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1.
Nat Commun ; 11(1): 5077, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033240

RESUMO

Although substantial progress has been made in cancer biology and treatment, clinical outcomes of bladder carcinoma (BC) patients are still not satisfactory. The tumor microenvironment (TME) is a potential target. Here, by single-cell RNA sequencing on 8 BC tumor samples and 3 para tumor samples, we identify 19 different cell types in the BC microenvironment, indicating high intra-tumoral heterogeneity. We find that tumor cells down regulated MHC-II molecules, suggesting that the downregulated immunogenicity of cancer cells may contribute to the formation of an immunosuppressive microenvironment. We also find that monocytes undergo M2 polarization in the tumor region and differentiate. Furthermore, the LAMP3 + DC subgroup may be able to recruit regulatory T cells, potentially taking part in the formation of an immunosuppressive TME. Through correlation analysis using public datasets containing over 3000 BC samples, we identify a role for inflammatory cancer-associated fibroblasts (iCAFs) in tumor progression, which is significantly related to poor prognosis. Additionally, we characterize a regulatory network depending on iCAFs. These results could help elucidate the protumor mechanisms of iCAFs. Our results provide deep insight into cancer immunology and provide an essential resource for drug discovery in the future.


Assuntos
Fibroblastos/patologia , Inflamação/patologia , Análise de Sequência de RNA , Análise de Célula Única , Bexiga Urinária/patologia , Área Sob a Curva , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Polaridade Celular , Proliferação de Células , Citocinas/metabolismo , Variações do Número de Cópias de DNA/genética , Células Dendríticas/metabolismo , Redes Reguladoras de Genes , Humanos , Ligantes , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Monócitos/patologia , Células Mieloides/patologia , Proteínas de Neoplasias/metabolismo , Linfócitos T Reguladores/imunologia , Microambiente Tumoral , Bexiga Urinária/imunologia
2.
Anticancer Res ; 40(11): 6473-6484, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109586

RESUMO

BACKGROUND/AIM: Glioblastoma multiforme (GBM) is an intractable tumor that has a very poor prognosis despite intensive treatment with temozolomide plus radiotherapy. PATIENTS AND METHODS: Sixteen newly diagnosed patients with high-grade gliomas were enrolled in a phase II study of the α-type-1 DC vaccine. Briefly, DCs obtained from the culture of enriched monocytes in the presence of a cytokine cocktail, were pulsed with a cocktail of 5 synthetic peptides and cryopreserved until injection into patients. RESULTS: The amount of IL-12 produced by activated DCs was higher than that previously reported. Among 15 evaluable patients, 10 showed positive CTL responses to any peptides in an ELISPOT assay. After 6 years of observation, five patients were still alive, and two of these patients were relapse-free. Moreover, a significant survival-prolonging effect was verified in DC-treated glioma patients. CONCLUSION: Peptide-cocktail-pulsed α-type-1 DC vaccines have a potential therapeutic effect on survival when used in combination with the standard regimen, which is partly based on IL-12-IFN-γ-mediated T-cell activation.


Assuntos
Vacinas Anticâncer/administração & dosagem , Células Dendríticas/imunologia , Glioma/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Adulto , Vacinas Anticâncer/imunologia , Polaridade Celular/imunologia , Intervalo Livre de Doença , Feminino , Glioma/imunologia , Glioma/patologia , Humanos , Interferon gama/genética , Interleucina-12/genética , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/patologia , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Linfócitos T/imunologia , Vacinação/métodos
3.
Proc Natl Acad Sci U S A ; 117(38): 23859-23868, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900945

