END_REF][START_REF] Neudauer | Distinct cellular effects and interactions of the Rho-family GTPase TC10[END_REF]. These data suggest that TC10 plays an important and specific role in these tissues. In the mouse muscle cell line C2C12, TC10 mRNA is moderately expressed in undifferentiated myoblasts and is strongly induced in myotubes, remaining at a high level in terminally differentiated myotubes [START_REF] Abe | Small GTPase Tc10 and its homologue RhoT induce N-WASP-mediated long process formation and neurite outgrowth[END_REF]. In agreement with these results, we find that TC10 is not detectable in proliferating human primary myoblasts. It appears after myoblast fusion and is maintained during differentiation.

This expression profile during differentiation suggests that TC10 does not play a role in myoblast fusion, as described for Rac [START_REF] Luo | Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion[END_REF] but rather is required for a later process. Consistent with this notion, TC10 is highly expressed in the heart where muscle cells do not fuse and are mononucleated. Surprisingly, while TC10 expression is stronger in striated muscle, its function remains unknown in this tissue.

In adipocytes, TC10 participates in glucose transport, regulating insulin-induced translocation of the GLUT4 glucose transporter protein from intracellular compartments to the plama membrane [START_REF] Chiang | Insulinstimulated GLUT4 translocation requires the CAP-dependent activation of TC10[END_REF][START_REF] Watson | Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation[END_REF].

However, adipocytes and muscle cells differ in their reliance on TC10 for insulininduced actin remodeling [START_REF] Jebailey | Skeletal muscle cells and adipocytes differ in their reliance on TC10 and Rac for insulin-induced actin remodeling[END_REF] et al., 2000), leaving TC10 without any known function in skeletal muscle.

Our studies initially highlighted the importance of TC10 for myofibrillogenesis in the prochordate Ciona intestinalis [START_REF] Coisy-Quivy | Identification of Rho GTPases implicated in terminal differentiation of muscle cells in ascidia[END_REF]. We expressed the dominant-negative mutants and constitutively active forms of each Rho family GTPase in striated muscle cells of the larva. Two Rho GTPases, RhoA and TC10

were required for myofibril assembly [START_REF] Coisy-Quivy | Identification of Rho GTPases implicated in terminal differentiation of muscle cells in ascidia[END_REF]. The mechanisms underlying these phenotypes have not been determined in ascidia. In the muscle cell line C2C12, RhoA is required for serum response factor (SRF)-mediated activation of several muscle-specific gene promoters [START_REF] Wei | RhoA signaling via serum response factor plays an obligatory role in myogenic differentiation[END_REF]. Consequently, RhoA positively regulates MyoD expression and skeletal muscle cell differentiation. Here, we suggest that TC10 plays a different role than RhoA in muscle differentiation.

Disruption of TC10 expression by shRNA in human primary myotube does not affect expression of the differentiation-specific proteins myogenin, obscurin and sarcomeric α-actinin. It leads to reduced levels of myosin heavy chain and troponin T, but overexpression of constitutively active TC10 does not induce the expression of these genes. Thus, our data implicate TC10 in sarcomere assembly and therefore in myofibrillogenesis in human muscle cells as in ascidia [START_REF] Coisy-Quivy | Identification of Rho GTPases implicated in terminal differentiation of muscle cells in ascidia[END_REF].

This strongly suggests an important conserved function for this Rho GTPase in muscle differentiation.

Identification of Obscurin as an RhoGEF for TC10

Although Rho GTPases may influence different stages of muscle differentiation, the regulation of their activity in mature myotubes is largely uncharacterized. In particular, the mechanisms underlying the cross-talk between myofibril assembly and the modulation of Rho GTPases activity are still unclear and no regulator (RhoGEFs or RhoGAPs) of Rho GTPases has been identified. In searching for a RhoGEF that might be involved in TC10 activation during myofibrillogenesis, we focused on obscurin, a sarcomere-associated protein containing a putative GDP-GTP exchange domain (RhoGEF) [START_REF] Young | Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly[END_REF]. This signaling domain is found in all orthologs of obscurin, namely in mammals, zebrafish, ascidia and nematode, suggesting an important conserved function in muscle differentiation [START_REF] Raeker | Obscurin is required for the lateral alignment of striated myofibrils in zebrafish[END_REF][START_REF] Sutter | Orthologous relationship of obscurin and Unc-89: phylogeny of a novel family of tandem myosin light chain kinases[END_REF][START_REF] Young | Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly[END_REF]. Obscurin wraps around myofibrils at the level of the M-line and Z-disk in striated muscle [START_REF] Kontrogianni-Konstantopoulos | Obscurin is a ligand for small ankyrin 1 in skeletal muscle[END_REF]. Disruption of its expression in vertebrates [START_REF] Borisov | Essential role of obscurin in cardiac myofibrillogenesis and hypertrophic response: evidence from small interfering RNAmediated gene silencing[END_REF][START_REF] Raeker | Obscurin is required for the lateral alignment of striated myofibrils in zebrafish[END_REF] like invertebrates [START_REF] Benian | The Caenorhabditis elegans gene unc-89, required fpr muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains[END_REF], leads to misorganization of myofibrillar structure. In consequence, Fukuzawa at al. suggest that obscurin signalling functions are necessary for sarcomere assembly [START_REF] Fukuzawa | Interactions with titin and myomesin target obscurin and obscurin-like 1 to the M-band -implications for hereditary myopathies[END_REF]. These results, together with our observations that implicate TC10 in myofibril organization, led us to test whether obscurin is a functional RhoGEF for TC10 during myofibrillogenesis. In human myotubes, TC10 binds directly to the RhoGEF domain of obscurin and, with a weaker affinity, a downstream region containing two immunoglobulin-like repeats that could stabilize the obscurin/TC10 interaction. Using Rho GTPase activation assays, we demonstrate that obscurin is a specific activator of TC10 but not Rac and Cdc42 in human primary myotubes. This important role of obscurin is confirmed by the observation that the expression of obscurin with TC10 increases the active phosphorylated form of PAK1 in human myotubes. These results show that obscurin is a functional and specific RhoGEF for TC10 in human skeletal muscle.

