RESUMO
While protein homeostasis is a hallmark of gene regulation, unraveling the hidden regulatory mechanisms that maintain homeostasis is difficult using traditional methods. To confront this problem, we CRISPR engineered a human cell line with multiple tags in the endogenous MYH9 gene, which encodes the essential and ubiquitous myosin-2A cytoskeletal motor. Using these cells, we imaged MYH9 transcription, translation, and mature mRNA and protein in distinct colors, enabling a full dissection of the central dogma. Our data show that MYH9 transcription is upregulated in an SRF-dependent manner in response to cytoskeletal cues and that MYH9 translation can either buffer or match the transcriptional response depending on context. Upon knockdown of actin-depolymerizing proteins like cofilin, translation efficiency drops by a factor of two to buffer strong transcriptional upregulation, likely to help prevent excessive myosin activity. In contrast, following serum stimulation, translation matches the transcriptional response to readily reestablish steady state. Our results identify contextual translational buffering as an important regulatory mechanism driving stable MYH9 expression. They also demonstrate the power and broad applicability of our cell line, which can now be used to accurately quantify central dogma dynamics in response to diverse forms of cellular perturbations.
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The translation of messenger RNA (mRNA) into proteins represents the culmination of gene expression. Recent technological advances have revolutionized our ability to investigate this process with unprecedented precision, enabling the study of translation at the single-molecule level in real time within live cells. In this review, we provide an overview of single-mRNA translation reporters. We focus on the core technology, as well as the rapid development of complementary probes, tags, and accessories that enable the visualization and quantification of a wide array of translation dynamics. We then highlight notable studies that have utilized these reporters in model systems to address key biological questions. The high spatiotemporal resolution of these studies is shedding light on previously unseen phenomena, uncovering the full heterogeneity and complexity of translational regulation.
Assuntos
Biossíntese de Proteínas , RNA Mensageiro , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Humanos , Animais , Sobrevivência Celular/genéticaRESUMO
During mitosis, kinetochore-microtubule attachments are monitored by a molecular surveillance system known as the spindle assembly checkpoint. The prevailing model posits that dynein evicts checkpoint proteins (e.g., Mad1, Mad2) from stably attached kinetochores by transporting them away from kinetochores, thus contributing to checkpoint silencing. However, the mechanism by which dynein performs this function, and its precise role in checkpoint silencing remain unresolved. Here, we find that dynein's role in checkpoint silencing is restricted to evicting checkpoint effectors from the fibrous corona, and not the outer kinetochore. Dynein evicts these molecules from the corona in a manner that does not require stable, end-on microtubule attachments. Thus, by disassembling the corona through indiscriminate microtubule encounters, dynein primes the checkpoint signaling apparatus so it can respond to stable end-on microtubule attachments and permit cells to progress through mitosis. Accordingly, we find that dynein function in checkpoint silencing becomes largely dispensable in cells in which checkpoint effectors are excluded from the corona.
