RESUMEN
Myriad physiological and pathogenic processes are governed by protein levels and modifications. Controlled protein activity perturbation is essential to studying protein function in cells and animals. Based on Trim-Away technology, we screened for truncation variants of E3 ubiquitinase Trim21 with elevated efficiency (ΔTrim21) and developed multiple ΔTrim21-based targeted protein-degradation systems (ΔTrim-TPD) that can be transfected into host cells. Three ΔTrim-TPD variants are developed to enable chemical and light-triggered programmable activation of TPD in cells and animals. Specifically, we used ΔTrim-TPD for (1) red-light-triggered inhibition of HSV-1 virus proliferation by degrading the packaging protein gD, (2) for chemical-triggered control of the activity of Cas9/dCas9 protein for gene editing, and (3) for blue-light-triggered degradation of two tumor-associated proteins for spatiotemporal inhibition of melanoma tumor growth in mice. Our study demonstrates that multiple ΔTrim21-based controllable TPD systems provide powerful tools for basic biology research and highlight their potential biomedical applications.
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Sistemas CRISPR-Cas , Edición Génica , Ratones , Animales , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteínas/metabolismo , Proteolisis , Mamíferos/metabolismoRESUMEN
In oocyte biology, the zona pellucida has long been known to operate three extracellular functions downstream of the secretory pathway, namely, encasing the oocytes in ovarian follicles, mediating sperm-oocyte interaction, and preventing premature embryo contact with oviductal epithelium. The present study uncovers a fourth function that is fundamentally distinct from the other three, being critical for embryonic cell survival in mice. Intriguingly, the three proteins of the mouse zona pellucida (ZP1, ZP2, ZP3) were found abundantly present also inside the embryo 4 days after fertilization, as shown by mass spectrometry, immunoblotting, and immunofluorescence. Contrary to current understanding of the roles of ZP proteins, ZP3 was associated more with the cytoskeleton than with secretory vesicles in the subcortical region of metaphase II oocytes and zygotes, and was excluded from regions of cell-cell contact in cleavage-stage embryos. Trim-away-mediated knockdown of ZP3 in fertilized oocytes hampered the first zygotic cleavage, while ZP3 overexpression supported blastocyst formation. Transcriptome analysis of ZP3-knockdown embryos pointed at defects of cytoplasmic translation in the context of embryonic genome activation. This conclusion was supported by reduced protein synthesis in the ZP3-knockdown and by the lack of cleavage arrest when Trim-away was postponed from the one-cell to the late two-cell stage. These data place constraints on the notion that zona proteins only operate in the extracellular space, revealing also a role during the oocyte-to-embryo transition. Ultimately, these data recruit ZP3 into the family of maternal factors that contribute to developmental competence of mouse oocytes.
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Semen , Zona Pelúcida , Femenino , Ratones , Masculino , Animales , Zona Pelúcida/metabolismo , Semen/metabolismo , Oocitos/metabolismo , Glicoproteínas de la Zona Pelúcida/genética , Glicoproteínas de la Zona Pelúcida/metabolismo , Folículo Ovárico/metabolismoRESUMEN
Tripartite motif-containing protein 21 (TRIM21) is a cytosolic antibody receptor and E3 ubiquitin ligase that promotes destruction of a broad range of pathogens. TRIM21 also underlies the antibody-dependent protein targeting method Trim-Away. Current evidence suggests that TRIM21 binding to antibodies leads to formation of a self-anchored K63 ubiquitin chain on the N terminus of TRIM21 that triggers the destruction of TRIM21, antibody, and target protein. Here, we report that addition of antibody and TRIM21 to Xenopus egg extracts promotes efficient degradation of endogenous target proteins, establishing cell-free Trim-Away as a powerful tool to interrogate protein function. Chemical methylation of TRIM21 had no effect on target proteolysis, whereas deletion of all lysine residues in targets abolished their ubiquitination and proteasomal degradation. These results demonstrate that target protein, but not TRIM21, polyubiquitination is required for Trim-Away, and they suggest that current models of TRIM21 function should be fundamentally revised.