In embryonic hearts, Z-disk association of obscurin is mainly observed in cells where the actin cytoskeleton is not yet regularly aligned in parallel myofibrils, and M-line localization is observed when myofibrils start to be arranged in parallel bundles [START_REF] Young | Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly[END_REF]. In mature cardiomyocytes, obscurin is found at the M-band of mature myofibrils [START_REF] Young | Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly[END_REF]. This coincidence of the redistribution of obscurin with remodelling of the actin cytoskeleton suggests that obscurin may be involved in this structural transition. The identification of a functional Rho GEF domain in obscurin supports this idea, because Rho GTPases, including TC10, are known to be key regulators of actin cytoskeletal reorganization [START_REF] Ridley | Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking[END_REF]. The precise molecular role of obscurin in the morphogenesis of the Z-disks, A-bands and M-lines is not known and is probably complex. Recent results suggest that the RhoGEF domain of obscurin interacts with RanBP9, a scaffolding protein, and that both can interact with the N-terminal region of titin to influence the formation of the Zdisk and A/I junction [START_REF] Bowman | The rho-guanine nucleotide exchange factor domain of obscurin regulates assembly of titin at the Z-disk through interactions with Ran binding protein 9[END_REF]. Given the moderate binding affinity of RanBP9 to obscurin, their interaction could play a transient role in myotube development [START_REF] Bowman | The rho-guanine nucleotide exchange factor domain of obscurin regulates assembly of titin at the Z-disk through interactions with Ran binding protein 9[END_REF]. In addition, RanBP9 might help stabilize the TC10/obscurin interaction. Since our results suggest that obscurin contains a functional RhoGEF motif, the functions of this protein likely involve signal transduction. Several mechanisms might link the obscurin/TC10-dependent signalling pathway to myofibril formation and organization. In terminally differentiated cardiac myocytes, desmin helps align myofibrils into striations and localizes at the Z-disk like obscurin. Desmin serves as an excellent substrate for PAK, which is an effector of TC10, and its phosphorylation by PAK dramatically inhibits its filament-forming ability [START_REF] Ohtakara | p21-activated kinase PAK phosphorylates desmin at sites different from those for Rho-associated kinase[END_REF]. Therefore, obscurin/TC10 pathways could control myofibril alignement via PAK and desmin. Obscurin, together with other interacting proteins like myomesin and titin [START_REF] Fukuzawa | Interactions with titin and myomesin target obscurin and obscurin-like 1 to the M-band -implications for hereditary myopathies[END_REF], is also involved in the organization of A-bands and the incorporation of myosin filaments into myofibrils. These processes could be mediated by the regulatory myosin light chain (R-MLC) and PAK1. Indeed, PAK1 modulates R-MLC function via direct phosphorylation and inhibition of myosin light chain kinase (MLCK) [START_REF] Bokoch | Biology of the p21-activated kinases[END_REF]. Since inhibition of MLCK disrupts thick filament assembly in embryonic myocytes in culture [START_REF] Li | Calcium transients regulate patterned actin assembly during myofibrillogenesis[END_REF], the phosphorylation of R-MLC seems essential for myofibril organization. The identification of targets of the obscurin/TC10-dependent signalling pathway will be essential to understand the mechanisms controling myofibril assembly.

In this study, we attribute a functional role to TC10 in human skeletal muscle and identify this RhoGTPase as an important signalling protein for sarcomere assembly.

Our identification of a functional RhoGEF domain in obscurin indicates that direct communication between the sarcomere and TC10 pathway leads to new sarcomere formation. Since mutations in RhoGEFs are already associated with pathologies, such as cancer, and, given the phenotype of obscurin and TC10 disruption in striated muscle, we expect that some human skeletal myopathies arise from defects in obscurin/TC10 pathway.

Materials and Methods

DNA constructs

cDNAs encoding differents fragments of the carboxyterminal region of human Obscurin were generated by PCR from cDNA DKFZp451F056 (from clone DKFZp451F056) which encodes the C-terminus of Obscurin between aa 5173-6620.

PCR products were subcloned into pDONR221 by Gateway technology (Invitrogen)

to generate pDONR221 ObsCT(ΔDH-PH) (aa 6007-6620), pDONR221ObsCT (aa 5679-6620), pDONR221ObsDH (aa 5679-5895). Expression vectors were obtained by recombination of entry vector with destination vector pGEX 2T-RfB or pCDNA3 myc-RfC. cDNAs encoding HA-TC10 wt and HA-TC10 CA (TC10/Q75L) were inserted into pDONR221 by Gateway technology and expression vectors were obtained by recombination of entry vector with lentiviral destination vector. ds oligonucleotides encoding a TC10-specific shRNA, as well as a negative control ds oligonucleotides were cloned into pENTR/U6 vector. Lentiviral vector expressing shRNA were obtained by recombinaison of entry vector with destination vector. All constructs was verified by sequence analysis by using an ABI Prism Sequencer.

Antibodies

Antibodies were obtained as follow: anti-sarcomeric α-actinin mAb, anti-MHC fast mAb, anti-TC10 pAb, anti-Troponin T mAb and anti-Tubulin mAb from Sigma; anti-Caveolin 3 mAb, anti-Rac mAb, anti-CD56 mAb and anti-Myogenin mAb from BD Transduction Laboratories; anti Cdc42 pAb and anti-HA mAb from Santa cruz Biotechnology, anti-HA pAb from Zymed Laboratories, anti-GFP mAb from Roche, anti-GFP pAb from Torrey Pines Biolabs, anti MBP mAb from New England Biolabs;

anti-myc pAb from Medical and Biological Laboratories; anti PAK pAb and anti P PAK pAb from Cell Signaling Technology. 9E10 anti-myc mAb was a gift from D. Matthieu and anti GST antiserum was a gift from A. Debant. The Alexa Fluor secondary antibodies were purchased from Molecular Probes, Invitrogen.

