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1.
Dev Biol ; 284(1): 112-25, 2005 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-15950215

RESUMO

A simple procedure to isolate neural stem cells would greatly facilitate direct studies of their properties. Here, we exploited the increase in EGF receptor (EGFR) levels, that occurs in late development stem cells or in younger precursors upon exposure to FGF-2, to isolate cells expressing high levels of EGFR (EGFR(high)) from the developing and the adult brain. Independently of age and region of isolation, EGFR(high) cells were highly enriched in multipotent precursors and displayed similar antigenic characteristics, with the exception of GFAP and Lex/SSEA-1 that were mainly expressed in adult EGFR(high) cells. EGFR levels did not correlate with neurogenic potential, indicating that the increase in EGFR expression does not directly affect differentiation. Instead, in the brain, many EGFR(high) precursors showed tangential orientation and, whether isolated from the cortex or striatum, EGFR(high) precursors displayed characteristics of cells originating from the ventral GZ such as expression Dlx and Mash-1 and the ability to generate GABAergic neurons and oligodendrocytes. Moreover, migration of EGFR(high) cells on telencephalic slices required EGFR activity. Thus, the developmentally regulated increase in EGFR levels may affect tangential migration of multipotent precursors. In addition, it can be used as a marker to effectively isolate telencephalic multipotent precursors from embryonic and adult tissue.


Assuntos
Encéfalo/embriologia , Movimento Celular/fisiologia , Receptores ErbB/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células-Tronco Multipotentes/fisiologia , Animais , Western Blotting , Encéfalo/metabolismo , Carbocianinas , Feminino , Fator 2 de Crescimento de Fibroblastos , Citometria de Fluxo , Imuno-Histoquímica , Camundongos
2.
J Cell Sci ; 118(Pt 3): 517-27, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15657075

RESUMO

Leishmania HASPB is a lipoprotein that is exported to the extracellular space from both Leishmania parasites and mammalian cells via an unconventional secretory pathway. Exported HASPB remains anchored in the outer leaflet of the plasma membrane mediated by myristate and palmitate residues covalently attached to the N-terminal SH4 domain of HASPB. HASPB targeting to the plasma membrane depends on SH4 acylation that occurs at intracellular membranes. How acylated HASPB is targeted to the plasma membrane and, in particular, the subcellular site of HASPB membrane translocation is unknown. In order to address this issue, we screened for clonal CHO mutants that are incapable of exporting HASPB. A detailed characterization of such a CHO mutant cell line revealed that the expression level of the HASPB reporter molecule is unchanged compared to CHO wild-type cells; that it is both myristoylated and palmitoylated; and that it is mainly localized to the plasma membrane as judged by confocal microscopy and subcellular fractionation. However, based on a quantitative flow cytometry assay and a biochemical biotinylation assay of surface proteins, HASPB transport to the outer leaflet of the plasma membrane is largely reduced in this mutant. From these data, we conclude that the subcellular site of HASPB membrane translocation is the plasma membrane as the reporter molecule accumulates in this location when export is blocked. Thus, these results allow us to define a two-step process of HASPB cell surface biogenesis in which SH4 acylation of HASPB firstly mediates intracellular targeting to the plasma membrane. In a second step, the plasma membrane-resident machinery, which is apparently disrupted in the CHO mutant cell line, mediates membrane translocation of HASPB. Intriguingly, the angiogenic growth factor FGF-2, another protein secreted by unconventional means, is shown to be secreted normally from the HASPB export mutant cell line. These observations demonstrate that the export machinery component defective in the export mutant cell line functions specifically in the HASPB export pathway.


