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1.
EMBO J ; 41(24): e111115, 2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36215693

RESUMO

Mitochondria and peroxisomes are closely related metabolic organelles, both in terms of origin and in terms of function. Mitochondria and peroxisomes can also be turned over by autophagy, in processes termed mitophagy and pexophagy, respectively. However, despite their close relationship, it is not known if both organelles are turned over under similar conditions, and if so, how this might be coordinated molecularly. Here, we find that multiple selective autophagy pathways are activated upon iron chelation and show that mitophagy and pexophagy occur in a BNIP3L/NIX-dependent manner. We reveal that the outer mitochondrial membrane-anchored NIX protein, previously described as a mitophagy receptor, also independently localises to peroxisomes and drives pexophagy. We show this process happens in vivo, with mouse tissue that lacks NIX having a higher peroxisomal content. We further show that pexophagy is stimulated under the same physiological conditions that activate mitophagy, including cardiomyocyte and erythrocyte differentiation. Taken together, our work uncovers a dual role for NIX, not only in mitophagy but also in pexophagy, thus illustrating the interconnection between selective autophagy pathways.


Assuntos
Macroautofagia , Mitofagia , Camundongos , Animais , Peroxissomos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo
2.
Haematologica ; 106(9): 2304-2311, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34042406

RESUMO

Patients with inherited anemia and hemoglobinopathies (such as sickle cell disease and ß-thalassemia) are treated with red blood cell (RBC) transfusions to alleviate their symptoms. Some of these patients may have rare blood group types or go on to develop alloimmune reactions, which can make it difficult to source compatible blood in the donor population. Laboratory-grown RBC represent a particularly attractive alternative which could satisfy an unmet clinical need. The challenge, however, is to produce - from a limited number of stem cells - the 2x1012 RBC required for a standard adult therapeutic dose. Encouraging progress has been made in RBC production from adult stem cells under good manufacturing practice. In 2011, the Douay group conducted a successful proof-of-principle mini-transfusion of autologous manufactured RBC in a single volunteer. In the UK, a trial is planned to assess whether manufactured RBC are equivalent to RBC produced naturally in donors, by testing an allogeneic mini-dose of laboratory-grown manufactured RBC in multiple volunteers. This review discusses recent progress in the erythroid culture field as well as opportunities for further scaling up of manufactured RBC production for transfusion practice.


Assuntos
Anemia Falciforme , Hemoglobinopatias , Anemia Falciforme/terapia , Transfusão de Sangue , Transfusão de Eritrócitos , Eritrócitos , Humanos
4.
Haematologica ; 101(9): 1018-27, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27247322

RESUMO

Ankyrin-R provides a key link between band 3 and the spectrin cytoskeleton that helps to maintain the highly specialized erythrocyte biconcave shape. Ankyrin deficiency results in fragile spherocytic erythrocytes with reduced band 3 and protein 4.2 expression. We use in vitro differentiation of erythroblasts transduced with shRNAs targeting ANK1 to generate erythroblasts and reticulocytes with a novel ankyrin-R 'near null' human phenotype with less than 5% of normal ankyrin expression. Using this model, we demonstrate that absence of ankyrin negatively impacts the reticulocyte expression of a variety of proteins, including band 3, glycophorin A, spectrin, adducin and, more strikingly, protein 4.2, CD44, CD47 and Rh/RhAG. Loss of band 3, which fails to form tetrameric complexes in the absence of ankyrin, alongside GPA, occurs due to reduced retention within the reticulocyte membrane during erythroblast enucleation. However, loss of RhAG is temporally and mechanistically distinct, occurring predominantly as a result of instability at the plasma membrane and lysosomal degradation prior to enucleation. Loss of Rh/RhAG was identified as common to erythrocytes with naturally occurring ankyrin deficiency and demonstrated to occur prior to enucleation in cultures of erythroblasts from a hereditary spherocytosis patient with severe ankyrin deficiency but not in those exhibiting milder reductions in expression. The identification of prominently reduced surface expression of Rh/RhAG in combination with direct evaluation of ankyrin expression using flow cytometry provides an efficient and rapid approach for the categorization of hereditary spherocytosis arising from ankyrin deficiency.