Cell cultures, transfection and lentiviral infection

Human primary myoblasts were isolated as described previously [START_REF] Kitzmann | Inhibition of Notch signaling induces myotube hypertrophy by recruiting a subpopulation of reserve cells[END_REF] from quadriceps muscle obtained from the ʻʻAFM-BTR Banque de tissus pour la recherche'' (Hôpital de la Pitié´-Salpêtrière, 75013 Paris). 30 000 myoblasts/ml were grown in GM (DMEM supplemented with 10% fetal calf serum (FCS) and 1%

UltroserG (Biosepra)) during five days. At the fifth day of culture, myoblasts fuse and cultures were switched to differentiation medium (DM) composed of DMEM supplemented with 10% FCS, 1µM human Insulin and Penicillin/Streptomycin. Thus, the sixth day of culture is the first "day post fusion". 293T cell lines were maintained in DMEM supplemented with 10% FCS and Penicillin/Streptomycin. 293T cells and human primary myotubes were transfected using jet PEI (Qbiogen) according to the manufacturer's instructions. 72 hours after plating, Myoblasts were infected with lentivirus in GM supplemented with 8µg/ml Hexadimethrine bromide (Sigma). The medium was replaced after 24 hours and cells were left 72 hours in GM before switching to DM.

Immunoprecipitation and immunoblotting

293T cells and human myotubes were washed with ice-cold PBS and lysed with lysis buffer containing 50mM Tris-HCl pH 7.5, 150mM NaCl, 5mM MgCl2, 1% TritonX-100 and protease inhibitors (Sigma). Lysates were clarified by centrifugation and the supernatants were incubated with the indicated antibodies and Protein G-Sepharose.

Immunocomplexes were washed with lysis buffer and bound proteins were analyzed by SDS/PAGE and immunoblotting. Analysis and quantification were performed with Kodak 1D3.6 software.

Recombinant protein purification and antibody production

GST and MBP fusions were expressed in E. coli strain BL21 using pGEX and pMAL vectors respectively, according to standard procedures. GST fusion proteins were eluted in buffer containing 10 mM Tris-HCl pH 7.5, 50mM reduced glutathione (Sigma) supplemented with 2mM MgCl2 and 10mM GDP for MBP-TC10 purification.

Recombinant GST-ObsDH was further used for biochemical studies and GST-ObsCT(ΔDH-PH) for raising antibodies. Rabbits were submitted to three rounds of injection of 0.1µg of GST-ObsCT(ΔDH-PH). Immunosera were depleted on GSTbound Sepharose and affinity-purified on GST-Obs-bound Sepharose.

In vitro interaction

For in vitro interaction studies, 5µg of MBP-TC10 bound to amylose resin was incubated with 5µg of GST or GST-ObsDH in binding buffer (10mM Tris-Hcl pH 7.5, 150mM NaCl, 0.1% casamino acids, 0.1% Tween 20). Amylose resin were washed three times in ice-cold binding buffer. Total and amylose-bound proteins were denatured in Laemmli buffer and analyzed by Western blotting using anti-MBP and anti-GST antibodies.

GST-PBD pull down assay

Myotubes were transfected with myc-ObsCT or myc-ObsCT(ΔDH-PH). 72h after transfection, cells were rapidly washed in ice-cold PBS and lysed with lysis buffer.

Lysates were centrifuged for 5min at 14,000g at 4°C, and samples were taken from the supernatant to estimate total protein concentration. 20mg of GST-PBD fusion bound to Sepharose beads were added to cell lysate and incubated for 1h at 4°C.

Beads were washed four times in lysis buffer, and bound proteins were eluted in Laemmli buffer. Total proteins and PBD affinity-purified proteins were analyzed by Western blotting and the specific bands were quantified with Kodak 1D3.6 software.

Immunofluorescence and microscopy

Cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS. Cells were incubated with primary antibodies for 2h, followed by Alexa Fluor secondary antibodies and DAPI staining. Cell imaging was performed at the "Centre Regional d'imagerie Cellulaire de Montpellier". Fluorescence was viewed with a Leica Microscope (Leica DM6000, Wetzlar, Germany) using a 63x/

Statistical analysis

All quantitative data are presented as means ± SEM. Statistical analysis to determine significance was performed using Student's t tests. Differences were considered to be statistically significant at the p < 0.05 level.

Figure 1 :

 1 Figure 1: Expression and activity of TC10 during differentiation of human

Figure 2 :

 2 Figure 2: Interaction between Obscurin and TC10 proteins overexpressed in

Figure 4 :

 4 Figure 4: Obscurin activates TC10

Figure 5 :

 5 Figure 5: Activation of TC10 by expression of carboxyterminal region of

Acknowledgments

We are grateful to A. Saltiel, A. Blangy, E. Bertrand, C. Mettling and P. Vaudin for the gifts of the TC10 vectors, MBP-TC10, pCDNA3myc-RfC and lentiviral destination vectors, respectively. We thank D. Mathieu and A. Debant for the gifts of anti-myc mAb and GST antiserum. We would like to thank the tissue bank for research (MYOBANK-AFM) of the Association Française contre les Myopathies (AFM) for generously providing human muscle biopsies. This work was supported by grants to A.P. from the AFM. M.C-Q. and O.T. were supported by the AFM and the Agence Nationale de Recherche contre le Sida (ANRS) respectively.

(C) The same experiment as in (B) except that the immunoprecipitation (IP) was performed with anti-obscurin polyclonal antiserum (Obs) or non-specific rabbit IgG (IgG). Binding of TC10 to the immunoprecipitated protein was detected by immunoblotting (IB) with an anti-HA monoclonal antibody. The lanes marked Lysate were loaded with 10% of the total cell lysates. The results shown are typical of four independent experiments. (D) 293T cells were transfected with expression vectors for either myc-tagged carboxyterminal region (myc-ObsCT) of Obscurin or same vector lacking the DH-PH domain (myc-ObsCT(ΔDH-PH)), along with either HA-TC10CA (TC10/Q75L) [START_REF] Chiang | Insulinstimulated GLUT4 translocation requires the CAP-dependent activation of TC10[END_REF] or HA-TC10DN (TC10/T31N) [START_REF] Chiang | Insulinstimulated GLUT4 translocation requires the CAP-dependent activation of TC10[END_REF] as indicated. The immunoprecipitation was performed with anti-obscurin polyclonal antiserum (IP Obs).

