extfieldsofstudy: Biology Medicine

text:
Distinct Biochemical Activities of Eyes absent During Drosophila Eye Development

Eyes absent (Eya) is a highly conserved transcriptional coactivator and protein phosphatase that plays vital roles in multiple developmental processes from Drosophila to humans. Eya proteins contain a PST (Proline-Serine-Threonine)-rich transactivation domain, a threonine phosphatase motif (TPM), and a tyrosine protein phosphatase domain. Using a genomic rescue system, we find that the PST domain is essential for Eya activity and Dac expression, and the TPM is required for full Eya function. We also find that the threonine phosphatase activity plays only a minor role during Drosophila eye development and the primary function of the PST and TPM domains is transactivation that can be largely substituted by the heterologous activation domain VP16. Along with our previous results that the tyrosine phosphatase activity of Eya is dispensable for normal Eya function in eye formation, we demonstrate that a primary function of Eya during Drosophila eye development is as a transcriptional coactivator. Moreover, the PST/TPM and the threonine phosphatase activity are not required for in vitro interaction between retinal determination factors. Finally, this work is the first report of an Eya-Ey physical interaction. These findings are particularly important because they highlight the need for an in vivo approach that accurately dissects protein function.

TPM, the PST domain alone, and the TPM alone, respectively. Eya and So bind to each other through the ED of Eya and the Six domain of So 8,11 to form a transcriptional activator complex. In addition, a series of Drosophila S2 cell-based transcriptional activation assays defined the PST/TPM domain as essential for Eya/So-mediated transactivation of a reporter. UAS-eya transgenes that lack both the PST-rich region and the TPM have drastically reduced ectopic eye-inducing capacity, with induction efficiency dropping from 98% to 1.5% 10 .
In addition to regulating transcription, Eya has predicted tyrosine and threonine phosphatase activities in the ED and TPM, respectively [17][18][19][22][23][24] . In Drosophila, tyrosine phosphatase-dead mutations lead to strongly reduced activities in ectopic eye induction and in vivo genetic rescue using the GAL4-UAS system 18,19,24 . In contrast to these studies, our previous findings revealed that eya genomic rescue (GR) constructs carrying mutations in two key tyrosine phosphatase active-site residues fully restore viability as well as eye formation and function in an eya null mutant background 25 . In mouse and Drosophila, the threonine phosphatase activity has been suggested to play an important role in the innate immune system 17 and a recent study using the GAL4-UAS system reported that Eya threonine phosphatase activity is not required for normal Drosophila eye development 24 .
Although previous cell culture and in vivo GAL4-UAS based expression studies have suggested specific functions for conserved Eya domains, we have shown that such assays may not always be reliable. In particular, we have developed a genomic rescue (GR) system that provides an accurate method for assessing the functional significance of individual protein domains in vivo 25,26 . In this study, we have used the GR strategy to conduct functional studies of Eya domains during Drosophila eye development. Interestingly, we found that a major function of Eya is transcriptional coactivation, while the threonine phosphates activity plays only a minor role during Drosophila development.

Results
The threonine phosphatase activity of Eya plays only a minor role in normal Drosophila development. To study eya function in vivo, we introduced a series of eya genomic rescue constructs (eyaGR) via site-specific transgenesis 27,28 to investigate the transcriptional activation and threonine phosphatase activity of Eya. A wild-type eya genomic rescue construct (eya + GR) is known to fully rescue viability and eye formation in an eya null mutant background, therefore serving as a positive control throughout our studies 25,26,29 . The eya Y4 GR construct has tyrosine-to-alanine substitutions for four key tyrosine residues known to be required for threonine phosphatase activity 17,24 (Fig. 1a). The eya ΔTPM GR construct has the entire TPM deleted but leaves the PST domain intact. Surprisingly, a single copy of each construct is able to substantially rescue eya 2 or eya cliIID mutant phenotypes, restoring viability and rescuing eye size to ~90% (Y4) or ~60% (ΔTPM) of wild-type, albeit with some mild disorganization (Fig. 1d,e and Fig. S1). While there appears to be a largely normal complement and arrangement of rhabdomeres in ommatidia of eya 2 ; eya Y4 GR/+ flies (Fig. 2e), eye discs from late third instar larvae (Fig. 2h) and 24 hrs after puparium formation (Fig. 2j) show defects in the number of cone cells and/or ommatidial fusion. Larval eye discs from eya Y4 GR and eya ΔTPM GR rescued animals are smaller and show a reduction in Eya and So staining anterior to and within the MF while expression levels are normal posteriorly (Fig. 2b' ,b",c' ,c"), suggesting that the threonine phosphatase activity does play a role during Drosophila eye development but this role is relatively minor as the eya Y4 GR construct can restore up to 90% of the eye size. The expression of the core RD genes Dachshund (Dac) and Eyeless (Ey) appear similar in eye discs of positive control and eya Y4 GR-rescued larvae (Figs 2a-c and 3a,b). In addition, we found no difference in photoreceptor axon projections between wild-type and eya 2 ; eya Y4 GR/+ flies (Fig. S2c), which show a regular pattern of projections in the lamina of the optic lobe.
eya plays an important role in the developmental events associated with morphogenetic furrow movement. Specifically, clonal analysis has shown that eya is required for the initiation and propagation of the MF and for regulation of the cell cycle 4,8,30,31 . Since loss of threonine phosphatase activity leads to a reduction of Eya expression anterior to and within the MF, we analyzed the effects of eya Y4 on both G1-arrest and induction of the proneural gene atonal (ato). We used the cell cycle marker Cyclin B to monitor G1 arrest. Normally, Cyclin B is exclusively expressed in cells in the G2 and M phases 32 . Immunohistochemistry shows eya Y4 GR rescued animals have largely normal Cyclin B and Ato expression patterns ( Fig. 3c-f), implying that the threonine phosphatase-inactive mutations do not adversely affect G1 arrest and initiation of retinal differentiation. This is not surprising since the loss of retinal cells in flies rescued with a single copy of eya Y4 GR is relatively mild; therefore, strong alterations in the expression of markers of cell cycle progression or photoreceptor differentiation are not expected.
The Eya threonine phosphatase-inactive mutation does not abolish interaction of Eya with Ey, So, or Dac. Phosphorylation is well known in other systems to regulate protein complex formation and protein stability via ubiquitin-mediated degradation 33,34 . Accordingly, we hypothesized that one or more of the RD proteins are direct substrates for Eya threonine phosphatase and that loss of this activity either disrupts the formation of RD protein complexes and/or destabilizes the RD proteins themselves. Furthermore, this effect may be specific to complexes involving Eyeless (Ey), thereby limiting effects anterior to the MF where Ey is expressed. We tested this hypothesis by doing co-immunoprecipitation (co-IP) in S2 cultured cells transiently transfected with epitope-tagged Eya, Ey, So, and Dac expression constructs. Similar amounts of RD proteins are expressed in transfected cells with or without Eya threonine phosphatase activity, and the Y4 mutation or the TPM deletion do not affect Eya protein expression levels in S2 cells (data not shown). As shown in Fig. 4, Eya Y4 and Eya ΔTPM co-IP with Ey, So, and Dac without obviously altered efficiency as wild-type Eya. Notably, this is the first report that Eya can bind to Ey. Previous studies also found that both Eya and Ey proteins interact with So 8,35 , suggesting Ey, Eya, and So may form a complex to mediate Drosophila eye development. Taken together, these observations suggest that the threonine phosphatase activity of Eya is not essential for interactions with other RD proteins.
The threonine phosphatase motif of Eya has transcriptional activation function. In addition to threonine phosphatase activity, previous cell culture transactivation reporter assays showed that the TPM Scientific RepoRts | 6:23228 | DOI: 10.1038/srep23228 has transcriptional activation function 10 . To test the hypothesis that this function is biologically relevant in vivo, we replaced the TPM only with VP16, a well-known heterologous transcriptional activation domain (Chasman et al., 1989). The resulting construct, eya ΔTPM+VP16 GR, was tested for rescue activity. Remarkably, while eya ΔTPM can restore about 60% of eye size, VP16 is able to largely complement loss of the TPM and restore eye development to approximately 90% of wild-type, both in eya 2 (Fig. 1a,e,f) and eya cliIID mutant backgrounds (Fig. S3a). The external eye morphology of eya ΔTPM+VP16 GR rescued eyes shows only minor disorganization compared to eya ΔTPM GR.
As shown in Fig. 2f, loss of the TPM causes abnormal ommatidial morphology in adult compound eyes. Flies rescued by one copy of eya ΔTPM GR have a reduced number and unusual arrangement of rhabdomeres compared with the normal trapezoidal array of photoreceptors in wild-type animals. Tangential sections of eya 2 ; eya ΔTPM+VP16 GR/+ adult eyes reveal ommatidia with the correct number and largely normal arrangement of rhabdomeres (Fig. S3b). Moreover, in contrast to wild-type ( Fig. S2a) and eya 2 ; eya ΔTPM+VP16 GR/+ (Fig. S2e) flies, axon terminations in the lamina plexus have irregular gaps and breaks (yellow arrows) in eya ΔTPM GR rescued flies, reminiscent of the photoreceptor axon defects in eya loss-of-function mutants 36 . These observations suggest that a major role of the TPM during Drosophila eye development is to provide transactivation function, that this activity is required for normal ommatidial development and photoreceptor axon projections, and that this function can be largely substituted by the VP16 domain.
The entire PST/TPM domain of Eya is critical for transcriptional activation during eye development. The PST/TPM domain of Eya is critical for transactivation in cell culture reporter assays 10 . In order to characterize the Drosophila Eya transcriptional activity in its native context in vivo, we generated four genomic rescue constructs: eya ΔPST/TPM GR (deletion of the PST/TPM domain), eya ΔPST GR (deletion of the PST domain alone), eya ΔPST/TPM +VP16 GR (substitution of both the PST and TPM domains with the VP16 activation domain) and eya ΔPST+VP16 GR (substitution of the PST domain alone with the VP16 activation domain) (Fig. 1a). We found that eya ΔPST/TPM GR completely fails to rescue eya 2 or eya cliIID mutant phenotypes, even when the transgene is present in two copies ( Fig. 1h and data not shown). We can readily detect the predicted, truncated eya ΔPST/TPM transcript and protein ( Fig. 5a-f) in late second instar eye discs prior to MF initiation, suggesting that although the transgene is expressed, at least initially, the Eya ΔPST/TPM protein is non-functional. While the eya ΔPST/TPM GR construct completely fails to rescue eya 2 mutant animals, the eya ΔPST GR retains slightly more function and can rescue about 5% of normal eye size (Fig. 1j). Previous S2 cell culture studies have suggested that both the PST and TPM domains contribute transcription activation function 10 and our GR data are consistent with these results. In addition to eya ΔTPM+VP16 GR, our other VP16 substitution genomic rescue results also confirm these findings. Specifically, the eya ΔPST/TPM +VP16 GR is sufficient to rescue about 5% of eye size in an eya 2 background (Fig. 1i), similar to that of the eya ΔPST GR construct alone. eya ΔPST+VP16 GR is able to restore eye development to ~30% of wild-type (Fig. 1k). Two copies of eya ΔPST/TPM +VP16 GR or eya ΔPST+VP16 GR consistently rescue eya 2 eye size better than one copy (Fig. S4). Moreover, eya ΔPST/TPM +VP16 GR, eya ΔPST GR, and eya ΔPST+VP16 GR fail to rescue eya cliIID mutants. These functional dissection studies reveal that the transactivation domain PST/TPM is essential for eye formation and viability in Drosophila. In addition, the PST domain is likely playing a more significant role than the TPM during Drosophila development since eya ΔTPM GR rescues 60% of the eye size compared to 5% of the eye size rescued by eya ΔPST GR and eya ΔTPM GR is able to restores viability to eya cliIID null mutants.
The PST/TPM domain regulates retinal determination gene expression. Eya can act as a transcriptional coactivator and physically interact with other RD proteins to regulate multiple developmental processes [7][8][9][10]37 . Therefore, we were interested in understanding the role of PST/TPM in RD gene regulation since it is critical for Eya function. Since eya ΔPST/TPM GR fails to rescue the eye phenotype of eya 2 animals and little Eya expression is detected at late third instar (data not shown), we used second instar larvae to assess the function of the PST/TPM when Eya ΔPST/TPM protein is still expressed (Fig. 5f). eya 2 flies rescued with two copies of eya ΔPST/TPM GR show slightly lower Eya expression compared to wild-type or eya GR-rescued animals at 68 hrs after egg laying (AEL) (Fig. 5c-f). We also found that Eya expression in eya 2 ; eya ΔPST/TPM GR eye discs is lower than that of wild-type discs at 56 hrs AEL (Fig. 5g-j). Similar reductions are observed for the expression of the retinal determination protein Dac, a known downstream target of Eya (Fig. 6a-h). In addition, in eya ΔPST/TPM clones (eya cliIID null clones rescued by a single copy of eya ΔPST/TPM GR) at 72 hrs AEL, Dac expression is reduced while the expression of Eya ΔPST/TPM is normal (Fig. 6i-l, yellow arrows). Taken together, these data imply that the PST/TPM domain of Eya is required for normal Dac expression.
Moreover, ey-Gal4 induced So expression in eya 2 animals rescued by one copy of eya ΔPST/TPM GR partially restores Dac expression (Fig. 7a-d), but has no effect on expression of Eya (Fig. 7e-h). These observations suggest that the PST/TPM positively regulates expression of Dac through the Eya binding partner So.
To test if the PST/TPM deletion affects Ey regulation and photoreceptor differentiation, we assayed Ey and Elav expression in eya ΔPST/TPM rescued eya null mutant clones. We found that eya ΔPST/TPM clones show a complete loss of Elav expression, a marker of photoreceptor differentiation 38 , posterior to the MF (Fig. 8a-d). In eya ΔPST/TPM clones posterior to the furrow, we found strong Ey expression (Fig. 8a'-d'), suggesting the PST/TPM domain of Eya is required for Ey suppression. Additionally, eya ΔPST/TPM clones result in the loss of photoreceptor development and black overgrowths in adults (Fig. S5d).