Assuntos
Anquirinas/deficiência , Proteínas Sanguíneas/metabolismo , Eritroblastos/metabolismo , Membrana Eritrocítica/metabolismo , Lisossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/química , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Citoesqueleto/genética , Citoesqueleto/metabolismo , Eritroblastos/química , Eritroblastos/citologia , Eritropoese/genética , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Mutação , Ligação Proteica , Multimerização Proteica , Proteólise , Esferocitose Hereditária/genética , Esferocitose Hereditária/metabolismo
5.
Neurosci Lett ; 590: 178-83, 2015 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-25668492

RESUMO

The small GTPase Rif is required for the early stages of dendritic spine formation in neurons, acting through the formin mDia2 to control actin polymerization. Rif is expressed at high levels in the brain, suggesting broader roles in neuronal function. We screened a yeast two-hybrid cDNA library to identify additional binding partners for Rif of potential relevance to neuronal function. We found that Rif interacts with FARP1, a neuronal activator of the RhoA GTPase. We show that Rif has two separate roles in FARP1 regulation-in controlling its association with plexinA4, and in releasing active RhoA from a plexinA4/FARP1 complex. The regulation of FARP1 by Rif promotes neurite retraction in cells stimulated with the semaphorin Sema6A.


Assuntos
Citoesqueleto/metabolismo , Neuritos/fisiologia , Receptores de Superfície Celular/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Células HEK293 , Humanos , Células Jurkat , Células PC12 , Ratos , Semaforinas/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo
6.
Haematologica ; 98(11): 1788-96, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23935019

RESUMO

Congenital dyserythropoietic anemia type II is an autosomally recessive form of hereditary anemia caused by SEC23B gene mutations. Patients exhibit characteristic phenotypes including multinucleate erythroblasts, erythrocytes with hypoglycosylated membrane proteins and an apparent double plasma membrane. Despite ubiquitous expression of SEC23B, the effects of mutations in this gene are confined to the erythroid lineage and the basis of this erythroid specificity remains to be defined. In addition, little is known regarding the stage at which the disparate phenotypes of this disease manifest during erythropoiesis. We employ an in vitro culture system to monitor the appearance of the defining phenotypes associated with congenital dyserythropoietic anemia type II during terminal differentiation of erythroblasts derived from small volumes of patient peripheral blood. Membrane protein hypoglycosylation was detected by the basophilic stage, preceding the onset of multinuclearity in orthochromatic erythroblasts that occurs coincident with the loss of secretory pathway proteins including SEC23A during erythropoiesis. Endoplasmic reticulum remnants were observed in nascent reticulocytes of both diseased and healthy donor cultures but were lost upon further maturation of normal reticulocytes, implicating a defect of ER clearance during reticulocyte maturation in congenital dyserythropoietic anemia type II. We also demonstrate distinct isoform and species-specific expression profiles of SEC23 during terminal erythroid differentiation and identify a prolonged expression of SEC23A in murine erythropoiesis compared to humans. We propose that SEC23A is able to compensate for the absence of SEC23B in mouse erythroblasts, providing a basis for the absence of phenotype within the erythroid lineage of a recently described SEC23B knockout mouse.


Assuntos
Anemia Diseritropoética Congênita/genética , Anemia Diseritropoética Congênita/patologia , Eritropoese/fisiologia , Fenótipo , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL
7.
PLoS One ; 7(6): e38356, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22723854

RESUMO

The availability of Erythropoietin (Epo) is essential for the survival of erythroid progenitors. Here we study the effects of Epo removal on primary human erythroblasts grown from peripheral blood CD34(+) cells. The erythroblasts died rapidly from apoptosis, even in the presence of SCF, and within 24 hours of Epo withdrawal 60% of the cells were Annexin V positive. Other classical hallmarks of apoptosis were also observed, including cytochrome c release into the cytosol, loss of mitochondrial membrane potential, Bax translocation to the mitochondria and caspase activation. We adopted a 2D DIGE approach to compare the proteomes of erythroblasts maintained for 12 hours in the presence or absence of Epo. Proteomic comparisons demonstrated significant and reproducible alterations in the abundance of proteins between the two growth conditions, with 18 and 31 proteins exhibiting altered abundance in presence or absence of Epo, respectively. We observed that Epo withdrawal induced the proteolysis of the multi-functional proteins Hsp90 alpha, Hsp90 beta, SET, 14-3-3 beta, 14-3-3 gamma, 14-3-3 epsilon, and RPSA, thereby targeting multiple signaling pathways and cellular processes simultaneously. We also observed that 14 proteins were differentially phosphorylated and confirmed the phosphorylation of the Hsp90 alpha and Hsp90 beta proteolytic fragments in apoptotic cells using Nano LC mass spectrometry. Our analysis of the global changes occurring in the proteome of primary human erythroblasts in response to Epo removal has increased the repertoire of proteins affected by Epo withdrawal and identified proteins whose aberrant regulation may contribute to ineffective erythropoiesis.