The binding of TC10 mutants to the immunoprecipitated protein was detected by immunoblotting (IB) with an anti-HA monoclonal antibody. The lanes marked Lysate were loaded with 10% of the total cell lysates. The results shown are typical of three independent experiments. GTPases (RhoA,Rac1,Cdc42,TCL and TC10). Expression levels of the different proteins were confirmed by SDS-PAGE, membrane transfer and immunoblotting of each cell lysate with anti-GFP antibody (GFP). The specificity of the anti-TC10 antibody (Sigma T8950) was also tested (TC10) in the same way.


MATERIAL AND METHODS

The studied samples come from offshore Ecuador. The samples set includes 12 washed residues (Table 1), represented by three fractions 50, 150 and 250 µm. All the samples are rich in microfossils, 7 of them (in red) were analysed for their planktonic and benthic foraminifer assemblages in order to provide biostratigraphy and paleoenvironmental reconstructions.

Table 1. Samples prepared and analysed (in red) for foraminifer content.

Microfossils were studied from all the samples and all the fractions under stereomicroscope, magnification 50-75x. For detailed morphological study and documentation of foraminifera, scanning electron microscope was used (Plate 1-7). Quantitative analysis is based on counting of 150-200 specimens of both planktonic and benthic foraminifera from each fraction. Results of quantitative analysis are summarized in Figures 1234567.

Taxonomical determination of foraminifera was done using [START_REF] Loeblich | Foraminiferal Genera and their Classification[END_REF] (genus level, benthic foraminifera), database Microtax (Young et al., 2021; planktonic foraminifera), atlases of Pacific foraminifera (benthic foraminifera; Cushman 1993[START_REF] Cushman | The foraminifera of the tropical Pacific collections of the "Albatross", 1899-1900. Part 3, Heterolicidae and buliminidae[END_REF][START_REF] Todd | The Foraminifera of the Tropical Pacific Collections of the Albatross , 1899-1900: part 4[END_REF]Debenay, 2012) and publication dealing with Ecuador to Peru Pliocene and Pleistocene foraminifera [START_REF] Malmgren | Benthic Foraminifera from Middle to Late Pleistocene, coastal upwelling sediments of ODP Hole 686B, Pacific Ocean, off Peru[END_REF][START_REF] Mallon | Benthic Foraminifera of the Peruvian & Ecuadorian Continental Margin[END_REF][START_REF] Erdem | Pleistocene to Holocene benthic foraminiferal assemblages from the Peruvian continental margin[END_REF].

Biostratigraphical correlation was based on planktonic foraminifera using data about stratigraphic ranges of species from [START_REF] Bolli | Oligocene to Holocene low latitude planktic foraminifera[END_REF], Berrgren et al. (1995), [START_REF] Petrizzo | 3G. Planktonic foraminifera[END_REF] and database Young et al. (2021) and references therein (see description of individual samples).

Paleoenvironmental interpretations of planktonic foraminifera were based on data from [START_REF] References Aze | A phylogeny of Cenozoic macroperforate planktonic foraminifera from fossil data[END_REF], [START_REF] Kennett | Neogene Planktonic Foraminifera[END_REF], and [START_REF] Schiebel | Planktic foraminifers in the modern ocean[END_REF]. Benthic foraminifera have been paleoecologically interpreted following the data from [START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF], [START_REF] Kaiho | Benthic foraminiferal dissolved-oxygen index and dissolved-oxygen levels in the Modern ocean[END_REF], and [START_REF] Gooday | Foraminifera associated with phytodetritus deposits at a bathyal site in the northern Rockall Trough (NE Atlantic): Seasonal contrasts and a comparison of stained and dead assemblages[END_REF]. [START_REF] Kennett | Neogene Planktonic Foraminifera[END_REF]Schiebel et al., 1997;[START_REF] References Aze | A phylogeny of Cenozoic macroperforate planktonic foraminifera from fossil data[END_REF]. In the fraction 250 µm, the abundance of large Globoratalia, Orbulina, Globigerinoides and Trilobus increases. These genera live in warm, stratified oligotrophic waters of subtropical and tropical zones (Hemleben et al., 1989;Schiebel & Hemleben, 2005).

Core

Benthic -Hypoxic, eutrophic markers as Bolivina, Uvigerina and Bulimina prevail in the smallest fraction that suggests oxygen depletion and high nutrient content in the sediment [START_REF] Kaiho | Benthic foraminiferal dissolved-oxygen index and dissolved-oxygen levels in the Modern ocean[END_REF][START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF]Murray, , 2014)). In addition, oxiphylic cibidoids, which prevail in the largest fraction, indicate oxic environment on the sea floor. Paleobathymetrically, all the recorded species are known from outer shelf to upper slope environment [START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF](Murray, , 2014)).

Summary: outer shelf to upper bathyal paleoenvironment, well communicating with open ocean, eutrophic. High nutrient input might cause decreasing in oxygen content within the sediment, however oxic environment was stayed on the sea floor. 1994).

Paleoecology (Fig. 2): P/B-ratio varies in individual fractions from 20-50%, in fractions 50 and 150 µm Globigerina bulloides prevails, which represents shallow-dwelling, herbivorous, opportunistic species blooming under high-productivity regimen (Schiebel et al., 1997) and Globigerina umbilicata representing open ocean mixed-layer tropical/subtropical [START_REF] References Aze | A phylogeny of Cenozoic macroperforate planktonic foraminifera from fossil data[END_REF], in larger fractions increased ratio of Globigerinoides and Orbulina (warm-water genera, shallow-dwellers occurring in stratified, oligotrophic subtropical and tropical areas; Hemleben et al., 1989, Schiebel & Hemleben, 2005).