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Proteínas , Ubiquitina-Proteína Ligasas , Ubiquitinación , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas/metabolismo , Ubiquitina/metabolismo , Anticuerpos/metabolismoRESUMEN
Trim-Away is a versatile intracellular protein degradation pathway that has been extensively explored in vitro. However, the in vivo application of Trim-Away is limited at oocyte and zygote stages due to the lack of an in vivo practical approach for intracellular antibody delivery. To broaden the application of Trim-Away, especially for clinical use, we developed a nanogel-based Nano-ERASER system. Here, we demonstrated that the intracellular delivery of anti-programmed cell death ligand 1 (PD-L1) antibody through Nano-ERASER could effectively deplete PD-L1 in triple negative breast cancer (TNBC) cells and induce cancer cell death. Furthermore, with the help of a tumor tissue-targeted nanogel, anti-PD-L1 antibody-loaded Nano-ERASER effectively inhibited tumor progression in a TNBC mouse model. These results confirmed that Nano-ERASER realized Trim-Away in adult animals for the first time, which could be an effective tool for disease treatment and studying gene/protein function both in vitro and in vivo.
RESUMEN
Nosema bombycis Naegeli (Dissociodihaplophasida: Nosematidae), an obligate intracellular parasite of the silkworm Bombyx mori, causes a devastating disease called pébrine. Every year pébrine will cause huge losses to the sericulture industry worldwide. Until now, there are no effective methods to inhibit the N. bombycis infection in silkworms. In this study, we first applied both the novel protein degradation Trim-Away technology and NSlmb (F-box domain-containing in the N-terminal part of supernumerary limbs from Drosophila melanogaster) to lepidopteran Sf9-III cells to check for specific degradation of a target protein in combination with a single-chain Fv fragment (scFv). Our results showed that the Trim-Away and NSlmb systems are both amenable to Sf9-III cells. We then created transgenic cell lines that overexpressed the protein degradation system and N. bombycis chimeric scFv targeting spore wall protein NbSWP12 and evaluated the effects of the insect transgenic cell lines on the proliferation of N. bombycis. Both methods could be applied to cell lines and both Trim-Away and NSlmb ubiquitin degradation systems effectively inhibited the proliferation of N. bombycis. Further, either of these degradation systems could be applied to individual silkworms through a transgenic platform, which would yield individual silkworms with high resistance to N. bombycis, thus greatly speeding up the process of acquiring resistant strains.
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Bombyx , Microsporidiosis , Nosema , Animales , Complejo de la Endopetidasa Proteasomal/metabolismo , Drosophila melanogaster , Bombyx/metabolismo , Animales Modificados Genéticamente , Ubiquitinas/metabolismoRESUMEN
In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.
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Origen de Réplica , Proteínas de Unión a Telómeros , Animales , Replicación del ADN , Momento de Replicación del ADN , Fase S/genética , Proteínas de Unión a Telómeros/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
"Trim-Away" technology enables rapid degradation of endogenous proteins without prior modification of protein-coding genes or mRNAs through delivery of antibodies that target proteins of interest. Although this approach can be readily applied to almost any cytosolic protein, strategies for cytosolic antibody delivery have been limited to microinjection or electroporation, which require skill-dependent operation or specialized equipment. Thus, the development of antibody delivery methods that are convenient, scalable, and preferably do not require detachment of adherent cells is required to extend the versatility of the Trim-Away method. Here, we developed a cell resealing technique optimized for Trim-Away degradation, which uses the pore-forming toxin streptolysin O (SLO) to permeabilize the cell membrane and delivered the antibodies of interest into HEK293T, HeLa, and HK-2 cell lines. We demonstrated the ability of Trim-Away protein degradation using IKKα and mTOR as targets, and we showed the availability of the developed system in antibody screening for the Trim-Away method. Furthermore, we effectively coupled Trim-Away with cyclic immunofluorescence and microscopic image-based analysis, which enables single-cell multiplexed imaging analysis. Taking advantage of this new analysis strategy, we were able to compensate for low signal-to-noise due to cell-to-cell variation, which occurs in the Trim-Away method because of the heterogenous contents of the introduced antibody, target protein, and TRIM21 in individual cells. Therefore, the reported cell resealing technique coupled with microscopic image analysis enables Trim-Away users to elucidate target protein function and the effects of target protein degradation on various cellular functions in a more quantitative and precise manner.
RESUMEN
The cytosolic antibody receptor and RING E3 ligase TRIM21 targets intracellular, antibody-coated immune complexes for degradation and activates the immune system. Here we review how TRIM21 degrades diverse targets and how this activity can be exploited in molecular biology and for the development of new therapeutics. In addition, we compare what is known about TRIM21's mechanism to other TRIM proteins and RING E3 ligases.
