RESUMEN
The Notch pathway is a widely utilized, evolutionarily conserved regulatory system that plays a central role in the fate decisions of multipotent precursor cells. Notch often acts by inhibiting differentiation along a particular pathway while permitting or promoting self-renewal or differentiation along alternative pathways. Haematopoietic cells and stromal cells express Notch receptors and their ligands, and Notch signalling affects the survival, proliferation, and fate choices of precursors at various stages of haematopoietic development, including whether haematopoietic stem cells self-renew or differentiate, common lymphoid precursors undergo T or B cell differentiation, or monocytes differentiate into macrophage or dendritic cells. These findings suggest that the Notch pathway plays a fundamental role in regulating haematopoietic development.
Asunto(s)
Hematopoyesis/fisiología , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Animales , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , Ligandos , Proteínas de la Membrana/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores NotchRESUMEN
The hematopoietic system is maintained by a rare population of hematopoietic stem cells (HSC) that are thought to undergo self-renewal as well as continuously produce progeny that differentiate into the various hematopoietic lineages. However, the mechanisms regulating cell fate choices by HSC and their progeny have not been understood. Results of most studies support a stochastic model of cell fate determination in which growth factors support only the survival or proliferation of the progeny specified along a particular lineage. In other developmental systems, however, Notch signaling has been shown to play a central role in regulating fate decisions of numerous types of precursors, often inhibiting a particular (default) pathway while permitting self-renewal or differentiation along an alternative pathway. There is also accumulating evidence that the Notch pathway affects survival, proliferation, and cell fate choices at various stages of hematopoietic cell development, including the decisions of HSC to self-renew or differentiate and of common lymphoid precursors to undergo T- or B-cell differentiation. These data suggest that the Notch pathway plays a fundamental role in the development and maintenance of the hematopoietic system.
Asunto(s)
Hematopoyesis/fisiología , Proteínas de la Membrana/metabolismo , Transactivadores/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , Ligandos , Proteínas de la Membrana/fisiología , Receptores Notch , Transducción de Señal , Transactivadores/fisiologíaRESUMEN
Notch-mediated cellular interactions are known to regulate cell fate decisions in various developmental systems. A previous report indicated that monocytes express relatively high amounts of Notch-1 and Notch-2 and that the immobilized extracellular domain of the Notch ligand, Delta-1 (Delta(ext-myc)), induces apoptosis in peripheral blood monocytes cultured with macrophage colony-stimulating factor (M-CSF), but not granulocyte-macrophage CSF (GM-CSF). The present study determined the effect of Notch signaling on monocyte differentiation into macrophages and dendritic cells. Results showed that immobilized Delta(ext-myc) inhibited differentiation of monocytes into mature macrophages (CD1a+/-CD14+/- CD64+) with GM-CSF. However, Delta(ext-myc) permitted differentiation into immature dendritic cells (CD1a+CD14-CD64-) with GM-CSF and interleukin 4 (IL-4), and further differentiation into mature dendritic cells (CD1a+CD83+) with GM-CSF, IL-4, and tumor necrosis factor-alpha (TNF-alpha). Notch signaling affected the differentiation of CD1a-CD14+ macrophage/dendritic cell precursors derived in vitro from CD34+ cells. With GM-CSF and TNF-alpha, exposure to Delta(ext-myc) increased the proportion of precursors that differentiated into CD1a+CD14- dendritic cells (51% in the presence of Delta(ext-myc) versus 10% in control cultures), whereas a decreased proportion differentiated into CD1a-CD14+ macrophages (6% versus 65%). These data indicate a role for Notch signaling in regulating cell fate decisions by bipotent macrophage/dendritic precursors.