Benthic foraminifera -in fractions 50 and 150 µm, outer shelf to upper bathyal, high-nutrient markers as Bolivina and Bulimina prevail [START_REF] Murray | Ecology and Palaeoecology of Benthic Foraminifera[END_REF][START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF]. Moreover, Epistominella, Cassidulina and Nonionella indicate seasonal phytodetritus pulses [START_REF] Gooday | Foraminifera associated with phytodetritus deposits at a bathyal site in the northern Rockall Trough (NE Atlantic): Seasonal contrasts and a comparison of stained and dead assemblages[END_REF]. In fraction >250 µm, inner shelf miliolids significantly increase [START_REF] Murray | Ecology and Palaeoecology of Benthic Foraminifera[END_REF][START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF].

Summary: outer shelf paleoenvironment, well communicating with open ocean, seasonally eutrophized (both marine and terrigenous source of nutrients are possible) with transported microfauna from the inner shelf. [START_REF] Kennett | Neogene Planktonic Foraminifera[END_REF]Schiebel et al., 1997[START_REF] References Aze | A phylogeny of Cenozoic macroperforate planktonic foraminifera from fossil data[END_REF]. In the fraction 250 µm, G. bulloides is missing, and Orbulina, Globigerinoides and Trilobus increase. These genera live in warm, stratified oligotrophic waters of subtropical and tropical belt (Hemleben et al., 1989;Schiebel & Hemleben, 2005).

Benthic -Hypoxic, eutrophic markers as Bolivina, Uvigerina and Bulimina prevail in the smallest fraction that suggested oxygen depletion and high nutrient content in the sediment [START_REF] Kaiho | Benthic foraminiferal dissolved-oxygen index and dissolved-oxygen levels in the Modern ocean[END_REF][START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF]Murray, , 2014)). Moreover, the Globocassidulina, common in fraction 50 µm, indicates seasonal phytodetritus pulses [START_REF] Gooday | Foraminifera associated with phytodetritus deposits at a bathyal site in the northern Rockall Trough (NE Atlantic): Seasonal contrasts and a comparison of stained and dead assemblages[END_REF]. Oxiphylic cibidoids, which prevail in the largest fraction, suggest oxic environment on the sea floor. Paleobathymetrically, all the recorded species indicate outer shelf to upper slope environment [START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF](Murray, , 2014)).

Summary: outer shelf to upper bathyal paleoenvironment, well communicating with open ocean, eutrophic, with seasonal phytodetritus pulses. Oxic environment is expected on the sea floor, while decreasing in oxygen content is considered within the sediment. Paleoecology (Fig. 4): Planktonic foraminifera are rare in the smallest fraction, abundant in larger fractions (P/B-ratio in fraction 50 µm 25%, elsewhere 85%), in fractions 50 and 150 µm, Globigerina bulloides dominates that is a high-productivity and mixed-layer marker (Schiebel et al., 1997). In larger fractions, abundance of Globigerina umbilicata gradually increases. This species indicates open ocean mixed-layer in tropical/subtropical climate zone [START_REF] Kennett | Neogene Planktonic Foraminifera[END_REF][START_REF] References Aze | A phylogeny of Cenozoic macroperforate planktonic foraminifera from fossil data[END_REF].

Benthic foraminifera -Globocassidulina and Cassidulina, common in fraction 50 µm, indicate seasonal phytodetritus pulses [START_REF] Gooday | Foraminifera associated with phytodetritus deposits at a bathyal site in the northern Rockall Trough (NE Atlantic): Seasonal contrasts and a comparison of stained and dead assemblages[END_REF]. This eco-group is accompanied by hypoxic, eutrophic markers as Bolivina, Uvigerina and Bulimina that suggests oxygen depletion and high nutrient content in the sediment [START_REF] Kaiho | Benthic foraminiferal dissolved-oxygen index and dissolved-oxygen levels in the Modern ocean[END_REF][START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF]Murray, , 2014)). Also common occurrence of lagenids might be related to increased nutrient content in the sediment. In addition, oxiphylic cibidoids suggest oxic environment on the sea floor. Paleobathymetrically, all the recorded species indicate outer shelf to upper slope environment [START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF](Murray, , 2014)).

Summary: outer shelf to upper bathyal paleoenvironment, well communicating with open ocean, eutrophic, with seasonal phytodetritus pulses. High nutrient input might cause decrease in oxygen content in the sediment, however oxic environment is suggested in the sea floor. [START_REF] Pearson | Late Paleocene to middle Miocene planktonic foraminifer biostratigraphy of the Ceara Rise[END_REF].

Paleoecology (Fig. 5): P/B-ratio varies in individual fractions from 60-80%, in fractions 50 and 150 µm strongly dominated Globigerina bulloides and Neogloboquadrina dutertrei. Globigerina bulloides represents high-productivity marker (Schiebel et al., 1997), and Neogloboquadrina dutertrei characterizes open ocean, thermocline, tropical to subtropical species (Shackleton & Vincent, 1978;[START_REF] Kennett | Neogene Planktonic Foraminifera[END_REF][START_REF] References Aze | A phylogeny of Cenozoic macroperforate planktonic foraminifera from fossil data[END_REF]. The relative abundances of Trilobus increase in the largest fractions, and indicate warm-oligotrophic waters of subtropical and tropical belt (Hemleben et al., 1989, Schiebel & Hemleben, 2005).

Benthic foraminifera -Variegated cibicidoids dominated in all fractions, and indicate outer shelf to upper bathyal oxic environment. The Globocassidulina and Cassidulina indicating seasonal phytodetritus pulses are common in fractions 50 and 150 µm [START_REF] Gooday | Foraminifera associated with phytodetritus deposits at a bathyal site in the northern Rockall Trough (NE Atlantic): Seasonal contrasts and a comparison of stained and dead assemblages[END_REF]. Highnutrient and hypoxic markers as Bolivina, Uvigerina and Bulimina suggest oxygen depletion in sediment [START_REF] Kaiho | Benthic foraminiferal dissolved-oxygen index and dissolved-oxygen levels in the Modern ocean[END_REF][START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF]Murray, , 2014)). [START_REF] Kucera | Biochronology and evolutionary implications of Late Neogene California margin planktonic foraminiferal events[END_REF]. However, the recorded specimens (Plate 6, Figs. 17, 20) do not fully correspond to the holotype of the species.