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Ribonucleoproteínas , Ubiquitina-Proteína Ligasas , Anticuerpos , Citosol/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Antibodies mediate the majority of their effects in the extracellular domain, or in intracellular compartments isolated from the cytosol. Under a growing list of circumstances, however, antibodies are found to gain access to the cytoplasm. Cytosolic immune complexes are bound by the atypical antibody receptor TRIM21, which mediates the rapid degradation of the immune complexes at the proteasome. These discoveries have informed the development of TRIM-Away, a technique to selectively deplete proteins using delivery of antibodies into cells. A range of related approaches that elicit selective protein degradation using intracellular constructs linking antibody fragments to degradative effector functions have also been developed. These methods hold promise for inducing the degradation of proteins as both research tools and as a novel therapeutic approach. Protein aggregates are a pathophysiological feature of neurodegenerative diseases and are considered to have a causal role in pathology. Immunotherapy is emerging as a promising route towards their selective targeting, and a role of antibodies in the cytosol has been demonstrated in cell-based assays. This review will explore the mechanisms by which therapeutic antibodies engage and eliminate intracellularly aggregated proteins. We will discuss how future developments in intracellular antibody technology may enhance the therapeutic potential of such antibody-derived therapies.
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Enfermedades Neurodegenerativas , Complejo Antígeno-Anticuerpo/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ribonucleoproteínas/metabolismoRESUMEN
Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin-proteasome and autophagy-lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.
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Epítopos/genética , Proteolisis , Ribonucleoproteínas/genética , Anticuerpos de Dominio Único/inmunología , Anticuerpos/genética , Anticuerpos/inmunología , Anticuerpos/farmacología , Epítopos/inmunología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Proteínas Fluorescentes Verdes/farmacología , Células HEK293 , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/inmunología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/inmunología , Ribonucleoproteínas/inmunología , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/farmacología , Ubiquitina/genética , Ubiquitina/inmunologíaRESUMEN
Investigations of genes required in early mammalian development are complicated by protein deposits of maternal products, which continue to operate after the gene locus has been disrupted. This leads to delayed phenotypic manifestations and underestimation of the number of genes known to be needed during the embryonic phase of cellular totipotency. Here we expose a critical role of the gene Cops3 by showing that it protects genome integrity during the 2-cell stage of mouse development, in contrast to the previous functional assignment at postimplantation. This new role is mediated by a substantial deposit of protein (94th percentile of the proteome), divided between an exceptionally stable cortical rim, which is prevalent in oocytes, and an ancillary deposit in the embryonic nuclei. Since protein abundance and stability defeat prospects of DNA- or RNA-based gene inactivation in oocytes, we harnessed a classical method next to an emerging method for protein inactivation: antigen masking (for functional inhibition) versus TRIM21-mediated proteasomal degradation, also known as 'Trim away' (for physical removal). Both resulted in 2-cell embryo lethality, unlike the embryos receiving anti-green fluorescent protein. Comparisons between COPS3 protein-targeted and non-targeted embryos revealed large-scale transcriptome differences, which were most evident for genes associated with biological functions critical for RNA metabolism and for the preservation of genome integrity. The gene expression abnormalities associated with COPS3 inactivation were confirmed in situ by the occurrence of DNA endoreduplication and DNA strand breaks in 2-cell embryos. These results recruit Cops3 to the small family of genes that are necessary for early embryo survival. Overall, assigning genes with roles in embryogenesis may be less safe than assumed, if the protein products of these genes accumulate in oocytes: the inactivation of a gene at the protein level can expose an earlier phenotype than that identified by genetic techniques such as conventional gene silencing.
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Blastómeros/metabolismo , Complejo del Señalosoma COP9/fisiología , Desarrollo Embrionario , Oocitos/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Animales , Blastómeros/ultraestructura , Complejo del Señalosoma COP9/biosíntesis , Complejo del Señalosoma COP9/genética , Supervivencia Celular , Roturas del ADN , Transferencia de Embrión , Desarrollo Embrionario/genética , Endorreduplicación , Femenino , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Histonas/biosíntesis , Histonas/genética , Proteínas Luminiscentes/análisis , Ratones , Microinyecciones , Oocitos/ultraestructura , Péptido Hidrolasas/biosíntesis , Péptido Hidrolasas/genética , Embarazo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteoma , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , Proteínas Recombinantes/análisis , Ribonucleoproteínas/fisiología , Transcriptoma , Cigoto/metabolismo , Proteína Fluorescente RojaRESUMEN
We previously identified the protein Lbh as necessary for cranial neural crest (CNC) cell migration in Xenopus through the use of morpholinos. However, Lbh is a maternally deposited protein and morpholinos achieve knockdowns through prevention of translation. In order to investigate the role of Lbh in earlier embryonic events, we employed the new technique "Trim-Away" to degrade this maternally deposited protein. Trim-Away utilizes the E3 ubiquitin ligase trim21 to degrade proteins targeted with an antibody and was developed in mammalian systems. Our results show that Xenopus is amenable to the Trim-Away technique. We also show that early knockdown of Lbh in Xenopus results in defects in gastrulation that present with a decrease in fibronectin matrix assembly, an increased in mesodermal cell migration and decrease in endodermal cell cohesion. We further show that the technique is also effective on a second abundant maternal protein PACSIN2. We discuss potential advantages and limit of the technique in Xenopus embryos as well as the mechanism of gastrulation inhibition.