Asunto(s)
Células Dendríticas/citología , Macrófagos/citología , Proteínas de la Membrana/fisiología , Monocitos/metabolismo , Receptores de Superficie Celular/fisiología , Factores de Transcripción , Antígenos de Diferenciación/análisis , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Interleucina-4/farmacología , Péptidos y Proteínas de Señalización Intracelular , Prueba de Cultivo Mixto de Linfocitos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Monocitos/citología , Monocitos/efectos de los fármacos , Estructura Terciaria de Proteína , Receptor Notch1 , Receptor Notch2 , Proteínas Recombinantes de Fusión/fisiología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Cell-cell interactions mediated by Notch and its ligands are known to effect many cell fate decisions in both invertebrates and vertebrates. However, the mechanisms involved in ligand induced Notch activation are unknown. Recently it was shown that, in at least some cases, endocytosis of the extracellular domain of Notch and ligand by the signaling cell is required for signal induction in the receptive cell. These results imply that soluble ligands (ligand extracellular domains) although capable of binding Notch would be unlikely to activate it. To test the potential activity of soluble Notch ligands, we generated monomeric and dimeric forms of the Notch ligand Delta-1 by fusing the extracellular domain to either a series of myc epitopes (Delta-1(ext-myc)) or to the Fc portion of human IgG-1 (Delta-1(ext-IgG)), respectively. Notch activation, assayed by inhibition of differentiation in C2 myoblasts and by HES1 transactivation in U20S cells, occurred when either Delta-1(ext-myc) or Delta-1(ext-IgG) were first immobilized on the plastic surface. However, Notch was not activated by either monomeric or dimeric ligand in solution (non-immobilized). Furthermore, both non-immobilized Delta-1(ext-myc) and Delta-1(ext-IgG) blocked the effect of immobilized Delta. These results indicate that Delta-1 extracellular domain must be immobilized to induce Notch activation in C2 or U20S cells and that non-immobilized Delta-1 extracellular domain is inhibitory to Notch function. These results imply that ligand stabilization may be essential for Notch activation.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Transducción de Señal/fisiología , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Células Inmovilizadas , Epítopos/metabolismo , Espacio Extracelular/química , Proteínas Fúngicas/metabolismo , Genes myc/fisiología , Humanos , Inmunoglobulina G , Péptidos y Proteínas de Señalización Intracelular , Ligandos , Proteínas de la Membrana/química , Proteínas de la Membrana/farmacología , Mieloma Múltiple , Fibras Musculares Esqueléticas/citología , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Receptores Notch , Solubilidad , Células Tumorales CultivadasRESUMEN
Hematopoietic stem cells give rise to progeny that either self-renew in an undifferentiated state or lose self-renewal capabilities and commit to lymphoid or myeloid lineages. Here we evaluated whether hematopoietic stem cell self-renewal is affected by the Notch pathway. Notch signaling controls cell fate choices in both invertebrates and vertebrates by inhibiting certain differentiation pathways, thereby permitting cells to either differentiate along an alternative pathway or to self-renew. Notch receptors are present in hematopoietic precursors and Notch signaling enhances the in vitro generation of human and mouse hematopoietic precursors, determines T- or B-cell lineage specification from a common lymphoid precursor and promotes expansion of CD8(+) cells. Here, we demonstrate that constitutive Notch1 signaling in hematopoietic cells established immortalized, cytokine-dependent cell lines that generated progeny with either lymphoid or myeloid characteristics both in vitro and in vivo. These data support a role for Notch signaling in regulating hematopoietic stem cell self-renewal. Furthermore, the establishment of clonal, pluripotent cell lines provides the opportunity to assess mechanisms regulating stem cell commitment and demonstrates a general method for immortalizing stem cell populations for further analysis.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Proteínas de la Membrana/fisiología , Receptores de Superficie Celular , Transducción de Señal , Factores de Transcripción , Animales , Linfocitos B/inmunología , Células de la Médula Ósea/citología , Línea Celular Transformada , Células Cultivadas , Citocinas/farmacología , Rayos gamma , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Interleucina-11/farmacología , Leucopoyesis , Ratones , Ratones Endogámicos C57BL , Receptor Notch1 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Timo/inmunología , TransfecciónRESUMEN
Notch signaling has been shown to play a key role in cell fate decisions in numerous developmental systems. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we reported the expression of human Notch-1 in CD34+ progenitors. In this study, we evaluated the expression of human Notch-1 and Notch-2 protein by hematopoietic cells. In immunofluoresence study, we detected low amounts of Notch-1 and Notch-2 protein in both CD34+ and CD34+Lin- cells, high amounts in CD14+ monocytes as well as B and T cells, but no expression in CD15+ granulocytes. We further found that an immobilized truncated form of the Notch ligand, Delta-1, induced apoptosis in monocytes in the presence of macrophage colony-stimulating factor (M-CSF), but not granulocyte-macrophage colony-stimulating factor (GM-CSF). The widespread expressions of Notch proteins suggest multiple functions for this receptor during hematopoiesis. These studies further indicate a novel role for Notch in regulating monocyte survival. (Blood. 2000;95:2847-2854)