Paleoecology: P/B-ratio varies in individual fractions from 60-90%, in fractions 50 and 150 µm Globigerina bulloides prevails, which is an eutrophic marker (Schiebel et al., 1997). G. umbilicata living in tropical/subtropical open-ocean mixed layer dominates in fraction 250 µm [START_REF] References Aze | A phylogeny of Cenozoic macroperforate planktonic foraminifera from fossil data[END_REF].

Benthic foraminifera -outer shelf to upper bathyal Globocassidulina and Cassidulina prevail in fraction 50 µm and indicate seasonal phytodetritus pulses [START_REF] Gooday | Foraminifera associated with phytodetritus deposits at a bathyal site in the northern Rockall Trough (NE Atlantic): Seasonal contrasts and a comparison of stained and dead assemblages[END_REF]. Variegated cibicidoids dominate in larger fractions (150 and 250 µm) that corresponds with outer shelf to upper bathyal oxic environment [START_REF] Kaiho | Benthic foraminiferal dissolved-oxygen index and dissolved-oxygen levels in the Modern ocean[END_REF][START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF]Murray, , 2014)). Paleoecology (Fig. 7): P/B-ratio varies in individual fractions from 70-80%; in fraction 50 only small globigerinas are recorded, in fraction 150 µm Globigerina bulloides prevails which represents a high-productivity marker (Schiebel et al., 1997) and Neogloboquadrina acostaensis representing open ocean, thermocline, tropical to warm subtropical species [START_REF] Kennett | Neogene Planktonic Foraminifera[END_REF][START_REF] References Aze | A phylogeny of Cenozoic macroperforate planktonic foraminifera from fossil data[END_REF]. The ratio of Globigerinoides and Orbulina increases in the larger fractions and indicates warm-oligotrophic waters of the subtropical and tropical zones (Hemleben et al., 1989;Schiebel & Hemleben, 2005).

Benthic foraminifera -Variegated cibicidoids dominate in all the fractions, indicating outer shelf to upper bathyal oxic environment. High-nutrient and hypoxic markers as Bolivina and Praeglobobulimina suggest high organic matter content and oxygen depletion in the sediment [START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF](Murray, , 2014)).

Summary: outer shelf paleoenvironment, well communicating with open ocean, well-ventilated on the sea floor and with decreasing oxygen content in the sediment.

CONCLUSIONS

Biostratigraphical correlation of samples was based on planktonic foraminifera. Because, I have no information about the geological relationship between the samples, each sample is separately interpreted. The majority of samples can be correlated with an interval from the Upper Pliocene (Piacenzian) to the Pleistocene (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) or slightly narrower interval (Piacenzian-Gelasian; sample KAT 12-33; Table 2.) Narrower interval (only Pleistocene-Holocene) can be determined for sample KAT 12-30. In addition, samples KAT 12-31 and 12-32 might be correlated only with an interval Zanclean-Holocene but Neogloboquadrina cf. inglei that is recorded in sample KAT 12-32 could indicate a narrower stratigraphical interval Piacenzian-Middle Pleistocene. However the recorded specimens do not fully fit with the holotype of the index taxon.

Paleoenvironmental interpretation suggests for all samples a broad communication with open ocean, samples [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] were deposited in outer shelf to upper bathyal, samples KAT 12-25 and 12-33 in outer shelf. Seasonal phytodetritus pulses are expected for all samples with exception sample KAT 12-33. While sea floor was well-ventilated, the oxygen depletion can be considered within the sediment (Table 2).

Sample Age Paleoecology

KAT 12-24/I/20-23 cm. Piacenzian -Pleistocene

Outer shelf to upper bathyal eutrophic, oxygen depletion in the sediment, oxic environment on the sea floor.

KAT 12-25/II/58-61 cm. Piacenzian -Pleistocene

Outer shelf, seasonally eutrophized (marine and/ or terrigenous source of nutrients) with transported microfauna from inner shelf.

KAT 12-29/I/76-79 cm Piacenzian -Pleistocene

Outer shelf to upper bathyal, eutrophic, with seasonal phytodetritus pulses, oxygen depletion in sediment, oxic environment on the sea floor.

KAT 12-30/I/50-53 cm. Pleistocene -Holocene Outer shelf to upper bathyal, eutrophic, with seasonal phytodetritus pulses, oxygen depletion in sediment, oxic environment on the sea floor.

KAT 12-31/I/35-38 cm. Zanclean -Holocene

Outer shelf to upper bathyal, eutrophic, with seasonal phytodetritus pulses, oxygen depletion in sediment, oxic environment on the sea floor.

KAT 12-32/I/60-63 cm.

Zanclean -Holocene (?Piacenzian -Middle Pleistocene)

Outer shelf to upper bathyal seasonally eutrophized during phytodetritus pulses.

KAT 12-33/I/42-45 cm. Piacenzian-Gelasian

Outer shelf paleoenvironment well-ventilated on the sea floor and with decreased oxygen content in the sediment.

  Figure 1. Sample KAT 12-24/I/20-23 cm. Quantified composition of foraminifer assemblages used for paleoenvironmental interpretations.

  Figure 2. Sample KAT 12-25/II/58-61 cm. Quantified composition of foraminifer assemblages used for paleoenvironmental interpretations.

  Figure 3. Sample KAT 12-29/I/76-79 cm. Quantified composition of foraminifer assemblages used for paleoenvironmental interpretations.

Figure 4 .

 4 Figure 4. Sample KAT 12-30/I/50-53 cm. Quantified composition of foraminifer assemblages used for paleoenvironmental interpretations.

Plate 4 .

