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1.
Int Immunol ; 35(7): 327-338, 2023 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-37052267

RESUMO

For cellular or tissue transplantation using induced pluripotent stem cells (iPSCs), from the viewpoint of time and economic cost, the use of allogeneic ones is being considered. Immune regulation is one of the key issues in successful allogeneic transplantation. To reduce the risk of rejection, several attempts have been reported to eliminate effects of the major histocompatibility complex (MHC) on the iPSC-derived grafts. On the other hand, we have shown that minor antigen-induced rejection is not negligible even when the MHC's impact is mitigated. In organ transplantation, it is known that donor-specific transfusion (DST) can specifically control immune responses to the donor. However, whether DST could control the immune response in iPSC-based transplantation was not clarified. In this study, using a mouse skin transplantation model, we demonstrate that infusion of donor splenocytes can promote allograft tolerance in the MHC-matched but minor antigen-mismatched conditions. When narrowing down the cell types, we found that infusion of isolated splenic B cells was sufficient to control rejection. As a mechanism, the administration of donor B cells induced unresponsiveness but not deletion in recipient T cells, suggesting that the tolerance was induced in the periphery. The donor B cell transfusion induced allogeneic iPSC engraftment. These results suggest for the first time a possibility that DST using donor B cells could induce tolerance against allogeneic iPSC-derived grafts.


Assuntos
Células-Tronco Pluripotentes Induzidas , Tolerância ao Transplante , Sobrevivência de Enxerto , Tolerância Imunológica , Complexo Principal de Histocompatibilidade , Transferência Adotiva , Rejeição de Enxerto
2.
J Biol Chem ; 298(2): 101579, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35031323

RESUMO

Rho family small GTPases (Rho) regulate various cell motility processes by spatiotemporally controlling the actin cytoskeleton. Some Rho-specific guanine nucleotide exchange factors (RhoGEFs) are regulated via tyrosine phosphorylation by Src family tyrosine kinase (SFK). We also previously reported that PLEKHG2, a RhoGEF for the GTPases Rac1 and Cdc42, is tyrosine-phosphorylated by SRC. However, the details of the mechanisms by which SFK regulates RhoGEFs are not well understood. In this study, we found for the first time that PLEKHG1, which has very high homology to the Dbl and pleckstrin homology domains of PLEKHG2, activates Cdc42 following activation by FYN, a member of the SFK family. We also show that this activation of PLEKHG1 by FYN requires interaction between these two proteins and FYN-induced tyrosine phosphorylation of PLEKHG1. We also found that the region containing the Src homology 3 and Src homology 2 domains of FYN is required for this interaction. Finally, we demonstrated that tyrosine phosphorylation of Tyr-720 and Tyr-801 in PLEKHG1 is important for the activation of PLEKHG1. These results suggest that FYN is a regulator of PLEKHG1 and may regulate cell morphology through Rho signaling via the interaction with and tyrosine phosphorylation of PLEKHG1.


Assuntos
Fatores de Troca de Nucleotídeo Guanina Rho , Proteínas rho de Ligação ao GTP , Quinases da Família src , Fosforilação , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Tirosina/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo
3.
J Cell Sci ; 133(15)2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32661090

RESUMO

CENP-B binds to CENP-B boxes on centromeric satellite DNAs (known as alphoid DNA in humans). CENP-B maintains kinetochore function through interactions with CENP-A nucleosomes and CENP-C. CENP-B binding to transfected alphoid DNA can induce de novo CENP-A assembly, functional centromere and kinetochore formation, and subsequent human artificial chromosome (HAC) formation. Furthermore, CENP-B also facilitates H3K9 (histone H3 lysine 9) trimethylation on alphoid DNA, mediated by Suv39h1, at ectopic alphoid DNA integration sites. Excessive heterochromatin invasion into centromere chromatin suppresses CENP-A assembly. It is unclear how CENP-B controls such different chromatin states. Here, we show that the CENP-B acidic domain recruits histone chaperones and many chromatin modifiers, including the H3K36 methylase ASH1L, as well as the heterochromatin components Suv39h1 and HP1 (HP1α, ß and γ, also known as CBX5, CBX1 and CBX3, respectively). ASH1L facilitates the formation of open chromatin competent for CENP-A assembly on alphoid DNA. These results indicate that CENP-B is a nexus for histone modifiers that alternatively promote or suppress CENP-A assembly by mutually exclusive mechanisms. Besides the DNA-binding domain, the CENP-B acidic domain also facilitates CENP-A assembly de novo on transfected alphoid DNA. CENP-B therefore balances CENP-A assembly and heterochromatin formation on satellite DNA.