Assuntos
Dineínas , Cinetocoros , Cinetocoros/metabolismo , Dineínas/metabolismo , Proteínas/metabolismo , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
Proteins of the actin depolymerizing factor (ADF)/cofilin family are ubiquitous among eukaryotes and are essential regulators of actin dynamics and function. Mammalian neurons express cofilin-1 as the major isoform, but ADF and cofilin-2 are also expressed. All isoforms bind preferentially and cooperatively along ADP-subunits in F-actin, affecting the filament helical rotation, and when either alone or when enhanced by other proteins, promotes filament severing and subunit turnover. Although self-regulating cofilin-mediated actin dynamics can drive motility without post-translational regulation, cells utilize many mechanisms to locally control cofilin, including cooperation/competition with other proteins. Newly identified post-translational modifications function with or are independent from the well-established phosphorylation of serine 3 and provide unexplored avenues for isoform specific regulation. Cofilin modulates actin transport and function in the nucleus as well as actin organization associated with mitochondrial fission and mitophagy. Under neuronal stress conditions, cofilin-saturated F-actin fragments can undergo oxidative cross-linking and bundle together to form cofilin-actin rods. Rods form in abundance within neurons around brain ischemic lesions and can be rapidly induced in neurites of most hippocampal and cortical neurons through energy depletion or glutamate-induced excitotoxicity. In ~20% of rodent hippocampal neurons, rods form more slowly in a receptor-mediated process triggered by factors intimately connected to disease-related dementias, e.g., amyloid-ß in Alzheimer's disease. This rod-inducing pathway requires a cellular prion protein, NADPH oxidase, and G-protein coupled receptors, e.g., CXCR4 and CCR5. Here, we will review many aspects of cofilin regulation and its contribution to synaptic loss and pathology of neurodegenerative diseases.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Degeneração Neural/patologia , Neurônios/metabolismo , Fatores de Despolimerização de Actina/química , Sequência de Aminoácidos , Animais , Humanos , Neuritos/metabolismo , NeurogêneseRESUMO
Nuclear envelope proteins influence cell cytoarchitecure by poorly understood mechanisms. Here we show that small interfering RNA-mediated silencing of lamin A/C (LMNA) promotes contrasting stress fiber assembly and disassembly in individual cells and within cell populations. We show that LMNA-deficient cells have elevated myosin-II bipolar filament accumulations, irregular formation of actin comet tails and podosome-like adhesions, increased steady state nuclear localization of the mechanosensitive transcription factors MKL1 and YAP, and induced expression of some MKL1/serum response factor-regulated genes such as that encoding myosin-IIA (MYH9). Our studies utilizing live cell imaging and pharmacological inhibition of myosin-II support a mechanism of deregulated myosin-II self-organizing activity at the nexus of divergent actin cytoskeletal aberrations resulting from LMNA loss. In light of our results, we propose a model of how the nucleus, via linkage to the cytoplasmic actomyosin network, may act to control myosin-II contractile behavior through both mechanical and transcriptional feedback mechanisms.
Assuntos
Citoesqueleto de Actina/metabolismo , Lamina Tipo A/metabolismo , Miosina Tipo II/metabolismo , Membrana Nuclear/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Células HeLa , Humanos , Lamina Tipo A/deficiência , Miosina Tipo II/genéticaRESUMO
Structural features of the nucleus including shape, size and deformability impact its function affecting normal cellular processes such as cell differentiation and pathological conditions such as tumor cell migration. Despite the fact that abnormal nuclear morphology has long been a defining characteristic for diseases such as cancer relatively little is known about the mechanisms that control normal nuclear architecture. Mounting evidence suggests close coupling between F-actin cytoskeletal organization and nuclear morphology however, mechanisms regulating this coupling are lacking. Here we identify that Cofilin/ADF-family F-actin remodeling proteins are essential for normal nuclear structure in different cell types. siRNA mediated silencing of Cofilin/ADF provokes striking nuclear defects including aberrant shapes, nuclear lamina disruption and reductions to peripheral heterochromatin. We provide evidence that these anomalies are primarily due to Rho kinase (ROCK) controlled excessive contractile myosin-II activity and not to elevated F-actin polymerization. Furthermore, we demonstrate a requirement for nuclear envelope LINC (linker of nucleoskeleton and cytoskeleton) complex proteins together with lamin A/C for nuclear aberrations induced by Cofilin/ADF loss. Our study elucidates a pivotal regulatory mechanism responsible for normal nuclear structure and which is expected to fundamentally influence nuclear function.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Forma do Núcleo Celular , Núcleo Celular/metabolismo , Miosina Tipo II/metabolismo , Linhagem Celular , Humanos , Mecanotransdução Celular , Quinases Associadas a rho/metabolismoRESUMO
