RESUMO
BACKGROUND: Heterozygous germline loss-of-function mutations in the aryl hydrocarbon receptor-interacting protein gene (AIP) predispose to childhood-onset pituitary tumours. The pathogenicity of missense variants may pose difficulties for genetic counselling and family follow-up. OBJECTIVE: To develop an in vivo system to test the pathogenicity of human AIP mutations using the fruit fly Drosophila melanogaster. METHODS: We generated a null mutant of the Drosophila AIP orthologue, CG1847, a gene located on the Xchromosome, which displayed lethality at larval stage in hemizygous knockout male mutants (CG1847exon1_3 ). We tested human missense variants of 'unknown significance', with 'pathogenic' variants as positive control. RESULTS: We found that human AIP can functionally substitute for CG1847, as heterologous overexpression of human AIP rescued male CG1847exon1_3 lethality, while a truncated version of AIP did not restore viability. Flies harbouring patient-specific missense AIP variants (p.C238Y, p.I13N, p.W73R and p.G272D) failed to rescue CG1847exon1_3 mutants, while seven variants (p.R16H, p.Q164R, p.E293V, p.A299V, p.R304Q, p.R314W and p.R325Q) showed rescue, supporting a non-pathogenic role for these latter variants corresponding to prevalence and clinical data. CONCLUSION: Our in vivo model represents a valuable tool to characterise putative disease-causing human AIP variants and assist the genetic counselling and management of families carrying AIP variants.
Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação de Sentido Incorreto , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Bioensaio , Drosophila melanogaster , Feminino , Expressão Gênica , Estudos de Associação Genética/métodos , Testes Genéticos , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Mutação com Perda de Função , Perda de Heterozigosidade , Masculino , Modelos Moleculares , Neoplasias Hipofisárias/diagnóstico , Neoplasias Hipofisárias/genética , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Forces controlling tissue morphogenesis are attributed to cellular-driven activities, and any role for extracellular matrix (ECM) is assumed to be passive. However, all polymer networks, including ECM, can develop autonomous stresses during their assembly. Here, we examine the morphogenetic function of an ECM before reaching homeostatic equilibrium by analyzing de novo ECM assembly during Drosophila ventral nerve cord (VNC) condensation. Asymmetric VNC shortening and a rapid decrease in surface area correlate with the exponential assembly of collagen IV (Col4) surrounding the tissue. Concomitantly, a transient developmentally induced Col4 gradient leads to coherent long-range flow of ECM, which equilibrates the Col4 network. Finite element analysis and perturbation of Col4 network formation through the generation of dominant Col4 mutations that affect assembly reveal that VNC morphodynamics is partially driven by a sudden increase in ECM-driven surface tension. These data suggest that ECM assembly stress and associated network instabilities can actively participate in tissue morphogenesis.
Assuntos
Drosophila , Matriz Extracelular , Animais , Drosophila/genética , Matriz Extracelular/fisiologia , Morfogênese/fisiologia , Sistema Nervoso CentralRESUMO
Protein turnover rate is difficult to obtain experimentally. This protocol shows how to mathematically model turnover rates in an intervention-free manner given the ability to quantify mRNA and protein expression from initiation to homeostasis. This approach can be used to calculate production and degradation rates and to infer protein half-life. This model was successfully employed to quantify turnover during Drosophila embryogenesis, and we hypothesize that it will be applicable to diverse in vivo or in vitro systems. For complete details on the use and execution of this protocol, please refer to Matsubayashi et al. (2020).
Assuntos
Biologia Computacional/métodos , Proteólise , RNA Mensageiro/metabolismo , Animais , Drosophila/metabolismo , Expressão Gênica/genética , Homeostase , Cinética , Modelos Teóricos , Proteínas/metabolismoRESUMO
The extracellular matrix (ECM) is a polymer network hypothesized to form a stable cellular scaffold. While the ECM can undergo acute remodeling during embryogenesis, it is experimentally difficult to determine whether basal turnover is also important. Most studies of homeostatic turnover assume an initial steady-state balance of production and degradation and measure half-life by quantifying the rate of decay after experimental intervention (e.g., pulse labeling). Here, we present an intervention-free approach to mathematically model basal ECM turnover during embryogenesis by exploiting our ability to live image de novo ECM development in Drosophila to quantify production from initiation to homeostasis. This reveals rapid turnover (half-life â¼7-10 h), which we confirmed by in vivo pulse-chase experiments. Moreover, ECM turnover is partially dependent on proteolysis and network interactions, and slowing turnover affects tissue morphogenesis. These data demonstrate that embryonic ECM undergoes constant replacement, which is likely necessary to maintain network plasticity to accommodate growth and morphogenesis.
Assuntos
Matriz Extracelular/metabolismo , Homeostase , Morfogênese , Animais , Membrana Basal/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Modelos TeóricosRESUMO
The most prominent developmental function attributed to the extracellular matrix (ECM) is cell migration. While cells in culture can produce ECM to migrate, the role of ECM in regulating developmental cell migration is classically viewed as an exogenous matrix presented to the moving cells. In contrast to this view, we show here that Drosophila embryonic hemocytes deposit their own laminins in streak-like structures to migrate efficiently throughout the embryo. With the help of transplantation experiments, live microscopy, and image quantification, we demonstrate that autocrine-produced laminin regulates hemocyte migration by controlling lamellipodia dynamics, stability, and persistence. Proper laminin deposition is regulated by the RabGTPase Rab8, which is highly expressed and required in hemocytes for lamellipodia dynamics and migration. Our results thus support a model in which, during embryogenesis, the Rab8-regulated autocrine deposition of laminin reinforces directional and effective migration by stabilizing cellular protrusions and strengthening otherwise transient adhesion states.
Assuntos
Proteínas de Drosophila/metabolismo , Laminina/metabolismo , Animais , Movimento Celular , Células Cultivadas , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Matriz Extracelular/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Hemócitos/citologia , Hemócitos/metabolismo , Microscopia de Fluorescência , Pseudópodes/fisiologiaRESUMO
The basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians [1]; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly [2], this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components appears to be hierarchical [3-5], yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types [6-9], and it is unclear how large, possibly insoluble protein complexes [10] are delivered into the matrix. Here we exploit our ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, we show that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components.