Deletion of the PST/TPM does not abolish interactions between Eya and Ey, So, or Dac. So
and Dac are known binding partners of Eya 7,8,11 . Since eya ΔPST/TPM GR rescued flies have no eyes, similar to the loss-of-function phenotypes of the core RD genes (ey, so, and dac), we hypothesized that the PST/TPM domain may mediate specific, essential interactions between Eya and Ey, So, or Dac. To test this hypothesis, we carried out co-immunoprecipitation (co-IP) experiments. As shown in Figs 4 and 9, both wild-type and Eya ΔPST/TPM can co-IP with Ey, So, and Dac, suggesting that deletion of the PST/TPM does not abolish the interactions between Eya and these three RD proteins. These observations are consistent with previous findings that Eya-So and Eya-Dac interaction is mediated via the ED of Eya 7,8,11 . The Eya domain that mediates Eya-Ey physical interaction remains to be determined.

Discussion
In this paper we report that loss of threonine phosphatase activity has little effect on Drosophila eye development, since eye development in eya Y4 GR rescued flies proceeds relatively normally. On the other hand, the essential function of the PST and the threonine phosphatase motif (TPM) is transcriptional activation that can be largely complemented by the heterologous activation domain VP16. Together with our findings that the PST and TPM are required for normal Drosophila eye development, we conclude that a major function of Eya during Drosophila eye development is as a transcriptional coactivator. Although the tyrosine phosphatase activity of the Eya Domain (ED) is dispensable for Eya function 25 , the specific role the ED plays in vivo has not been reported.
The retinal determination (RD) network is a small group of highly conserved transcriptional regulators that are both necessary for eye development and sufficient to trigger ectopic eye formation when overexpressed (a,b) Co-immunoprecipitation (co-IP) studies between wild-type and threonine phosphatase-dead Eya (Eya Y4 and Eya ΔTPM ) and Ey or So are shown. Flag-tagged Eya was co-expressed with HA-Ey or Myc-So in S2 cells and co-IP with anti-Flag beads followed by western blotting (WB) was performed. Ey, So, and Eya (wild-type and mutants) were detected by anti-HA, anti-Myc, and anti-Flag antibodies, respectively. Lanes 1, 2, and 3 show that Eya WT , Eya Y4 and Eya ΔTPM can pull down So and Ey, respectively. Empty vector is the negative control ( in other imaginal discs [1][2][3][4][5][6][7][8]14,21,39 . As a vital member of the RD network, a unique feature of the Eya proteins is that they have several distinct biochemical activities. In Drosophila, previous cell culture reporter assays and cDNA-based Gal4-UAS genetic rescue studies suggested that the PST-rich region is a transactivation domain and plays a role in ectopic eye induction, while the TPM and ED possess threonine and tyrosine phosphatase activity, respectively 10,18,19,24 . Intriguingly, our results using genomic rescue constructs are consistent with previous studies of the PST/TPM transactivation domain, but are contrary to previous reports that the tyrosine phosphatase domain, but not the threonine phosphatase domain, governs Drosophila eye development.
In our work, we have found that both the TPM and PST contribute transcriptional activation for normal eye development. Substituting the heterologous activation domain VP16 for the TPM and PST domain substantially restores Eya function. Two reasons could account for the failure of complete rescue by VP16. First, the TPM or PST have other, distinct functions. Although we have excluded the possibility that the TPM and PST are required for Eya binding with Ey, So, or Dac in this report, we cannot rule out other possibilities. For example, previous findings identified the PST/TPM domain of Eya as the primary target of Nmo and Abl-mediated phosphorylation in kinase assays 36,40 . Second, there may be insufficient activation function provided by VP16 -perhaps due to an inability to make specific contacts with other proteins, or that the fusion proteins do not have the proper conformation to interact properly via other domains.
The transcriptional role of Eya has been studied in Drosophila through genetic and/or biochemical interaction with the transcription factors So and Dac 7,8 . In this paper, we further indicate that the PST/TPM domain positively regulates Dac expression and this regulation may be mediated via So. Moreover, the PST/TPM is required to suppress Ey expression posterior to the furrow. These observations are consistent with previous reports that dac expression requires both so and eya 7,14,39,41 and both Eya and So are necessary to mediate Ey repression posterior to the MF 42 . Our studies localize these functions of Eya to the PST/TPM domain.
Although genetic interactions between Eya and Ey have been widely reported, physical interactions between these two RD proteins have not. In this paper, we report that Eya physically interacts with Ey for the first time. Previous studies also found physical interactions between Eya-So 8 and Ey-So 35 , suggesting that Ey-Eya-So may form a ternary complex. In addition, previous findings show that ectopic eye induction by Ey requires the presence of Eya and So 43 , and the expression patterns of all three genes overlap extensively and are nearly identical anterior to the MF 43 . Moreover, misexpression of Eya and So induces the formation of ectopic eyes; however, this effect is lost in an ey mutant background 8,21 . Finally, ey is a direct target of Eya and So 11,44 and vice versa -eya and so are direct targets of Ey 45,46 . Since Groucho is a repressor of the Eya-So complex 10 , Ey may act as an activator of  1-3 show RT-PCR on RNA prepared from eyes discs 68 hrs after egg laying (AEL). Lane 1: wild-type; Lane 2: eya 2 ; eya ΔPST/TPM GR; Lane 3: eya 2 ; Lane 4: water. A truncated ΔPST/TPM transcript is readily detected (Lane 2). (b) An anti-Eya Western blot on extracts prepared from 68 hrs after egg laying (AEL) eye discs (n = 40/lane) from either eya cliIID /CyO; eya + GR/+ or eya cliIID /CyO; eya ΔPST/TPM GR/+ animals shows a readily detectable, truncated ΔPST/TPM protein (*). Heterozygous eya cliIID animals were used to obtain enough tissue for the experiment. Western blot presented in b is cropped to improve clarity and full-length blot is presented in Supplementary Fig. S10. (c-f) Eye discs prepared from larvae 68 hrs AEL are stained for Eya expression. (g-j) Eya staining of 56 hrs AEL eye discs.  Eya-So to increase transcriptional output of Dac. Consistent with this hypothesis, loss of ey, eya, or so function causes loss of Dac expression, suggesting that Ey, So, and Eya are primary regulators of Dac 7,8,47 . Similar relationships have been observed with Pax6, Eya1/2 and Six3, mouse orthologs of ey, eya, and so, respectively. Specifically, mouse Pax6 mutants have reduced levels of Eya1 and eya 2 in the optic vesicle and overlying ectoderm 48,49 and Pax6 induces expression of Six3 when ectopically expressed in mice 50 . In addition, we used STRING 51 , a database of known and predicted protein interactions, to predict protein-protein interactions for Ey, Eya and So. As expected, we found equally high associations for all three pairs of complexes (Fig. S6), providing further evidence of strong interactions among these RD proteins, which may act together in a ternary complex.
In addition, our genomic rescue assays show that the threonine phosphatase activity is largely but not entirely dispensable for Drosophila eye development. Our threonine-phosphatase inactive GRs can robustly rescue eye formation in eya null mutants, but the rescued eyes show disorganized external and internal morphology as compared to wild-type rescue controls. This result is in contrast to another report based on the GAL4-UAS system that finds the threonine phosphatase activity of Eya to be dispensable during eye development 24 . The reason for this difference is that our GR system offers higher resolution thereby allowing detection of more subtle defects in morphology, while the GAL4-UAS system is a less accurate approach. In particular, Liu et al. did in fact observe a disorganized eye phenotype in eya 2 flies rescued by UAS-eya Y4 . However, this phenotype appeared similar to the imperfect rescue achieved with the wild-type UAS-eya transgene. For this reason, they could not uncover the requirement for the threonine phosphatase activity during differentiation. This report highlights the need for careful interpretation of results based on the GAL4-UAS system and the superior sensitivity of the GR method. Although the threonine phosphatase activity of Eya plays only a minor role during eye development, it has been reported to be involved in the innate immune response in both Drosophila and mouse 17,24 .
In summary, we have shown that both the transcriptional activation and threonine phosphatase activity of Eya are required for normal Drosophila eye development. However, a primary function of Eya during this process is transcriptional coactivation, while the phosphatase activity plays only a minor role. Our study provides an accurate approach to assess the functional significance of individual protein domains in vivo, highlighting the importance of the transactivation function of Eya during Drosophila development. As Eya is conserved and plays important roles in retinal development throughout the metazoa, the underlying mechanisms of Eya function are likely to be conserved in vertebrates as well.