The contractile actin cortex is important for diverse fundamental cell processes, but little is known about how the assembly of F-actin and myosin II motors is regulated. We report that depletion of actin depolymerizing factor (ADF)/cofilin proteins in human cells causes increased contractile cortical actomyosin assembly. Remarkably, our data reveal that the major cellular defects resulting from ADF/cofilin depletion, including cortical F-actin accumulation, were largely due to excessive myosin II activity. We identify that ADF/cofilins from unicellular organisms to humans share a conserved activity to inhibit myosin II binding to F-actin, indicating a mechanistic rationale for our cellular results. Our study establishes an essential requirement for ADF/cofilin proteins in the control of normal cortical contractility and in processes such as mitotic karyokinesis. We propose that ADF/cofilin proteins are necessary for controlling actomyosin assembly and intracellular contractile force generation, a function of equal physiological importance to their established roles in mediating F-actin turnover.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Miosina Tipo II/metabolismo , Células HeLa , Humanos , Ligação ProteicaRESUMO
Thermal stability is important for the manufacture, distribution and administration of vaccines, especially in tropical developing countries, where particularly adverse field conditions exist. Current live-attenuated flavivirus vaccines exhibit relatively poor liquid stability in clinical settings, and clinicians are instructed to discard the yellow fever vaccine 1h after reconstitution. We have identified novel combinations of excipients that greatly enhance the thermal stability of live-attenuated DEN-2 PDK-53-based flavivirus vaccine candidates. Liquid formulations comprising a sugar, albumin and a pluronic polymer minimized the loss of flavivirus infectious titer to less than 0.5 log(10)pfu after storage for at least 8h at 37°C, 7 days at room temperature or at least 11 weeks at 4°C. Additionally, these formulations prevented reduction of viral infectivity after two freeze-thaw cycles of virus. Formulated candidate vaccines were readily lyophilized and reconstituted with minimal loss of viral titers. In mice, the formulations were safe and did not hinder the ability of the vaccine virus to generate a potent, protective immune response. These formulations provided significantly greater liquid-phase stability than has been reported previously for other flavivirus vaccine formulations. The enhanced thermal stability provided by the formulations described here will facilitate the effective distribution of flavivirus vaccines worldwide.
Assuntos
Vacinas contra Dengue , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Vacinas Virais , Vírus do Nilo Ocidental/imunologia , Vacina contra Febre Amarela , Albuminas , Animais , Química Farmacêutica , Chlorocebus aethiops , Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Camundongos , Testes de Neutralização , Polímeros , Estabilidade Proteica , Temperatura , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Células Vero , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Vacina contra Febre Amarela/administração & dosagem , Vacina contra Febre Amarela/imunologia , Vírus da Febre Amarela/imunologiaRESUMO
Formins are well-known for promoting actin assembly, but they also play a lesser-studied role in microtubule stabilization. In this issue of Developmental Cell, Cheng et al. (2011) demonstrate that the formin homology protein mDia3 is regulated by Aurora B Kinase and contributes to the generation of kinetochore-microtubule attachments in mitosis.
RESUMO
Dephosphorylation (activation) of cofilin, an actin binding protein, is stimulated by initiators of neuronal dysfunction and degeneration including oxidative stress, excitotoxic glutamate, ischemia, and soluble forms of beta-amyloid peptide (Abeta). Hyperactive cofilin forms rod-shaped cofilin-saturated actin filament bundles (rods). Other proteins are recruited to rods but are not necessary for rod formation. Neuronal cytoplasmic rods accumulate within neurites where they disrupt synaptic function and are a likely cause of synaptic loss without neuronal loss, as occurs early in dementias. Different rod-inducing stimuli target distinct neuronal populations within the hippocampus. Rods form rapidly, often in tandem arrays, in response to stress. They accumulate phosphorylated tau that immunostains for epitopes present in "striated neuropil threads," characteristic of tau pathology in Alzheimer disease (AD) brain. Thus, rods might aid in further tau modifications or assembly into paired helical filaments, the major component of neurofibrillary tangles (NFTs). Rods can occlude neurites and block vesicle transport. Some rod-inducing treatments cause an increase in secreted Abeta. Thus rods may mediate the loss of synapses, production of excess Abeta, and formation of NFTs, all of the pathological hallmarks of AD. Cofilin-actin rods also form within the nucleus of heat-shocked neurons and are cleared from cells expressing wild type huntingtin protein but not in cells expressing mutant or silenced huntingtin, suggesting a role for nuclear rods in Huntington disease (HD). As an early event in the neurodegenerative cascade, rod formation is an ideal target for therapeutic intervention that might be useful in treatment of many different neurological diseases.
