RESUMO
Epithelial tissues can be polarized along two axes: in addition to apical-basal polarity they are often also polarized within the plane of the epithelium, known as planar cell polarity (PCP). PCP depends upon the conserved Wnt/Frizzled (Fz) signaling factors, including Fz itself and Van Gogh (Vang/Vangl in mammals). Here, taking advantage of the complementary features of Drosophila wing and mouse skin PCP establishment, we dissect how Vang/Vangl phosphorylation on a specific conserved tyrosine residue affects its interaction with two cytoplasmic core PCP factors, Dishevelled (Dsh/Dvl1-3 in mammals) and Prickle (Pk/Pk1-3). We demonstrate that Pk and Dsh/Dvl bind to Vang/Vangl in an overlapping region centered around this tyrosine. Strikingly, Vang/Vangl phosphorylation promotes its binding to Prickle, a key effector of the Vang/Vangl complex, and inhibits its interaction with Dishevelled. Thus phosphorylation of this tyrosine appears to promote the formation of the mature Vang/Vangl-Pk complex during PCP establishment and conversely it inhibits the Vang interaction with the antagonistic effector Dishevelled. Intriguingly, the phosphorylation state of this tyrosine might thus serve as a switch between transient interactions with Dishevelled and stable formation of Vang-Pk complexes during PCP establishment.
Assuntos
Polaridade Celular , Proteínas Desgrenhadas , Proteínas de Drosophila , Proteínas de Membrana , Animais , Camundongos , Polaridade Celular/genética , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Receptores Frizzled/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , FosforilaçãoRESUMO
Efficient migration on adhesive surfaces involves the protrusion of lamellipodial actin networks and their subsequent stabilization by nascent adhesions. The actin-binding protein lamellipodin (Lpd) is thought to play a critical role in lamellipodium protrusion, by delivering Ena/VASP proteins onto the growing plus ends of actin filaments and by interacting with the WAVE regulatory complex, an activator of the Arp2/3 complex, at the leading edge. Using B16-F1 melanoma cell lines, we demonstrate that genetic ablation of Lpd compromises protrusion efficiency and coincident cell migration without altering essential parameters of lamellipodia, including their maximal rate of forward advancement and actin polymerization. We also confirmed lamellipodia and migration phenotypes with CRISPR/Cas9-mediated Lpd knockout Rat2 fibroblasts, excluding cell type-specific effects. Moreover, computer-aided analysis of cell-edge morphodynamics on B16-F1 cell lamellipodia revealed that loss of Lpd correlates with reduced temporal protrusion maintenance as a prerequisite of nascent adhesion formation. We conclude that Lpd optimizes protrusion and nascent adhesion formation by counteracting frequent, chaotic retraction and membrane ruffling.This article has an associated First Person interview with the first author of the paper.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Pseudópodes , Citoesqueleto de Actina , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/genética , Adesão Celular , Movimento CelularRESUMO
AKAP200 is a Drosophila melanogaster member of the "A Kinase Associated Protein" family of scaffolding proteins, known for their role in the spatial and temporal regulation of Protein Kinase A (PKA) in multiple signaling contexts. Here, we demonstrate an unexpected function of AKAP200 in promoting Notch protein stability. In Drosophila, AKAP200 loss-of-function (LOF) mutants show phenotypes that resemble Notch LOF defects, including eye patterning and sensory organ specification defects. Through genetic interactions, we demonstrate that AKAP200 interacts positively with Notch in both the eye and the thorax. We further show that AKAP200 is part of a physical complex with Notch. Biochemical studies reveal that AKAP200 stabilizes endogenous Notch protein, and that it limits ubiquitination of Notch. Specifically, our genetic and biochemical evidence indicates that AKAP200 protects Notch from the E3-ubiquitin ligase Cbl, which targets Notch to the lysosomal pathway. Indeed, we demonstrate that the effect of AKAP200 on Notch levels depends on the lysosome. Interestingly, this function of AKAP200 is fully independent of its role in PKA signaling and independent of its ability to bind PKA. Taken together, our data indicate that AKAP200 is a novel tissue specific posttranslational regulator of Notch, maintaining high Notch protein levels and thus promoting Notch signaling.