Assuntos
Cromatina , Heterocromatina , Autoantígenos/genética , Centrômero , Proteína Centromérica A/genética , Cromatina/genética , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Epigênese Genética , Heterocromatina/genética , Humanos
4.
Mol Cell Biochem ; 459(1-2): 83-93, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31089935

RESUMO

It is well known that Rho family small GTPases (Rho GTPase) has a role of molecular switch in intracellular signal transduction. The switch cycle between GTP-bound and GDP-bound state of Rho GTPase regulates various cell responses such as gene transcription, cytoskeletal rearrangements, and vesicular trafficking. Rho GTPase-specific guanine nucleotide exchange factors (RhoGEFs) are regulated by various extracellular stimuli and activates Rho GTPase such as RhoA, Rac1, and Cdc42. The molecular mechanisms that regulate RhoGEFs are poorly understood. Our studies reveal that Dbl's big sister (DBS), a RhoGEF for Cdc42 and RhoA, is phosphorylated at least on tyrosine residues at 479, 660, 727, and 926 upon stimulation by SRC signaling and that the phosphorylation at Tyr-660 is particularly critical for the serum response factor (SRF)-dependent transcriptional activation of DBS by Ephrin type-B receptor 2 (EPHB2)/SRC signaling. In addition, our studies also reveal that the phosphorylation of Tyr-479 and Tyr-660 on DBS leads to the actin cytoskeletal reorganization by EPHB2/SRC signaling. These findings are thought to be useful for understanding pathological conditions related to DBS such as cancer and non-syndromic autism in future.


Assuntos
Receptor EphB2/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Células HEK293 , Humanos , Receptor EphB2/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética , Quinases da Família src/genética
5.
Biochim Biophys Acta ; 1793(3): 506-15, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19321127

RESUMO

Protein 4.1B is a membrane skeletal protein expressed in various organs, and is associated with tumor suppressor in lung cancer-1 (TSLC1) in vitro. Although involvement of 4.1B in the intercellular junctions and tumor-suppression was suggested, some controversial results posed questions to the general tumor-suppressive function of 4.1B and its relation to TSLC1 in vivo. In this study, the expression of 4.1B and its interaction with TSLC1 were examined in rodent adrenal gland, and the involvement of 4.1B in tumorigenesis and the effect of 4.1B deficiency on TSLC1 distribution were also investigated using rodent pheochromocytoma and 4.1B-knockout mice. Although plasmalemmal immunolocalization of 4.1B was shown in chromaffin cells of rodent adrenal medulla, expression of 4.1B was maintained in developed pheochromocytoma, and morphological abnormality or pheochromocytoma generation could not be found in 4.1B-deficient mice. Furthermore, molecular interaction and colocalization of 4.1B and TSLC1 were observed in mouse adrenal gland, but the immunolocalization of TSLC1 along chromaffin cell membranes was not affected in the 4.1B-deficient mice. These results suggest that the function of 4.1B as tumor suppressor might significantly differ among organs and species, and that plasmalemmal retention of TSLC1 would be maintained by molecules other than 4.1B interacting in rodent chromaffin cells.