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Citoesqueleto de Actina/metabolismo , Cofilina 1/metabolismo , Corpos de Inclusão/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Citoesqueleto de Actina/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Humanos , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Corpos de Inclusão/patologia , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Neurônios/patologia , Estresse Oxidativo/fisiologiaRESUMO
We previously showed that the transcription factor Pax3 regulates mesenchymal-to-epithelial transition (MET) in cultured osteogenic Saos-2 cells. Herein we demonstrate that Pax3 induced MET in these cells requires intact Pax3 DNA binding motifs and is associated with the altered expression and activity of numerous proteins involved in signal transduction pathways that regulate cytoskeleton remodeling, the majority of which were not previously detected by mRNA expression array analysis. Proper levels of active Rho GTPases are essential for Pax3 induced MET. Rac activity and actomyosin contractility via Rho/ROCK signaling are required for the formation of circumferential actin bundles, epithelial discoid cell shape and the regulation of membrane protrusions. Precise spatial activation of Rho GTPase signaling components is also paramount for MET. Endogenous PAK2, Rac1 and PIX, a Rac/Cdc42-GEF, localize to focal adhesions. Dynamic localization of PAK and PIX to focal adhesions is required for Pax3 induced MET and is dependent on full PAK activity because kinase dead or GTPase-binding deficient mutants of PAK sequester PIX at focal adhesions and disrupt Pax3 induced phenotypic MET. All together, our results define roles for Rho GTPases and their effectors in MET and newly identify proteins and signal transduction cascades regulated by Pax3.
Assuntos
Epitélio/fisiologia , Mesoderma/fisiologia , Morfogênese , Fatores de Transcrição Box Pareados/metabolismo , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Forma Celular , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miosinas/metabolismo , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Ativadas por p21 , Proteínas rho de Ligação ao GTP/genética , Quinases Associadas a rhoRESUMO
Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3zeta, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3zeta binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.
Assuntos
Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Fatores de Despolimerização de Actina , Actinas/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Galinhas , Destrina , Humanos , Técnicas In Vitro , Quinases Lim , Proteínas dos Microfilamentos/genética , Modelos Biológicos , Complexos Multiproteicos , Neurônios/metabolismo , Fosfoproteínas Fosfatases/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Quinases Ativadas por p21Assuntos
Actinas/metabolismo , Hidrolases/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fatores de Despolimerização de Actina , Animais , Humanos , Fosforilação , Fosfotransferases/metabolismo , Ligação Proteica/fisiologia , Fosfato de Piridoxal/metabolismoRESUMO
Entry of Salmonella into mammalian cells is strictly dependent on the reorganization of actin cytoskeleton induced by a panel of Salmonella type III secreted proteins. Although several factors have been identified to be responsible for inducing the actin polymerization and stability, little is known about how the actin depolymerization contributes to Salmonella-induced actin rearrangements. We report here that activity cycles of host actin depolymerizing factor (ADF and cofilin) are modulated by Salmonella during bacterial entry. Efficient Salmonella internalization involves an initial dephosphorylation of ADF and cofilin followed by phosphorylation, suggesting that ADF and cofilin activities are increased briefly. Expression of a kinase dead form of an ADF/cofilin kinase (LIM kinase 1) or a catalytically inactive ADF/cofilin phosphatase (Slingshot), but not constitutively active LIM kinase 1 or wild-type Slingshot, resulted in decreased invasion. These data suggest that ADF/cofilin activities play a key role in the actin polymerization/depolymerization process induced by Salmonella. The activation of ADF/cofilin is brief and has to be reversed to facilitate efficient bacterial entry. Surprisingly, co-expression of constitutive active ADF and cofilin prevented efficient Salmonella entry, whereas expression of either one alone had no effect. We propose that ADF and cofilin actin-dynamizing activities and their activity cycling via phosphorylation are required for efficient Salmonella internalization.
