RESUMO
Differential cell motility, which plays a key role in many developmental processes, is perhaps most evident in examples of pattern formation in which the different cell types arise intermingled before sorting out into discrete tissues. This is thought to require heterogeneities in responsiveness to differentiation-inducing signals that result in the activation of cell type-specific genes and 'salt and pepper' patterning. How differential gene expression results in cell sorting is poorly defined. Here we describe a novel gene (hfnA) that provides the first mechanistic link between cell signalling, differential gene expression and cell type-specific sorting in Dictyostelium. HfnA defines a novel group of evolutionarily conserved HECT ubiquitin ligases with an N-terminal filamin domain (HFNs). HfnA expression is induced by the stalk differentiation-inducing factor DIF-1 and is restricted to a subset of prestalk cells (pstO). hfnA(-) pstO cells differentiate but their sorting out is delayed. Genetic interactions suggest that this is due to misregulation of filamin complex activity. Overexpression of filamin complex members phenocopies the hfnA(-) pstO cell sorting defect, whereas disruption of filamin complex function in a wild-type background results in pstO cells sorting more strongly. Filamin disruption in an hfnA(-) background rescues pstO cell localisation. hfnA(-) cells exhibit altered slug phototaxis phenotypes consistent with filamin complex hyperactivity. We propose that HfnA regulates filamin complex activity and cell type-specific motility through the breakdown of filamin complexes. These findings provide a novel mechanism for filamin regulation and demonstrate that filamin is a crucial mechanistic link between responses to differentiation signals and cell movement in patterning based on 'salt and pepper' differentiation and sorting out.
Assuntos
Proteínas Contráteis/metabolismo , Dictyostelium/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Protozoários/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Proteínas Contráteis/química , Proteínas Contráteis/classificação , Proteínas Contráteis/genética , Dictyostelium/citologia , Dictyostelium/genética , Filaminas , Humanos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/classificação , Proteínas dos Microfilamentos/genética , Filogenia , Proteínas de Protozoários/química , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
To support effective process development there is a requirement for rapid analytical methods that can identify and quantitate adenoviral particles throughout the manufacturing process, from cellular lysate through to purified adenovirus. An anion-exchange high-performance liquid chromatography method for the analysis of adenovirus type 5 (Ad5) particles has been developed using a novel quaternary amine monolithic column (Bio-Monolith QA, Agilent). The developed method separates intact Ad5 from contaminating proteins and DNA, thus allowing analysis of non-purified samples during process development. Regeneration conditions were incorporated to extend the functional life of the column. Once developed, the method was qualified according to performance criteria of repeatability, intermediate precision and linearity. The linear working range of analysis was established between 7.5 x 10(8) to at least 2.4 x 10(10) viral particles (3 x 10(10) to 9.6 x 10(11) viral particles/mL), with a correlation coefficient of 0.9992. Relative standard deviations (RSDs) for intra- and inter-day repeatability and precision for retention time and peak area were less than 1 and 2.5%, respectively.