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Development of cryogenic correlated light electron microscopy methods to study mechanisms of intracellular trafficking and their relationships to the secretory pathway

Carter, S. D. and Mageswaran, S. K. and Farino, Z. J. and Mamede, J. I. and Hope, T. J. and Frank, J. and Freyberg, Z. and Jensen, G. J. (2016) Development of cryogenic correlated light electron microscopy methods to study mechanisms of intracellular trafficking and their relationships to the secretory pathway. Molecular Biology of the Cell, 27 . Art. No. P30. ISSN 1059-1524. http://resolver.caltech.edu/CaltechAUTHORS:20170331-131655781

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Abstract

The application of cryogenic electron microscopy (cryo‐EM) to the study of cellular ultrastructure provides a resolution several orders of magnitude better than light microscopy. Although this approach is increasingly applied in situ, it suffers from limitations in our ability to target imaging to specific intracellular features including the subcellular localization of specific events of interest. Cryogenic correlated light and electron microscopy (cryo‐CLEM) helps to overcome this problem by spatially locating areas of interest inside cells using fluorescence from genetically tagged or stained cellular molecules and allows for the visualization of localized fluorescently‐tagged proteins down to the level of individual organelles. Here, we attempted to study the secretory pathway in a specialized mammalian cell line, insulin‐secreting INS‐1E cells, expressing genetically‐encoded fluorophores as a model system to develop a cryo‐CLEM methodology. We discovered that there are many bright sources of autofluorescence in frozen cells. Based on our initial observations and the current understanding in the field, we hypothesized that autofluorescence from endogenous cellular substrates exhibits a broader spectrum of fluorescence than the fluorescence range of our expressed fluorescent proteins. To test this, we developed a quantitative approach to discriminate between autofluorescence and the fluorescent signal from genetically‐encoded fluorophores by measuring fluorescent intensities across different bandwidths. To validate this new methodology, we visualized multiple fluorophore‐tagged organelle markers in our experimental cell system. We found that DsRed2‐cytochrome c oxidase and chromogranin A‐GFP proteins were targeted in INS‐1E cells to mitochondria and secretory granules by cryo‐CLEM, consistent with their respective well‐established intracellular localizations. Moreover, these fluorescent signals were clearly distinguishable from autofluorescence emanating from endogenous structures including insulin crystals and multilamellar bodies. Overall, our novel cryo‐CLEM methods open the door to the study of cellular phenomena and structures with a new degree of specificity.


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ORCID:
AuthorORCID
Jensen, G. J.0000-0003-1556-4864
Additional Information:© 2016 American Society for Cell Biology.
Record Number:CaltechAUTHORS:20170331-131655781
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20170331-131655781
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:75593
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:01 Apr 2017 04:50
Last Modified:01 Apr 2017 04:50

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