Methods
Fly strains and maintenance. All flies were maintained with standard corn meal and yeast extract medium at 25 °C. Canton-S was used as a wild-type control. Heat shocks were performed at 37 °C as described previously 52 . To test the function of the mutant eyaGR during eye development, we crossed transgenes into the following mutant backgrounds: eya 2 , which completely lack eyes due to a deletion of an enhancer required for eya expression during eye development 4 , and eya cliIID , which is a null allele caused by a premature stop codon that causes recessive embryonic lethality 53 . Wild-type clones and eya ΔPST/TPM clones were generated by crossing w/Y; FRT40A and w/Y; eya clillD FRT40A/CyO; eya ΔPST/TPM GR with ywhs-flp; w+ ubiGFP, FRT40A animals, respectively.

Recombineering-induced mutagenesis of eya + GR and Drosophila transgenesis.
A two-step recombineering method was used to create the Y4, ΔTPM, ΔTPM+ VP16, ΔPST/TPM, ΔPST/TPM+ VP16, ΔPST and ΔPST+ VP16 mutations in the eya + GR construct as described previously 54 . Recombineering products were verified by DNA sequencing and restriction enzyme fingerprint digestion prior to transgenesis. Constructs were inserted into the attP2 docking site on the third chromosome using PhiC31-mediated transgenesis and site-specific integration was confirmed by genomic PCR with attP/attB primers 28 . Transgenic flies were confirmed by genomic DNA PCR sequencing. Primer sequences are available on request.
Construction of cell culture expression plasmids. We used the Q5 Site-Directed Mutagenesis Kit (NEB) to introduce a series of mutations in cell culture expression plasmids which were confirmed by DNA sequencing. These mutations include: pMT-Flag-Eya Y4 , pMT-Flag-Eya ΔTPM , pMT-Flag-Eya ΔPST/TPM and pMT-HA-Dac. pAHW-Ey was generated from destination vector pAHW and pUAST-Ey (a gift from Dr. Rui Chen, Houston, TX) according to the Gateway protocol provided by the Drosophila Genomics Resource Center. pMT-Flag-Eya, pMT-Myc-So, pMT-dac, and pAHW were kindly provided by Dr. Ilaria Rebay (Chicago, IL). Primers used in this report are listed in Table S1.  Co-IP and western blots. Transfected cells were lysed by rocking at 4 °C for 30 min in Pierce IP lysis buffer (Thermo Fisher Scientific) with a Roche Complete, Mini, EDTA-free protease inhibitor cocktail tablet. The lysates were subjected to immunoprecipitation with anti-Flag-conjugated agarose beads (Sigma) for 2 h at 4 °C. After washing three times with lysis buffer, immunoprecipitates were boiled in 4× NuPAGE LDS sample buffer (Novex), and western blotting was carried out according to the NuPAGE electrophoresis (Novex) protocol with rabbit anti-Flag (1:1000, Sigma), rabbit anti-MYC (1:100, Santa Cruz Biotechnology), and rabbit anti-HA (1:200, Santa Cruz Biotechnology) antibodies.
For tissue preparation, 68 hrs AEL eye discs (n = 40) were collected in cold RIPA lysis buffer (Thermo Fisher Scientific). After centrifuge at 20000 g for 10 min at 4 °C, the supernatant was transferred to a new tube and ready for western blot analysis.
Histology and immunohistochemistry. Staining of eye discs and imaging of the adult eye were conducted as described previously 42 . Immunohistochemistry on 48 hr pupal eye discs and tangential sections of adult eyes were generated as previously described 55 . For antibodies used, please reference Table S2.
RT-PCR. RNA was extracted from 56 hrs AEL eye discs using PureLink RNA Mini Kit (Ambion). Reverse transcription was performed according to the instructions of SuperScript One-Step RT-PCR kit (Invitrogen).


extfieldsofstudy:
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COSMIC: somatic cancer genetics at high-resolution

COSMIC, the Catalogue of Somatic Mutations in Cancer (http://cancer.sanger.ac.uk) is a high-resolution resource for exploring targets and trends in the genetics of human cancer. Currently the broadest database of mutations in cancer, the information in COSMIC is curated by expert scientists, primarily by scrutinizing large numbers of scientific publications. Over 4 million coding mutations are described in v78 (September 2016), combining genome-wide sequencing results from 28 366 tumours with complete manual curation of 23 489 individual publications focused on 186 key genes and 286 key fusion pairs across all cancers. Molecular profiling of large tumour numbers has also allowed the annotation of more than 13 million non-coding mutations, 18 029 gene fusions, 187 429 genome rearrangements, 1 271 436 abnormal copy number segments, 9 175 462 abnormal expression variants and 7 879 142 differentially methylated CpG dinucleotides. COSMIC now details the genetics of drug resistance, novel somatic gene mutations which allow a tumour to evade therapeutic cancer drugs. Focusing initially on highly characterized drugs and genes, COSMIC v78 contains wide resistance mutation profiles across 20 drugs, detailing the recurrence of 301 unique resistance alleles across 1934 drug-resistant tumours. All information from the COSMIC database is available freely on the COSMIC website.

INTRODUCTION
A large proportion of human cancer is caused by the acquisition of somatic mutations across an individual's lifetime, and large-scale sequencing of patient cohorts has now described millions of such mutations across the human genome. The Catalogue of Somatic Mutations in Cancer (COSMIC) is a database system that collects these somatic mutation data from a variety of public sources into one standardized repository, and make it easily explorable in a variety of graphical, tabulated and downloadable ways. To provide the greatest support in cancer research, COSMIC encompasses all forms of human cancer, from the most frequent cancers in lung, breast and colon, to extremely rare forms of blood cancer, observed by a clinician only once or twice in a career.
Begun in 2004 with curations across only four human genes (1) COSMIC has grown into a large genome-wide system to explore patterns of somatic mutations in all cancers; substantial genetic data are now generated routinely across human tumours and this is captured by expert, standardized curation procedures. Additionally, recent studies have characterized particular mutations in the evolution of genetic resistance to clinical therapeutics. While ensuring that COSMIC encompasses the full coverage of human cancer genetics, these resistance mutations are emphasised in a new section to highlight their impact in clinical oncology.

DATABASE CONTENT
As described previously (2,3), curation of somatic mutation data into COSMIC proceeds via two parallel pathways. Expert manual literature curation addresses the most important cancer genes, emphasizing full and exhaustive curation of existing literature before release, followed by regular updates. These key cancer genes are selected from the Cancer Gene Census (4), a listing of over 600 genes with substantial evidence describing their strong role in oncology. High quality control results in the rejection of over 30% of papers due to inconsistency or insufficient detail. In parallel, expert curation of genome-wide tumour analyses requires manual assignment of tumour classifications and clinical details, but large files of genetic variant data are annotated and uploaded via a semi-automated system using Ensembl as a source of transcriptome data. Total contents in the v78 release (Sept 2016) are described in Table 1.
Somatic mutation data are collected across all cancer diseases, currently 1335 disease descriptions across more than 5000 detailed classifications. Manual literature curation Emphasizing the effectiveness of the literature curation approach, over 60% of COSMIC's genome-wide content is curated from scientific literature, whilst less than a third is from consortium sources. Once curated into COSMIC, all data are standardized and combined in a single database. Every mutation is assigned a co-ordinate on the human reference genome. Since 2015, the default reference is GRCh38, but an archive system on GRCh37 is maintained. Each mutation is either assigned a coding or non-coding annotation. Non-coding descriptions are simple statements of DNA sequence change at specified genomic locations. Coding mutations are additionally annotated according to their impact on the gene they affect, describing coding nucleotide sequence change and peptide sequence change. Manually curated mutations, on key Cancer Census genes, are annotated to transcripts which are most supported in the literature (to best support the community scrutinizing these genes). However, all other gene variations are annotated via the Ensembl database (7) to the longest CCDS transcript (Consensus Coding Domain Sequences; 8). Gene Fusions are described in terms of their exon content from each partner gene. Again, the transcripts that are selected to describe fusions are those most used in the literature, ensuring best support to this community. Gene expression variants are additional numeric values (Z-scores) linked to named genes in COSMIC, referencing expression differences in each sample from a population norm (calculated per-disease cohort). Copy Number changes are annotated to describe a ploidy value for each gene across each tested tumour sample, and described as gain or loss when compared to the average ploidy value across each tumour. Hyper/hypo-methylated CpG dinucleotides are described in a sample when the beta-value for each probe differs substantially from the per-disease pop-ulation norm. Finally, structural breakpoints are given basic annotations to reference genome co-ordinates, and interpreted into more descriptive consequence annotations when supported by additional published information.
Tumour classification is standardized and described in several nomenclatures to ensure high-resolution, but also to facilitate integrative analyses. Each tumour sample is primarily classified in COSMIC according to a custom vocabulary developed by a team of leading pathologists; available and described here: http://cancer.sanger.ac.uk/cosmic/ classification. This classification is now translated into the National Cancer Institute thesaurus ("NCIt"; 9; https: //ncit.nci.nih.gov/ncitbrowser), selected as the highestresolution public ontology across cancer diseases. In parallel, the NCIt classifications are also translated into Experimental Factor Ontology classifications ("EFO"; 10) supporting multiple ways to integrate COSMIC data into wider analyses across cancer genetics.
Increased genomic curation has led to a larger range of variants annotated across the genome, with every human gene now showing a spread of somatic mutation across its length. In order to reduce the complexity of these data and support studies identifying disease drivers, all point mutations in COSMIC is tagged with a single nucleotide polymorphism (SNP) status and a pathogenicity value. The SNP status defines whether each variant has been previously described in the 1000 genomes study (11), or in a panel of normal samples used as controls in ICGC genomic profiling experiments. Pathogenicity scores are determined by the algorithm FATHMM-MKL (12) with scores interpreted to specify each variant as 'pathogenic' or 'neutral'.
A new topic for curation was introduced in 2016, encompassing the genetics of drug resistance. As described, millions of mutations have been observed across cancer, with thousands of alleles implicated in disease causation. Of these, a small number are additionally described as causing clinical resistance to pharmaceutical therapies. These details, when published, are also captured in the COSMIC curation processes, with the latest release (v78; Sept 2016) describing the range of mutations conferring resistance to 20 anti-cancer treatments.