RESUMO

Bacteria can move across surfaces using type IV pili (T4P), which undergo cycles of extension, adhesion, and retraction. The T4P localization pattern varies between species; however, the underlying mechanisms are largely unknown. In the rod-shaped Myxococcus xanthus cells, T4P localize at the leading cell pole. As cells reverse their direction of movement, T4P are disassembled at the old leading pole and then form at the new leading pole. Thus, cells can form T4P at both poles but engage only one pole at a time in T4P formation. Here, we address how this T4P unipolarity is realized. We demonstrate that the small Ras-like GTPase MglA stimulates T4P formation in its GTP-bound state by direct interaction with the tetratricopeptide repeat (TPR) domain-containing protein SgmX. SgmX, in turn, is important for polar localization of the T4P extension ATPase PilB. The cognate MglA GTPase activating protein (GAP) MglB, which localizes mainly to the lagging cell pole, indirectly blocks T4P formation at this pole by stimulating the conversion of MglA-GTP to MglA-GDP. Based on these findings, we propose a model whereby T4P unipolarity is accomplished by stimulation of T4P formation at the leading pole by MglA-GTP and SgmX and indirect inhibition of T4P formation at the lagging pole by MglB due to its MglA GAP activity. During reversals, MglA, SgmX, and MglB switch polarity, thus laying the foundation for T4P formation at the new leading pole and inhibition of T4P formation at the new lagging pole.


Assuntos
Proteínas de Bactérias , Proteínas de Fímbrias , Fímbrias Bacterianas , Polaridade Celular , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/química , Fímbrias Bacterianas/metabolismo , Repetições de Tetratricopeptídeos
4.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(9): 782-787, 2020 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-32967761

RESUMO

Objective To investigate the effects of Kruppel like factor 4 (KLF4) gene knockdown on the polarization of RAW264.7 macrophages. Methods KLF4 knockdown lentiviral vector was constructed by RNA interfering. The lentiviral vector was transfected into RAW264.7 cells to realize stable KLF4 gene silencing in RAW264.7 cells. Interleukin-4 (IL-4) was used to stimulate macrophages in wild type group, KLF4 knockdown group and negative control group. The mRNA expression of inducible nitric oxide synthase (iNOS), IL-1ß, tumor necrosis factor α (TNF-α) and Arg1, IL-10, transforming growth factor ß (TGF-ß) of the cells was detected by reverse transcription-PCR. Immunocytochemical staining was used to detect and localize iNOS and Arg1 protein in RAW264.7 cells. Results Levels of iNOS and IL-1ß mRNA in RAW264.7 cells were significantly raised, while levels of Arg1, IL-10 and TGF-ß mRNA were significantly reduced after KLF4 gene knockdown. Levels of KLF4, Arg1, IL-10 and TGF-ß mRNA went up, while the relative levels of iNOS, IL-1ß and TNF-α mRNA went down in wild-type RAW264.7 cells after IL-4 intervention. After shKLF4 group was intervened by IL-4, levels of iNOS, IL-1ß and TNF-α mRNA in shKLF4 group (lentivirus group) were lower than those in wild-type group and higher than those in negative control group. Levels of Arg1, IL-10 and TGF-ß mRNA in shKLF4 group after IL-4 treatment were higher than those in wild-type group, while Arg1 and IL-10 were lower than those in negative control group. Compared with wide-type group, the expression of iNOS protein significantly decreased, while Arg1 protein significantly increased in shKLF4 group 12 hours after IL-4 treatment. Conclusion Knockdown of KLF4 promotes the polarization of RAW264.7 macrophages into M1 as well as inhibits their polarization into M2.


Assuntos
Macrófagos , Animais , Polaridade Celular , Fatores de Transcrição Kruppel-Like , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Fator de Crescimento Transformador beta/genética , Fator de Necrose Tumoral alfa/metabolismo
5.
PLoS Genet ; 16(9): e1008912, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946434