Assuntos
Apoptose/efeitos dos fármacos , Eritroblastos/efeitos dos fármacos , Eritroblastos/metabolismo , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/metabolismo , Eritropoetina/farmacologia , Proteoma , Caspases/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Eritroblastos/citologia , Células Precursoras Eritroides/citologia , Humanos , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Proteínas/metabolismo , Proteólise , Proteômica/métodos , Fatores de Tempo
8.
Blood ; 118(1): 182-191, 2011 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-21527529

RESUMO

Band 3, the major anion transport protein of human erythrocytes, forms the core of a multiprotein complex in the erythrocyte membrane. Here we studied the spatiotemporal mechanisms of band 3 multiprotein complex assembly during erythropoiesis. Significant pools of intracellular band 3 and Rh-associated glycoprotein (RhAG) were found in the basophilic erythroblast. These intracellular pools decreased in the polychromatic erythroblast, whereas surface expression increased and were lowest in the orthochromatic erythroblast and reticulocytes. Protease treatment of intact cells to remove extracellular epitopes recognized by antibodies to band 3 and RhAG was used to study surface delivery kinetics and intracellular complex composition from the proerythroblast stage to the enucleated reticulocyte. Newly synthesized band 3 and protein 4.2 interact initially in the early stages of the secretory pathway and are found associated at the plasma membrane from the basophilic stage of erythropoiesis. Although we could successfully coimmunoprecipitate Rh with RhAG from plasma membrane pools at a similar stage, no intracellular interaction between these proteins was detectable. Knockdown of RhAG during early erythropoiesis was accompanied by a concomitant drop in membrane expression of Rh polypeptides. These data are consistent with assembly of major components of the band 3 macrocomplex at an early stage during erythropoiesis.


Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Eritroblastos/metabolismo , Membrana Eritrocítica/metabolismo , Eritropoese/fisiologia , Complexos Multiproteicos/metabolismo , Reticulócitos/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Retículo Endoplasmático/metabolismo , Eritroblastos/citologia , Complexo de Golgi/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/metabolismo , Ligação Proteica/fisiologia , RNA Interferente Pequeno , Reticulócitos/citologia
9.
Haematologica ; 95(9): 1594-8, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20378567

RESUMO

The study of human erythropoiesis in health and disease requires a robust culture system that consistently and reliably generates large numbers of immature erythroblasts that can be induced to differentiate synchronously. We describe a culture method modified from Leberbauer et al. (2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34(+) cells. This pure population of immature erythroblasts can be expanded to obtain 4x10(8) erythroblasts from 1x10(8) PBMC after 13-14 days in culture. Upon synchronized differentiation, high levels of enucleation (80-90%) and low levels of cell death (<10%) are achieved. We compared the yield of erythroblasts obtained from PBMC, CD34(+) cells or PBMC depleted of CD34(+) cells and show that CD34(-) cells represent the most significant early erythroid progenitor population. This culture system may be particularly useful for investigating the pathophysiology of anemic patients where only small blood volumes are available.