DATA ACCESS
COSMIC is most easily explored using its public website (http://cancer.sanger.ac.uk). This has been custom-built to make the many annotations in COSMIC easy to explore in user-friendly graphical ways, whilst also providing large tabulated data sets which underpin each presentation. Beyond this website, described below, opportunities have been pursued to make COSMIC available to other resources enabling them to add broad cancer context to further public genomic resources. For instance, COSMIC directly supports pediatric cancer research with full data sets in St. Jude ProteinPaint (13) (https://pecan.stjude.org/#/proteinpaint), provides genome-wide context of cancer somatic variation in Ensembl (7) (http://www.ensembl.org) and highlights oncology targets across the Cancer Gene Census in OpenTargets (https://www.opentargets.org). A COSMIC beacon (http://cancer.sanger.ac.uk/cosmic/beacon) is also available, conforming to GA4GH standards (http://ga4gh. org/#/beacon), allowing COSMIC to be queried within large federated analyses. Full database contents in a variety of useful formats, including Oracle dump files, can be obtained via download (http://cancer.sanger.ac.uk/cosmic/ download), requiring registration (https://cancer.sanger.ac. uk/cosmic/register).

Website overview
The COSMIC website is available online at http://cancer. sanger.ac.uk. The front page (Figure 1) offers multiple ways to explore the database ('Resources', 'Tools'), and a range of pages describing the database content, how the data were generated, and details on its access ('Expert Curation', 'Data'). On the right-hand side, a Circos diagram summarizes mutation recurrence across all tumour genomes in COSMIC (version 78, Sept 2016, contains 28,366 genomes and exomes). With a zoomed version for closer exploration (http://cancer.sanger.ac.uk/cosmic/landscape) of recurrent peaks, this image is clickable and responds with a detailed genomic perspective across the region chosen. Primarily, however, COSMIC is navigated via the central 'Search' box that will accept any gene name or synonym, mutation syntax, sample/cell line name or paper author and respond with a list of options matching the input search term alongside a few details to aid selection. Once a selection is clicked, a summary page will be shown, overviewing the gene, sample, mutation or publication selected, with further links to specialize a query across COSMIC; in this way somatic mutation trends can be explored in detail across multiple genes and disease types.
Mutation trends across COSMIC are most easily explored when starting with a specific gene or cancer disease. Primarily driven by Human Genome Organisation (HUGO) gene names (http://www.genenames.org/), gene searches will also search all gene synonyms (as well as other terms, including sample/mutation/author) and return any entries that include the characters specified. For instance, searching for 'PTEN' will return one gene, since this gene name is unique. However, searching for 'RAS' returns 70 entries, since this term occurs in multiple gene names and synonyms; choosing a RAS family member (e.g. KRAS) returns a more specific result.
Once a gene is selected, COSMIC begins gene analysis by showing the distribution of mutations across the gene's length in a histogram format. In this graphic, the x-axis represents the linear peptide sequence (from Methionine to Stop), with an option to represent nucleotides instead of amino acids (in the right-hand-side Filters box). The histogram graphic summarizes mutation content across several different mechanisms, encompassing several Y axes, vertically aligned, with peak height representing the number of samples in which each mutation was observed (scale bar on left side shows numbers of samples mutated at each position). From top to bottom, mini-histograms show mutation recurrence peaks across the gene sequence for: single nucleotide substitutions, multinucleotide substitutions, small insertions, small deletions, copy number gain/loss, gene overexpression/underexpression and hyper/hypo methylated CpG dinucleotides. For instance, Figure 2 shows the mutation profile for the ABL1 gene, a major driver of blood cancers. Clearly most cancer mutations cluster within and around the tyrosine kinase domain of this peptide, a characteristic of driver mutations in this broad gene family. Using the left-hand scale bars to judge the impact of each mutation type, ABL1 primarily drives cancer development by kinase-domain point mutations and over-expression. Only small numbers of other mutation types are observed. The graphic is zoomable, requiring a simple click-and-drag of the mouse cursor across the region of interest to display it in more detail.
The right-hand side Filters box allows this histogram (together with all information in other tabs on this page) to be sifted and explored in multiple ways, with options to select mutations from a particular tissue or disease, or a particular gene region, specify only confirmed somatic mutations, or only cell lines or patient samples, as well as a variety of other more mechanistic mutation characteristics.
The webpage shows different perspectives across a gene's mutation content in separate 'tabs', arranged at the top of the page, offering several ways to scrutinize the large amount of information available. Particularly useful to explore the impact of selected genes across hundreds of cancer types curated, 'Tissue' shows a breakdown of mutations across tissues and diseases arranged in a matrix. Four mutation types are shown on the x-axis, and primary tissue types on the y-axis. Between these, small red singlebar histograms show the impact of each mutation type on each tissue, larger bars indicating a higher impact, allowing very rapid evaluation of which tissues are affected most by the gene selected ('n' is also shown to evaluate the significance of the red bar; if the number is small, the significance is low). For ABL1, the longest bar is 'Haematopoietic & Lymphoid', highlighting its impact in blood cancers. Clicking on the phrase 'Haematopoietic & Lymphoid' will show a breakdown of the cancer disease classifications under this tissue, indicating that ABL1 mutations primarily drive 28% of evaluated Chronic Myeloid Leukaemias, and 32% of Acute Lymphoblastic Leukaemias (in COSMIC v78).

Cancer browser
Cancer diseases are most easily navigated using the Cancer Browser (http://cancer.sanger.ac.uk/cosmic/browse/tissue), where a selection can be made from a list, avoiding the need to type often complex pathology terms. Selection of a primary tissue is essential, then a choice is offered across related subtissue, histology and subhistology terms. These are all clickable, and all choices may be skipped using the top 'include all' option. Once a selection is made, clicking 'Go' will calculate mutation profiles across all genes for the disease selected. For instance, choosing simply 'Lung' cancer and pressing Go, immediately shows the most frequently mutated top 20 genes in Lung cancer (ordered by mutation frequency), primarily highlighting well-characterized genes such as TP53, EGFR and KRAS). Similar to the Gene Analysis pages, additional tabs offer different perspectives on the mutation burden of the selected disease.

Genome browser
Somatic mutations in cancer are now annotated across the entire human genome. With over 4 million coding point mutations and over 13 million non-coding point mutations, a method to evaluate the genomic context across this information is essential. The COSMIC Genome Browser (http: //cancer.sanger.ac.uk/cosmic/browse/genome) achieves this, showing all COSMIC mutation data aligned with a range of genomic annotations, using an instance of the Jbrowse platform (14) that makes enormous data easily and rapidly explorable. The front page to this system offers Search functionality, described above, but returns a genomic (rather than genic) view on the selection made. Initial views show gene structure, point mutations and genome structure breakpoints for the chosen region. Zooming in and out allows exploration of details and trends across a genomic region, with mutation details transforming into histograms when the data becomes too crowded onscreen. On the left side of the screen, a number of additional tracks are offered, divided into annotations of genomic structure/function from the Ensembl database (7) and multiple annotations of somatic mutation from the COSMIC database. These allow the investigation of all somatic mutation and variation data in alignment with structures of coding genes, non-coding RNAs (including microRNAs) and regulatory regions. In addition, the content of dbSNP (15) (https://www.ncbi.nlm. nih.gov/snp/) is made available, to allow the exploration of relationships between somatic mutations in COSMIC and wider human variation encompassed by dbSNP.

Genetics of drug resistance
As precision medicine progresses, an increasing literature is emerging which describes genetic responses to targeted therapies. Somatic mutations acquired during tumour evolution are often under selective pressure when monotherapies are applied to cancer patients, leading to emergence of new clones containing resistant alleles and causing therapeutic failure. COSMIC now curates this information (overviewed at http://cancer.sanger.ac.uk/cosmic/ drug resistance), describing the range of somatic mutations Nucleic Acids Research, 2017, Vol. 45, Database issue D781 Figure 2. The main Gene Analysis histogram summarises all mutation content across a single gene, in this case gene ABL1. From top-to-bottom, histograms represent mutation recurrence, at each nucleotide/amino acid position, divided into separate segments for single nucleotide substitutions, multinucleotide substitutions, insertions, deletions, copy number gain/loss, gene over/under-expression and CpG hyper/hypo-methylation. Mutation recurrence across the tyrosine kinase domain (highlighted in purple) indicates that this, when mutated, is the key region driving cancer. causing resistance to selected therapeutics. In v78 of COS-MIC (September 2016), 20 drugs are detailed, describing 301 unique sequence variants that have caused drug resistance in 1934 tumour samples. This information is presented in the COSMIC website in graphical form with pie charts and histograms describing the recurrence of genes and mutations in the evolution of therapeutic resistance. For example, tumours driven by ABL1 mutations have been treated with four named therapies (Imatinib, Dasatinib, Bosutinib and Nilotinib) as well as other unnamed kinase inhibitors. The landscape of resistance to these drugs is presented at http://cancer.sanger.ac.uk/cosmic/gene/analysis? ln=ABL1#dt, with options to see the range of genes involved, or the much larger range of sequence variants (Figure 3).

COSMIC cell lines
In parallel to the COSMIC curations shown on the main COSMIC website, 'COSMIC Cell Lines' (http://cancer. sanger.ac.uk/cell lines) makes available the molecular profiling of 1015 cancer cell lines commonly used in laboratory research, particularly in evaluations of pharmaceutical activity and efficacy. This is a separate but parallel database, overviewing updates across the information published in (16), including full exome sequencing of each cell line, copy number analysis and gene expression analysis. Additional information is added as further experiments are completed. The data are hosted in an analytic website with identical functionality to the curated COSMIC system (described above), with additional links to raw data files and drug sensitivity evaluations (hosted at http://www.cancerrxgene. org/).

Downloads
In addition to the free and open presentation of COS-MIC in its public website, COSMIC is also available for download in multiple formats (https://cancer.sanger.ac.uk/ cosmic/download). Multiple files in TSV (Tab-separated format) or VCF (Variant call format) formats provide complete data sets across each COSMIC release, and the entire database is provided as a full Oracle export file. Downloading complete COSMIC data sets requires registration, but subsequent access is full and free to all academic and non-profit institutes (https://cancer.sanger.ac. uk/cosmic/register).

FUTURE WORK
COSMIC is built primarily via curation of published literature by expert scientists. This is still a major focus, and as this team expands, COSMIC has been able to encompass greater breadth of annotations, recently resulting in the new focus on Genetics of Drug Resistance. All aspects of cancer genetic curation will continue, with an emphasis on gathering details for deep descriptive annotations across key cancer genes, and broad molecular profiles across cancer genomes. The COSMIC database has grown considerably over the last few releases, and it is anticipated this growth will continue (Figure 4).
The information in the COSMIC database is made available in a number of different ways, and these will be expanded. By encompassing analytic navigation of large data across multiple diseases and mechanisms, the COSMIC website is increasingly complex, and redesigning some of its presentations will make it easier to explore. Supporting external integration of COSMIC is also a priority. A programmatic API will overcome much of the current need to download large data files, and will be very helpful in supporting studies needing to bring large data sets together. In these ways, COSMIC will continue to support a wide range of cancer research investigations, from those exploring primary tumour genetics, through diagnostic and pharmaceutical target identification, to those seeking to understand responses to clinical intervention.