RESUMO

The mechanism(s) through which mammalian kinase MELK promotes tumorigenesis is not understood. We find that the C. elegans orthologue of MELK, PIG-1, promotes apoptosis by partitioning an anti-apoptotic factor. The C. elegans NSM neuroblast divides to produce a larger cell that differentiates into a neuron and a smaller cell that dies. We find that in this context, PIG-1 MELK is required for partitioning of CES-1 Snail, a transcriptional repressor of the pro-apoptotic gene egl-1 BH3-only. pig-1 MELK is controlled by both a ces-1 Snail- and par-4 LKB1-dependent pathway, and may act through phosphorylation and cortical enrichment of nonmuscle myosin II prior to neuroblast division. We propose that pig-1 MELK-induced local contractility of the actomyosin network plays a conserved role in the acquisition of the apoptotic fate. Our work also uncovers an auto-regulatory loop through which ces-1 Snail controls its own activity through the formation of a gradient of CES-1 Snail protein.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Actomiosina/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/fisiologia , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Morte Celular/fisiologia , Polaridade Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/genética , Miosina Tipo II/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Transcrição/genética
6.
PLoS One ; 15(9): e0239083, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32970752

RESUMO

Genes in the noncanonical WNT signaling pathway controlling planar cell polarity have been linked to the neural tube defect myelomeningocele. We hypothesized that some genes in the WNT signaling network have a higher mutational burden in myelomeningocele subjects than in reference subjects in gnomAD. Exome sequencing data from 511 myelomeningocele subjects was obtained in-house and data from 29,940 ethnically matched subjects was provided by version 2 of the publicly available Genome Aggregation Database. To compare mutational burden, we collapsed rare deleterious variants across each of 523 human WNT signaling genes in case and reference populations. Ten WNT signaling genes were disrupted with a higher mutational burden among Mexican American myelomeningocele subjects compared to reference subjects (Fishers exact test, P ≤ 0.05) and seven different genes were disrupted among individuals of European ancestry compared to reference subjects. Gene ontology enrichment analyses indicate that genes disrupted only in the Mexican American population play a role in planar cell polarity whereas genes identified in both populations are important for the regulation of canonical WNT signaling. In summary, evidence for WNT signaling genes that may contribute to myelomeningocele in humans is presented and discussed.


Assuntos
Meningomielocele/genética , Mutação , Via de Sinalização Wnt , Polaridade Celular , Ontologia Genética , Humanos , Taxa de Mutação , Proteínas Wnt/genética
7.
Nat Commun ; 11(1): 4035, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788578

RESUMO

Polyphosphates are linear polymers and ubiquitous metabolites. Bacterial polyphosphates are long chains of hundreds of phosphate units. Here, we report that mouse survival of peritoneal Escherichia coli sepsis is compromised by long-chain polyphosphates, and improves with bacterial polyphosphatekinase deficiency or neutralization using recombinant exopolyphosphatase. Polyphosphate activities are chain-length dependent, impair pathogen clearance, antagonize phagocyte recruitment, diminish phagocytosis and decrease production of iNOS and cytokines. Macrophages bind and internalize polyphosphates, in which their effects are independent of P2Y1 and RAGE receptors. The M1 polarization driven by E. coli derived LPS is misdirected by polyphosphates in favor of an M2 resembling phenotype. Long-chain polyphosphates modulate the expression of more than 1800 LPS/TLR4-regulated genes in macrophages. This interference includes suppression of hundreds of type I interferon-regulated genes due to lower interferon production and responsiveness, blunted STAT1 phosphorylation and reduced MHCII expression. In conclusion, prokaryotic polyphosphates disturb multiple macrophage functions for evading host immunity.