Assuntos
Endocitose/fisiologia , Proteínas dos Microfilamentos/metabolismo , Infecções por Salmonella/metabolismo , Salmonella/metabolismo , Fatores de Despolimerização de Actina , Actinas/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Extensões da Superfície Celular/metabolismo , Citoesqueleto/metabolismo , Destrina , Células HeLa , Humanos , Quinases Lim , Proteínas dos Microfilamentos/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Adenoviruses infect a wide range of cell types, do not require integration into the host cell genome, and can be produced as replication-deficient viruses capable of expressing transgenes behind any desired promoter. Thus, they are ideal for use in expressing transgenes in the postmitotic neuron. This chapter describes simplifications in the protocols for making recombinant adenoviruses and their use in expressing transgenes in primary neurons of several different types.
Assuntos
Adenoviridae/genética , Vetores Genéticos/genética , Neurônios/virologia , Transfecção/métodos , Transgenes/genética , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas/citologia , Células Cultivadas/fisiologia , Células Cultivadas/virologia , Regulação da Expressão Gênica/genética , Humanos , Neurônios/citologia , Neurônios/fisiologia , Replicação Viral/genéticaRESUMO
ADF/cofilins are key regulators of actin dynamics in normal cells. Recent findings suggest that, under cellular stress, the wild-type proteins might form complexes with actin that can alter cell function. Owing to their rapid formation, these complexes might initiate or aid in the progression of diseases as diverse as Alzheimer's disease and ischemic kidney disease. Although evidence for their involvement in diseases other than Alzheimer's and ischemic kidney disease is tenuous, recent studies suggest that altered production, regulation or localization of these proteins might lead to cognitive impairment, inflammation, infertility, immune deficiencies and other pathophysiological defects.
Assuntos
Actinas/metabolismo , Infertilidade/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neoplasias/metabolismo , Fatores de Despolimerização de Actina , Actinas/química , Animais , Quimiotaxia , Destrina , Humanos , Proteínas dos Microfilamentos/química , Sinapses/metabolismoRESUMO
Paired box-containing transcription factors play fundamental roles in pattern formation during embryonic development of diverse organisms ranging from Drosophila to mammals. Although mutations to Pax3 and other Pax-family genes in both mice and humans result in numerous tissue-specific morphological defects, little is known about the cellular processes that Pax genes regulate. We show that extopic Pax3 expression in two distinct phenotypically mesenchymal mammalian cell lines induces the formation of multi-layered condensed cell aggregates with epithelial characteristics. For one of these lines, we showed further that Pax3-induced cell aggregation is accompanied by specific morphological changes, including a significant reduction in cell size, altered cell shape and dramatic alterations to both membrane and cytoskeleton architecture. In addition to mediating a phenotypic mesenchymal-to-epithelial transition, Pax3 also establishes the conditions in these cells for a subsequent hepatocyte growth factor/scatter factor (HGF/SF)-induced phenotypic epithelial-to-mesenchymal transition. Thus, our data show a novel morphogenetic activity for Pax3 which, when absent in vivo, is predicted to give rise to the observed structural defects in somites and the neural tube during embryonic development.
Assuntos
Agregação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Epitélio/metabolismo , Mesoderma/metabolismo , Fatores de Transcrição/metabolismo , Actinas/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Células COS , Caderinas/metabolismo , Polaridade Celular , Tamanho Celular , Meios de Cultivo Condicionados , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Genes Reporter , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Osteossarcoma , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Fenótipo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Rabdomiossarcoma , Células Tumorais Cultivadas , Proteína da Zônula de Oclusão-1RESUMO
Mutations to Pax3 and other Pax family genes in both mice and humans result in numerous tissue-specific morphological defects. Little is known, however, about the cellular and molecular mechanisms by which Pax genes regulate morphogenesis. We previously showed that Pax3 induces cell aggregation and a mesenchymal-to-epithelial transition in Saos-2 cells. We show here that Pax3-induced aggregates arise through the formation of distinct structures involving cell rearrangements and cell behaviors resembling those that occur during gastrulation and neurulation known as convergent extension. During these Pax3-induced processes, Dishevelled and Frizzled are localized to the actin cytoskeleton and both proteins coimmunoprecipitate focal adhesion components from detergent-insoluble cell fractions. We show further that these Pax3-induced cell movements are associated with activation of a Wnt-signaling cascade, resulting in induction and activation of c-Jun-N-terminal kinase/stress activated protein kinase (JNK/SAPK). All of these Wnt-signaling factors exhibit altered subcellular distribution in Pax3-expressing cells. In particular, we show the localization of JNK/SAPK to both the nucleus and to cytoplasmic multi-vesicular structures. These data show that Pax3 regulates morphogenetic cell behavior and that regulation of a conserved, planar cell polarity/noncanonical Wnt-signaling cascade entailing JNK activation is a function of Pax3 activity.