Asunto(s)
Apoptosis/fisiología , Citocinas/farmacología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Monocitos/fisiología , Transactivadores/metabolismo , Antígenos CD/análisis , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/citología , Células Cultivadas , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ligandos , Monocitos/citología , Monocitos/efectos de los fármacos , Receptores Notch , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transactivadores/genéticaRESUMEN
We examined the expression of two members of the Notch family, Notch-1 and Notch-2, and one Notch ligand, Jagged-1, in hematopoietic cells. Both Notch-1 and Notch-2 were detected in murine marrow precursors (Lin-Sca-1+c-kit+). The Notch ligand, Jagged-1, was not detected in whole marrow or in precursors. However, Jagged-1 was seen in cultured primary murine fetal liver stroma, cultured primary murine bone marrow stroma, and in stromal cell lines. These results indicate a potential role for Notch-Notch ligand interactions in hematopoiesis. To further test this possibility, the effect of Jagged-1 on murine marrow precursor cells was assessed by coculturing sorted precursor cells (Lin-Sca-1+c-kit+) with a 3T3 cell layer that expressed human Jagged-1 or by incubating sorted precursors with beads coated with the purified extracellular domain of human Jagged-1 (Jagged-1(ext)). We found that Jagged-1, presented both on the cell surface and on beads, promoted a twofold to threefold increase in the formation of primitive precursor cell populations. These results suggest a potential use for Notch ligands in expanding precursor cell populations in vitro.
Asunto(s)
Células Madre Hematopoyéticas/citología , Proteínas de la Membrana/biosíntesis , Factores de Transcripción , Células 3T3 , Animales , Proteínas de Unión al Calcio , Diferenciación Celular , Fibroblastos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular , Proteína Jagged-1 , Ligandos , Proteínas de la Membrana/metabolismo , Ratones , Unión Proteica , Receptor Notch1 , Receptor Notch2 , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/metabolismo , Proteínas Serrate-Jagged , TransfecciónRESUMEN
This paper identifies a neuronal receptor for tenascin-C (tenascin/cytotactin), an extracellular matrix protein that has previously been detected in developing sensory and motor neuron pathways and has been shown to regulate cell migration in the developing CNS. Antibodies specific for each subunit of the integrin alpha 8 beta 1 are used to demonstrate that alpha 8 beta 1 mediates neurite outgrowth of embryonic sensory and motor neurons on this extracellular matrix protein. In addition, expression of alpha 8 in K562 cells results in surface expression of alpha 8 beta 1 heterodimers that are shown to promote attachment of this cell line to tenascin. The major domain in tenascin that mediates neurite outgrowth is shown to be localized to fibronectin type III repeats 6-8.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Cadenas alfa de Integrinas , Neuronas Motoras/metabolismo , Neuronas Aferentes/metabolismo , Receptores de Antígenos/fisiología , Animales , Moléculas de Adhesión Celular Neuronal/farmacología , Células Cultivadas , Embrión de Pollo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/farmacología , Fibronectinas/química , Ganglios Espinales/ultraestructura , Técnicas de Inmunoadsorción , Integrinas/análisis , Integrinas/química , Sustancias Macromoleculares , Neuronas Motoras/ultraestructura , Neuritas/fisiología , Neuritas/ultraestructura , Neuronas Aferentes/ultraestructura , Receptores de Antígenos/análisis , Secuencias Repetitivas de Ácidos Nucleicos , TenascinaRESUMEN
We have studied the role of vinculin in regulating integrin-dependent neurite outgrowth in PC12 cells, a neuronal cell line. Vinculin is a cytoskeletal protein believed to mediate interactions between integrins and the actin cytoskeleton. In differentiated PC12 cells, the cell body, the neurite, and the growth cone contain vinculin. Within the growth cone, both the proximal region of "consolidation" and the more distal region consisting of lamellipodia and filopodia contain vinculin. To study the role of vinculin in neurite outgrowth, we generated vinculin-deficient isolates of PC12 cell lines by transfection with vectors expressing antisense vinculin RNA. In some of these cell lines, vinculin levels were reduced to 18-23% of normal levels. In the vinculin-deficient cell lines, neurite outgrowth on laminin was significantly reduced. In time-lapse analysis, growth cones advanced much more slowly than normal. Further analysis indicated that this deficit could be explained in large part by changes in the behaviors of filopodia and lamellipodia. Filopodia were formed in normal numbers, extended at normal rates, and extended to approximately normal lengths, but were much less stable in the vinculin deficient compared to control PC12 cells. Similarly, lamellipodia formed and grew nearly normally, but were dramatically less stable in the vinculin-deficient cells. This can account for the reduction in rate of growth cone advance. These results indicate that interactions between integrins and the actin-based cytoskeleton are necessary for stability of both filopodia and lamellipodia.