Assuntos
Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Polifosfatos/metabolismo , Animais , Apresentação do Antígeno/imunologia , Polaridade Celular , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferon Tipo I/metabolismo , Lipopolissacarídeos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Fenótipo , Sepse/imunologia , Análise de Sobrevida , Transcriptoma/genética
8.
PLoS Genet ; 16(8): e1008820, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32750048

RESUMO

The core planar polarity proteins are essential mediators of tissue morphogenesis, controlling both the polarised production of cellular structures and polarised tissue movements. During development the core proteins promote planar polarisation by becoming asymmetrically localised to opposite cell edges within epithelial tissues, forming intercellular protein complexes that coordinate polarity between adjacent cells. Here we describe a novel protein complex that regulates the asymmetric localisation of the core proteins in the Drosophila pupal wing. DAnkrd49 (an ankyrin repeat protein) and Bride of Doubletime (Bdbt, a non-canonical FK506 binding protein family member) physically interact, and regulate each other's levels in vivo. Loss of either protein results in a reduction in core protein asymmetry and disruption of the placement of trichomes at the distal edge of pupal wing cells. Post-translational modifications are thought to be important for the regulation of core protein behaviour and their sorting to opposite cell edges. Consistent with this, we find that loss of DAnkrd49 or Bdbt leads to reduced phosphorylation of the core protein Dishevelled and to decreased Dishevelled levels both at cell junctions and in the cytoplasm. Bdbt has previously been shown to regulate activity of the kinase Discs Overgrown (Dco, also known as Doubletime or Casein Kinase Iε), and Dco itself has been implicated in regulating planar polarity by phosphorylating Dsh as well as the core protein Strabismus. We demonstrate that DAnkrd49 and Bdbt act as dominant suppressors of Dco activity. These findings support a model whereby Bdbt and DAnkrd49 act together to modulate the activity of Dco during planar polarity establishment.


Assuntos
Caseína Quinase Iépsilon/metabolismo , Polaridade Celular , Proteínas de Drosophila/metabolismo , Morfogênese , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Caseína Quinase Iépsilon/genética , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Mutação com Perda de Função , Ligação Proteica , Transporte Proteico , Proteínas de Ligação a Tacrolimo/genética , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento
9.
Proc Natl Acad Sci U S A ; 117(36): 22193-22203, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32839317

RESUMO

The establishment of axon/dendrite polarity is fundamental for neurons to integrate into functional circuits, and this process is critically dependent on microtubules (MTs). In the early stages of the establishment process, MTs in axons change dramatically with the morphological building of neurons; however, how the MT network changes are triggered is unclear. Here we show that CAMSAP1 plays a decisive role in the neuronal axon identification process by regulating the number of MTs. Neurons lacking CAMSAP1 form a multiple axon phenotype in vitro, while the multipolar-bipolar transition and radial migration are blocked in vivo. We demonstrate that the polarity regulator MARK2 kinase phosphorylates CAMSAP1 and affects its ability to bind to MTs, which in turn changes the protection of MT minus-ends and also triggers asymmetric distribution of MTs. Our results indicate that the polarized MT network in neurons is a decisive factor in establishing axon/dendritic polarity and is initially triggered by polarized signals.


Assuntos
Polaridade Celular/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/fisiologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Imunoprecipitação , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Neurônios , Paclitaxel , Ligação Proteica
10.
Nat Commun ; 11(1): 3914, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764676

RESUMO

Cell polarity is fundamental to the development of both eukaryotes and prokaryotes, yet the mechanisms behind its formation are not well understood. Here we found that, phytohormone auxin-induced, sterol-dependent nanoclustering of cell surface transmembrane receptor kinase 1 (TMK1) is critical for the formation of polarized domains at the plasma membrane (PM) during the morphogenesis of cotyledon pavement cells (PC) in Arabidopsis. Auxin-induced TMK1 nanoclustering stabilizes flotillin1-associated ordered nanodomains, which in turn promote the nanoclustering of ROP6 GTPase that acts downstream of TMK1 to regulate cortical microtubule organization. In turn, cortical microtubules further stabilize TMK1- and flotillin1-containing nanoclusters at the PM. Hence, we propose a new paradigm for polarity formation: A diffusive signal triggers cell polarization by promoting cell surface receptor-mediated nanoclustering of signaling components and cytoskeleton-mediated positive feedback that reinforces these nanodomains into polarized domains.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Polaridade Celular/fisiologia , Ácidos Indolacéticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Membrana Celular/metabolismo , Polaridade Celular/genética , Metabolismo dos Lipídeos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Biológicos , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Reguladores de Crescimento de Planta/metabolismo , Plantas Geneticamente Modificadas , Agregados Proteicos , Estabilidade Proteica , Proteínas Serina-Treonina Quinases/química , Transdução de Sinais
11.
BMC Infect Dis ; 20(1): 488, 2020 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-32646445