Assuntos
Agregação Celular/fisiologia , Movimento Celular/fisiologia , Polaridade Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição , Proteínas de Peixe-Zebra , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Tamanho Celular , Proteínas de Ligação a DNA/genética , Proteínas Desgrenhadas , Ativação Enzimática , Adesões Focais/metabolismo , Receptores Frizzled , Genes Reporter , Humanos , Imuno-Histoquímica , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteossarcoma , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Células Tumorais Cultivadas , Vinculina/metabolismo , Proteínas WntRESUMO
We have recently demonstrated that the retinoblastoma family of negative cell cycle regulators can form complexes with a class of developmental factors which contain paired-like (PL) homeodomains (Wiggan et al. [1998] Oncogene 16:227-236). Our screens led to the isolation of a novel PL-homeodomain protein which had been isolated independently by another group and called Alx-4 (Qu et al. [1997] Development 124:3999-4008). Mice homozygous for a targeted null mutation of Alx-4 have several abnormalities, including preaxial polydactyly, suggesting that Alx-4 plays a role in pattern formation in limb buds. In data that we present here, we show that Alx-4 is expressed in mesenchymal condensations of a diverse group of tissues whose development is dependent on epithelial-mesenchymal interactions, many of which are additionally dependent on expression of the HMG-box-containing protein, LEF-1. Alx-4-expressing tissues include osteoblast precursors of most bones, the dermal papilla of hair and whisker follicles, the dental papilla of teeth, and a subset of mesenchymal cells in pubescent mammary glands. We show further that Alx-4 strongly activates transcription from a promoter containing the homeodomain binding site, P2. Optimal activation requires specific sequences in the N-terminal portion of Alx-4 as well as a proline-rich region downstream of the PL-homeodomain, but not the paired-tail at the C terminus. Taken together, our results demonstrate that Alx-4 is a potent transcriptional activator that is expressed at sites of epithelial-mesenchymal interactions during murine embryonic development.
Assuntos
Proteínas de Homeodomínio/genética , Transativadores/genética , Animais , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Células COS , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário e Fetal/genética , Epitélio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Folículo Piloso/embriologia , Folículo Piloso/metabolismo , Hibridização In Situ , Fator 1 de Ligação ao Facilitador Linfoide , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Animais/metabolismo , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Gravidez , Regiões Promotoras Genéticas , Dente/embriologia , Dente/metabolismo , Fatores de Transcrição/genética , TransfecçãoRESUMO
The specific loss of pRB or p107 together with p130 disrupts the normal development of only a very limited spectrum of tissues. These developmental defects have been attributed primarily to deregulation of E2F activity and consequent uncontrolled proliferation. We hypothesized, however, that the tissue-specific nature of these defects may also reflect deregulation of pRB-family associated factors that are specifically involved in determining cell fate. We report here that the pRB-family members interact with transcription factors which contain paired-like homeodomains such as MHox, Chx10 and Pax-3. The interaction between the pRB-family and the paired-like homeodomain proteins was initially identified in a yeast two-hybrid screen where the N-terminal portion of p130 was used to isolate interacting factors from an embryonic mouse library. This interaction was confirmed by in vitro binding and co-immunoprecipitation assays. We show further that co-expression of Pax-3 dependent pRB, p107 or p130 with Pax-3 causes repression of activated transcription from the c-met promoter. These data demonstrate that the pRB-family proteins can modulate the activity of factors which specifically control cell fate and/or differentiation as well as controlling cell cycle regulators.