Assuntos
Neoplasias das Glândulas Suprarrenais/metabolismo , Medula Suprarrenal/metabolismo , Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Feocromocitoma/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Neoplasias das Glândulas Suprarrenais/patologia , Animais , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular , Imunoglobulinas/análise , Hibridização In Situ , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos , Microscopia Eletrônica , Feocromocitoma/patologia , Proteínas Supressoras de Tumor/análise
6.
Biochem Biophys Res Commun ; 388(4): 689-94, 2009 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-19695231

RESUMO

The Krüppel-associated box-containing zinc finger gene family (KRAB-ZNF) is one of the largest gene families of transcriptional factors in the human genome. Although the functions of most of these genes remain to be determined, it is known that KRAB-mediated transcriptional repression requires a direct interaction with the KAP1 co-repressor. By mammalian one- or two-hybrid experiments in HEK293 cells, we compared transcriptional repression activities of 61 human KRAB-ZNFs. The results showed that six SCAN-KRAB-containing ZNFs are KAP1-independent transcriptional repressors whose SCAN-KRAB domain is unable to associate with KAP1 despite retaining transcriptional repression activity. Transcriptional repression activities of the SCAN-KRAB domain of KAP1-independent KRAB-ZNFs are not influenced by depletion of endogenous KAP1 levels by small interfering RNA. Although the mechanism by which KAP1-independent KRAB-ZNFs repress transcriptional activity remains to be elucidated, it appears that there may be a pathway for transcriptional repression that does not involve KAP1. These results provide new insight into the functions of the members of the KRAB-ZNF family.


Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Transcrição Gênica , Dedos de Zinco/genética , Linhagem Celular , Humanos , Proteínas Repressoras/metabolismo , Proteína 28 com Motivo Tripartido
7.
Genes Cells ; 13(5): 483-95, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18429820

RESUMO

Myosin VI is involved in a wide range of endocytic and exocytic membrane trafficking pathways; clathrin-mediated endocytosis, intracellular transport of clathrin-coated and -uncoated vesicles, AP-1B-dependent basolateral sorting in polarized epithelial cells and secretion from the Golgi complex to the cell surface. In this study, using a yeast two-hybrid screen, we identified brain-enriched kinase/lemur tyrosine kinase 2 (BREK/LMTK2), a transmembrane serine/threonine kinase with previously unknown cellular functions, as a myosin VI-interacting protein. Several binding experiments confirmed the interaction of myosin VI with BREK in vivo and in vitro. Immunocytochemical analyses revealed that BREK localizes to cytoplasmic membrane vesicles and to perinuclear recycling endosomes. Notably, cells in which BREK was depleted by siRNA were still able to internalize transferrin molecules and to transport them to early endosomes, but were unable to transport them to perinuclear recycling endosomes. Our results show that BREK is critical for the transition of endocytosed membrane vesicles from early endosomes to recycling endosomes and also suggest an involvement of myosin VI in this pathway.


Assuntos
Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Linhagem Celular , Citoplasma/química , Citoplasma/metabolismo , Regulação para Baixo , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Cadeias Pesadas de Miosina/química , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/metabolismo , Transferrina/metabolismo , Técnicas do Sistema de Duplo-Híbrido
8.
Small GTPases ; 10(5): 361-366, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-28489964

RESUMO

PLEKHG2 is a Gßγ- and Gαs-dependent guanine nucleotide exchange factor for Rac1 and Cdc42 small GTPases and has been shown to mediate signaling pathways such as those for actin cytoskeletal reorganization and serum response element (SRE)-dependent gene transcription. We have shown that the four-and-a-half LIM domains (FHL) 1 acts as a positive regulator of PLEKHG2. Here, we evaluated the other FHL family members and found that the FHL1A specifically regulate the PLEKHG2 activity. Moreover, FHL1A further enhanced Gßγ- and PLEKHG2-induced SRE-dependent gene transcription, whereas FHL1A partially restored the attenuated PLEKHG2-induced SRE-dependent gene transcription by Gαs. Our results suggest that FHL1A specifically interacts with PLEKHG2 to regulate a function of PLEKHG2 that is modified by the interaction of Gßγ and Gαs.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/metabolismo , Elemento de Resposta Sérica , Transcrição Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Proteínas Musculares/genética , Domínios Proteicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Cell Signal ; 61: 93-107, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31100317

