RESUMO
Within a relatively short period of time, single cell transcriptome analyses (SCT) have become increasingly ubiquitous with transcriptomic research, uncovering plentiful details that boost our molecular understanding of various biological processes. Stemming from SCT analyses, the ever-growing number of newly assigned genetic markers increases our understanding of general function and development, while providing opportunities for identifying genes associated with disease. SCT analyses have been carried out using tissue from numerous organisms. However, despite the great potential of zebrafish as a model organism, other models are still preferably used. In this mini review, we focus on eye research as an example of the advantages in using zebrafish, particularly its usefulness for single cell transcriptome analyses of developmental processes. As studies have already shown, the unique opportunities offered by zebrafish, including similarities to the human eye, in combination with the possibility to analyze and extract specific cells at distinct developmental time points makes the model a uniquely powerful one. Particularly the practicality of collecting large numbers of embryos and therefore isolation of sufficient numbers of developing cells is a distinct advantage compared to other model organisms. Lastly, the advent of highly efficient genetic knockouts methods offers opportunities to characterize target gene function in a more cost-efficient way. In conclusion, we argue that the use of zebrafish for SCT approaches has great potential to further deepen our molecular understanding of not only eye development, but also many other organ systems.
RESUMO
In human, pathogenic variants in smad3 are one cause of familial aortopathy. We describe a novel SMAD3 variant of unknown significance (VUS), V244F, in a patient who presented with aortic root dilation, right coronary artery ectasia, abdominal aortic aneurysm, right vertebral artery atresia, and cavernoma. Determination of variant pathogenicity impacted multiple aspects of the patient's care, including the most appropriate surgical threshold for which to recommend a valve-sparing aortic root replacement. To determine whether the newly identified SMAD3 variant, and whether SMAD3 induced aortopathy in general, can be assayed in a zebrafish embryo model, we injected smad3a mRNA into Tg[kdrl:mCherry] zebrafish embryos. By measuring the size of the dorsal aorta at 48hpf we found a correlation between pathogenic SMAD3 variants and increased dorsal aortic diameter. The newly identified V244F variant increased dorsal aortic diameter (p < 0.0001) similar to that of the pathogenic control variant T261I (p < 0.0084). In addition, we examined several previously identified variants of uncertain significance and found P124T (p < 0.0467), L296P (p < 0.0025) and A349P (p < 0.0056) to behave like T261I. These results demonstrate that the zebrafish embryo assay was successful in validating known pathogenic variants, classifying our newly identified variant V244F as likely pathogenic, and classifying previously identified variants P124T, L296P, and A349P as likely pathogenic. Overall, our findings identify a novel SMAD3 variant that is likely pathogenic as well as offer a new mechanism to model SMAD3 VUSs in vivo.
RESUMO
Developmental regulation of the vertebrate visual system has been a focus of investigation for generations as understanding this critical time period has direct implications on our understanding of congenital blinding disease. The majority of studies to date have focused on transcriptional regulation mediated by morphogen gradients and signaling pathways. However, recent studies of post translational regulation during ocular development have shed light on the role of the ubiquitin proteasome system (UPS). This rather ubiquitous yet highly diverse system is well known for regulating protein function and localization as well as stability via targeting for degradation by the 26S proteasome. Work from many model organisms has recently identified UPS activity during various milestones of ocular development including retinal morphogenesis, retinal ganglion cell function as well as photoreceptor homeostasis. In particular work from flies and zebrafish has highlighted the role of the E3 ligase enzyme family, Seven in Absentia Homologue (Siah) during these events. In this review, we summarize the current understanding of UPS activity during Drosophila and vertebrate ocular development, with a major focus on recent findings correlating Siah E3 ligase activity with two major developmental stages of vertebrate ocular development, retinal morphogenesis and photoreceptor specification and survival.
