RESUMO
Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.
Assuntos
Nucléolo Celular/metabolismo , Gânglios Espinais/metabolismo , Cinesinas/metabolismo , Neurônios/metabolismo , Fosfoproteínas/química , Proteínas de Ligação a RNA/química , Nervo Isquiático/metabolismo , Sequência de Aminoácidos , Animais , Transporte Axonal/genética , Linhagem Celular Tumoral , Nucléolo Celular/ultraestrutura , Gânglios Espinais/citologia , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Cinesinas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Neurônios/citologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Cultura Primária de Células , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Nervo Isquiático/citologia , NucleolinaRESUMO
Tropomyosin-related tyrosine kinase B (TrkB) is the receptor for brain-derived neurotrophic factor (BDNF) and provides critical signaling that supports the development and function of the mammalian nervous system. Like other receptor tyrosine kinases (RTKs), TrkB is thought to signal as a dimer. Using cell imaging and biochemical assays, we found that TrkB acted as a monomeric receptor at the plasma membrane regardless of its binding to BDNF and initial activation. Dimerization occurred only after the internalization and accumulation of TrkB monomers within BDNF-containing endosomes. We further showed that dynamin-mediated endocytosis of TrkB-BDNF was required for the effective activation of the kinase AKT but not of the kinase ERK1/2. Thus, we report a previously uncharacterized mode of monomeric signaling for an RTK and a specific role for the endosome in TrkB homodimerization.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Membrana Celular/metabolismo , Dinaminas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Multimerização Proteica , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Animais , Endocitose , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Bidirectional molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. The molecular mechanisms underlying such communication are of keen interest and could provide new targets for intervention in motoneuron disease. Here, we developed a microfluidic platform with motoneuron cell bodies on one side and muscle cells on the other, connected by motor axons extending through microgrooves to form functional NMJs. Using this system, we were able to differentiate between the proximal and distal effects of oxidative stress and glial-derived neurotrophic factor (GDNF), demonstrating a dying-back degeneration and retrograde transmission of pro-survival signaling, respectively. Furthermore, we show that GDNF acts differently on motoneuron axons versus soma, promoting axonal growth and innervation only when applied locally to axons. Finally, we track for the first time the retrograde transport of secreted GDNF from muscle to neuron. Thus, our data suggests spatially distinct effects of GDNF--facilitating growth and muscle innervation at axon terminals and survival pathways in the soma.
Assuntos
Axônios/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Microfluídica , Neurônios Motores/metabolismo , Músculo Esquelético/metabolismo , Junção Neuromuscular/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Técnicas de Cocultura , Técnicas Imunoenzimáticas , Microscopia de Fluorescência , Neurônios Motores/citologia , Músculo Esquelético/citologia , Estresse Oxidativo , Fosforilação , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell's height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity.
Assuntos
Escherichia coli Enteropatogênica/patogenicidade , Células Epiteliais/citologia , Células Epiteliais/microbiologia , Infecções por Escherichia coli/fisiopatologia , Interações Hospedeiro-Patógeno/fisiologia , Ressonância de Plasmônio de Superfície/métodos , Animais , Adesão Celular/fisiologia , Técnicas de Cultura de Células , Tamanho Celular , Cães , Endocitose/fisiologia , Raios Infravermelhos , Células Madin Darby de Rim Canino , Microscopia Confocal , RefratometriaRESUMO
Enterohaemorrhagic Escherichia coli and enteropathogenic E. coli are enteropathogens characterized by their ability to induce the host cell to form actin-rich structures, termed pedestals. A type III secretion system, through which the pathogens deliver effector proteins into infected host cells, is essential for their virulence and pedestal formation. Enterohaemorrhagic E. coli encodes two similar effectors, EspM1 and EspM2, which activate the RhoA signalling pathway and induce the formation of stress fibres upon infection of host cells. We confirm these observations and in addition show that EspM inhibits the formation of actin pedestals. Moreover, we show that translocation of EspM into polarized epithelial cells induces dramatic changes in the tight junction localization and in the morphology and architecture of infected polarized monolayers. These changes are manifested by altered localization of the tight junctions and 'bulging out' morphology of the cells. Surprisingly, despite the dramatic changes in their architecture, the cells remain alive and the epithelial monolayer maintains a normal barrier function. Taken together, our results show that the EspM effectors inhibit pedestal formation and induce tight junction mislocalization as well as dramatic changes in the architecture of the polarized monolayer.