Assuntos
Proteínas de Ancoragem à Quinase A/fisiologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster , Lisossomos/metabolismo , Proteínas de Membrana/fisiologia , Proteólise , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptores Notch/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Larva , Proteínas de Membrana/genética , Estabilidade Proteica , Transdução de Sinais/genética , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
Vaccinia virus enhances its cell-to-cell spread by inducing Arp2/3-dependent actin polymerisation. This process is initiated by Src- and Abl-mediated phosphorylation of the viral transmembrane protein A36, leading to recruitment of a signalling network consisting of Grb2, Nck, WIP and N-WASP. Nck is a potent activator of N-WASP-Arp2/3-dependent actin polymerisation. However, recent observations demonstrate that an interaction between Nck and N-WASP is not required for vaccinia actin tail formation. We found that Cdc42 cooperates with Nck to promote actin tail formation by stabilising N-WASP beneath the virus. Cdc42 activation is mediated by the Rho guanine-nucleotide-exchange factor (GEF) intersectin-1 (ITSN1), which is recruited to the virus prior to its actin-based motility. Moreover, Cdc42, ITSN1 and N-WASP function collaboratively in a feed-forward loop to promote vaccinia-induced actin polymerisation. Outside the context of infection, we demonstrate that ITSN1 also functions together with Cdc42, Nck and N-WASP during phagocytosis mediated by the Fc gamma receptor. Our observations suggest that ITSN1 is an important general regulator of Cdc42-, Nck- and N-WASP-dependent actin polymerisation.
Assuntos
Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Oncogênicas/metabolismo , Vaccinia virus/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/ultraestrutura , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Linhagem Celular , Proteína Adaptadora GRB2/genética , Humanos , Fosforilação , Transdução de Sinais/genética , Vaccinia virus/patogenicidade , Vaccinia virus/ultraestrutura , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
Transport of cargoes by kinesin-1 is essential for many cellular processes. Nevertheless, the number of proteins known to recruit kinesin-1 via its cargo binding light chain (KLC) is still quite small. We also know relatively little about the molecular features that define kinesin-1 binding. We now show that a bipartite tryptophan-based kinesin-1 binding motif, originally identified in Calsyntenin is present in A36, a vaccinia integral membrane protein. This bipartite motif in A36 is required for kinesin-1-dependent transport of the virus to the cell periphery. Bioinformatic analysis reveals that related bipartite tryptophan-based motifs are present in over 450 human proteins. Using vaccinia as a surrogate cargo, we show that regions of proteins containing this motif can function to recruit KLC and promote virus transport in the absence of A36. These proteins interact with the kinesin light chain outside the context of infection and have distinct preferences for KLC1 and KLC2. Our observations demonstrate that KLC binding can be conferred by a common set of features that are found in a wide range of proteins associated with diverse cellular functions and human diseases.