RESUMO

BACKGROUND: Washington University polyomavirus (WUPyV) is a novel human polyomavirus detected in childwith acute respiratory infection in 2007. However, the relationship between WUPyV and respiratory diseases has yet to be established for lacking of a suitable in vitro culture system. METHODS: To isolate WUPyV with human airway epithelial (HAE) cells, the positive samples were incubated in HAE, and then the nucleic acid, VP1 protein and virions were detected using real-time PCR, immunofluorescence and electron microscopy respectively. RESULTS: The result showed that WUPyV could replicate effectively in HAE cells and virions with typical polyomavirus characteristics could be observed. Additionally, the entire genome sequence of the isolated strain (BJ0771) was obtained and phylogenetic analysis indicated that BJ0771 belongs to gene cluster I. CONCLUSIONS: Our findings demonstrated clinical WUPyV strain was successfully isolated for the first time in the world and this will help unravel the etiology and pathogenic mechanisms of WUPyV in respiratory infection diseases.


Assuntos
Células Epiteliais/virologia , Infecções por Polyomavirus/diagnóstico , Infecções por Polyomavirus/virologia , Polyomavirus/genética , Polyomavirus/isolamento & purificação , Mucosa Respiratória/patologia , Infecções Respiratórias/diagnóstico , Adolescente , Proteínas do Capsídeo/genética , Polaridade Celular , Células Cultivadas , Criança , Pré-Escolar , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Família Multigênica , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Infecções Respiratórias/virologia , Vírion/genética , Replicação Viral , Sequenciamento Completo do Genoma
12.
Nat Commun ; 11(1): 3516, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665580

RESUMO

It is unclear whether the establishment of apical-basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane.


Assuntos
Membrana Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/metabolismo , Polaridade Celular/genética , Polaridade Celular/fisiologia , Células Epiteliais/metabolismo , Feminino , Humanos , Morfogênese/genética , Morfogênese/fisiologia , Proteínas do Tecido Nervoso/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Proteínas rab de Ligação ao GTP/genética
13.
Life Sci ; 258: 118137, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32712299

RESUMO

AIMS: Chagas disease is a neglected tropical disease. The ability of Trypanosoma cruzi to survive within phagocytes is likely a critical factor for T. cruzi dissemination in the host. For control of the parasite load and host survival, macrophage action is required. Concanavalin-A (Con-A) presents properties that modulate immune functions and protect hosts from several experimental infectious diseases. Here, we evaluated the effects of Con-A on peritoneal macrophages as well as on the course of experimental infection by T. cruzi. MAIN METHODS: BALB/c mice, a susceptible model for T. cruzi infection, were treated with Con-A via the intraperitoneal route and 3 days later infected with T. cruzi. We quantified parasitemia, cytokines and nitric oxide (NO). Peritoneal exudate and macrophages were collected for macrophage phenotyping and cell viability, NO and cytokine detection, as well as for T. cruzi internalization and release index determination. KEY FINDINGS: Con-A treatment induced IL-17a and NO production by cells from the peritoneal cavity, and M1 marker expression predominated on peritoneal macrophages. These cells are also more prone to producing TNF-α, IL-6 and NO when infected by T. cruzi and show high trypanocidal capacity. Due to a hostile peritoneal microenvironment caused by Con-A, which induces macrophage cNOS and iNOS expression, infected BALB/c mice showed reduced parasitemia and an increased survival rate. SIGNIFICANCE: We conclude that Con-A can induce peritoneal M1 macrophage polarization to increase trypanocidal activity, resulting in ameliorated systemic infection in a susceptible experimental model.


