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1.
Sci Signal ; 15(719): eabg9782, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35104163

RESUMO

Superresolution techniques have advanced our understanding of complex cellular structures and processes but require the attachment of fluorophores to targets through tags or antibodies, which can be bulky and result in underlabeling. To overcome these limitations, we developed a technique to visualize the nanoscale binding locations of signaling proteins by taking advantage of their native interaction domains. Here, we demonstrated that pPAINT (protein point accumulation in nanoscale topography) is a new, single-molecule localization microscopy (SMLM) technique and used it to investigate T cell signaling by visualizing the Src homology 2 (SH2) domain, which is common in signaling molecules. When SH2 domain-containing proteins relocate to the plasma membrane, the domains selectively, transiently, and reversibly bind to preferred phosphorylated tyrosine residues on receptors. This transient binding yields the stochastic blinking events necessary for SMLM when observed with total internal reflection microscopy and enables quantification of binding coefficients in intact cells. We used pPAINT to reveal the binding sites of several T cell receptor-proximal signaling molecules, including Zap70, PI3K, Grb2, Syk, Eat2, and SHP2, and showed that the probes could be multiplexed. We showed that the binding half-life of the tandem SH2 domain of PI3K correlated with binding site cluster size at the immunological synapses of T cells, but that longer binding lifetimes were associated with smaller clusters for the monovalent SH2 domain of Eat2. These results demonstrate the potential of pPAINT for investigating phosphotyrosine-mediated signaling processes at the plasma membrane.


Assuntos
Microscopia , Domínios de Homologia de src , Sequência de Aminoácidos , Sítios de Ligação , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica
2.
Nat Cell Biol ; 20(2): 162-174, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29335528

RESUMO

Mitochondria are subcellular organelles that are critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, and on their coordinated translation, import and respiratory complex assembly. Here, we characterize EXD2 (exonuclease 3'-5' domain-containing 2), a nuclear-encoded gene, and show that it is targeted to the mitochondria and prevents the aberrant association of messenger RNAs with the mitochondrial ribosome. Loss of EXD2 results in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of the Drosophila melanogaster EXD2 orthologue (CG6744) causes developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that is reversed with an antioxidant diet. Our results define a conserved role for EXD2 in mitochondrial translation that influences development and ageing.


Assuntos
Proteínas de Drosophila/fisiologia , Exonucleases/genética , Longevidade/genética , Proteínas Mitocondriais/fisiologia , Ribossomos Mitocondriais/metabolismo , Biossíntese de Proteínas , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Exonucleases/fisiologia , Células Germinativas/metabolismo , Homeostase , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco/metabolismo
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