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Gastrulación , Proteínas de Xenopus/fisiología , Xenopus laevis/embriología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Movimiento Celular , Ectodermo/citología , Ectodermo/embriología , Ectodermo/patología , Inducción Embrionaria , Endodermo/citología , Endodermo/embriología , Endodermo/fisiología , Fibronectinas/metabolismo , Mesodermo/citología , Mesodermo/embriología , Mesodermo/fisiología , Morfolinos , Cresta Neural/citología , Cresta Neural/embriología , Proteolisis , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/inmunología , Proteínas de Xenopus/metabolismoRESUMEN
Human islet amyloid polypeptide (hIAPP, also known as amylin) is a co-secreting protein of insulin in human pancreatic ß-cells. It is encapsulated in vesicles and secreted out of the cells with insulin. hIAPP can promote insulin secretion and regulate blood glucose homeostasis in the body under the normal physiological conditions. However, hIAPP misfolding or excessive accumulation can cause toxic effects on the ß cells, which in turn affect cell function, resulting in type 2 diabetes mellitus (T2DM) for the affected individuals. In order to eliminate the excessive accumulation of hIAPP in the cell and to maintain its normal synthetic function, we have adopted a new protein degradation technology called Trim-Away, which can degrade the target protein in a short time without affecting the mRNA transcription and translation synthesis function of the target protein. First, we overexpressed hIAPP in the rat insulinoma cells (INS1) to simulate its excessive accumulation and analyzed its effect in INS1 cells by measuring the release of LDH (lactate dehydrogenase), CCK8 activity and PI-Annexin V positive ratio. Results showed that excessive accumulation of hIAPP caused ß cell apoptosis. Second, real-time quantitative PCR analysis and ELISA detection showed that the synthesis and secretion of insulin were hindered. We used Trim-Way technology to specifically eliminate the excessive accumulation of hIAPP protein in hIAPP overexpressing INS1 cells. Cell activity experiments confirmed that clearance of hIAPP reduced the cell death phenotype. Further ELISA experiments confirmed that INS1 cells restored insulin secretion ability. This study examined the toxic effect of hIAPP excessive accumulation in INS1 cells and demonstrated the cytotoxicity clearance effect of Trim-Way technology in pancreatic ß-cells. Our research has provided a new strategy for using Trim-Away technology for treatment of diabetes.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Insulinoma , Neoplasias Pancreáticas , Animales , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos , Neoplasias Pancreáticas/genética , Pliegue de Proteína , RatasRESUMEN
RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID.
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Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Factor de Transcripción TFIID/metabolismo , Transcripción Genética , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Células HCT116 , Humanos , Unión Proteica , ARN Polimerasa II/metabolismo , Factor de Transcripción TFIID/genéticaRESUMEN
Chromosome segregation errors during female meiosis are a leading cause of pregnancy loss and human infertility. The segregation of chromosomes is driven by interactions between spindle microtubules and kinetochores. Kinetochores in mammalian oocytes are subjected to special challenges: they need to withstand microtubule pulling forces over multiple hours and are built on centromeric chromatin that in humans is decades old. In meiosis I, sister kinetochores are paired and oriented toward the same spindle pole. It is well established that they progressively separate from each other with advancing female age. However, whether aging also affects the internal architecture of centromeres and kinetochores is currently unclear. Here, we used super-resolution microscopy to study meiotic centromere and kinetochore organization in metaphase-II-arrested eggs from three mammalian species, including humans. We found that centromeric chromatin decompacts with advancing maternal age. Kinetochores built on decompacted centromeres frequently lost their integrity and fragmented into multiple lobes. Fragmentation extended across inner and outer kinetochore regions and affected over 30% of metaphase-II-arrested (MII) kinetochores in aged women and mice, making the lobular architecture a prominent feature of the female meiotic kinetochore. We demonstrate that a partial cohesin loss, as is known to occur in oocytes with advancing maternal age, is sufficient to trigger centromere decompaction and kinetochore fragmentation. Microtubule pulling forces further enhanced the fragmentation and shaped the arrangement of kinetochore lobes. Fragmented kinetochores were frequently abnormally attached to spindle microtubules, suggesting that kinetochore fragmentation could contribute to the maternal age effect in mammalian eggs.