Assuntos
Polaridade Celular , Doença de Chagas/patologia , Concanavalina A/farmacologia , Interleucina-17/metabolismo , Macrófagos Peritoneais/patologia , Macrófagos Peritoneais/parasitologia , Óxido Nítrico/metabolismo , Trypanosoma cruzi/fisiologia , Animais , Polaridade Celular/efeitos dos fármacos , Doença de Chagas/metabolismo , Feminino , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/metabolismo , Parasitemia/metabolismo , Parasitemia/patologia , Trypanosoma cruzi/efeitos dos fármacos
14.
Proc Natl Acad Sci U S A ; 117(32): 19310-19320, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32727892

RESUMO

Fat, Fat-like, and Dachsous family cadherins are giant proteins that regulate planar cell polarity (PCP) and cell adhesion in bilaterians. Their evolutionary origin can be traced back to prebilaterian species, but their ancestral function(s) are unknown. We identified Fat-like and Dachsous cadherins in Hydra, a member of phylum Cnidaria a sister group of bilaterian. We found Hydra does not possess a true Fat homolog, but has homologs of Fat-like (HyFatl) and Dachsous (HyDs) that localize at the apical membrane of ectodermal epithelial cells and are planar polarized perpendicular to the oral-aboral axis of the animal. Using a knockdown approach we found that HyFatl is involved in local cell alignment and cell-cell adhesion, and that reduction of HyFatl leads to defects in tissue organization in the body column. Overexpression and knockdown experiments indicate that the intracellular domain (ICD) of HyFatl affects actin organization through proline-rich repeats. Thus, planar polarization of Fat-like and Dachsous cadherins has ancient, prebilaterian origins, and Fat-like cadherins have ancient roles in cell adhesion, spindle orientation, and tissue organization.


Assuntos
Caderinas/metabolismo , Polaridade Celular , Hydra/citologia , Animais , Caderinas/genética , Adesão Celular , Hydra/classificação , Hydra/genética , Hydra/metabolismo , Filogenia , Fuso Acromático/genética , Fuso Acromático/metabolismo
15.
Clin Sci (Lond) ; 134(12): 1433-1448, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32478392

RESUMO

Recent identification of an RNA-binding protein (HuR) that regulates mRNA turnover and translation of numerous transcripts via binding to an ARE in their 3'-UTR involved in inflammation and is abnormally elevated in varied kidney diseases offers a novel target for the treatment of renal inflammation and subsequent fibrosis. Thus, we hypothesized that treatment with a selective inhibition of HuR function with a small molecule, KH-3, would down-regulate HuR-targeted proinflammatory transcripts thereby improving glomerulosclerosis in experimental nephritis, where glomerular cellular HuR is elevated. Three experimental groups included normal and diseased rats treated with or without KH-3. Disease was induced by the monoclonal anti-Thy 1.1 antibody. KH-3 was given via daily intraperitoneal injection from day 1 after disease induction to day 5 at the dose of 50 mg/kg BW/day. At day 6, diseased animals treated with KH-3 showed significant reduction in glomerular HuR levels, proteinuria, podocyte injury determined by ameliorated podocyte loss and podocin expression, glomerular staining for periodic acid-Schiff positive extracellular matrix proteins, fibronectin and collagen IV and mRNA and protein levels of profibrotic markers, compared with untreated disease rats. KH-3 treatment also reduced disease-induced increases in renal TGFß1 and PAI-1 transcripts. Additionally, a marked increase in renal NF-κB-p65, Nox4, and glomerular macrophage cell infiltration observed in disease control group was largely reversed by KH-3 treatment. These results strongly support our hypothesis that down-regulation of HuR function with KH-3 has therapeutic potential for reversing glomerulosclerosis by reducing abundance of pro-inflammatory transcripts and related inflammation.