Asunto(s)
Adhesión Celular , Membrana Celular/fisiología , Neuritas/fisiología , Vinculina/fisiología , Actinas/análisis , Actinas/biosíntesis , Animales , Membrana Celular/ultraestructura , Clonación Molecular , Expresión Génica , Vectores Genéticos , Cinética , Células PC12 , Ratas , Factores de Tiempo , Transfección , Vinculina/biosíntesis , Vinculina/deficienciaRESUMEN
An automated, video-driven system was used to measure approximately 30 parameters of cell motion and accompanying changes in shape. This "Dynamic Morphology System" is based upon the Expertvision Motion Analysis System and is driven by a SUN computer. With the aid of this system, amoebic movement and shape changes were compared for vegetative wild-type Dictyostelium discoideum amoebae and a motility mutant, Mo-1. The measured parameters included speed, angle change, bearing, length, width, roundness, boundary flow, and curvature; and cell behavior was visualized monitoring amoebic tracks, difference pictures, and a newly developed ring expansion plot. Wild-type cells remained elongated, moved continuously and retained polarity throughout migration. In contrast, Mo-1 did not translocate, was round rather than elongated, formed bulges rather than elongated pseudopods, and exhibited no polarity. In contrast to the anterior f-action distribution in wild-type cells, f-actin in Mo-1 was distributed evenly as a shell just under the entire plasma membrane, a distribution consistent with the lack of polar cytoplasmic expansion.
Asunto(s)
Dictyostelium/fisiología , Actinas/análisis , Animales , Movimiento Celular , Dictyostelium/ultraestructura , Procesamiento de Imagen Asistido por Computador , Faloidina , Seudópodos/fisiología , Seudópodos/ultraestructura , Coloración y Etiquetado , Factores de Tiempo , Grabación de Cinta de VideoRESUMEN
When developing amebae of Dictyostelium discoideum are treated with constant concentrations of cAMP above 10(-8)M, the average rate of motility is depressed, with maximum inhibition at roughly 10(-6)M. It is demonstrated that shifting the concentration of cAMP from 0 M to concentrations ranging from 10(-8) to 10(-6)M in a perfusion chamber results in the immediate inhibition of motility. After shifting from 0 M to 10(-8) or 10(-7)M, the rate of cell motility remains low, then rebounds to a higher level, exhibiting a standard adaptation response. No adaptation is exhibited after a shift from 0 M to 10(-6)M, a concentration resulting in maximum inhibition. It is demonstrated that the level of inhibition and the extent of the adaptation period are dependent upon the concentration of cAMP after the shift, and that submaximal inhibition is additive. The characteristics of adaptation in this motility response are very similar to the characteristics of adaptation for the relay system and phosphorylation of the putative cAMP receptor.
Asunto(s)
Quimiotaxis , AMP Cíclico/farmacología , Dictyostelium/fisiología , Dictyostelium/efectos de los fármacos , Dictyostelium/crecimiento & desarrollo , Cinética , Factores de TiempoRESUMEN
The preaggregative period of Dictyostelium discoideum is composed of two sequential rate-limiting components. The timing mutant FM-1 exhibits a decrease in the length of the preaggregative period and the interval between the maxifinger and early culminate II stage. In contrast, it is normal in all aspects of growth, in the sequence of morphogenetic stages, in spore formation, in the capacity to rapidly recapitulate morphogenesis, and in the erasure event and subsequent program of dedifferentiation. By the reciprocal shift experiment, it is demonstrated that FM-1 is completely missing the first of the two rate-limiting components comprising the preaggregative period. The FM-1 mutation is heritable and behaves as a single mutation mapping to linkage group II. However, the FM-1 variant switches at relatively high frequency to several other timing phenotypes with longer preaggregative periods which in turn switch at high frequency. The FM-1 phenotype is considered in terms of timing regulation, and the process of high frequency switching between timing phenotypes is compared to other newly discovered switching systems.