RESUMO

The Rho family small GTPases mediate cell responses through actin cytoskeletal rearrangement. We previously reported that PLEKHG2, a Rho-specific guanine nucleotide exchange factor, is regulated via interaction with several proteins. We found that PLEKHG2 interacted with non-receptor tyrosine kinase ABL1, but the cellular function remains unclear. Here, we show that the interaction between PLEKHG2 and ABL1 attenuated the PLEKHG2-induced serum response element-dependent gene transcription in a tyrosine phosphorylation-independent manner. PLEKHG2 and ABL1 were co-localized and accumulated within cells co-expressing PLEKHG2 and ABL1. The cellular fractionation analysis suggested that the accumulation involved actin cytoskeletal reorganization. We also revealed that the co-expression of PLEKHG2 with ABL1, but not BCR-ABL, suppressed cell growth and synergistically enhanced NF-κB-dependent gene transcription. The cell growth suppression was canceled by co-expression with IκBα, a member of the NF-κB inhibitor protein family. This study suggests that the interaction between PLEKHG2 and ABL1 suppresses cell growth through intracellular protein accumulation via the NF-κB signaling pathway.


Assuntos
Proliferação de Células/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Transdução de Sinais/genética , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Inibidor de NF-kappaB alfa/metabolismo , Fosforilação/genética , Agregados Proteicos/genética , Ligação Proteica/genética , Proteínas Proto-Oncogênicas c-abl/genética , Elemento de Resposta Sérica/genética , Transcrição Gênica/genética , Transfecção
10.
DNA Res ; 15(3): 137-49, 2008 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-18316326

RESUMO

In this study, we established new systematic protocols for the preparation of cDNA clones, conventionally termed open reading frame (ORF) clones, suitable for characterization of their gene products by adopting a restriction-enzyme-assisted cloning method using the Flexi cloning system. The system has following advantages: (1) preparation of ORF clones and their transfer into other vectors can be achieved efficiently and at lower cost; (2) the system provides a seamless connection to the versatile HaloTag labeling system, in which a single fusion tag can be used for various proteomic analyses; and (3) the resultant ORF clones show higher expression levels both in vitro and in vivo. With this system, we prepared ORF clones encoding 1,929 human genes and characterized the HaloTag-fusion proteins of its subset that are expressed in vitro or in mammalian cells. Results thus obtained have demonstrated that our Flexi ORF clones are efficient for the production of HaloTag-fusion proteins that can provide a new versatile set for a variety of functional analyses of human genes.


Assuntos
Clonagem Molecular/métodos , Fases de Leitura Aberta/genética , Proteoma/análise , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Células Cultivadas , Chlorocebus aethiops , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Biossíntese de Proteínas , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
11.
J Histochem Cytochem ; 55(12): 1199-206, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17712176

RESUMO

Our recent studies demonstrated the localization of protein 4.1B, a member of the 4.1 skeletal membrane proteins, to the basolateral membranes of the S1-S2 renal proximal tubules. In the present studies, we investigated the presence of binding partners that could form a molecular complex with the 4.1B protein. Immunohistochemistry revealed the localization of p55, a membrane-associated guanylate kinase, and the sodium bicarbonate cotransporter1 (NBC1), to the basolateral membrane domain of S1-S2 in mouse renal proximal tubules. Using immunoprecipitation of kidney lysates with anti-p55 antibody, a positive band was blotted with anti-4.1B antibody. GST fusion proteins including the NBC1 and 4.1B regions were confirmed to bind with each other by electrophoresis after mixing. Both NBC1- and 4.1B-specific bands were detected in renal protein mixtures immunoprecipated by either anti-4.1B- or NBC1-specific antibodies. It is likely that NBC1, 4.1B, and p55 form a molecular complex in the basolateral membrane of the kidney S1-S2 proximal tubules. We propose that the 4.1B-containing membrane skeleton may play a role in regulating the Na(+) and HCO(3)(-) reabsorption in S1-S2 proximal tubules.


