Correlative all-optical quantification of mass density and mechanics of sub-cellular compartments with fluorescence specificity
- Technische Universität Dresden, Germany
- Max Planck Institute for the Science of Light, Germany
- University of Rostock, Germany
Abstract
Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples - so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample - a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.
Data availability
The data sets generated during and/or analyzed during the current study are available from figshare under the following link: https://doi.org/10.6084/m9.figshare.c.5347778
-
Combined fluorescence, optical diffraction tomography and Brillouin microscopyFigshare repository, doi:10.6084/m9.figshare.c.5347778.
Article and author information
Author details
Funding
Deutsche Forschungsgemeinschaft (419138906)
- Simon Alberti
- Jochen Guck
Volkswagen Foundation (92847)
- Simon Alberti
- Jochen Guck
Alexander von Humboldt-Stiftung
- Jochen Guck
NOMIS Stiftung
- Andreas Hermann
Hermann und Lilly Schilling-Stiftung
- Andreas Hermann
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Rohit V Pappu, Washington University in St Louis, United States
Publication history
- Preprint posted: October 30, 2020 (view preprint)
- Received: March 17, 2021
- Accepted: January 8, 2022
- Accepted Manuscript published: January 10, 2022 (version 1)
- Version of Record published: February 4, 2022 (version 2)
Copyright
© 2022, Schlüßler et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,975
- Page views
-
- 388
- Downloads
-
- 12
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Correlative all-optical quantification of mass density and mechanics of sub-cellular compartments with fluorescence specificity
eLife 11:e68490.
https://doi.org/10.7554/eLife.68490
Further reading
-
- Cell Biology
- Neuroscience
Prolonged exposure to loud noise has been shown to affect inner ear sensory hair cells in a variety of deleterious manners, including damaging the stereocilia core. The damaged sites can be visualized as ‘gaps’ in phalloidin staining of F-actin, and the enrichment of monomeric actin at these sites, along with an actin nucleator and crosslinker, suggests that localized remodeling occurs to repair the broken filaments. Herein, we show that gaps in mouse auditory hair cells are largely repaired within 1 week of traumatic noise exposure through the incorporation of newly synthesized actin. We provide evidence that Xin actin binding repeat containing 2 (XIRP2) is required for the repair process and facilitates the enrichment of monomeric γ-actin at gaps. Recruitment of XIRP2 to stereocilia gaps and stress fiber strain sites in fibroblasts is force-dependent, mediated by a novel mechanosensor domain located in the C-terminus of XIRP2. Our study describes a novel process by which hair cells can recover from sublethal hair bundle damage and which may contribute to recovery from temporary hearing threshold shifts and the prevention of age-related hearing loss.
-
- Biochemistry and Chemical Biology
- Cell Biology
Autophagy is an essential catabolic pathway which sequesters and engulfs cytosolic substrates via autophagosomes, unique double-membraned structures. ATG8 proteins are ubiquitin-like proteins recruited to autophagosome membranes by lipidation at the C-terminus. ATG8s recruit substrates, such as p62, and play an important role in mediating autophagosome membrane expansion. However, the precise function of lipidated ATG8 in expansion remains obscure. Using a real-time in vitro lipidation assay, we revealed that the N-termini of lipidated human ATG8s (LC3B and GABARAP) are highly dynamic and interact with the membrane. Moreover, atomistic MD simulation and FRET assays indicate that N-termini of LC3B and GABARAP associate in cis on the membrane. By using non-tagged GABARAPs, we show that GABARAP N-terminus and its cis-membrane insertion are crucial to regulate the size of autophagosomes in cells irrespectively of p62 degradation. Our study provides fundamental molecular insights into autophagosome membrane expansion, revealing the critical and unique function of lipidated ATG8.