BIOCHEMICAL AND ELECTRON MICROSCOPIC CHANGES INDUCED BY GIARDIA IN EXPERIMENTALLY INFECTED LAMBS

The present study was conducted to evaluate the effect of Giardia on the biochemical serum constituents of experimentally infected lambs, in addition to studying the observed alterations accompanying Giardia infection in the intestinal mucosa using scanning electron microscopy. Twenty lambs were allotted into two equal groups, Group A ( non infected) was kept as control negative and Group B (infected) was orally inoculated by 10 Giardia cysts. The biochemical changes were assessed in both groups on 7. 14, 21 and 30 days post inoculation (dpi). The study disclosed that Giardia induced a significant drop in the levels of serum electrolytes (chloride,sodium, potassium and calcium), blood glucose, different enzymes (lipase, amylase and alkaline phosphatase). Furthermore, the levels of urea, liver enzymes (alanine aminotransferase and aspartate aminotransferase), inflammatory marker (C-reactive protein) and oxidative stress marker (malondialdehyde) were elevated, but nitric oxide was declined by 30 dpi. The scanning electron microscopy of the intestinal mucosa of the infected lambs revealed a notable alteration which was fully explained. All the presented results interpret the pathophysiological effect of Giardia which adversely affects the health status of lambs.

INTRODUCTION
Giardia sp. is the most common and important intestinal parasite and it is a frequent common cause of diarrheal diseases through the world. It localized and multiplies in the small intestines of human and domestic animals [1,2]. In ruminant, Giardia causes impairment in feed conversion efficiency which adversely affects the health status and causes enormous economic losses to livestock industry, greatly affects reproductive performance and causes significant production losses [3,4]. Giardiasis outbreak in sheep mostly showed no clinical sign or mortality, but Giardia-infected lambs had malabsorption, decreased weight gain, and reduced feed efficiency [5]. The pathophysiological changes associated with Giardia infection include: malabsorption of electrolytes and nutrients, intestinal microvillus atrophy, loss of epithelial barrier function due to the produced ulceration and infarction , increased permeability and narrowing of the lumen [6,7]. The Changes in the sero-biochemical constituents and the hypothesis of decreased activity of the defense system for protecting tissues from the free radical damage associated with Giardia infection in lambs need further investigation. This was explained in the current study by detecting the alteration in the sero-biochemical parameters, electrolytes and digestive enzymes and by assessing

MATERIALS AND METHODS
Cysts. Giardia cysts were collected from naturally infected sheep. They were identified after lugol's iodine and Giemsa staining. All the collected cysts were liquefied in distilled water and stored at 4 o c . Concentration of fecal specimens was practiced by zinc sulfate floatation technique, followed by twice washing of the concentrated cysts by centrifugation at 1000 rpm for 10 minutes. Lastly, the supernatant was decanted and the sediment was resuspended in a saline solution. Haemocytometer was used to quantify the density of the cysts inoculum. The final inoculum were adjusted to 10 4 Cysts/ lamb. Animal groups. Lambs used in this study were of both sexes and aged 6-8 months. They were kept in wellventilated bin cages and provided with standard food and water. Twenty lambs were equally allocated into two groups; group A ( non-infected group) and Group B (infected group). The lambs were kept in acclimatized conditions for two weeks. During this period, the feces of the lambs were examined daily by direct wet smear and flotation techniques to exclude the existence of any parasites before the experiment.
Ethical approval. Lambs in this work were permitted by the local committee of the Faculty of Veterinary Medicine, Benha University and according to the guidelines of the National Institute of Health in Egypt.

Giardia cyst inoculation.
Each lamb in group B was inoculated orally by a single feeding of Giardia cysts (10 4 Cysts/ lamb).

Cysts detection.
Because of the intermittent shedding of Giardia cyst is, the lambs feces were examined 3 times daily for ten days till appearance of Giardia cysts and establishment of the infection among all lambs.
Serum collection. Ten ml of blood was collected from the jugular vein of each lamb in group A and B into non heparinized tubes. The blood samples were left to stand for 30-60 minutes at room temperature for clotting. The clotted blood were centrifuged at 3,000 rpm for 10 min for serum separation, all the sera were stored at −20°C till further analysis [8].
Biochemical assessments. Estimation of biochemical changes was done by analyzing the serum samples of the two groups for chloride, sodium, potassium, calcium, creatinine (mg/dl), urea , alanine aminotransferase (ALT), aspartate aminotransferase (AST), C-reactive protein(CRP), glucose, amylase, lipase, alkaline phosphatase (ALP), malondialdehyde (MDA) and nitric oxide (NO) using a high performance liquid chromatography method (HPLC, Sykam 1125 pump system, Germany) [9][10][11]. The changes in the biochemical parameters were assessed on 7 th , 14 th , 21 th ,30 th dpi by comparing the mean values ± SE of lambs in A and B groups.

Electron Microscopy
Two infected lambs were sacrificed at the end the experiment and small portions of theirduodenum, proximal part of the jejunum and the lower digestive tract were obtained to identify the changes associated with Giardia trophozoites and cysts. The specimens were kept in glutraldehyde (2.5%) at 4 o c for scanning electron microscope (SEM). They were then rinsed in distilled water and dehydrated in acetone alcohol series (25%, 50%, 75%, and

Clinical Symptoms
The prepatent period of Giardia ranged between 8-10 days in group B (infected group). The cysts shedding continued all through the period of the experimental study ( Fig. 1. a, b). The infected lambs suffered from decreased weight gain, loss of feed efficacy and diarrhea. The onset of diarrhea was on 14 th dpi and continued intermittently throughout the study period.

Biochemical Assessment
The

Electron Microscopy
In the infected lambs, scanning electron microscopy of the duodenum and the proximal part of the jejunum revealed varying degrees of destruction in the intestinal villi. Large number of pear shaped trophozoites measuring 12-18 X 7-10 µm were seen clasped in situ by their convex sucking discs and bulging above the brush border of the microvilli. Some intestinal villi showed absence of the microvillus border with their basic epithelium.
The connective tissue of the intestinal mucosa and submucosa showed fibrosis. (Fig. 1. d,e). Correspondingly, oval shaped Giardia cysts measuring 9-12 X 8-10 µm were noticed located in the lower digestive tract , without evident changes in the intestinal structure. (Fig 1. c).

DISCUSSION
In the current study, a recorded significant decrease in the electrolyte level ( CL, Na, K and Ca) was encountered among infected lambs as compared with the non-infected ones. This aforesaid results came in agreement with other reports [1,14,15]. Previous studies demonstrated that the reduced absorption of Na + is due to the loss of the absorptive power of the intestinal epithelium [16,17]. In contrast, Ayaz, et al. [18] reported that K and Na levels didn't significantly alter in animals infected with many endoparasites. Moreover, [19,20]   The recorded change in the electrolyte balance could be explained by the intestinal inflammation produced due to the activity of Giardia trophozoites Such inflammation and the consequential impairment in electrolytes secretion and absorption often lead to acid, base and electrolyte imbalance which may cause diverse complications in body [21]. This may also interpret the recorded cases of diarrhea which were noticed during the experiment.
The non-significant effect of Giardia on creatinine level was formerly revealed by Rosa, et al. [22]. Though, Giardia showed a significant decreasing effect on urea level as previously encountered by Ragbetli, et al. [1]. In our opinion, this elevation may be created as a result of the increase of nitrogen metabolism in Giardia infection.
The malnutrition effect of Giardia among infected lambs was associated with a low ALP level. Since, the hepatobiliary integrity is measured by the serum ALP [23] so the low ALP level accuses the indirect negative effect of Giardia on liver tissues and bile duct .Accordingly, this effect on the liver tissues could explain the recorded elevation of the liver enzymes ( ALT and AST) among infected lambs in the study.
Mostly, the low levels of amylase and lipase among infected lambs was in accordance with the result of Buret [6]. This outcome was explained by the drawback effect of Giardia trophozoites on the pancreatic function which inhibits pancreatic lipase and amylase activities [24]. The impairment of serum glucose level was also observed among infected lambs. This observation may reflect the malabsorption and maldigestion effects accompanying Giardia infection [25].
The significant elevation of CRP among infected than non infected lambs may be accredited to the damage produced by the attachment of Giardia trophozoites to the mucosa of the intestine which continuously stimulated the hepatocytes for elevating the synthesis of CRP as innate defense mechanism. Mainly, CRP cooperates with the raised mucosal phagocytic activities along with IgA which acts as a humoral defense mechanism against Giardia [26,27].
The high level of MDA among infected lambs is an indication of the oxidative stress produced in the infected tissues [28] due to Giardia. The decreased activity of the defense mechanism for protecting the tissues from the damage produced by Giardia might be one of the reasons of elevation the level of MDA among infected lambs [29].
As regard, the significant decrease in the level of serum NO among infected lambs was consistent with another report [1]. Nitric oxide formation has been encountered in many parasitic infections including Giardia [30,31]. It is considered as only part of an immunopathological series against infection [32]. The low NO level may be credited to the release of arginine deiminase and flavohemoglobins by Giardia trophozoites which consumes all the local arginine (precursor of nitric acid formation) [33].
The intetstinal changes scanned by the electron microscopy came in agreement with other reports on Giardia [7,34]. Such changes ensured that the colonization of the intestine with Giardia trophozoites caused diffuse destruction in the microvillus brush border which in turn resulted in maldigestion and malabsorption effects.
Accordingly, a combination of electrolyte hyper secretion and malabsorption appears to be accountable for accumulation of fluid in the intestine, which aggravate diarrhoea [35,36]. All the above mentioned alterations mainly had a drawback effect on the biochemical serum constituents specially electrolytes [21].

CONCLUSION
The study advanced our standing of the pathophysiological effect of Giardia on lambs. It induced a significant declining effect on serum electrolytes (CL, Na, K, and Ca), digestive enzymes (Lipase, amylase and ALP) and glucose. Likewise, it significantly elevated the level of urea, Liver enzymes (ALT, AST), libertaed inflammatory


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The expression of Lamin A mutant R321X leads to endoplasmic reticulum stress with aberrant Ca2+ handling

Abstract Mutations in the Lamin A/C gene (LMNA), which encodes A‐type nuclear Lamins, represent the most frequent genetic cause of dilated cardiomyopathy (DCM). This study is focused on a LMNA nonsense mutation (R321X) identified in several members of an Italian family that produces a truncated protein isoform, which co‐segregates with a severe form of cardiomyopathy with poor prognosis. However, no molecular mechanisms other than nonsense mediated decay of the messenger and possible haploinsufficiency were proposed to explain DCM. Aim of this study was to gain more insights into the disease‐causing mechanisms induced by the expression of R321X at cellular level. We detected the expression of R321X by Western blotting from whole lysate of a mutation carrier heart biopsy. When expressed in HEK293 cells, GFP‐ (or mCherry)‐tagged R321X mislocalized in the endoplasmic reticulum (ER) inducing the PERK‐CHOP axis of the ER stress response. Of note, confocal microscopy showed phosphorylation of PERK in sections of the mutation carrier heart biopsy. ER mislocalization of mCherry‐R321X also induced impaired ER Ca2+ handling, reduced capacitative Ca2+ entry at the plasma membrane and abnormal nuclear Ca2+ dynamics. In addition, expression of R321X by itself increased the apoptosis rate. In conclusion, R321X is the first LMNA mutant identified to date, which mislocalizes into the ER affecting cellular homeostasis mechanisms not strictly related to nuclear functions.