Assuntos
Olho/embriologia , Proteínas Nucleares/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Drosophila , Olho/crescimento & desenvolvimento , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/fisiologia , Vertebrados , Peixe-ZebraRESUMO
Congenital retinal dystrophies are a major cause of unpreventable and incurable blindness worldwide. Mutations in CDHR1, a retina specific cadherin, are associated with cone-rod dystrophy. The ubiquitin proteasome system (UPS) is responsible for mediating orderly and precise targeting of protein degradation to maintain biological homeostasis and coordinate proper development, including retinal development. Recently, our lab uncovered that the seven in absentia (Siah) family of E3 ubiquitin ligases play a role in optic fissure fusion and identified Cdhr1a as a potential target of Siah. Using two-color whole mount in situ hybridization and immunohistochemistry, we detected siah1 and cdhr1a co-expression as well as protein localization in the retinal outer nuclear layer (ONL), and more precisely in the connecting cilium of rods and cones between 3-5 days post fertilization (dpf). We confirmed that Siah1 targets Cdhr1a for proteasomal degradation by co-transfection and co-immunoprecipitation in cell culture. To analyze the functional importance of this interaction, we created two transgenic zebrafish lines that express siah1 or an inactive siah1 (siah1ΔRING) under the control of the heat shock promoter to modulate Siah activity during photoreceptor development. Overexpression of siah1, but not siah1ΔRING, resulted in a decrease in the number of rods and cones at 72 h post fertilization (hpf). The number of retinal ganglion cells, amacrine and bipolar cells was not affected by Siah1 overexpression, and there was no significant reduction of proliferating cells in the Siah1 overexpressing retina. We did, however, detect increased cell death, confirmed by an increase in the number of TUNEL + cells in the ONL, which was proteasome-dependent, as proteasome inhibition rescued the cell death phenotype. Furthermore, reduction in rods and cones resulting from increased Siah1 expression was rescued by injection of cdhr1a mRNA, and to an even greater extent by injection of a Siah1-insensitive cdhr1a variant mRNA. Lastly, CRISPR induced loss of Cdhr1a function phenocopied Siah1 overexpression resulting in a significant reduction of rods and cones. Taken together, our work provides the first evidence that Cdhr1a plays a role during early photoreceptor development and that Cdhr1a is regulated by Siah1 via the UPS.
RESUMO
Vertebrate retinal development requires timely and precise fusion of the optic fissure (OF). Failure of this event leads to congenital vision impairment in the form of coloboma. Recent studies have suggested hyaloid vasculature to be involved in OF fusion. In order to examine this link, we analyzed OF fusion and hyaloid vasculogenesis in the zebrafish pax2a noi mutant line. We first determined that pax2a-/- embryos fail to accumulate F-actin in the OF prior to basement membrane (BM) degradation. Furthermore, using 3D and live imaging we observed reduced OF hyaloid vascularization in pax2a-/- embryos. When examining the connection between pax2a loss of function and hyaloid vasculature, we observed significant reduction of talin1 expression, a regulator of hyaloid vasculature. In addition, cranial VEGF expression was found to be reduced in pax2a-/- embryos. Pharmacological inhibition of VEGF signaling phenocopied the pax2a-/- vasculature, F-actin and BM degradation phenotypes. Lastly, we determined that OF associated hyaloid vasculature is a source of mmp2, mmp14a and mmp14b expression and showed that mmp2 is functionally necessary for degradation of OF BM. Taken together we propose a pax2a driven mechanism that ensures proper and timely hyaloid vasculature invasion of the OF in order to facilitate availability of the BM remodeler mmp2.
Assuntos
Embrião não Mamífero , Olho/irrigação sanguínea , Olho/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/fisiologia , Morfogênese/genética , Retina/embriologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Membrana Basal/metabolismo , Coloboma/etiologia , Expressão Gênica , Metaloproteinase 2 da Matriz/metabolismo , Mutação , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Assembly of the ocular anterior segment (AS) is a critical event during development of the vertebrate visual system. Failure in this process leads to anterior segment dysgenesis (ASD), which is characterized by congenital blindness and predisposition to glaucoma. The anterior segment is largely formed via a neural crest-derived population, the Periocular Mesenchyme (POM). In this study, we aimed to characterize POM behaviors and transcriptional identities during early establishment of the zebrafish AS. Two-color fluorescent in situ hybridization suggested that early AS associated POM comprise of a heterogenous population. In vivo and time-course imaging analysis of POM distribution and migratory dynamics analyzed using transgenic zebrafish embryos (Tg[foxc1b:GFP], Tg[foxd3:GFP], Tg[pitx2:GFP], Tg[lmx1b.1:GFP], and Tg[sox10:GFP]) revealed unique AS distribution and migratory behavior among the reporter lines. Based on fixed timepoint and real-time analysis of POM cell behavior a comprehensive model for colonization of the zebrafish AS was assembled. Furthermore, we generated single cell transcriptomic profiles (scRNA) from our POM reporter lines and characterized unique subpopulation expression patterns. Based on scRNA clustering analysis we observed cluster overlap between neural crest associated (sox10/foxd3), POM (pitx2) and finally AS specified cells (lmx1b, and foxc1b). scRNA clustering also revealed several novel markers potentially associated with AS development and/or function including lum, fmoda, adcyap1b, tgfbi, and hmng2. Taken together, our data indicates that AS-associated POM, or Anterior Segment Mesenchyme (ASM), is not homogeneous but rather comprised of several subpopulations with differing colonization patterns, migration behavior, and transcriptomic profiles.