Assuntos
Guanilato Quinases/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas de Membrana/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Animais Recém-Nascidos , Imuno-Histoquímica , Imunoprecipitação , Túbulos Renais Proximais/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos , Microscopia Imunoeletrônica
12.
Anat Sci Int ; 80(2): 61-70, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15960311

RESUMO

The polarized architecture of epithelial cells is a fundamental determinant of cell structures and functions. Both formation and orientation of proper epithelial polarity are needed for cell-cell or cell-matrix adhesion, signal transduction and cytoskeletal interactions of multimolecular complexes at apical, lateral and basal cell membranes. These cell membrane domains are usually segregated by some junctional complexes. Recent molecular genetic studies on the anchor structure between myelin sheaths and axons have indicated the specific molecular organization for polarization of axolemma and the myelin sheaths at paranodes, termed 'septate-like junctions'. It was also speculated that other mammalian organs may use a similar junctional system. The protein 4.1 B was originally found to be localized in paranodes and juxtaparanodes of myelinated nerve fibers. Our recent immunohistochemical studies on protein 4.1B have indicated its significance for the cell-cell and/or cell-matrix adhesion in various rodent organs. The protein 4.1 family of proteins have been supposed to possess variable molecular domains relating to cell adhesion, ion balance, receptor responses and signal transduction. Therefore, more precise studies on the molecular structure and the functional domains of protein 4.1B, as well as on its changes under physiological and pathological conditions, may provide a clue for organogenesis in various mammalian organs.


Assuntos
Estruturas da Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Vísceras/metabolismo , Animais , Adesão Celular/fisiologia , Estruturas da Membrana Celular/ultraestrutura , Polaridade Celular/fisiologia , Células Epiteliais/ultraestrutura , Humanos , Proteínas dos Microfilamentos , Organogênese/fisiologia , Vísceras/ultraestrutura
13.
DNA Res ; 11(5): 353-60, 2004 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-15747583

RESUMO

DNA array technology has made remarkable progress in recent years and has become an indispensable tool in molecular biology research. However, preparing high-quality custom-made DNA arrays at a reasonable cost is still an important concern because we cannot abandon the use of DNA array systems designed for specific purposes. To address these problems, we here report the use of rolling circle amplification products of cDNA plasmids dissolved in 80% formamide as DNA probes immobilized on a nylon membrane. First, because formamide is practically non-volatile under ambient conditions and nucleic acids are easily dissolved in it, the use of formamide as a DNA solvent ensures that the DNA concentration of the solution will not change during arraying, which often takes several hours to a day depending on the number of DNA spots and arrays to produce. Secondly, the use of rolling circle amplification technology greatly reduced the labor needed to prepare the spotted DNA. The results in this study demonstrate that the introduction of these two modifications in preparation of nylon DNA array greatly improved its quality.


Assuntos
Perfilação da Expressão Gênica , Nylons , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Controle de Custos , Sondas de DNA , Formamidas/química , Humanos , Membranas Artificiais , Análise de Sequência com Séries de Oligonucleotídeos/economia , Plasmídeos , Controle de Qualidade , Manejo de Espécimes
14.
DNA Res ; 9(3): 99-106, 2002 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-12168954

RESUMO

We have accumulated information on protein-coding sequences of uncharacterized human genes, which are known as KIAA genes, through cDNA sequencing. For comprehensive functional analysis of the KIAA genes, it is necessary to prepare a set of cDNA clones which direct the synthesis of functional KIAA gene products. However, since the KIAA cDNAs were derived from long mRNAs (> 4 kb), it was not expected that all of them were full-length. Thus, as the first step toward preparing these clones, we evaluated the integrity of protein-coding sequences of KIAA cDNA clones through comparison with homologous protein entries in the public database. As a result, 1141 KIAA cDNAs had at least one homologous entry in the database, and 619 of them (54%) were found to be truncated at the 5' and/or 3' ends. In this study, 290 KIAA cDNA clones were tailored to be full-length or have considerably longer sequences than the original clones by isolating additional cDNA clones and/or connected parts of additional cDNAs or PCR products of the missing portion to the original cDNA clone. Consequently, 265, 8, and 17 predicted CDSs of KIAA cDNA clones were increased in the amino-, carboxy-, and both terminal sequences, respectively. In addition, 40 cDNA clones were modified to remove spurious interruption of protein-coding sequences. The total length of the resultant extensions at amino- and carboxy-terminals of KIAA gene products reached 97,000 and 7,216 amino acid residues, respectively, and various protein domains were found in these extended portions.