Introduction
Lamins are the only intermediate filaments localized in the nucleus, underlying the inner nuclear membrane [1]. They play a crucial role in maintaining cellular as well as nuclear integrity and, together with other nuclear envelope proteins, Lamins work as a scaffold for proteins that regulate DNA synthesis, responses to DNA damage, chromatin organization, gene transcription, cell cycle progression, cell differentiation and cell migration [2,3]. Lamins have been classified into two types based on biochemical properties, expression patterns and encoding genes. B-type Lamins are expressed in most cells and encoded by two separate genes. A-type Lamins, known as Lamin A and C, are detected primarily in differentiated cell types and result from the alternative splicing of the Lamin A gene, LMNA [4].
To date, more than 450 different mutations have been identified in the LMNA and collected once published in the UMD-LMNA mutation database at http://www.umd.be/LMNA/. Mutations in LMNA cause a group of inheritable disease phenotypes identified as Laminopathies. Most of these diseases affect specifically the striated muscle with a persistent involvement of the heart that develops dilated cardiomyopathy (DCM), conduction system disorders (CD), and arrhythmias [5]. Many LMNA mutation carriers have a poor prognosis [6], because of a high rate of major cardiac events, such as sudden cardiac death (SD), life-threatening ventricular arrhythmias, extreme bradycardia because of high-degree atrio-ventricular block and progression to end-stage heart failure [5]. In addition to DCM-CD, some atypical forms of LMNA-related cardiac diseases were reported [7,8].
Recently, severe forms of arrhythmogenic right ventricular cardiomyopathy (ARVC) have been linked to LMNA mutations [9] with genetic and phenotypic overlap between DCM and ARVC [9][10][11][12][13]. Although the physiological role of Lamins in diverse cell functions has been precisely investigated, the molecular mechanisms induced by LMNA mutations and leading to the cardiac phenotypes described above are not yet fully understood [14][15][16].
This study is focused on a representative LMNA nonsense mutation that introduces a premature termination codon within the 6th of 12 LMNA exons producing a truncated protein isoform in the central a-helical coiled-coil rod domain (coil 2B) of the Lamin A protein. The resulting mutant variant of Lamin A, R321X, misses the nuclear localization signal (NLS), which is located downstream in the Lamin A protein (aa 417-422) and co-segregates with DCM and cardiac rhythm disturbances in affected family members [17,18].
However, no molecular mechanisms other than Nonsense Mediated Decay of the Messenger (NMD) and haploinsufficency were proposed to explain the cardiac phenotype [17,18]. Interestingly, Geiger and collaborators showed that the efficiency of NMD seems to be tissue-dependent since only a modest reduction of the mutant transcript was observed in the myocardium compared to skin fibroblasts, suggesting that haploinsufficiency could not be the only DCM-causing molecular mechanism. Of note, when expressed in HeLa cells R321X has abnormal nucleoplasmic localization and a peculiar cytoplasmic distribution, with obscure impact on cell homeostasis [17].
We identify this mutation in several members of an Italian family with a frequent history of sudden death, confirming that this mutation is associated with a very severe cardiac phenotype and poor prognosis. We have been able to detect the expression of R321X both in the left and right ventricles of heart biopsies from a patient carrying this particular mutation. Thus, we tried to get more insights into the disease-causing mechanisms first by a detailed analysis of R321X expression and localization in HEK293 cells. Interestingly, we found that R321X was not targeted to the nuclear envelope rather it accumulates in the endoplasmic reticulum (ER) and into the nucleoplasm. Functional studies showed that the presence of R321X into the ER caused the onset of the ER stress response that was in turn accompanied by ER Ca 2+ handling abnormalities and thus increased susceptibility to apoptosis. In conclusion, this is the first Lamin A mutant identified so far which mislocalizes into the ER and affects cellular homeostasis mechanisms not strictly related to nuclear functions.

Patients
The Lamin A mutant R321X, identified for the first time in 2006 [19], has been also found in several members of an Italian family screened in our Clinical Unit dedicated to cardiomyopathies. All participants underwent clinical workup, including medical history, physical examination, 12-lead electrocardiogram (ECG), transthoracic echocardiography and 24-hr ECG recording. All participants provided written informed consent. This study conforms to the principles outlined in the Declaration of Helsinki and was approved by the Ethics Committee of University Hospital Consortium, Policlinico of Bari, Italy. Formalin-fixed paraffin-embedded heart biopsies of patients IV-1 and a healthy control were obtained from the Hospital Niguarda Ca'Granda, Unit of Pathological Anatomy (Milan, Italy) and the Frederico II University Hospital, Unit of Pathological Anatomy (Naples, Italy) respectively.

Generation of the Lamin A constructs
The non-synonymous C961T nucleotide substitution was performed with Stratagene's Quik Change II XL site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA), using the PCR product of LMNA cDNA cloned in the pcDNA TM 6.2/N-EmGFP-DEST (Thermo Fisher Scientific, Waltham, MA, USA) vector system as a template [13]. Mutagenic primers were designed using Quik Change Primer Design Program available online at www.agilent.com/genomics/qcpd/. The mutation was verified by sequencing. The synthetic gene mCherry (mCh) was designed with XbaI and PspXI flanking restriction sites, inserted into pMA-RQ (ampR) and synthesized by Life technologies TM (Thermo Fisher Scientific). For the generation of Lamin A m-Cherry tagged constructs, EmGFP was excised from EmGFP-tagged Lamin A constructs and substituted with mCherry excised from pMA-RQ vector by XbaI and PspXI (both from New England Biolabs â Inc., Ipswich, MA, USA) double digestion. The final constructs were purified using QIAfilter TM Plasmid Maxi Kit (QIAgen, Valencia, CA, USA) and then verified by sequencing. The generation of the FLAG-tagged R321X is described in the Supporting Information.
Cell culture and transient transfection HEK293 cell line was maintained in culture in DMEM high glucose, 2 mM L-glutamine, 10% foetal bovine serum, penicillin (50 U/ml) and streptomycin (50 U/ml) at 37°C, 5% CO 2 in a humidified incubator, and seeded for transfection. HEK293 cells were transfected with ether GFP-(or mCh)-Lamin A-or GFP-(or mCh)-R321X constructs using Lipofectamine 2000 reagent according to the manufacturer's instructions (Thermo Fisher Scientific). For the analysis of Ca 2+ dynamics in the ER or in the nucleus, cells were transiently transfected either with, Cameleon D1ER [20] or nuclear Ratiometric Pericam respectively [21].

Immunofluorescence confocal analysis
For immunofluorescence confocal analysis, cells were grown on coverslips and 24 hr after transfection were fixed in PFA with 0. For immunohistochemical confocal analysis, serial 10 lm thin sections from formalin-fixed paraffin-embedded heart biopsies were attached on coverslips and deparaffinized in Histolemon (Carlo Erba, Milano, Italy) and rehydrated in a graded ethanol series. Antigen retrieval was performed by boiling sections in 50 mM glycine-HCl (pH 3) for 30 min. Nonspecific binding sites were blocked with 1% BSA in phosphate-buffered saline (PBS) plus SDS 0.01% for 30 min. at room temperature. Sections were then incubated with the primary antibodie (anti-phospho PERK, 1:200; Bioss Antibodies) over night at 4°C. After washes in PBS, sections were incubated with the appropriate AlexaFluor-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 hr at room temperature. After washes in PBS, nuclei were stained with propidium iodide (Sigma-Aldrich) and sections were finally mounted in PBS/glycerol (1:1) containing 1% n-propylgallate, pH 8.0.
Evaluation of cytosolic and ER Ca 2+ levels with either Fura-2 or FRET-based probe D1ER Steady-state-or real time-FRET experiments were performed as described [23]. Briefly, HEK293 cells seeded on poly-L-lysine-coated glass coverslips were transiently cotransfected with plasmids encoding either mCh-Lamin A or mCh-R321X and the FRET-based probe D1ER, (gift from Prof. Roger Tsien) [20]. Details are available in the Supporting Information.
For intracellular Ca 2+ measurements mCh-Lamin A or mCh-R321X-HEK293 cells were loaded with 5 lM Fura-2 (Thermo Fisher Scientific) for 25 min. at 37°C in DMEM. Coverslips with dye-loaded cells were mounted in a perfusion chamber (FCS2 Closed Chamber System; BIOP-TECHS, Butler, PA, USA) and measurements were performed with an inverted microscope (Nikon Eclipse TE2000-S microscope) equipped for single cell fluorescence measurements and imaging analysis. Details are available in the Supporting Information.

Apoptosis assays
Detection of apoptotic cells was performed by flow cytometry using 7amino-actinomycin (7-AAD) staining (BD Pharmingen kit, San Jose, CA, USA). In brief, HEK293 cells were seeded at a density of 10 5 cells/well in 24-well plates, transfected either with GFP-Lamin A or GFP-R321X. Forty-eight hours post-transfection, cells were harvested, washed twice with cold PBS, resuspended in binding buffer and incubated with 7-AAD for 15 min. at room temperature accordingly to manufacturer's instruction. The stained cells were analysed on a FACS Canto II flow cytometer.

Results
Clinical and experimental data from an Italian family carrying R321X mutant As shown in Figure 1A, the pedigree of the family was consistent with autosomal dominant transmission. The genetic screening has been performed in 14 members and 7 of them resulted positive for the mutation (+/À in the pedigree). The clinical features co-segregating with the R321X, collected in Table 1, were consistent with different cardiac phenotypes such as arrhythmogenic cardiomyopathy, DCM, conduction disturbances, supraventricular and ventricular arrhythmias, and sudden cardiac death (SD). On the basis of family history and clinical features, it is most likely that the family members who suddenly died were carrying the same mutation indicating that the R321X induces a peculiar and severe form of cardiolaminopathy with a very poor prognosis.
Western blotting was performed to explore the expression of Lamin A and R321X in samples of left and right ventricular myocardium obtained from the index patient (IV-1) at the time of cardiac transplantation. As control, we used cardiac muscle tissue from a patient who experienced heart transplantation due to an ischaemic heart disease not related to LMNA mutations (Fig. 1B).
Three different procedures for protein extraction from formalinfixed paraffin-embedded heart biopsies were used [22,24,25]. In our hands, only the protocol reported by Nirmalan et al. [22] enabled efficient extraction of enough immunoreactive protein to be detectable by Western blot analysis. Figure 1B shows a representative Western blotting using an antibody detecting both Lamin A and Lamin C. A protein band with an expected molecular weight of approximately 37 KD, representing the truncated mutant protein, was detected in the left and right ventricle of the R321X mutation carrier, although at a visible reduced expression levels compared to Lamin A (Pt IV-I). This result was consistent with the transcript analyses reported by Geiger et al. [17], in the heart of the patient characterized in their work. On the other hand, no protein band with similar molecular weight was detectable in the cardiac muscle tissue from the control patient (Pt ctr). As expected, both the 72 KD Lamin A band and the 64 KD Lamin C band were expressed in the mutation carrier and in the control patient. The Lamin A/Lamin C protein ratio was not statistically different in the two heart samples (data not shown).
This prompted us to functionally characterize this mutation to gain more insights into disease-causing mechanisms at cellular level.