Assuntos
Antígenos CD34/análise , Proliferação de Células , Eritroblastos/citologia , Eritropoese , Técnicas de Cultura de Células/métodos , Humanos
10.
J Cell Sci ; 123(Pt 8): 1247-52, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20233848

RESUMO

Actin stress fibers are fundamental components of the actin cytoskeleton that produce contractile force in non-muscle cells. The formation of stress fibers is controlled by the small GTPase RhoA and two highly related proteins, RhoB and RhoC. Together, this subgroup of actin-regulatory proteins represents the canonical pathway of stress-fiber formation. Here, we show that the Rif GTPase is an alternative trigger of stress-fiber formation in epithelial cells. Rif is distantly related to RhoA; however, we show that the two proteins share a common downstream partner in stress-fiber formation--the Diaphanous-related formin mDia1. Rif-induced stress fibers also depend on the activity of the ROCK protein kinase. Unlike RhoA, Rif does not raise ROCK activity in cells, instead Rif appears to regulate the localization of myosin light chain phosphorylation. This study establishes Rif as a general regulator of Diaphanous-related formins and shows how non-classical Rho family members can access classical Rho pathways to create new signaling interfaces in cytoskeletal regulation.


Assuntos
Actinas/metabolismo , Células Epiteliais/enzimologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fibras de Estresse/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Extensões da Superfície Celular/enzimologia , Células Epiteliais/citologia , Forminas , Células HeLa , Humanos , Ligação Proteica , Quinases Associadas a rho/metabolismo
11.
Haematologica ; 95(8): 1278-86, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20179084

RESUMO

BACKGROUND: Protein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47, CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex. DESIGN AND METHODS: We used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis. RESULTS: We report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47, higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(-) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression, arguing against a direct reciprocal relationship. CONCLUSIONS: We have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated, in part, by increased CD44-cytoskeleton binding.


Assuntos
Proteínas do Citoesqueleto/deficiência , Membrana Eritrocítica/metabolismo , Eritropoese , Proteínas de Membrana/metabolismo , Adulto , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Anquirinas/metabolismo , Sequência de Bases , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Antígeno CD47/genética , Antígeno CD47/metabolismo , Diferenciação Celular , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Eritroblastos/citologia , Eritroblastos/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Immunoblotting , Imunoprecipitação , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Ligação Proteica , Homologia de Sequência do Ácido Nucleico
12.
Curr Protoc Cell Biol ; Chapter 14: Unit 14.8, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18360815

RESUMO

The Rho GTPase family of signaling proteins controls a wide range of highly dynamic cellular processes. Activation of Rho GTPases can be investigated and quantified in cell extracts using so-called pull-down assays. Proteins that bind specifically to the activated form of the Rho GTPase are used to capture it onto a bead support. Western blotting of the captured samples with specific antibodies then allows for quantification of the level of Rho GTPase activation in the sample. This unit describes the techniques for preparing the reagents required for assays of RhoA, Rac, and Cdc42 and gives practical tips for the successful application of the assay in a range of situations.


Assuntos
Bioensaio , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Células HeLa , Humanos , Camundongos , Plasmídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Células Swiss 3T3 , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/isolamento & purificação , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/isolamento & purificação , Proteínas rac de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/isolamento & purificação
13.
J Cell Sci ; 120(Pt 20): 3491-9, 2007 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-17928305

RESUMO

Animal cell movement is effected through a combination of protrusive and contractile events. Non-muscle cells contain stress fibres - bundles of actomyosin that are the major mediators of cell contraction and that can be compared to the highly organised actomyosin arrays of muscle cells. Recent studies have defined regulatory mechanisms that control stress fibre formation, placing the ROCK protein kinase at the centre of a complex signalling network controlling actomyosin contractility and stress fibre assembly. As we uncover the details of stress fibre construction, it is becoming clear that different categories of stress fibres exist. Some of these structures are less suited for cell motility and more suited to static contraction. In keeping with this, many specialised contractile cell types use stress fibres to remodel tissues and extracellular matrix.


Assuntos
Fibras de Estresse/fisiologia , Quinases Associadas a rho/metabolismo , Animais , Movimento Celular , Proteínas Contráteis/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/fisiologia , Fibroblastos/fisiologia , Adesões Focais/fisiologia , Redes e Vias Metabólicas , Proteínas dos Microfilamentos/metabolismo , Transdução de Sinais , Fibras de Estresse/ultraestrutura
14.
Curr Protoc Cell Biol ; Chapter 4: Unit4.17, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18228517

RESUMO

The surface of metazoan cells is a landscape not clearly visualized by light microscopy. Many cells elaborate protrusive structures such as microvilli, filopodia, lamellipodia, and surface ruffles that play important roles in the interaction between the cell and its environment. The high resolution of scanning electron microscopy makes it an ideal technique for studies of the cell surface; however, preservation of fine surface structure can be problematic. Here we highlight the critical factors in sample preparation to ensure optimal high-resolution imaging of the surface of mammalian cells and tissues.


