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<p>Small subcellular organelles orchestrate key cellular functions. How biomolecules are spatially organized within these assemblies is poorly understood. Here, we report an automated super-resolution imaging and analysis workflow that integrates confocal microscopy, morphological object screening, targeted 3D super-resolution STED microscopy and quantitative image analysis. Using this smart microscopy workflow, we target the 3D organization of <i>NEAT1</i>, an architectural RNA that constitutes the structural backbone of paraspeckles, a membraneless nuclear organelle. Using site-specific labeling, morphological sorting and particle averaging, we reconstruct the morphological space of paraspeckles along their development cycle from over 10,000 individual particles. Applying spherical harmonics analysis, we report so-far unknown heterotypes of <i>NEAT1</i> RNA organization. By integrating multi-positional labeling, we determine the coarse conformation of <i>NEAT1</i> within the organelle and show that the 3’ end forms a loop-like structure at the surface of the paraspeckle. Our study reveals key structural features of paraspeckle structure and growth, as well as the molecular organization of its scaffolding RNA.</p>

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Smart 3D super-resolution microscopy reveals the architecture of the RNA scaffold in a nuclear body

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月27日

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  • Enya S. Berrevoets,
  • Laurell F. Kessler,
  • Ashwin Balakrishnan,
  • Ellen Kazumi Okuda,
  • Michaela Müller-McNicoll,
  • Bernd Rieger,
  • Sjoerd Stallinga,
  • Mike Heilemann

摘要

Small subcellular organelles orchestrate key cellular functions. How biomolecules are spatially organized within these assemblies is poorly understood. Here, we report an automated super-resolution imaging and analysis workflow that integrates confocal microscopy, morphological object screening, targeted 3D super-resolution STED microscopy and quantitative image analysis. Using this smart microscopy workflow, we target the 3D organization of NEAT1, an architectural RNA that constitutes the structural backbone of paraspeckles, a membraneless nuclear organelle. Using site-specific labeling, morphological sorting and particle averaging, we reconstruct the morphological space of paraspeckles along their development cycle from over 10,000 individual particles. Applying spherical harmonics analysis, we report so-far unknown heterotypes of NEAT1 RNA organization. By integrating multi-positional labeling, we determine the coarse conformation of NEAT1 within the organelle and show that the 3’ end forms a loop-like structure at the surface of the paraspeckle. Our study reveals key structural features of paraspeckle structure and growth, as well as the molecular organization of its scaffolding RNA.