Assuntos
Proteína Semelhante a ELAV 1/antagonistas & inibidores , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Nefrite/metabolismo , Nefrite/patologia , Animais , Biomarcadores/metabolismo , Peso Corporal , Polaridade Celular , Colágeno/genética , Colágeno/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrose , Humanos , Inflamação/patologia , Testes de Função Renal , Glomérulos Renais/fisiopatologia , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , NADPH Oxidase 4/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Antígenos Thy-1 , Fator de Transcrição RelA/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
16.
PLoS Biol ; 18(6): e3000687, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32520957

RESUMO

In the tumor microenvironment, local immune dysregulation is driven in part by macrophages and dendritic cells that are polarized to a mixed proinflammatory/immune-suppressive phenotype. The unfolded protein response (UPR) is emerging as the possible origin of these events. Here we report that the inositol-requiring enzyme 1 (IRE1α) branch of the UPR is directly involved in the polarization of macrophages in vitro and in vivo, including the up-regulation of interleukin 6 (IL-6), IL-23, Arginase1, as well as surface expression of CD86 and programmed death ligand 1 (PD-L1). Macrophages in which the IRE1α/X-box binding protein 1 (Xbp1) axis is blocked pharmacologically or deleted genetically have significantly reduced polarization and CD86 and PD-L1 expression, which was induced independent of IFNγ signaling, suggesting a novel mechanism in PD-L1 regulation in macrophages. Mice with IRE1α- but not Xbp1-deficient macrophages showed greater survival than controls when implanted with B16.F10 melanoma cells. Remarkably, we found a significant association between the IRE1α gene signature and CD274 gene expression in tumor-infiltrating macrophages in humans. RNA sequencing (RNASeq) analysis showed that bone marrow-derived macrophages with IRE1α deletion lose the integrity of the gene connectivity characteristic of regulated IRE1α-dependent decay (RIDD) and the ability to activate CD274 gene expression. Thus, the IRE1α/Xbp1 axis drives the polarization of macrophages in the tumor microenvironment initiating a complex immune dysregulation leading to failure of local immune surveillance.


Assuntos
Antígeno B7-H1/metabolismo , Polaridade Celular , Endorribonucleases/metabolismo , Macrófagos/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Antígeno CD11b/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/patologia , Modelos Lineares , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Neoplasias/metabolismo , Fenótipo , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box/metabolismo
17.
PLoS Genet ; 16(6): e1008890, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32579558

RESUMO

The Drosophila apical photoreceptor membrane is defined by the presence of two distinct morphological regions, the microvilli-based rhabdomere and the stalk membrane. The subdivision of the apical membrane contributes to the geometrical positioning and the stereotypical morphology of the rhabdomeres in compound eyes with open rhabdoms and neural superposition. Here we describe the characterization of the photoreceptor specific protein PIP82. We found that PIP82's subcellular localization demarcates the rhabdomeric portion of the apical membrane. We further demonstrate that PIP82 is a phosphorylation target of aPKC. PIP82 localization is modulated by phosphorylation, and in vivo, the loss of the aPKC/Crumbs complex results in an expansion of the PIP82 localization domain. The absence of PIP82 in photoreceptors leads to misshapped rhabdomeres as a result of misdirected cellular trafficking of rhabdomere proteins. Comparative analyses reveal that PIP82 originated de novo in the lineage leading to brachyceran Diptera, which is also characterized by the transition from fused to open rhabdoms. Taken together, these findings define a novel factor that delineates and maintains a specific apical membrane domain, and offers new insights into the functional organization and evolutionary history of the Drosophila retina.