RESUMO
The eye primordium arises as a lateral outgrowth of the forebrain, with a transient fissure on the inferior side of the optic cup providing an entry point for developing blood vessels. Incomplete closure of the inferior ocular fissure results in coloboma, a disease characterized by gaps in the inferior eye and recognized as a significant cause of pediatric blindness. Here, we identify eight patients with defects in tissues of the superior eye, a congenital disorder that we term superior coloboma. The embryonic origin of superior coloboma could not be explained by conventional models of eye development, leading us to reanalyze morphogenesis of the dorsal eye. Our studies revealed the presence of the superior ocular sulcus (SOS), a transient division of the dorsal eye conserved across fish, chick, and mouse. Exome sequencing of superior coloboma patients identified rare variants in a Bone Morphogenetic Protein (Bmp) receptor (BMPR1A) and T-box transcription factor (TBX2). Consistent with this, we find sulcus closure defects in zebrafish lacking Bmp signaling or Tbx2b. In addition, loss of dorsal ocular Bmp is rescued by concomitant suppression of the ventral-specific Hedgehog pathway, arguing that sulcus closure is dependent on dorsal-ventral eye patterning cues. The superior ocular sulcus acts as a conduit for blood vessels, with altered sulcus closure resulting in inappropriate connections between the hyaloid and superficial vascular systems. Together, our findings explain the existence of superior coloboma, a congenital ocular anomaly resulting from aberrant morphogenesis of a developmental structure.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Coloboma/embriologia , Coloboma/genética , Citocromo P-450 CYP1B1/genética , Olho/embriologia , Adulto , Animais , Animais Geneticamente Modificados , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Embrião de Galinha , Embrião não Mamífero , Fator 6 de Diferenciação de Crescimento/genética , Fator 6 de Diferenciação de Crescimento/metabolismo , Humanos , Lactente , Camundongos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Kinetochore (KT) localization of mitotic checkpoint proteins is essential for their function during mitosis. hSpindly KT localization is dependent on the RZZ complex and hSpindly recruits the dynein-dynactin complex to KTs during mitosis, but the mechanism of hSpindly KT recruitment is unknown. Through domain-mapping studies we characterized the KT localization domain of hSpindly and discovered it undergoes farnesylation at the C-terminal cysteine residue. The N-terminal 293 residues of hSpindly are dispensable for its KT localization. Inhibition of farnesylation using a farnesyl transferase inhibitor (FTI) abrogated hSpindly KT localization without affecting RZZ complex, CENP-E, and CENP-F KT localization. We showed that hSpindly is farnesylated in vivo and farnesylation is essential for its interaction with the RZZ complex and hence KT localization. FTI treatment and hSpindly knockdown displayed the same mitotic phenotypes, indicating that hSpindly is a key FTI target in mitosis. Our data show a novel role of lipidation in targeting a checkpoint protein to KTs through protein-protein interaction.