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Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/fisiología , Cinetocoros/metabolismo , Oocitos/metabolismo , Envejecimiento , Animales , Proteínas de Ciclo Celular/metabolismo , Centrómero/fisiología , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Femenino , Células HEK293 , Humanos , Cinetocoros/fisiología , Meiosis/fisiología , Ratones , Microtúbulos/metabolismo , Células 3T3 NIH , Oocitos/fisiología , Óvulo/metabolismo , Óvulo/fisiología , Huso Acromático/fisiología , Porcinos , CohesinasRESUMEN
Aneuploidy caused by abnormal chromosome segregation during early embryo development leads to embryonic death or congenital malformation. Centromere protein F (CENPF) is a member of centromere protein family that regulates chromosome segregation during mitosis. However, its necessity in early embryo development has not been fully investigated. In this study, expression and function of CENPF was investigated in mouse early embryogenesis. Detection of CENPF expression and localization revealed a cytoplasm, spindle and nuclear membrane related dynamic pattern throughout mitotic progression. Farnesyltransferase inhibitor (FTI) was employed to inhibit CENPF farnesylation in zygotes. The results showed that CENPF degradation was inhibited and its specific localization on nuclear membranes in morula and blastocyst vanished after FTI treatment. Also, CAAX motif mutation leads to failure of CENPF-C630 localization in morula and blastocyst. These results indicate that farnesylation plays a key role during CENPF degradation and localization in early embryos. To further assess CENPF function in parthenogenetic or fertilized embryos development, morpholino (MO) and Trim-Away were used to disturb CENPF function. CENPF knockdown in Metaphase II (MII) oocytes, zygotes or embryos with MO approach resulted in failure to develop into morulae and blastocysts, revealing its indispensable role in both parthenogenetic and fertilized embryos. Disturbing of CENPF with Trim-Away approach in zygotes resulted in impaired development of 2-cell and 4-cell, but did not affect the morula and blastocyst formation because of the recovered expression of CENPF. Taken together, our data suggest CENPF plays an important role during early embryonic development in mice. Abbreviation: CENPF: centromere protein F; MO: morpholino; FTI: Farnesyltransferase inhibitor; CENPE: centromere protein E; IVF: in vitro fertilization; MII: metaphase II; SAC: spindle assembly checkpoint; Mad1: mitotic arrest deficient 1; BUB1: budding uninhibited by benzimidazole 1; BUBR1: BUB1 mitotic checkpoint serine/threonine kinase B; Cdc20: cell division cycle 20.
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Blastocisto/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Proteínas de Microfilamentos/metabolismo , Oocitos/metabolismo , Cigoto/metabolismo , Animales , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Embrión de Mamíferos/embriología , Farnesiltransferasa/antagonistas & inhibidores , Femenino , Técnicas de Silenciamiento del Gen , Metafase/genética , Ratones , Ratones Endogámicos ICR , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Morfolinos/genética , Morfolinos/farmacología , Mórula/metabolismo , Oocitos/crecimiento & desarrollo , Partenogénesis/genética , Piperidinas/farmacología , Embarazo , Prenilación , Piridinas/farmacología , Cigoto/crecimiento & desarrolloRESUMEN
Trim-Away is a recent technique to rapidly deplete a protein from any cell type. Guided by antibodies, TRIM21 selects proteins for destruction. However, the applicability of this method in model organisms has not been investigated. Here, we show that Trim-Away can degrade proteins in zebrafish embryos. Trim-Away depletes proteins faster than morpholinos, which enables analysis of protein function during early embryogenesis. Furthermore, Trim-Away can be applied to evaluate the role of maternally contributed proteins in zebrafish embryos. Our findings indicate that Trim-Away is a powerful tool to perform functional analysis of proteins during zebrafish development.