 4 Foraminifer species identified in sample KAT 12-30/I/50-53 cm. 1, Pseudobrizalina lobata (Brady, 1881);

  Summary: outer shelf to upper bathyal paleoenvironment, well communicating with open ocean, well-ventilated on the sea floor with decreased oxygen content in the sediment probably due to increased seasonal phytodetritus pulses.

Figure 5 .

 5 Figure 5. Sample KAT 12-31/I/ 35-38 cm. Quantified composition of foraminifer assemblages used for paleoenvironmental interpretations.

  Summary: outer shelf to upper bathyal paleoenvironment, well communicating with open ocean, seasonally eutrophized during phytodetritus pulses.

2 ,

 2 Figure 6 Sample KAT 12-32/I/60-63 cm. Quantified composition of foraminifer assemblages used for paleoenvironmental interpretations.

Figure 7 . 2 ,

 72 Figure 7. Sample KAT 12-33/I/42-45 cm. Quantified composition of foraminifer assemblages used for paleoenvironmental interpretations.

); 12-13, Globigerinoides tenellus Parker; 14, Globigerinita glutinata

(Egger, 1893)

; 15, Globoturborotalita rubescens

(Hofker, 1956)

; 16, Verneuilina sp.; 17, Uvigerina auberiana d'Orbigny, 1839; 18, Uvigerina peregrina Cushman, 1923; 19, Fursenkoina sp.; 20, Reussella spinulosa (Reuss, 1850); 21, Bolivina spissa Cushman, 1926; 22, Pseudobrizalina lobata (Brady, 1881); 23, Bolivina alata (Seguenza, 1862); 24, Cibicides pachyderma; 25, Mississippina pacifica Parr, 1950; 26, Hanzawaia sp.; 27, Cibicides mckannai Galloway and Wissler, 1927;

28, diatom; 29, Radiolaria. Scale bar 50 µm. Sample KAT 12-25/ II/58-61 cm/80 g Planktonic foraminifera: Globigerina

	bulloides, Globigerina umbilicata, Trilobatus trilobus,
	Globigerinoides ruber, Globorotalia tumida, Globoconella inflata, Orbulina universa (Plate 2, Figs.
	21-28).
	Benthic foraminifera: Textularia sp., Quinqueloculina div. sp., Spirosigmoilina sp., Epistominella
	pacifica, Cibicidoides div. sp., Elphidium excavatum, Porosononion granosum, Nonionella auris,
	Bolivina div. sp., Bulimina div. sp., Uvigerina peregrina, Globocassidulina oblonga, (Plate 2, Figs. 1-
	21).
	Other microfossils: pyritized diatoms, Radiolaria, Pteropoda, Ostracoda, fragments of molluscs,
	sponge spicules, fish bones and scales.
	Biostratigraphy: Piacenzian-Pleistocene based on ranges of Globigerina umbilicata (FO at the base of
	Piacenzian, LO) within the Holocene; Kennett & Srinivasan 1983); Globorotalia tumida (FO at the
	base of Pliocene; Wade et al. 2011); Globoconella inflata (FO base during the Piacenzian; Wei,

Sample KAT 12-29/ I / 76-79 cm/ 67 g Planktonic foraminifera:

  Globigerina bulloides, Globigerina umbilicata, Globigerinita glutinata, Neogloboquadrina acostaensis, Neogloboquadrina sp., Orbulina universa, Globigerinoides tenellus, Globigerinoides ruber, Trilobatus trilobus, Trilobatus. sacculifer, Globorotalia tumida (Plate 3,. Globocassidulina oblonga, Cassidulina sp., Bolivina div. sp., Bulimina pagoda, Bulimina elongata, Uvigerina div.sp., Cancris oblongus, cibicidoids (Lobatula lobatula, Cibicidoides sp., Hanzawaia sp., Anomalina sp.), Lenticulina sp. Nonion sp. (Plate 3, Figs. 1-10). Pleistocene-Piacenzian based on co-occurrence of Neogloboquadrina acostaensis (LO within Pleistocene;[START_REF] Pearson | Late Paleocene to middle Miocene planktonic foraminifer biostratigraphy of the Ceara Rise[END_REF]; Globigerinoides tenellus (FO at the base of Piacenzian stage;[START_REF] Pearson | Late Paleocene to middle Miocene planktonic foraminifer biostratigraphy of the Ceara Rise[END_REF]; Globorotalia tumida (FO at the base of Pliocene;[START_REF] References Aze | A phylogeny of Cenozoic macroperforate planktonic foraminifera from fossil data[END_REF]; Globigerina umbilicata (FO at the base of Piacenzian, LO within the Holocene;[START_REF] Kennett | Neogene Planktonic Foraminifera[END_REF].

	Benthic foraminifera: Other microfossils: Radiolaria, sponge spicules, echinoid spines, fish bones and scales, otoliths,
	mollusk fragments, coral fragments.

Biostratigraphy:

Paleoecology (Fig.

3

): Planktonic foraminifera are rare in the smallest fraction (40 %), but abundant in larger fractions (P/B-ratio ¯65% and 90%). Globigerina bulloides and G. umbilicata markedly dominate in fractions 50 and 150 µm. Both species indicate mixed water, G. bulloides moreover eutrophic environment. Globigerina umbilicata suggests open-ocean mixed layer in tropical/subtropical climate belt

Sample KAT 12-30/ I/50-53 cm/68g Planktonic foraminifera:

  Globigerina bulloides, Globigerina umbilicata, Trilobatus quadrilobatus, Globigerinoides ruber, Globorotalia hirsuta, Globorotalia tumida, Neogloboquadrina sp. (Plate 4,. Globocassidulina oblonga, Cassidulina sp., Bolivina div. sp., Bulimina pagoda, Uvigerina peregrina, Cancris oblongus, lagenids (Lenticulina sp., Nodosaria sp., Dentalina sp. Planularia sp. Procerolagena cylindrocostata), cibicidoids (Lobatula lobatula, Cibicidoides sp., Hanzawaia sp., Anomalina sp.), Pseudononion granuloumbilicatum(Plate 4,. Pleistocene-Holocene based on co-occurrence of Globorotalia hirsuta(FO in Pleistocene;[START_REF] References Aze | A phylogeny of Cenozoic macroperforate planktonic foraminifera from fossil data[END_REF]; Globorotalia tumida (FO at the base of Pliocene; Wade et al., 2011; Globigerina umbilicata (FO at the base of Piacenzian, LO within Holocene;[START_REF] Kennett | Neogene Planktonic Foraminifera[END_REF].