Assuntos
DNA Complementar/genética , Genes , Fases de Leitura Aberta/genética , Proteínas/genética , Clonagem Molecular , Perfilação da Expressão Gênica , Genoma Humano , Humanos , RNA Mensageiro/metabolismo
15.
J Histochem Cytochem ; 52(6): 769-77, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15150285

RESUMO

Cell-cell adhesion is crucial not only for mechanical adhesion but also for tissue morphogenesis. Protein 4.1B, a member of the protein 4.1 family named from an erythrocyte membrane protein, is a potential organizer of an adherens system. In adult mouse seminiferous tubules, protein 4.1B localized in the basal compartment, especially in the attaching region of spermatogonia and Sertoli cells. Protein 4.1B localization and appearance were not different in each spermatogenic stage. Developmentally, protein 4.1B was not detected at postnatal day 3 (P3), was diffusely localized at P15, and was found in the basal compartment during the third week. By double staining for protein 4.1B and F-actin, their localizations were shown to be different, indicating that protein 4.1B was localized in a region lower than the basal ectoplasmic specialization that formed the Sertoli-Sertoli junction. By electron microscopy, immunoreactive products were seen mainly on the membranes of Sertoli cells. In the W/W(v) mutant mouse, the seminiferous epithelium had few germ cells. Protein 4.1B and beta-catenin were not detected, although the basal ectoplasmic specialization was retained. These results indicate that protein 4.1B may be related to the adhesion between Sertoli cells and germ cells, especially the spermatogonium.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Epitélio Seminífero/metabolismo , Animais , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/genética , Imuno-Histoquímica , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Epitélio Seminífero/crescimento & desenvolvimento , Epitélio Seminífero/ultraestrutura , Células de Sertoli/metabolismo , Transativadores/metabolismo , beta Catenina
16.
J Mol Histol ; 35(4): 347-53, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15503808

RESUMO

Protein 4.1 family proteins are thought to interact with membrane proteins and also membrane skeletons. In this study, immunohistochemical studies by light and electron microscopy were performed with a specific antibody against protein 4.1B. Specific protein 4.1B immunolabeling was observed in simple columnar epithelium in the adult rat large intestine, small intestine and stomach. Protein 4.1B immunolabeling was localized along the membranes facing the adjacent cells (lateral portion) and also facing the extracellular matrix (basal portion). Moreover, a spatial protein 4.1B expression gradient was observed along the crypt-villus axis of the rat small and large intestinal epithelium: strong protein 4.1B expression was present within the villus, with the crypt showing barely any detectable protein 4.1B. The expression of protein 4.1B was not detected in the stratified squamous epithelium in the forestomach or the esophagus. By immunoelectron microscopy, the immunolabeling of the cells was observed to be restricted to the cytoplasmic side just beneath the plasma membrane, including the membranes adjacent to the next cells, except for the tight junctions. We conclude that the protein 4.1B expression pattern is related to the maturation of simple columnar epithelium in the rat digestive system, probably by the effect of adhesion.