Assuntos
Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Vaccinia virus/química , Vaccinia virus/fisiologia , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Motivos de Aminoácidos , Transporte Biológico , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Genoma Humano , Genótipo , Células HeLa , Humanos , Cinesinas/química , Cinesinas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Triptofano/química , Vaccinia virus/genética , Proteínas da Matriz Viral/genética , Liberação de VírusRESUMO
BACKGROUND: Fabry Disease (FD) is a rare X-linked metabolic lysosomal disorder. FD has a broad range of symptoms which vary markedly between patients. The heterogenous nature of the disease makes diagnosis difficult for health care professionals (HCPs), which in turn has a significant effect on the patient's quality of life (QoL). As few adolescent patients are eligible for treatment, to date there has been little published data on the burden of disease and impact of treatment on these patients and their caregivers. This study was developed to provide some insight into these groups. METHODS: An online-based survey was performed to gather further insights on the burden of FD in 14 adolescents aged 12-15 years old across three European countries, from the perspective of the patients, caregivers and HCPs. RESULTS: Symptom burden was found to be high in the adolescent population, with 'pain' and 'intolerance to heat or cold' commonly reported symptoms, both by patients and to HCPs. Eleven of the 14 patients surveyed were receiving enzyme replacement therapy (ERT), with their post-ERT symptomology showing improvement when compared to symptoms before receiving ERT. The majority of caregivers believe their child's overall health has improved since starting ERT. While there was a positive outlook towards ERT noted by the patients and caregivers, 4/5 HCPs believed there is 'a need for more efficacious treatment options' and all HCPs noted that there is 'a need for more manageable treatment options'. FD was shown to place a burden on caregivers, who reported feelings of guilt and absences from work. CONCLUSIONS: Data show there is a significant symptom burden for the adolescent, which affects their QoL and mental health, as well as placing a burden on the wider family. While ERT is an effective treatment and provides symptom relief for many of the respondents in the survey, they still reported symptom burden. Additionally, there was reporting of reluctance to engage in treatment or difficulties associated with the treatment. Heterogeneity in symptom presentation suggests that the treatment regimen needs to be tailored to the individual. Physicians therefore need to have a choice of treatment options available to help them manage symptoms and disease where the benefit to risk ratio is in favour of undergoing treatment.
Assuntos
Doença de Fabry , Adolescente , Cuidadores/psicologia , Criança , Terapia de Reposição de Enzimas , Doença de Fabry/tratamento farmacológico , Humanos , Qualidade de Vida , Inquéritos e QuestionáriosRESUMO
Planar cell polarity (PCP) and neural tube defects (NTDs) are linked, with a subset of NTD patients found to harbor mutations in PCP genes, but there is limited data on whether these mutations disrupt PCP signaling in vivo. The core PCP gene Van Gogh (Vang), Vangl1/2 in mammals, is the most specific for PCP. We thus addressed potential causality of NTD-associated Vangl1/2 mutations, from either mouse or human patients, in Drosophila allowing intricate analysis of the PCP pathway. Introducing the respective mammalian mutations into Drosophila Vang revealed defective phenotypic and functional behaviors, with changes to Vang localization, post-translational modification, and mechanistic function, such as its ability to interact with PCP effectors. Our findings provide mechanistic insight into how different mammalian mutations contribute to developmental disorders and strengthen the link between PCP and NTD. Importantly, analyses of the human mutations revealed that each is a causative factor for the associated NTD.
As an embryo develops, its cells must work together to build mature tissues and organs. During the formation of the nervous system, for example, a sheet of cells destined to become the brain and spinal cord folds up into a tube spanning the length of the embryo. Normally, this tube known as the 'neural tube' zips up, and the cells that will eventually become skin and other surrounding tissues close in over it. If the neural tube does not close completely, different parts of the spinal cord or brain can remain unprotected. This can cause diseases called neural tube defects, such as spina bifida, which is characterized by holes in the backbone exposing the spinal cord and surrounding membranes. Patients with neural tube defects can have similar genetic mutations, for example, in the genes controlling a process called "planar cell polarity", or PCP for short. Cells arranged in flat sheets use the PCP process to sense direction, and it is this process that allows structures, such as the scales on a fish or the hairs on a mouse, to all point in the same direction. PCP is also important in embryonic development: sheets of cells that can sense direction correctly can therefore move collectively to complete complex tasks (such as closing the neural tube). However, no-one knew whether the specific PCP gene mutations implicated in neural tube defects in humans actually affected the cells' ability to sense direction, or indeed whether they were even involved in causing the diseases. Humphries et al. set out to find out more about these mutations using fruit flies as a model system. The fruit fly is widely used to study the genes and signals involved in direction sensing, especially PCP. Problems with PCP produce easily measurable changes in the wing and eye, showing what went wrong and how badly. Humphries et al. genetically engineered fruit flies to have the same mutations as human patients and revealed that these mutations did indeed alter cells' ability to sense direction. These experiments also showed that each mutation did so in a different way, and with varying severity. This explained why the same mutations caused different levels of neural defects in mice (which are commonly used to study human diseases) and suggests that they might contribute to neural tube disorders in humans. These results show potential connections between neural tube defects and direction sensing in cells. In the future, this study and follow-up work could help researchers to understand what types of mutation have the most impact, which may eventually allow doctors to better predict who is most at risk of being affected by these conditions.
