RESUMEN
Cells communicate with their environment via surface receptors, but nanoscopic receptor organization with respect to complex cell surface morphology remains unclear. This is mainly due to a lack of accessible, robust and high-resolution methods. Here, we present an approach for mapping the topography of receptors at the cell surface with nanometer precision. The method involves coating glass coverslips with glycine, which preserves the fine membrane morphology while allowing immobilized cells to be positioned close to the optical surface. We developed an advanced and simplified algorithm for the analysis of single-molecule localization data acquired in a biplane detection scheme. These advancements enable direct and quantitative mapping of protein distribution on ruffled plasma membranes with near isotropic 3D nanometer resolution. As demonstrated successfully for CD4 and CD45 receptors, the described workflow is a straightforward quantitative technique to study molecules and their interactions at the complex surface nanomorphology of differentiated metazoan cells.
Asunto(s)
Nanotecnología , Receptores de Superficie Celular , Animales , Membrana Celular/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Neuroblastoma cell line SH-SY5Y, due to its capacity to differentiate into neurons, easy handling, and low cost, is a common experimental model to study molecular events leading to Alzheimer's disease (AD). However, it is prevalently used in its undifferentiated state, which does not resemble neurons affected by the disease. Here, we show that the expression and localization of amyloid-ß protein precursor (AßPP), one of the key molecules involved in AD pathogenesis, is dramatically altered in SH-SY5Y cells fully differentiated by combined treatment with retinoic acid and BDNF. We show that insufficient differentiation of SH-SY5Y cells results in AßPP mislocalization.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Factor Neurotrófico Derivado del Encéfalo , Diferenciación Celular/fisiología , Neuronas/fisiología , Tretinoina , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Línea Celular Tumoral , Humanos , Microscopía Intravital/métodos , Modelos Biológicos , Neuroblastoma , Estrés Oxidativo , Proteolisis , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
Linker for activation in T cells (LAT) is a critical regulator of T-cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane, is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and super-resolution imaging and flow cytometry, we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics are further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of nonpalmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Membrana Celular/metabolismo , Glicina/metabolismo , Proteínas de la Membrana/metabolismo , Prolina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Calcio/metabolismo , Glicina/genética , Humanos , Células Jurkat , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía Confocal , Microscopía de Interferencia , Simulación de Dinámica Molecular , Mutación , Prolina/genética , Dominios Proteicos , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Linfocitos T/metabolismoRESUMEN
TEAD transcription factors regulate gene expression through interactions with DNA and other proteins. They are crucial for the development of eukaryotic organisms and to control the expression of genes involved mostly in cell proliferation and differentiation; however, their deregulation can lead to tumorigenesis. To study the interactions of TEAD1 with M-CAT motifs and their inverted versions, the KD of each complex was determined, and H/D exchange, quantitative chemical cross-linking, molecular docking, and smFRET were utilized for structural characterization. ChIP-qPCR was employed to correlate the results with a cell line model. The results obtained showed that although the inverted motif has 10× higher KD, the same residues were affected by the presence of M-CAT in both orientations. Molecular docking and smFRET revealed that TEAD1 binds the inverted motif rotated 180°. In addition, the inverted motif was proven to be occupied by TEAD1 in Jurkat cells, suggesting that the low-affinity binding sites present in the human genome may possess biological relevance.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Proteínas Nucleares/química , Factores de Transcripción/química , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Células Jurkat , Simulación del Acoplamiento Molecular , Proteínas Nucleares/metabolismo , Motivos de Nucleótidos , Unión Proteica , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismoRESUMEN
Plasma membrane proteins synthesised at the endoplasmic reticulum are delivered to the cell surface via sorting pathways. Hydrophobic mismatch theory based on the length of the transmembrane domain (TMD) dominates discussion about determinants required for protein sorting to the plasma membrane. Transmembrane adaptor proteins (TRAP) are involved in signalling events which take place at the plasma membrane. Members of this protein family have TMDs of varying length. We were interested in whether palmitoylation or other motifs contribute to the effective sorting of TRAP proteins. We found that palmitoylation is essential for some, but not all, TRAP proteins independent of their TMD length. We also provide evidence that palmitoylation and proximal sequences can modulate sorting of artificial proteins with TMDs of suboptimal length. Our observations point to a unique character of each TMD defined by its primary amino acid sequence and its impact on membrane protein localisation. We conclude that, in addition to the TMD length, secondary sorting determinants such as palmitoylation or flanking sequences have evolved for the localisation of membrane proteins.