Assuntos
Proteínas de Transporte/genética , Segregação de Cromossomos/genética , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Prenilação de Proteína/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Farnesiltranstransferase/antagonistas & inibidores , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Mutação , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno , Deleção de Sequência , Fuso Acromático/genéticaRESUMO
Axial patterning of the developing eye is critically important for proper axonal pathfinding as well as for key morphogenetic events, such as closure of the optic fissure. The dorsal retina is initially specified by the actions of Bone Morphogenetic Protein (BMP) signaling, with such identity subsequently maintained by the Wnt-ß catenin pathway. Using zebrafish as a model system, we demonstrate that Secreted frizzled-related protein 1a (Sfrp1a) and Sfrp5 work cooperatively to pattern the retina along the dorso-ventral axis. Sfrp1a/5 depleted embryos display a reduction in dorsal marker gene expression that is consistent with defects in BMP- and Wnt-dependent dorsal retina identity. In accord with this finding, we observe a marked reduction in transgenic reporters of BMP and Wnt signaling within the dorsal retina of Sfrp1a/5 depleted embryos. In contrast to studies in which canonical Wnt signaling is blocked, we note an increase in BMP ligand expression in Sfrp1a/5 depleted embryos, a phenotype similar to that seen in embryos with inhibited BMP signaling. Overexpression of a low dose of sfrp5 mRNA causes an increase in dorsal retina marker gene expression. We propose a model in which Sfrp proteins function as facilitators of both BMP and Wnt signaling within the dorsal retina.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Retina/embriologia , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Animais , Sequência de Bases , Primers do DNA , Hibridização In Situ , Morfogênese , Reação em Cadeia da Polimerase em Tempo Real , Peixe-ZebraRESUMO
PURPOSE: To characterize the molecular mechanisms underlying retinal apoptosis induced by loss of Gdf6, a TGFß ligand. METHODS: The role of Gdf6 in regulating apoptosis was studied using a zebrafish gdf6a(-/-) mutant, which encodes a truncated, nonfunctional protein. To investigate whether intrinsic or extrinsic apoptotic mechanisms were involved, morpholino antisense oligonucleotides targeting baxa, baxb, and p53 were employed. Caspase-3 immunohistochemistry (IHC) was performed to assay apoptosis. Pharmacologic inhibition (using SB203580) and IHC were used to investigate the role of p38 mitogen activated protein (MAP) kinase activation in gdf6a(-/-)-induced apoptosis. To assess the role of Gdf6a in transcriptional regulation of TGFß signal transducers, in situ hybridization (ISH) was performed using probes to smad1, 5, 7, and 8. RESULTS: Results indicate maximal ocular apoptosis occurs 28 hours post fertilization (hpf) in gdf6a(-/-) mutants that is mediated independently of p53 by intrinsic mechanisms involving Bax proteins. Also, gdf6a(-/-) mutants exhibit markedly increased p38 MAP kinase activation that can be inhibited to significantly reduce retinal apoptosis. A reduction in retinal smad1 expression was also noted in gdf6a(-/-) mutants. CONCLUSIONS: gdf6a(-/-)-induced apoptosis is characterized by the involvement of intrinsic apoptotic pathways, p38 MAP kinases, and dysregulated smad expression. Modulation of key mediators can inhibit retinal apoptosis offering potential avenues of therapy. However, the efficacy of pharmacomodulation in improvement of visual function needs to be further examined.
Assuntos
Apoptose/fisiologia , Fator 6 de Diferenciação de Crescimento/fisiologia , Retina/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Caspase 3/metabolismo , Fator 6 de Diferenciação de Crescimento/genética , Imuno-Histoquímica , Retina/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad1/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Synthetic targeted endonucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have recently emerged as powerful tools for targeted mutagenesis, especially in organisms that are not amenable to embryonic stem cell manipulation. Both ZFNs and TALENs consist of DNA-binding arrays that are fused to the nonspecific FokI nuclease domain. In an effort to improve targeted endonuclease mutagenesis efficiency, we enhanced their catalytic activity using the Sharkey FokI nuclease domain variant. All constructs tested display increased DNA cleavage activity in vitro. We demonstrate that one out of four ZFN arrays containing the Sharkey FokI variant exhibits a dramatic increase in mutagenesis frequency in vivo in zebrafish. The other three ZFNs exhibit no significant alteration of activity in vivo. Conversely, we demonstrate that TALENs containing the Sharkey FokI variant exhibit absent or severely reduced in vivo mutagenic activity in zebrafish. Notably, Sharkey ZFNs and TALENs do not generate increased toxicity-related defects or mortality. Our results present Sharkey ZFNs as an effective alternative to conventional ZFNs, but advise against the use of Sharkey TALENs.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Mutagênese Sítio-Dirigida/métodos , Peixe-Zebra/genética , Animais , Dedos de ZincoRESUMO
During histogenesis of the vertebrate central nervous system (CNS), neuronal progenitors must interact with germinal zone (GZ) niches, differentiate, and morphologically mature, and neurons must migrate to their final positions. The extrinsic cues that control neurogenesis, specify neurons, and guide their movement are relatively well understood. However, less is known about how neurons spatiotemporally modify cell-cell interactions and cell polarization to navigate through complex, distinct cellular environments during neuronal circuit formation. Here we examine the parallels between the mechanisms controlling epithelial morphogenesis and the cell adhesion events by which neural cells organize GZ niches and direct neuronal migration. We focus on the emerging relationship between neuronal adhesive interactions and conserved cell-polarity signaling cascades.