Assuntos
Proteínas de Transporte/genética , Polaridade Celular/fisiologia , Proteínas de Drosophila/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Mutação , Defeitos do Tubo Neural/genética , Animais , Modelos Animais de Doenças , Drosophila , Humanos , Camundongos , Defeitos do Tubo Neural/etiologiaRESUMO
During its egress, vaccinia virus transiently recruits AP-2 and clathrin after fusion with the plasma membrane. This recruitment polarizes the viral protein A36 beneath the virus, enhancing actin polymerization and the spread of infection. We now demonstrate that three NPF motifs in the C-terminus of A36 recruit AP-2 and clathrin by interacting directly with the Epsin15 homology domains of Eps15 and intersectin-1. A36 is the first identified viral NPF motif containing protein shown to interact with endocytic machinery. Vaccinia still induces actin tails in the absence of the A36 NPF motifs. Their loss, however, reduces the cell-to-cell spread of vaccinia. This is due to a significant reduction in virus release from infected cells, as the lack of intersectin-1 recruitment leads to a loss of Cdc42 activation, impairing N-WASP-driven Arp2/3-mediated actin polymerization. Our results suggest that initial A36-mediated virus release plays a more important role than A36-driven super-repulsion in promoting the cell-to-cell spread of vaccinia.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Motivos de Aminoácidos , Vaccinia virus/fisiologia , Proteínas Estruturais Virais/metabolismo , Liberação de Vírus , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Clatrina/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Vaccinia virus/química , Vaccinia virus/genética , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
The role of clathrin in pathogen entry has received much attention and has highlighted the adaptability of clathrin during internalization. Recent studies have now uncovered additional roles for clathrin and have put the spotlight on its role in pathogen spread. Here, we discuss the manipulation of clathrin by pathogens, with specific attention to the processes that occur at the plasma membrane. In the majority of cases, both clathrin and the actin cytoskeleton are hijacked, so we also examine the interplay between these two systems and their role during pathogen internalization, egress and spread.
Assuntos
Actinas/metabolismo , Clatrina/metabolismo , Endocitose , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/microbiologia , Citoesqueleto de Actina/virologia , Animais , Aderência Bacteriana , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Membrana Celular/virologia , Interações Hospedeiro-Patógeno , Transporte Proteico , Shigella flexneri/fisiologia , Vaccinia virus/fisiologia , Internalização do Vírus , Liberação de Vírus , Replicação ViralRESUMO
During their egress, newly assembled vaccinia virus particles fuse with the plasma membrane and enhance their spread by inducing Arp2/3-dependent actin polymerization. Investigating the events surrounding vaccinia virus fusion, we discovered that vaccinia transiently recruits clathrin in a manner dependent on the clathrin adaptor AP-2. The recruitment of clathrin to vaccinia dramatically enhances the ability of the virus to induce actin-based motility. We demonstrate that clathrin promotes clustering of the virus actin tail nucleator A36 and host N-WASP, which activates actin nucleation through the Arp2/3 complex. Increased clustering enhances N-WASP stability, leading to more efficient actin tail initiation and sustained actin polymerization. Our observations uncover an unexpected role for clathrin during virus spread and have important implications for the regulation of actin polymerization.