Assuntos
Proteínas do Citoesqueleto/biossíntese , Sistema Digestório/metabolismo , Epitélio/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/biossíntese , Animais , Sistema Digestório/ultraestrutura , Epitélio/ultraestrutura , Imuno-Histoquímica , Microscopia Imunoeletrônica , Ratos , Ratos Sprague-Dawley
17.
Neurosci Lett ; 366(1): 15-7, 2004 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-15265581

RESUMO

Recent molecular studies on anchoring structures between myelin sheaths by glial cells (oligodendrocytes and Schwann cells (Sc) in the central (CNS) and peripheral nervous system (PNS), respectively) and axons indicated protein-protein interaction for the polarization of paranodes in the axons. The protein 4.1 (4.1) family was originally found in erythrocytes as a component of membrane skeletons, and genetic approaches revealed the precise family members. One of them, 4.1B, has been reported to be localized in paranodes and juxtaparanodes of myelinated axons. In this study, in addition to the myelinated axons, we also present the localization of 4.1B in nerve fibers in the adult mouse enteric nervous system, a subpopulation of mature unmyelinated nerve fibers in PNS. Ultrastructurally, 4.1B localized along the membranes of unmyelinated axons. Such unmyelinated axons were surrounded only by Sc, suggesting that the 4.1B may also have a role in direct Sc-axon interactions and maturation of the axons, as well as myelinating glial cell-axon interactions.


Assuntos
Axônios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Sistema Nervoso Entérico/metabolismo , Proteínas de Membrana/metabolismo , Fibras Nervosas Amielínicas/metabolismo , Animais , Axônios/ultraestrutura , Sistema Nervoso Entérico/ultraestrutura , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fibras Nervosas Amielínicas/ultraestrutura
18.
Brain Res ; 1307: 53-62, 2010 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-19853587

RESUMO

Protein 4.1 (band 4.1 or 4.1R) was originally identified as an abundant protein of the human erythrocyte, in which it stabilizes the spectrin/actin cytoskeleton. Subsequently, several new family members, 4.1N, 4.1G and 4.1B, have been identified, which are expressed in many cell types, in particular at cell-cell junctions. We previously reported that 4.1R and 4.1N are expressed in the inner ear hair cells with specific localization patterns, and that 4.1R forms a complex with the membrane-associated guanylate kinase (MAGUK) protein p55 and two deafness gene products, myosin XV and whirlin. To determine the functions of the other family members, 4.1G and 4.1B, we observed their expression patterns in developing stereocilia in mice inner ear hair cells. 4.1G is expressed in the basal tapers of the stereocilia bundle in early postnatal stages. 4.1B was specifically and constantly expressed in the stereocilia tips during postnatal development. Additionally, we found that 4.1B is ablated in the hair cells of both myosin XV and whirlin mutant mice at all stages in hair cell development. These results suggest that 4.1 family members play important roles in the development and maintenance of the inner ear hair cells, and that 4.1B may be a member of the myosin XV-whirlin complex that is important for stereocilia maturation.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Orelha Interna/citologia , Orelha Interna/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Ciliadas Auditivas Internas/metabolismo , Proteínas de Membrana/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Mutantes , Proteínas dos Microfilamentos , Mutação/genética , Miosinas/genética
20.
Methods Mol Biol ; 577: 25-39, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19718506

RESUMO

Expression-ready cDNA clones, where the open reading frame (ORF) of the gene of interest is placed under the control of an appropriate promoter, are critical for functional characterization of the gene products. To create a resource of human gene products, we attempted to systematically convert original cDNA clones to expression-ready forms for recombinant proteins. For this purpose, we adopted a rare-cutting restriction enzyme-based system, the Flexi cloning system, to construct ORF clones. Taking advantage of the fully sequenced cDNA clones we accumulated to date, a number of sets of Flexi ORF clones in a 96-well format have been prepared. In this section, two methods for the preparation of Flexi ORF clones in a 96-well format are described. A protocol for transferring ORFs between Flexi vectors in a 96-well format is also described. We believe that the resultant clone set could be successfully used as a versatile reagent for functional characterization of human proteins.


Assuntos
Clonagem Molecular/métodos , Ensaios de Triagem em Larga Escala/métodos , Fases de Leitura Aberta , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Primers do DNA/genética , Escherichia coli/genética , Vetores Genéticos , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Biologia Molecular/instrumentação , Biologia Molecular/métodos , Reação em Cadeia da Polimerase , Proteômica/instrumentação , Proteômica/métodos
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