Expression of R321X mutant in HEK293 cells
R321X mutant derives from a LMNA nonsense mutation, which introduces a premature termination codon, upstream the NLS, producing a truncated protein isoform in the coil2b of Lamin A ( Fig. 2A). Here, GFP-tagged Lamin A wild-type (GFP-Lamin A) and R321X (GFP-R321X) were transiently expressed in HEK293 cells and identified by Western blotting using an anti-GFP antibody. Figure 2B shows two immunoreactive bands of approximately 95 and 65 kD corresponding to the wild-type (68 kD for Lamin A plus 27 kD for GFP) and the truncated protein (37 kD for R321X plus 27 kD for GFP) respectively. Image analysis by laser-scanning confocal microscopy showed that R321X was mainly localized in the nucleoplasm and in a cytoplasmatic vesicular compartment when compared to Lamin A, which was selectively expressed along the nuclear rim (Fig. 2B, Lamin A, R321X). Colocalization of both GFP-Lamin A and GFP-R321X with calnexin, a well-known ER marker, supports the conclusion that most of R321X is localized in to the ER. As indicated by white arrows, a fraction of the R321X staining (Fig. 2C), does not overlap with calnexin staining, indicating a possible expression of R321X also in proteasomes. To test this hypothesis, we treated GFP-R321X expressing HEK-cells with a well-known proteasome inhibitor (MG-132, 25 lM for 5 hr) and we found that the expression of GFP-R321X was completely confined to the ER. These results likely indicate that R321X accumulated within the ER, undergoes the proteosomal degradation pathway. Of note, the expression of GFP-R321X into the ER did not  alter the localization of other nuclear envelope proteins (such as emerin and the nuclear pores, see Fig. S1), as observed for other LMNA mutations producing truncated version of Lamin A [26].
ER localization of R321X mutant was also evaluated in a cardiac cell line, HL-1. As shown in Fig. S2A, when expressed in HL-1 cells, R321X colocalized with the cardiac SERCA2 pump clearly indicating

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ER mislocalization of the truncated protein also in cardiac cells. In addition, ER localization of the truncated protein seems to be not related to the altered folding of the native protein induced by its fusion with GFP or mCherry tags. When R321X was tagged with a FLAG marker (of about 1 kD) at the free N-terminus and immunolocalized with an anti-FLAG antibody we did not find significant differences regarding R321X expression/localization (see Fig. S2B).

The mislocalization of R321X mutant into ER induces ER stress
We verified whether the inappropriate localization of a nuclear envelope protein such as Lamin A into the ER might induce the so-called unfolded protein response (UPR). This is an adaptive mechanism involving a complex transcriptional program that induces the phosphorylation of PERK, a kinase localized on the ER membrane. PERK phosphorylates eIF2 leading to expression of transcription factors such as GADD153 (CHOP) ultimately promoting the expression of proapoptotic genes [27]. We analysed the expression of phosphorylated PERK (p-PERK) by Western blotting (Fig. 3A) in cells transfected with either GFP-Lamin A or GFP-R321X in control conditions or after experimental manoeuvre known to induce ER stress such as pre-treatment with cyclopiazonic acid (CPA, 100 lM for 5 hr), a wellknown inhibitor of the sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump [28]. As shown in the densytometric analysis the expression of GFP-R321X by itself significantly increased the expression of p-PERK when compared with cells expressing GFP-Lamin A (566 AE 28.4% versus 100 AE 20.1% P < 0.001). The expression of p-PERK in GFP-R321X expressing cells was maximal and unaffected by pre-treatment with CPA (580 AE 12% versus 566 AE 28.4% P = n.s.), whose effect was significant only in cells expressing GFP-Lamin A (100 AE 20.1% versus 509 AE 31% P < 0.001). These data suggest that the expression of GFP-R321X is able, by itself, to induce phosphorylation of PERK and thus ER stress also in the absence of any further ER stress-inducing manoeuvre. Similar results were obtained when we analysed the expression of CHOP under the same experimental conditions (Fig. S3). Next, we used scanning confocal fluorescence microscopy to evaluate the expression of p-PERK in formalin-fixed paraffin-embedded heart samples obtained from both the index (IV) and control patients. Of note, a clear perinuclear staining was evident only in the cardiac cells of the index patient indicating phosphorylation of PERK in the ER and thus ER stress (Fig. 3B).

Expression of R321X decreases ER Ca 2+ levels by impairing both ER Ca 2+ uptake and leak
To verify whether the observed ER stress was is turn impacting the proper functionality of the ER, we measured intraluminal ER Ca 2+ Fig. 3 Effect of the expression of GFP-R321X on the activation of the ER stress marker p-PERK. (A) HEK 293 cells expressing either GFP-Lamin A or GFP-R321X were left untreated (À) or treated with the ER stress inductor CPA 100 lM for 5 hr (+). Cell lysates were subjected to Western blot analysis to detect the phosphorylation of PERK (p-PERK). The statistical analysis graph of relative expression levels of p-PERK was normalized to that of GAPDH. Statistical analysis was performed on three independent experiments and significance calculated by Student's t-test for unpaired samples. ***P < 0.001 versus GFP-Lamin A -CPA. (B) Formalin-fixed paraffin-embedded heart sections of the mutation carrier (right panel, Pt IV-I) and the control patient (left panel, Pt ctr) were analysed by confocal laser-scanning microscopy using an anti-p-PERK antibody (green) as ER stress marker and propidium iodide (red). Colocalization of green/red signals is showed in the merge panels. levels with a genetically encoded FRET-based probe, D1ER [20], in cells transfected with either m-Cherry (mCh)-Lamin A or mCh-R321X. Steady-state FRET experiments showed that ER Ca 2+ levels measured as NetFRET were significantly reduced in cells expressing mCh-R321X when compared with cells expressing the mCh-Lamin A (ER Ca 2+ levels as % of control: 58.2 AE 4.36%, n = 72 cells versus 97.7 AE 4.91%, n = 74 cells, P < 0.001) or not transfected cells (ER Ca 2+ levels as % of control: 58.2 AE 4.36%, n = 72 cells versus 100 AE 5.6%, n = 64 cells, P < 0.001) (Fig. 4A). Since basal ER Ca 2+ levels depends on the dynamic equilibrium between active Ca 2+ accumulation by the SERCA pump and passive leak through calcium channels, mCh-R321X expression could affect either process. On one hand, expression of R321X could reduce Ca 2+ uptake by the SERCA, either by reducing the resting cytosolic [Ca 2+ ] ([Ca 2+ ] cyt ) or by a direct inhibitory effect on the pump. To clarify this critical issue, we investigated these possibilities independently. At first, the possibility that mCh-R321X affected primarily [Ca 2+ ] cyt (and hence ER Ca 2+ levels only indirectly) was verified with Fura-2. In these experiments the cells were transiently transfected with either mCh-Lamin A or mCh-R321X and positive cells were distinguished from controls by the typical red fluorescence emitted upon illumination with green light. As shown in Fig. 4B no difference in the resting [Ca 2+ ] cyt were observed between mCh-R321X-transfected and -control cells ([Ca 2+ ] cyt : 108.08 AE 4.09 nM, n = 114 cells versus 115.54 AE 5.09 nM, n = 107 cells, P = n.s.). We then analysed whether the expression of mCh-R321X might directly affect the activity of the SERCA pump (Fig. 5A). ER Ca 2+ stores were first depleted with 20 lM CPA in the absence of external Ca 2+ . The rate of Ca 2+ reuptake into the ER was measured as the initial slope of the FRET ratio increase after 2 mM Ca 2+ re-addition. Under these experimental conditions, the rate of Ca 2+ entry into the ER resulted significantly lower in cells expressing R321X when compared with cells expressing the mCh-Lamin A [Slope (ratio/min): 0.054 AE 0.008, n = 49 cells versus 0.094 AE 0.0147, n = 46 cells, P < 0.01], suggesting the functional impairment of SERCA activity in cells expressing the mutant (Fig. 5A  right panel). In addition, when SERCA pump was blocked by CPA in the absence of extracellular Ca 2+ , we are able to unmask the passive ER Ca 2+ leak (Fig. 5B). The rate of Ca 2+ leak was measured as the initial slope of the FRET ratio decrease after CPA addition. Surprisingly, the rate of Ca 2+ leak from the ER was significantly greater in mCh-R321X than in mCh-Lamin A-expressing cells (Fig. 5B right panel), [Slope (ratio/min.): 0.056 AE 0.006, n = 47 cells versus 0.028 AE 0.007, n = 46 cells, P < 0.001], although the ER/cytosol Ca 2+ gradient was lower in the cells expressing the mutant. Thus, we were depicting a scenario in which the expression and ER mislocalization of R321X caused generalized ER stress dramatically decreasing the Ca 2+ levels into the ER and impairing both ER refill and leak processes. Of note, neither ER Ca 2+ -levels nor the refill/leak rate were affected by the expression of another Lamin A mutant (DUP) expressed in the nuclear rime [13], clearly indicating that the ER mislocalization of R321X is the key event leading the cells to ER stress and abnormal Ca 2+ handling by the ER (Fig. S4).

ER expression of R321X affects the overall cellular Ca 2+ dynamics
It was previously reported that a reduction of~30% in steady-state ER Ca 2+ levels results in a substantial activation (>50%) of the socalled capacitative Ca 2+ entry (CCE) [29]. The reduction in steady- state ER Ca 2+ levels caused by R321X expression is thus expected to cause an activation of this pathway. Therefore, we measured CCE in mCh-R321X-and mCh-Lamin A-transfected cells loaded with Fura-2 AM (Fig. 6A). The cells were treated with the SERCA blocker (20 lM CPA) while perfused with Ca 2+ -free medium. This procedure first evoked a transient increase in [Ca 2+ ] cyt due to release of Ca 2+ from the stores. As expected by the lower ER Ca 2+ levels in mCh-R321X-expressing cells (Fig. 4B), CPA-induced ER Ca 2+ release was significantly reduced when compared with cells expressing the mCh-Lamin A (Fig. 6B, Ca 2+ release, D[Ca 2+ ] cyt : 71.63 AE 5.08 nM, n = 57 versus 212.72 AE 5.08, n = 60, P < 0.001). When the release of stored Ca 2+ was complete, 2 mM Ca 2+ was re-added to the medium. This manoeuvre evoked a second, larger [Ca 2+ ] cyt increase via CCE, which was markedly lower in mCh-R321X-transfected cells (Fig. 6B, CCE, D[Ca 2+ ] cyt : 155.71 AE 10.69 nM, n = 57 versus 254.29 AE 21.83, n = 60, P < 0.001). This evidence indicates that, together with the mechanisms identified so far, the specific expression of R321X into the ER is also able to impinge a mechanism important to the Ca 2+ influx from the extracellular space. Interestingly, the expression levels of the proteins involved in the Ca 2+ handling were not significantly different under GFP-R321X expression when compared with GFP-Lamin A-expressing cells (Fig. S5), suggesting that the ER mislocalization of R321X is the key event leading the cells to ER stress and abnormal Ca 2+ handling. Of note, the impaired ER Ca 2+ mobilization observed in mCh-R321X-expressing cells was also mirrored into the nucleoplasm. We elicited an IP3R-mediated Ca 2+ release form the ER using ATP, a physiological agonist of the purynergic receptors endogenously expressed in HEK293 cells [30] and we monitored either cytoplasmic or nucleoplasmic Ca 2+ dynamics using Fura-2AM and nuclear ratiometric Pericam (nu-Pericam) [21] respectively. The increases in [Ca 2+ ] cyt and in nucleoplasmic Ca 2+ levels were both significantly lower in cells expressing mCh-R321X when compared to mCh-Lamin A-expressing cells (Fig. 7A Fig. 7B, right panel, nu-Pericam DR/R 0 : 0.142 AE 0.02, n = 30 versus 0.222 AE 0.034, n = 28, P < 0.05). Given the continuity between the ER and the nuclear envelope, it may also possible that the impairment of ER Ca 2+ homeostasis results from a general weakening of the nuclear envelope induced by the expression of R321X. We ruled out this possibility by measuring nuclear Ca 2+ permeability that resulted unaffected under expression of mCh-R321X (Fig. S6). Therefore, the impairment of Ca 2+ handling in the nucleoplasm might be of extreme physiological importance since its potential effect on genes regulation.