Assuntos
Membrana Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Fotorreceptoras de Invertebrados/metabolismo , Retina/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Evolução Biológica , Diferenciação Celular/genética , Membrana Celular/genética , Membrana Celular/ultraestrutura , Polaridade Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação com Perda de Função , Masculino , Microscopia Eletrônica de Transmissão , Fosforilação , Células Fotorreceptoras de Invertebrados/citologia , Células Fotorreceptoras de Invertebrados/ultraestrutura , Filogenia , Proteína Quinase C/metabolismo , Retina/citologia , Retina/ultraestrutura , Transcrição Genética
18.
Proc Natl Acad Sci U S A ; 117(24): 13571-13579, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32482850

RESUMO

Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a "9 + 2" microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), Camsap3 tm1a/tm1a , exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination of Camsap3 tm1a/tm1a mice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, in Camsap3 tm1a/tm1a , MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs of Xenopus embryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia.


Assuntos
Corpos Basais/metabolismo , Cílios/metabolismo , Transtornos da Motilidade Ciliar/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Axonema/metabolismo , Polaridade Celular , Transtornos da Motilidade Ciliar/genética , Células Epiteliais/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Xenopus
19.
Nat Commun ; 11(1): 2952, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528053

RESUMO

The formation and maintenance of subcellular structures and organelles with a well-defined size is a key requirement for cell function, yet our understanding of the underlying size control mechanisms is limited. While budding yeast cell polarization and subsequent assembly of a septin ring at the site of bud formation has been successfully used as a model for biological self-assembly processes, the mechanisms that set the size of the septin ring at the bud neck are unknown. Here, we use live-cell imaging and genetic manipulation of cell volume to show that the septin ring diameter increases with cell volume. This cell-volume-dependence largely accounts for modulations of ring size due to changes in ploidy and genetic manipulation of cell polarization. Our findings suggest that the ring diameter is set through the dynamic interplay of septin recruitment and Cdc42 polarization, establishing it as a model for size homeostasis of self-assembling organelles.


Assuntos
Saccharomycetales/citologia , Saccharomycetales/metabolismo , Biologia Celular , Divisão Celular/fisiologia , Crescimento Celular , Polaridade Celular/fisiologia , Tamanho Celular
20.
Life Sci ; 256: 117989, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32565250

RESUMO

AIMS: The beneficial effects of cannabinoid type 2 receptor (CB2R) activation have been verified in various tissue repair processes. Our recent study revealed CB2R activation promotes myogenesis partly through Nrf2 signaling in a mouse skeletal muscle ischemia-reperfusion (IR) injury model. Other relevant mechanisms need to be further elucidated. Macrophages orchestrate tissue regeneration mainly by changing their phenotype and function. The aim of this study was to investigate the role of CB2R in IR-induced skeletal muscle regeneration, focusing on its impact on macrophage polarization and the consequences on myogenesis. MAIN METHODS: The effects of CB2R on skeletal muscle regeneration, and the macrophage infiltration and M1/M2 polarization were tested with the IR injury model in wild type (WT) and CB2R knockout (CB2R-KO) mice. The effect of CB2R on peritoneal macrophage polarization, and its impact on the myoblasts differentiation was evaluated by co-culture experiments in vitro. KEY FINDINGS: The present study revealed the myofiber regeneration was hindered in the CB2R-KO mice. The infiltration of M1 macrophages and relevant markers' protein expression were enhanced in the CB2R-KO mice, while that of M2 macrophages was decreased compared with the WT mice. The in vitro studies further demonstrated that the absence of CB2R promoted M1 polarization while inhibited M2 polarization. The promoted M1 polarization and retarded M2 polarization in CB2R-KO macrophages hindered myoblasts differentiation. SIGNIFICANCE: Overall, these results suggested CB2R plays a beneficial effect on skeletal muscle regeneration partly by regulating macrophage M1/M2 polarization after IR injury in mice.


Assuntos
Polaridade Celular/fisiologia , Macrófagos/fisiologia , Músculo Esquelético/fisiologia , Receptor CB2 de Canabinoide/deficiência , Regeneração/fisiologia , Traumatismo por Reperfusão/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/irrigação sanguínea
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