Assuntos
Antígenos de Protozoários/metabolismo , Membrana Celular/metabolismo , Mutação , Proteínas de Protozoários/metabolismo , Acilação , Animais , Antígenos de Protozoários/genética , Biotinilação , Células CHO , Membrana Celular/química , Cricetinae , Cricetulus , Citosol/química , Doxiciclina/farmacologia , Ácidos Graxos/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Membranas Intracelulares/química , Leishmania/fisiologia , Proteínas de Membrana/análise , Mutagênese Insercional , Parasitos/fisiologia , Fragmentos de Peptídeos/genética , Transporte Proteico , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/genética
3.
J Cell Sci ; 117(Pt 9): 1727-36, 2004 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15075234

RESUMO

Endoplasmic reticulum/Golgi-dependent protein secretion depends on signal peptides that mediate membrane translocation of nascent secretory proteins into the lumen of the endoplasmic reticulum. Classical secretory proteins are transported across the membrane of the endoplasmic reticulum in an unfolded conformation, which is similar to protein import into mitochondria. This process is mediated by Sec61, the protein-conducting channel of the endoplasmic reticulum. Employing both FACS-based in vivo transport assays and confocal microscopy, we now show that fibroblast growth factor 2 (FGF-2), a pro-angiogenic mediator exported from mammalian cells by an unconventional secretory pathway, does not need to be unfolded in order to be released into the extracellular space. These findings suggest that the molecular apparatus mediating export of FGF-2 is not only distinct from classical translocation machineries in terms of molecular identity but also operates in a mechanistically distinct manner that allows membrane translocation of FGF-2 in a folded conformation.


Assuntos
Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Membranas Intracelulares/metabolismo , Dobramento de Proteína , Aminopterina/farmacologia , Animais , Células CHO , Cricetinae , Fator 2 de Crescimento de Fibroblastos/genética , Citometria de Fluxo , Camundongos , Microscopia Confocal , Mitocôndrias/metabolismo , Modelos Biológicos , Conformação Proteica , Desnaturação Proteica , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Transfecção
4.
J Cell Sci ; 115(Pt 18): 3619-31, 2002 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-12186948

RESUMO

Basic fibroblast growth factor (FGF-2) is a secretory protein that lacks a signal peptide. Consistently, FGF-2 has been shown to be secreted by an ER-Golgi-independent mechanism; however, the machinery mediating this process remains to be established at the molecular level. Here we introduce a novel experimental system based on flow cytometry that allows the quantitative assessment of non-classical FGF-2 secretion in living cells. Stable cell lines have been created by retroviral transduction that express various kinds of FGF-2-GFP fusion proteins in a doxicyclin-dependent manner. Following induction of protein expression, biosynthetic FGF-2-GFP is shown to translocate to the outer surface of the plasma membrane as determined by both fluorescence activated cell sorting (FACS) and confocal microscopy. Both N- and C-terminal GFP tagging of FGF-2 is compatible with FGF-2 export, which is shown to occur in a controlled fashion rather than through unspecific release. The experimental system described has strong implications for the identification of both FGF-2 secretion inhibitors and molecular components involved in FGF-2 secretion. In the second part of this study we made use of the FGF-2 export system described to analyze the fate of biosynthetic FGF-2-GFP following export to the extracellular space. We find that secreted FGF-2 fusion proteins accumulate in large heparan sulfate proteoglycan (HSPG)-containing protein clusters on the extracellular surface of the plasma membrane. These microdomains are shown to be distinct from caveolae-like lipid rafts known to play a role in FGF-2-mediated signal transduction. Since CHO cells lack FGF high-affinity receptors (FGFRs), it can be concluded that FGFRs mediate the targeting of FGF-2 to lipid rafts. Consistently, FGF-2-GFP-secreting CHO cells do not exhibit increased proliferation activity. Externalization and deposition of biosynthetic FGF-2 in HSPG-containing protein clusters are independent processes, as a soluble secreted intermediate was demonstrated. The balance between intracellular FGF-2 and HSPG-bound secreted FGF-2 is shown not to be controlled by the availability of cell surface HSPGs, indicating that the FGF-2 secretion machinery itself is rate-limiting.


Assuntos
Células CHO/metabolismo , Membrana Celular/metabolismo , Células Eucarióticas/metabolismo , Espaço Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Células CHO/citologia , Comunicação Celular/genética , Membrana Celular/genética , Cricetinae , Células Eucarióticas/citologia , Espaço Extracelular/genética , Fator 2 de Crescimento de Fibroblastos/genética , Vetores Genéticos , Proteínas de Fluorescência Verde , Proteoglicanas de Heparan Sulfato/metabolismo , Proteínas Luminescentes , Glicoproteínas de Membrana/genética , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Microscopia Confocal , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução Genética
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