Expression of R321X increases the rate of cell apoptosis
The events described so far may in turn increase the ER stress condition finally inducing cell death by apoptosis. To verify this hypothesis we measured the rate of cell apoptosis in either GFP-Lamin A or GFP-R321X expressing cells by cytofluorimetry. As shown in Figure 8A, Lamin A and R321X-expressing cells were stained with 7-AAD to assess the apoptotic cell populations. We showed that the apoptotic rate significantly increased up to 45% in GFP-R321X transfected cells compared to control cells (Fig. 8A, right panel, % of apoptotic cells: 45 AE 5.2% versus 18 AE 3.2%, P < 0.001). One of the most common signalling cascades involved in apoptosis is the activation of a highly specialized family of cysteinyl-aspartate proteases (caspases). Once activated, caspases initiate cell death by cleaving and activating effectors such as the Poly (ADP-ribose) polymerase (PARP) [31]. Once cleaved, an 85 kD fragment was excited from the 116 kD intact PARP molecule. As shown in Figure 8B, in GFP-R321X-expressing cells the product of caspases-dependent PARP cleavage (85 kD band) was clearly overexpressed compared to the full length

Discussion
It has been extensively reported that mutations in LMNA are able to mislocalize either Lamin A/C or well-known Lamin A/C-binding partners [13,16,17,32]. However, LMNA mutations described so far mislocalized only within the nuclear compartment, thus compromising both nuclear envelope stiffness/resistance and gene profile expression. On the basis of this observation, two opposing 'structural' and 'gene expression' models have been postulated to explain how mutations within LMNA gene give rise to distinct diseases. Loss of Lamin A/C function, for example, may result in nuclear fragility leading to the death of individual cells upon mechanical stress or other environmental factors. Alternatively, abnormal Lamin A/C function could alter tissue/cell type-specific gene expression either through impaired interaction between Lamin A/C and transcription factors/regulators or through changes in the spatial organization of chromatin [15].

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R321X mutant derives from a LMNA nonsense mutation that introduce a premature termination codon within the 6th of 12 LMNA exons before the NLS producing a truncated protein isoform in the central a-helical coiled-coil rod domain of Lamin A ( Fig. 2A).
In patients carrying the same mutation, the messenger coding the mutated version of R321X was largely degraded by NMD in skin fibroblasts [17,18] and to a lesser extent in the heart [17,18]. Accordingly, the truncated R321X protein was not easily detectable in patient tissues, suggesting the LMNA haploinsufficency as the only pathomechanism underlying the cardiomyopathy observed in the mutant carriers. However, only a modest reduction of the mutant transcript was observed in the myocardium of these carriers compared to skin fibroblasts [17] clearly suggesting that the NMD may not be efficient enough to avoid the harmful effects of the expressed R321X in affected tissues, especially in the heart, which is constantly under intense mechanical stress and thus prone to disorders by even weak cellular imbalance.
Moreover, several findings from meta-analysis of previous literature, suggest that in humans severe early phenotypes are associated with dominant negative or toxic gain of function mechanisms because of the presence of mutated Lamin A. In contrast, late onset and/or milder phenotypes may arise through loss of function secondary to haploinsufficiency [33]. In addition, the mild cardiac phenotype of heterozygous LMNA +/À knock-out mice confirms that the Lamin A haploinsufficiency per se cannot account for severe phenotypes co-segregating with several LMNA mutations [34]. We found that the expression of R321X is associated, in an Italian family, with several cases of SD at early age and severe cardiomyopathies, clearly suggesting the dominant negative effect of this Lamin A mutation.
Of note, we have been able to detect by Western blotting the truncated mutant protein in the left and right ventricle of the R321X mutation carrier (Pt IV-I) at the expected molecular weight, although at a visible reduced expression levels compared to the wild-type Lamin A. This result was consistent with the transcript analyses reported by Geiger et al. [17] in the heart of the patient characterized in their work.
As expected, both the 72 KD Lamin A band and the 64 KD Lamin C band were also expressed in the mutation carrier. A skewed ratio of Lamin A/Lamin C proteins expression has been reported in fibroblasts from skin biopsies of DCM patients carrying the same mutation [18]. Although we found the same trend in heart of patient IV-I, we noticed that this result was highly dependent on both the region of the heart analysed (left versus right ventricles) and on the experimental variability. Therefore, the involvement of skewed Lamin A to lamin C ratio in the onset of DCM needs further validation, especially in the cardiac tissue.
We then functionally characterized this Lamin A mutant and we demonstrated that R321X profoundly affects the ER functions when expressed in cells. We found that this mutant accumulates into ER, consistent with a previous study in which an in vitro Lamin A construct lacking the NLS sequence was found to accumulate into the ER compartment [35]. In addition, we found that R321X accumulates into the nucleoplasm via either a passive diffusion process of the protein through the nuclear pores or by a 'piggybacking'-mediated mechanism, in other words, by a preassembled complex with a protein containing a NLS [36].
However, a pool of A-type Lamins within the nucleoplasm, which is distinct from nuclear envelope peripheral Lamin A, has been already identified in both physiological [37] and pathological [38] conditions. Thus, the ER localization of this LMNA mutant represents the novel feature of this Lamin A mutated variant. We depicted a scenario in which the introduction of the premature stop codon in the LMNA gene, before the NLS nuclear localization signature, produces a truncated and unfolded version of Lamin A, which likely is unable to leave the ER and to continue its biosynthetic pathway. The accumulation of R321X into the ER impairs the ability of this organelle to properly handle Ca 2+ . Finally, we found that the overall cellular response to both misfolded Lamin A accumulation into the ER and luminal Ca 2+ depletion is to increase cell apoptosis rate, via activation of cellular caspases and the PERK-CHOP cascade, involved in transcriptional responses to cellular stress. We dissected all the ER mechanisms involved in the luminal and cytosolic Ca 2+ homeostasis in both Lamin A and R321X-expressing cells. Notably, ER severely lost the ability to both release and re-uptake Ca 2+ as well as to functionally couple to the plasma membrane to allow the CCE from the extracellular space in R321X-expressing cells. We found that the ER stress induced by R321X impaired CCE, SERCA activity and ER Ca 2+ leak. Lack of proper Ca 2+ homeostasis caused both failure in restoring normal ER Ca 2+ levels and impaired agonist-induced Ca 2+ changes both in the cytosol and in the nuclear compartment. Interestingly, neither the expression of SERCA nor the expression of STIM1 and ORAI1 resulted affected in R321X-expressing cells, rather suggesting a functional impairment of these proteins. We can speculate that defective STIM1-ORAI1 coupling occurs because of the loss of ER physiological morphology and redistribution of STIM1 to ER sheets. Accordingly, a direct correlation between ER morphology and Store Operated Calcium Entry (SOCE) functionality has been recently reported [39]. Although the increase in Ca 2+ leak from ER has been demonstrated during ER stress [40], the decrease in SERCA activity has never been reported under this condition. It is possible, however, that a decrease in ATP production in mitochondria, secondary to a decrease in ER Ca 2+ dynamics [41,42], may impair the SERCA activity in R321X expressing cells. Depending on the duration and severity of the ER stress, cells can re-establish normal ER function or lead to cell death by apoptosis. We found that the expression of R321X significantly increased the rate of cell apoptosis in resting state, suggesting a severe and un-compensated ER stress in R321X-expressing cells. It is known that ER Ca 2+ store depletion by itself can induce ER stress and cell apoptosis [43,44]. It is indeed possible that the loss of luminal Ca 2+ caused by ER stress further reinforces the ER stress response leading to cell apoptosis. Moreover, the impairment in Ca 2+ dynamics into the nucleus may shift the transcription profile towards pro-apoptotic genes [45]. Downstream consequences of impaired ER functions are expected and reported in cardiomyopathy. Disruption of ER homeostasis has been linked to several processes of cardiovascular diseases including ischaemia/reperfusion injury [46], DCM [47] and HF [48], often associated to ryanodine receptors mutations [49]. The disruption of ER homeostasis in cardiomyocytes has, as expected, consequence on the impairment of cardiac contractility. The consequent cell apoptosis may in turn account for conduction defects, arrhythmia and sudden death as previously discussed [13]. A schematic drawing summarizing our working hypothesis is shown in Figure 9.  9 Schematic diagram illustrating the effects induced by expression of R321X in HEK293 cells. ER mis-localization of R321X elicits the ER stress response leading to: (i) activation of the PERK-CHOP signalling pathway leading to apoptosis; (ii) ER abnormal Ca 2+ handling leading to impaired cytosolic and nuclear Ca 2+ signalling which in turn might impact Ca 2+ -mediated process such as gene regulation and contraction in cardiac cells. Advances in the design of novel compounds and therapeutic strategies to manipulate levels of ER stress in disease have been recently reported [50] and most importantly some pharmacological agents used in clinical settings such as pravastatin, pioglitazone, Tempol were reported to affect UPR pathways and ER disfunction [51], suggesting novel therapeutic strategies for this severe form of cardiac laminopathy induced by R321X mutant expression.
In addition, we have shown that R321X likely undergoes degradation via proteasome-mediated mechanism just like another abnormal Lamin A protein termed progerin [52]. Gabriel et al. showed that treatment with the activating proteasomal activity, sulforaphane, enhanced the degradation of progerin, decreased the formation of insoluble progerin aggregates and induced clearance through proteasomal mechanisms in affected and normal fibroblasts. This evidence might provide a potential therapeutic strategy also for other mutants of Lamin A, such as R321X.