Assuntos
Extensões da Superfície Celular/ultraestrutura , Microscopia Eletrônica de Varredura/instrumentação , Microscopia Eletrônica de Varredura/métodos , Preservação de Tecido/métodos , Animais , Comunicação Celular , Células Epiteliais/ultraestrutura , Microscopia Eletrônica de Varredura/tendências , Microvilosidades/ultraestrutura , Pseudópodes , Preservação de Tecido/instrumentação , Preservação de Tecido/tendências
15.
Exp Cell Res ; 306(1): 216-29, 2005 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15878346

RESUMO

Regeneration of the urothelium is rapid and effective in order to maintain a barrier to urine following tissue injury. Whereas normal human urothelial (NHU) cells are mitotically quiescent and G0 arrested in situ, they rapidly enter the cell cycle upon seeding in primary culture and show reversible growth arrest at confluency. We have used this as a model to investigate the role of EGF receptor signaling in urothelial regeneration and wound-healing. Transcripts for HER-1, HER-2, and HER-3 were expressed by quiescent human urothelium in situ. Expression of HER-1 was upregulated in proliferating cultures, whereas HER-2 and HER-3 were more associated with a growth-arrested phenotype. NHU cells could be propagated in the absence of exogenous EGF, but autocrine signaling through HER-1 via the MAPK and PI3-kinase pathways was essential for proliferation and migration during urothelial wound repair. HB-EGF was expressed by urothelium in situ and HB-EGF, epiregulin, TGF-alpha, and amphiregulin were expressed by proliferating NHU cells. Urothelial wound repair in vitro was attenuated by neutralizing antibodies against HER-1 ligands, particularly amphiregulin. By contrast, the same ligands applied exogenously promoted migration, but inhibited proliferation, implying that HER-1 ligands provoke differential effects in NHU cells depending upon whether they are presented as soluble or juxtacrine ligands. We conclude that proliferation and migration during wound healing in NHU cells are mediated through an EGFR autocrine signalling loop and our results implicate amphiregulin as a key mediator.


Assuntos
Comunicação Autócrina/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Urotélio/citologia , Anfirregulina , Anticorpos/farmacologia , Ciclo Celular/genética , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Família de Proteínas EGF , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/imunologia , Fator de Crescimento Epidérmico/farmacologia , Epirregulina , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/fisiologia , Expressão Gênica/genética , Glicoproteínas/genética , Glicoproteínas/imunologia , Glicoproteínas/farmacologia , Substâncias de Crescimento/deficiência , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Quinazolinas/farmacologia , Regeneração/efeitos dos fármacos , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/imunologia , Fator de Crescimento Transformador alfa/farmacologia , Urotélio/fisiologia , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia
16.
Curr Biol ; 15(2): 129-33, 2005 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-15668168

RESUMO

Eukaryotic cells produce a variety of specialized actin-rich surface protrusions. These include filopodia-thin, highly dynamic projections that help cells to sense their external environment. Filopodia consist of parallel filaments of actin, bundled by actin crosslinking proteins. The filaments are oriented with their rapidly growing "barbed" ends at the protruding tip and their slowly growing "pointed" ends at the base. Extension occurs by polymerization at the tip and is controlled by regulation of filament capping. The Rho GTPase Cdc42 is a key mediator of filopodia formation, which it regulates through binding CRIB domain-containing effectors. Cdc42 binds and activates the WASP proteins, which in turn activate the actin-nucleating complex Arp2/3. It also binds and activates IRSp53, which recruits the Ena/WASP family protein Mena to the filopodial tip and protects elongating actin filaments from capping. Previously, we identified another Rho family GTPase, Rif, as a potent stimulator of filopodial protrusion through a mechanism that does not require Cdc42. Here we characterize the differences between filopodia induced by these two small GTPases and show that the Rif effector in this pathway is the Diaphanous-related formin mDia2. Thus, Rif and Cdc42 represent two distinct routes to the induction of filopodia-producing structures with both shared and unique properties.