Asunto(s)
Dictyostelium/citología , Factores de Tiempo , Tiempo , Animales , Agregación Celular , Diferenciación Celular , Células Clonales/citología , Dictyostelium/efectos de los fármacos , Dictyostelium/genética , Farmacorresistencia Microbiana/genética , Ácido Edético/farmacología , Morfogénesis , Mutación , Nistatina , FenotipoRESUMEN
A conjugate of hippuryllysine (HP) and adenylic acid was synthesized and purified. The structure of the conjugate, hippuryllysyl(N-epsilon-5'-phospho)adenosine (HLAMP) was established using 31P nuclear magnetic resonance, UV spectroscopy, acid/base lability, and enzyme digestion with AMP deaminase, alkaline phosphatase, 5'-nucleotidase, and a phosphoamidase activity recently identified in Dictyostelium discoideum. The results indicate that HLAMP contains a phosphoamide bond between the phosphate of AMP and the epsilon amino group of HL. Employing a microdroplet assay to assess chemotactic activity, HLAMP was found to be a potent chemoattractant of 7-h developing amoebae of D. discoideum. Other conjugates, including lysine-AMP (LAMP), tuftsin-AMP (TAMP) and avidin-AMP (AVAMP), as well as the degradation products of HLAMP (HL, AMP, and lysine) exhibited no chemotactic activity. The molecular structure of HLAMP is compared to that of other known chemoattractants of the cellular slime molds, and possible chemotactic receptors for HLAMP are considered.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Dictyostelium/fisiología , Dictyostelium/efectos de los fármacos , Hidrolasas/metabolismo , Relación Estructura-ActividadRESUMEN
Amebae of Dictyostelium discoideum normally chemotax to aggregation centers by assessing the direction of outwardly moving, nondissipating waves of the chemoattractant cAMP. However, D. discoideum amebae can also assess the direction of a relatively stable spatial gradient. We demonstrate that amebae migrating towards the "source" of a stable, spatial gradient move faster, extend fewer pseudopodia, and turn less frequently than amebae migrating away from the "source" in the same spatial gradient. In addition, amebae extend lateral pseudopods in a polarized fashion from the anterior half of the cell, and do so as frequently towards the source as away from the source. However, those formed towards the source more often produce a turn than those formed away from the source. These results suggest that there may be two decision-making systems, one localized in the pseudopods, and one along the entire cell body; they support the suggestion that Dictyostelium amebae may employ a temporal mechanism to assess the direction of a spatial gradient of chemoattractant.
Asunto(s)
Quimiotaxis , AMP Cíclico/farmacología , Dictyostelium/fisiología , Seudópodos/fisiología , Movimiento Celular , Quimiotaxis/efectos de los fármacos , Dictyostelium/citología , Dictyostelium/efectos de los fármacos , Cinética , Seudópodos/efectos de los fármacosRESUMEN
In an aggregation territory of Dictyostelium discoideum, outwardly moving, nondissipating waves of the chemoattractant cAMP sweep across each ameba. At the front of each wave, an ameba experiences an increasing temporal and a positive spatial gradient of cAMP. At the back of a wave, an ameba experiences a decreasing temporal and a negative spatial gradient of cAMP. Employing a perfusion chamber, we have mimicked the temporal dynamics of these waves in the absence of a spatial gradient and demonstrated that the frequency of lateral pseudopod formation and the frequency of turning are dramatically affected by the direction and dynamics of the temporal gradient. In addition, since an ameba will move in a directed fashion up a shallow, nonpulsatile gradient of cAMP, we also mimicked the increasing temporal gradient generated by an ameba moving up a shallow spatial gradient. The frequency of lateral pseudopod formation and the frequency of turning were depressed. Together, these results demonstrate that amebae can assess the direction of a temporal gradient of chemoattractant in the absence of a spatial gradient and alter both the frequency of pseudopod extension and turning, accordingly. Although these results do not rule out the involvement of a spatial mechanism in assessing a spatial gradient, they strongly suggest that the temporal dynamics of a cAMP wave or the temporal gradient generated by an ameba moving through a spatial gradient may play a major role in chemotaxis.