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Mapping Absolute DNA Density in Cell Nuclei using Single-molecule Localization Microscopy
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Una plataforma para mejorar la microscopia cuantitativa de localización de moléculas únicas

Ottavia Golfetto1, Devin L Wakefield1, Eliedonna E Cacao1

  • 1Department of Molecular Medicine , Beckman Research Institute, City of Hope , 1500 East Duarte Road , Duarte , California 91010 , United States.

Journal of the American Chemical Society
|September 27, 2018
PubMed
Resumen
Este resumen es generado por máquina.

Un nuevo ensayo de superficie para el aislamiento molecular (SAMI) mejora la microscopía cuantitativa de localización de una sola molécula (qSMLM) para la cuantificación precisa de proteínas. Este método mejora el estudio de la organización endógena de las proteínas y sus cambios en respuesta a las terapias.

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Área de la Ciencia:

  • La biofísica
  • Biología celular
  • Microscopía

Sus antecedentes:

  • La microscopia cuantitativa de localización de una sola molécula (qSMLM) es crucial para los estudios de organización de proteínas in situ.
  • La interpretación precisa de los datos de qSMLM se ve obstaculizada por las incertidumbres en las propiedades fotofísicas del reportero fluorescente.
  • La detección de proteínas endógenas es un desafío debido a la heterogeneidad de la etiqueta y las limitaciones del tamaño del reportero.

Objetivo del estudio:

  • Desarrollar y validar un nuevo ensayo de superficie para el aislamiento molecular (SAMI) para qSMLM.
  • Caracterizar las propiedades fotofísicas de los reporteros fluorescentes utilizando SAMI-qSMLM.
  • Determinar la densidad y la nanoorganización de las proteínas endógenas de la membrana, incluyendo EGFR, HER2 y HER3.

Principales métodos:

  • Desarrollo del ensayo de superficie para el aislamiento molecular (SAMI) para qSMLM.
  • Caracterización de las propiedades fotofísicas de las proteínas fluorescentes y los tintes.
  • Se utilizaron ligandos fluorescentes con fragmentos de anticuerpos diseñados para la detección de proteínas endógenas.
  • Empleado SAMI, ingeniería de anticuerpos y análisis de correlación de pares para la cuantificación de receptores.
  • Investigó la nanoorganización del receptor en las líneas celulares del cáncer de mama.

Principales resultados:

  • SAMI-qSMLM proporcionó una cuantificación sólida de las propiedades moleculares.
  • Se determinó con éxito la densidad y la nanoorganización de EGFR, HER2 y HER3 unidos a la membrana.
  • Se observaron cambios distintos en la densidad de los receptores y la nanoorganización después del tratamiento con agentes terapéuticos en las células de cáncer de mama.

Conclusiones:

  • La plataforma SAMI desarrollada mejora significativamente la cuantificación molecular en qSMLM.
  • Este enfoque permite la detección eficiente de proteínas endógenas con alta especificidad.
  • La plataforma tiene potencial para estudiar el entorno proteico local en células intactas y comprender los efectos terapéuticos.