Assuntos
Proteínas de Transporte/metabolismo , Pseudópodes/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Forminas , Células HeLa , Humanos , Imunoprecipitação , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Técnicas do Sistema de Duplo-Híbrido , Proteína cdc42 de Ligação ao GTP/metabolismo
17.
Cell Microbiol ; 5(11): 773-83, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14531893

RESUMO

Enteropathogenic Escherichia coli (EPEC) are a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens. Enteropathogenic Escherichia coli encode a type III secretion system (TTSS) to transfer effector proteins into host cells, a process which is essential for virulence. In addition to generation of A/E lesions, the TTSS is also implicated in the ability of EPEC to invade cultured cells but the effector proteins responsible for promoting invasion have not been identified. In this paper we confirm the requirement of TTSS in EPEC invasion and demonstrate important roles for the Map and Tir effector molecules. Whereas in trans expression of Tir in the tir mutant restored invasion to wild-type levels, similar complementation of the map mutation by in trans expression of Map results in a hyperinvasive phenotype. The Map effector protein has two distinct functions within host cells, mediating Cdc42-dependent filopodia formation and targeting mitochondria to elicit dysfunction. The former function appears to be related to Map's ability to promote invasion as this was inhibited by interference with Cdc42 signalling. Conversely, Map targeting to mitochondria is not necessary for invasion. Promotion of EPEC invasion by Tir appears to involve interaction with intimin but is independent of pedestal formation, and intimin-Tir interaction is neither necessary nor sufficient for invasion. Comparison of the invasiveness of strains lacking Tir and/or Map with wild-type or mutant strains expressing the effectors in trans provides evidence that Map and Tir stimulate invasion by synergistic mechanisms. This synergism, which is in stark contrast to the antagonistic actions of Map and Tir in regulating filopodia and pedestal formation, further illustrates the complex interplay between EPEC effectors.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Fagócitos/metabolismo , Receptores de Superfície Celular/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Proteínas/genética , Proteínas/metabolismo , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , RNA Interferente Pequeno/metabolismo , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína da Síndrome de Wiskott-Aldrich , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
18.
Am J Pathol ; 163(2): 493-504, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12875970

RESUMO

Studies investigating changes in gene expression in urothelial carcinoma have generally compared tumors of different stages and grades but comparisons between low-grade, noninvasive tumors and normal urothelium are needed to identify genes involved in early tumor development. We isolated the urothelium from a low-grade tumor and corresponding normal mucosa by laser capture microdissection on frozen sections. The RNA extracted was amplified to generate suppressive subtractive cDNA libraries. Random sequencing of cDNA clones identified approximately 100 unique species. Of these 83% were known genes, 15% had homology to genes with an unknown function in humans, and 2% did not show homology to any published gene sequence. Two of the known genes, the 67-kd laminin receptor (67LR) and tumor-associated trypsin inhibitor (TATI), had previously been associated with metastatic progression in many tumor types, although 67LR has not been investigated in urothelial tumors. Immunolabeling of the original tissue with antibodies against these two genes confirmed overexpression, validating our strategy: 67LR was not expressed in the normal urothelium but was present in the tumor, whereas TATI expression was confined to umbrella cells in the normal urothelium, but extended to all cell layers in the tumor. We investigated both markers further in a separate series of tumors of different stages and grades. TATI was more consistently overexpressed than 67LR in all tumor grades and stages. Levels of secreted TATI were significantly higher in urine samples from patients with tumors compared to controls. Our strategy, combining laser capture microdissection and cDNA library construction, has identified genes that may be involved in the early phases of urothelial tumor development rather than with disease progression, highlighting the importance of comparing tumor with normal rather than just tumors of different stages and grades.


Assuntos
Carcinoma/genética , Regulação Neoplásica da Expressão Gênica , Receptores de Laminina/genética , Inibidor da Tripsina Pancreática de Kazal/genética , Neoplasias da Bexiga Urinária/genética , Urotélio/metabolismo , Urotélio/patologia , Carcinoma/patologia , Perfilação da Expressão Gênica , Biblioteca Gênica , Humanos , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Queratina-20 , Lasers , Receptores de Laminina/metabolismo , Inibidor da Tripsina Pancreática de Kazal/urina , Regulação para Cima , Neoplasias da Bexiga Urinária/patologia
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