Assuntos
Adesão Celular/fisiologia , Sistema Nervoso Central/embriologia , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Animais , Moléculas de Adesão Celular/fisiologia , Moléculas de Adesão Celular Neuronais/fisiologia , Comunicação Celular , Linhagem da Célula , Movimento Celular , Polaridade Celular , Transdiferenciação Celular , Microambiente Celular , Sistema Nervoso Central/citologia , Humanos , Modelos Neurológicos , Morfogênese , Células Neuroepiteliais/citologia , Células Neuroepiteliais/fisiologia , Neurônios/citologia , Transdução de SinaisRESUMO
Accurate chromosome segregation in mitosis is required to maintain genetic stability. hZwint-1 [human Zw10 (Zeste white 10)-interacting protein 1] is a kinetochore protein known to interact with the kinetochore checkpoint protein hZw10. hZw10, along with its partners Rod (Roughdeal) and hZwilch, form a complex which recruits dynein-dynactin and Mad1-Mad2 complexes to the kinetochore and are essential components of the mitotic checkpoint. hZwint-1 localizes to the kinetochore in prophase, before hZw10 localization, and remains at the kinetochore until anaphase, after hZw10 has dissociated. This difference in localization timing may reflect a role for hZwint-1 as a structural kinetochore protein. In addition to hZw10, we have found that hZwint-1 interacts with components of the conserved Ndc80 and Mis12 complexes in yeast two-hybrid and GST (glutathione transferase) pull-down assays. Furthermore, hZwint-1 was found to have stable FRAP (fluorescence recovery after photobleaching) dynamics similar to hHec1, hSpc24 and hMis12. As such, we proposed that hZwint-1 is a structural protein, part of the inner kinetochore scaffold and recruits hZw10 to the kinetochore. To test this, we performed mutagenesis-based domain mapping to determine which regions of hZwint-1 are necessary for kinetochore localization and which are required for interaction with hZw10. hZwint-1 localizes to the kinetochore through the N-terminal region and interacts with hZw10 through the C-terminal coiled-coil domain. The two domains are at opposite ends of the protein as expected for a protein that bridges the inner and outer kinetochore.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Proteínas do Citoesqueleto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Domínios e Motivos de Interação entre Proteínas , Fuso AcromáticoRESUMO
The mitotic checkpoint functions to ensure accurate chromosome segregation by regulating the progression from metaphase to anaphase. Once the checkpoint has been satisfied, it is inactivated in order to allow the cell to proceed into anaphase and complete the cell cycle. The minus end-directed microtubule motor dynein/dynactin has been implicated in the silencing of the mitotic checkpoint by "stripping" checkpoint proteins off kinetochores. A recent study suggested that Nordihydroguaiaretic acid (NDGA) stimulates dynein/dynactin-mediated transport of its cargo including ZW10 (Zeste White 10). We analyzed the effects of NDGA on dynein/dynactin dependent transport of the RZZ (Zeste White 10, Roughdeal, Zwilch) complex as well as other kinetochore components from kinetochores to spindle poles. Through this approach we have catalogued several kinetochore and centromere components as dynein/dynactin cargo. These include hZW10, hZwilch, hROD, hSpindly, hMad1, hMad2, hCENP-E, hCdc27, cyclin-B and hMps1. Furthermore, we found that treatment with NDGA induced a robust accumulation and complete stabilization of hZW10 at spindle poles. This finding suggests that NDGA may not induce dynein/dynactin transport but rather interfere with cargo release. Lastly, we determined that NDGA induced accumulation of checkpoint proteins at the poles requires dynein/dynactin-mediated transport, hZW10 kinetochore localization and kinetochore-microtubule attachments but not tension or Aurora B kinase activity.
Assuntos
Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Antioxidantes , Transporte Biológico/efeitos dos fármacos , Proteínas de Ciclo Celular , Complexo Dinactina , Células HeLa , Humanos , Cinetocoros/metabolismo , Inibidores de Lipoxigenase , Masoprocol/farmacologia , Microtúbulos , Fuso Acromático/metabolismoRESUMO
The brain's circuitry is established by directed migration and synaptogenesis of neurons during development. Although neurons mature and migrate in specific patterns, little is known about how neurons exit their germinal zone niche. We found that cerebellar granule neuron germinal zone exit is regulated by proteasomal degradation of Pard3A by the Seven in Absentia homolog (Siah) E3 ubiquitin ligase. Pard3A gain of function and Siah loss of function induce precocious radial migration. Time-lapse imaging using a probe to measure neuronal cell contact reveals that Pard3A promotes adhesive interactions needed for germinal zone exit by recruiting the epithelial tight junction adhesion molecule C to the neuronal cell surface. Our findings define a Siah-Pard3A signaling pathway that controls adhesion-dependent exit of neuronal progenitors or immature neurons from a germinal zone niche.
Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular , Movimento Celular , Cerebelo/citologia , Cerebelo/metabolismo , Neurônios/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Moléculas de Adesão Celular/química , Proteínas de Ciclo Celular , Linhagem Celular , Polaridade Celular , Cerebelo/embriologia , Cães , Humanos , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Camundongos , Morfogênese , Neurônios/citologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Interferência de RNA , Transdução de Sinais , Células-Tronco/fisiologia , Transfecção , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The mitotic checkpoint is an essential surveillance mechanism that ensures high fidelity chromosome segregation during mitosis. Mitotic checkpoint function depends on numerous kinetochore proteins, including ZW10, ROD, and Zwilch (the ROD-ZW10-Zwilch complex). Through an extensive mutagenesis screen of hZW10, we have mapped the kinetochore localization domain of hZW10 as well as the hZwint-1 interaction domain. We find that hZwint-1-noninteracting mutants still localize to kinetochores. In addition, using fluorescence recovery after photobleaching, we have found that hZW10 residency at metaphase kinetochores is brief (half-time of 13 s). However, during prometaphase or at unattached kinetochores, enhanced green fluorescent protein-hZW10 becomes a stable component of the kinetochore. Moreover, we find that stable hZW10 kinetochore residency at prometaphase kinetochores is dependent on its interaction with hZwint-1, and is essential for mitotic checkpoint arrest.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Genes cdc/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/genética , Proteínas Nucleares/metabolismo , Fuso Acromático/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Recuperação de Fluorescência Após Fotodegradação , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinetocoros/ultraestrutura , Metáfase/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Proteínas Nucleares/genética , Fuso Acromático/genética , Fuso Acromático/ultraestrutura , Fatores de TempoRESUMO
The mitotic checkpoint ensures proper chromosome segregation by monitoring two critical events during mitosis. One is kinetochore attachment to the mitotic spindle, and the second is the alignment of chromosomes at the metaphase plate, resulting in tension across sister kinetochores (reviewed in [1, 2]). Mitotic-checkpoint proteins are known to accumulate at unaligned chromosomes that have not achieved proper kinetochore-microtubule attachments or established an adequate level of tension across sister kinetochores. Here, we report that hZW10 and hROD, two components of the evolutionarily conserved RZZ complex, accumulate at kinetochores in response to the loss of tension. By using live-cell imaging and FRAP, we showed that the accumulation of hZW10 at tensionless kinetochores stems from a 4-fold reduction of kinetochore turnover rate. We also found that cells lacking hZW10 escape loss-of-tension-induced mitotic-checkpoint arrest more rapidly than those arrested in response to the lack of kinetochore-microtubule attachments. Furthermore, we show that pharmacological inhibition of Aurora B kinase activity with ZM447439 in the absence of tension, but not in the absence of kinetochore-microtubule attachments, results in the loss of hZW10, hROD, and hBub1 from kinetochores. We therefore conclude that Aurora B kinase activity is required for the accumulation of tension-sensitive mitotic-checkpoint components, such as hZW10 and hROD, in order to maintain mitotic-checkpoint arrest.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinase B , Aurora Quinases , Complexo Dinactina , Dineínas/metabolismo , Células HeLa , Humanos , Paclitaxel , Moduladores de TubulinaRESUMO
The assembly of the centromere, a specialized region of DNA along with a constitutive protein complex which resides at the primary constriction and is the site of kinetochore formation, has been puzzling biologists for many years. Recent advances in the fields of chromatin, microscopy, and proteomics have shed a new light on this complex and essential process. Here we review recently discovered mechanisms and proteins involved in determining mammalian centromere location and assembly. The centromeric core protein CENP-A, a histone H3 variant, is hypothesized to designate centromere localization by incorporation into centromere-specific nucleosomes and is essential for the formation of a functional kinetochore. It has been found that centromere localization of centromere protein A (CENP-A), and therefore centromere determination, requires proteins involved in histone deacetylation, as well as base excision DNA repair pathways and proteolysis. In addition to the incorporation of CENP-A at the centromere, the formation of heterochromatin through histone methylation and RNA interference is also crucial for centromere formation. The assembly of the centromere and kinetochore is complex and interdependent, involving epigenetics and hierarchical protein-protein interactions.