	Benthic foraminifera: Other microfossils: Radiolaria (Plate 4, Figs. 14), sponge spicules, fish bones and scales.

Biostratigraphy:

  Globigerina bulloides, Neogloboquadrina dutertrei, Trilobatus trilobus, Trilobatus sacculifer, Trilobatus immaturus, Trilobatus cf. quadrilobatus, Globorotalia tumida, Globigerinita glutinata (Plate 5,. Quinqueloculina div. sp., Spiroloculina sp., Lenticulina sp. Epistominella pacifica, Bolivina div. sp., Bulimina elongata, Praeglobobulimina sp., Uvigerina sp., Trifarina sp., cibicidoids (Cibicides sp., Cibididoides div.sp., Hanzawaia sp.), Melonis sp., Nonion sp., Globocassidulina oblonga, Cassidulina sp. (Plate 5, Figs. 1-13). Zanclean-Holocene based on stratigraphical ranges of Globorotalia tumida (FO at the base of Pliocene; Wade et al., 2011); Neogloboquadrina dutertrei (FO at base of Zanclean stage;

	Sample KAT 12-31/ I/ 35-38 cm/ 52g
	Planktonic foraminifera: Benthic foraminifera: Other microfossils: Pteropoda, Ostracoda, sponge spicules.

2, Bolivina spissa Cushman, 1926; 3, Bolivina costata d'Orbigny, 1839; 4, Bulimina pagoda Cushman, 1927; 5, Uvigerina peregrina Cushman, 1923; 6, Globocassidulina oblonga (Reuss, 1850); 7, Cancris oblongus (Williamson, 1858); 8, Planularia sp.; 9, Pseudononion granuloumbilicatum Zheng, 1979; 10, Procerolagena cylindrocostata Albani & Yassini, 1989; 11-12, lobatula (Walker & Jacob, 1798), 12 -specimens with microborings; 13, Cibicides mckannai Galloway and Wissler, 1927; 14, Radiolaria; 15, Globigerina umbilicata Orr & Zaitzeff 1971; 16, Trilobatus quadrilobatus (d'Orbigny, 1846); 17, Globigerinoides ruber (d'Orbigny, 1839); 18, Globorotalia hirsuta (d'Orbigny, 1839); 19-20; Globorotalia tumida (Brady, 1877). Scale bar 50 µm.

Biostratigraphy:

Sample KAT 12-32 /I/60-63 cm. Planktonic foraminifera:

  Globigerina bulloides, G. umbilicata, Trilobatus trilobus, T. immaturus, Globigerinoides tenellus, G. ruber, Globorotalia tumida, Pulleniatina obliquiloculata , Orbulina universa, Globoturborotalita rubescens, Globigerinella calida, Neogloboquadrina cf. inglei and biserial Streptochilus globulosum (Plate 6,. Zaninettia conica, Textularia sp., Quinqueloculina div. sp., Epistominella pacifica, cibicidoids, Lenticulina sp., Nonionella auris, Bolivina div. sp., Globocassidulina oblonga, Cassidulina sp. In small fraction (50 µm) Globocassidulina prevails, in larger fractions (150 and 250 µm) cibididoids prevail(Plate 6,. Zanclean-Holocene based on co-occurrence of Streptochilus globulosum (FO at the base of Zanclean;[START_REF] Resig | Stratigraphic distribution of late Neogene species of the planktonic foraminifer Streptochilus in the Indo-Pacific[END_REF]; Globigerinella calida (FO within Zanclean; Kennet & Srinivasan, 1983); Globorotalia tumida (FO at the base of Pliocene; Wade et al., 2011); Pulleniatina obliquiloculata (FO at the base of Zanclean; Pearson & Chaisson, 1997); Globoturborotalita rubescens (FO at the base of Zanclean; Pearson & Chaisson, 1997). Based on Neogloboquadrina cf. inglei, this sample could correlate with a narrower stratigraphical interval such as Piacenzian-Middle Pleistocene

	Benthic foraminifera: Other microfossils: Pteropoda, abundant mollusk fragments, coral fragments, Bryozoan, sponge
	spicules, fish bones, scales and otoliths.

Biostratigraphy:

Sample KAT 12-33/I/42-45 cm. Planktonic foraminifera:

  Globigerina bulloides, Neogloboquadrina acostaensis, Trilobatus trilobus, Globigerinoides div.sp., Globorotalia tumida, Globorotalia scitula, Globigerinita sp., Globoturborotalita cf. woodi, Orbulina universa (Plate 7,. Quinqueloculina div. sp., Spiroloculina sp., Epistominella pacifica, Bolivina spissa, Praeglobobulimina sp., Trifarina sp., cibicidoids (Cibicides sp., Cibididoides div.sp., Hanzawaia sp., Anomalina sp., Anomalinoides sp.), Melonis sp., Nonion sp., Globocassidulina oblonga, Cassidulina(Plate 7,. Piacenzian-Gelasian from co-occurrence of Globigerinoides tenellus (FO at the base of Piacenzian;[START_REF] Pearson | Late Paleocene to middle Miocene planktonic foraminifer biostratigraphy of the Ceara Rise[END_REF]; Globigerinoides extremus (LO during the Gelasian stage; Wade et al., 2011); Globoturborotalita cf. woodi (LO during the Gelasian stage; Wade et al., 2011).

	Benthic foraminifera: Other microfossils: Pteropoda, Ostracoda, fragments of molluscs, sponge spicules, echinoderm
	spines.
	Biostratigraphy:

Table 2. Summary of biostratigraphic and paleoenvironment interpretation of the analysed samples.

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