Cytoskeleton: Capturing intermediate filament networks
Cells are highly structured systems that contain a network of filaments known as the cytoskeleton. This scaffolding structure helps cells to maintain their shape, internal organization, and integrity under stress. It also plays important roles in intracellular transport, cell signaling and in cell proliferation, growth, differentiation and death. To fulfill their purpose, cytoskeletal filaments must be spatially organized, regulated and integrated in a manner that meets the rapidly changing needs of a cell (Kim and Coulombe, 2007; Block et al., 2015).
Recent advances in microscopy and image analysis have provided transformative insight into the three-dimensional (3D) architecture of cellular components in tissues, organs and entire organisms. Now, in eLife, Rudolf Leube and colleagues at RWTH Aachen University – including Reinhard Windoffer as first author – report on new insights into networks of intermediate filaments in three different types of epithelial cells (Windoffer et al., 2022).
The researchers used confocal microscopy and newly developed image analysis tools to describe the 3D organization of the entire intermediate filament network in three types of epithelial cells: MDCK cells, which are derived from canine kidneys, HaCaT keratinocytes derived from human skin, and retinal pigment epithelial (RPE) cells from mice. To visualize the network of keratin intermediate filaments in all three cell types, a specific keratin, known as Keratin 8 (K8), was tagged with a green fluorescence marker. The 3D models were generated based on microscopy images of filaments containing fluorescent K8, and the digitized representations of these images were analyzed at different scales to quantitatively describe the properties of the filaments and the networks they form.
From a methodology standpoint alone, the study by Windoffer et al. breaks new ground and sets the stage for an atlas-type collection holding information on the organization and architecture of intermediate filaments for a plethora of cell types, under various biological conditions. Among the myriad findings reported in this article, three stand out because they help capture the breadth of the study.
First, on a cellular scale, intermediate filaments show a specific spatial organization in the three cell types analyzed. For instance, MDCK kidney cells feature distinct apical and basal keratin intermediate filament networks that are interconnected but each possess unique features (Figure 1). HaCat cells are comparatively very flat and are densely packed with keratin filaments that enclose the nucleus laterally and include long bundles that run parallel to the cell’s longest axis. Retinal pigment epithelial (RPE) cells, which were analyzed in the natural context of the eye in situ, exhibit a comparatively less dense keratin intermediate filament network that is surprisingly prominent in the cytoplasmic apical domain, which faces photoreceptor cells in the retina. The mechanisms underlying these differences, and others reported by the researchers, are certainly worth investigating.
Second, at a subcellular level, Windoffer et al. provide a thorough account of several key properties of intermediate filaments, which help inform the reciprocal interplay between mechanical forces and network architecture at both local and cell-wide levels. Again, an interesting mix of convergence and divergence are reported for the three cell types analyzed.
Third, at a molecular level, conversions of digital representations of the intermediate filaments into biochemical quantities revealed that the mass of keratin in skin keratinocytes is similar to mass measurements obtained using quantitative western blotting (Feng et al., 2013). Such detailed knowledge can help guide future efforts to identify and characterize the mechanisms that regulate the amount of keratin proteins and filaments present in several types of epithelial cells.
With this rigorous, innovative and elegant study, Windoffer et al. provide a methodological framework to probe the architecture and organizing principles of intermediate filament networks for any cell type in 3D. This contribution is timely in that it complements emerging evidence about the high-resolution structure of mature, intermediate filaments emanating from cryo-electron tomography imaging (e.g., Turgay et al., 2017; Weber et al., 2021) and also from crystallographic studies (e.g., Lee et al., 2020; Eldirany et al., 2021).
High resolution information about both core architecture of individual intermediate filaments and their spatial organization as intricate 3D networks within cells is sorely needed to foster a deeper understanding of their mechanical and non-mechanical roles in epithelial cells, and their disruption in disease. As we await additional quantitative data of intermediate filaments networks in other cell types and/or biological circumstances, the study by Windoffer et al. sets the stage for follow-up analyzes regarding the principles and mechanisms underlying the spatial organization of intermediate filaments in vivo.
References
-
Physical properties of cytoplasmic intermediate filamentsBiochimica et Biophysica Acta 1853:3053–3064.https://doi.org/10.1016/j.bbamcr.2015.05.009
-
Recent insight into intermediate filament structureCurrent Opinion in Cell Biology 68:132–143.https://doi.org/10.1016/j.ceb.2020.10.001
-
Keratin intracellular concentration revisited: implications for keratin function in surface epitheliaThe Journal of Investigative Dermatology 133:850–853.https://doi.org/10.1038/jid.2012.397
Article and author information
Author details
Publication history
Copyright
© 2022, Coulombe
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,199
- views
-
- 117
- downloads
-
- 0
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
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)
Further reading
-
- Cell Biology
Classical G-protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how well internalized receptors are supplied with G proteins. To address this gap, we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20–30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial-trans Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our findings provide a subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments.
-
- Cell Biology
Anionic lipid molecules, including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), are implicated in the regulation of epidermal growth factor receptor (EGFR). However, the role of the spatiotemporal dynamics of PI(4,5)P2 in the regulation of EGFR activity in living cells is not fully understood, as it is difficult to visualize the local lipid domains around EGFR. Here, we visualized both EGFR and PI(4,5)P2 nanodomains in the plasma membrane of HeLa cells using super-resolution single-molecule microscopy. The EGFR and PI(4,5)P2 nanodomains aggregated before stimulation with epidermal growth factor (EGF) through transient visits of EGFR to the PI(4,5)P2 nanodomains. The degree of coaggregation decreased after EGF stimulation and depended on phospholipase Cγ, the EGFR effector hydrolyzing PI(4,5)P2. Artificial reduction in the PI(4,5)P2 content of the plasma membrane reduced both the dimerization and autophosphorylation of EGFR after stimulation with EGF. Inhibition of PI(4,5)P2 hydrolysis after EGF stimulation decreased phosphorylation of EGFR-Thr654. Thus, EGFR kinase activity and the density of PI(4,5)P2 around EGFR molecules were found to be mutually regulated.