RESUMO
The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here, we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state, followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Luond et al. (2021).
Assuntos
Transição Epitelial-Mesenquimal , Neoplasias , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Camundongos , Camundongos Transgênicos , TranscriptomaRESUMO
mRNA localization is an evolutionarily widespread phenomenon that can facilitate subcellular protein targeting. Extensive work has focused on mRNA targeting through 'zip-codes' within untranslated regions (UTRs), whereas much less is known about translation-dependent cues. Here, we examine mRNA localization in Caenorhabditis elegans embryonic epithelia. From an smFISH-based survey, we identified mRNAs associated with the cell membrane or cortex, and with apical junctions in a stage- and cell type-specific manner. Mutational analyses for one of these transcripts, dlg-1/discs large, revealed that it relied on a translation-dependent process and did not require its 5' or 3' UTRs. We suggest a model in which dlg-1 transcripts are co-translationally localized with the nascent protein: first the translating complex goes to the cell membrane using sequences located at the C-terminal/3' end, and then apically using N-terminal/5' sequences. These studies identify a translation-based process for mRNA localization within developing epithelia and determine the necessary cis-acting sequences for dlg-1 mRNA targeting.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Membrana Celular/genética , Desenvolvimento Embrionário/genética , Guanilato Quinases/genética , Biossíntese de Proteínas/genética , Junções Aderentes/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Polaridade Celular/genética , Embrião não Mamífero/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Junções Intercelulares/genética , Proteínas de Membrana/genética , Transporte Proteico/genética , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
Loss of innervation of skeletal muscle is a determinant event in several muscle diseases. Although several effectors have been identified, the pathways controlling the integrated muscle response to denervation remain largely unknown. Here, we demonstrate that PKB/Akt and mTORC1 play important roles in regulating muscle homeostasis and maintaining neuromuscular endplates after nerve injury. To allow dynamic changes in autophagy, mTORC1 activation must be tightly balanced following denervation. Acutely activating or inhibiting mTORC1 impairs autophagy regulation and alters homeostasis in denervated muscle. Importantly, PKB/Akt inhibition, conferred by sustained mTORC1 activation, abrogates denervation-induced synaptic remodeling and causes neuromuscular endplate degeneration. We establish that PKB/Akt activation promotes the nuclear import of HDAC4 and is thereby required for epigenetic changes and synaptic gene up-regulation upon denervation. Hence, our study unveils yet-unknown functions of PKB/Akt-mTORC1 signaling in the muscle response to nerve injury, with important implications for neuromuscular integrity in various pathological conditions.