In this second Editorial of 2020, we provide a brief synopsis of three Original Papers nicely illustrating the breadth and variety of histochemical and cell biological investigations. We begin by highlighting a new one-step method for staining semithin sections from tissues prepared for transmission electron microscopy, and then cover detailed cell biological and high-resolution imaging approaches to investigate mechanisms involved in the trafficking of a cell surface receptor to the nucleus, and the organellar origin of the membrane of phagophores responsible for selective autophagy of insoluble cytoplasmic protein aggregates. We hope you enjoy reading about these highlighted studies, as well as the other interesting offerings in this issue.
One-step polychromatic staining of semithin sections of epoxy-embedded tissues
Stained semithin sections prepared from double aldehyde- and osmium-fixed and resin-embedded tissues and cells provide high structural resolution for light microscopy, and importantly the same specimens can be subsequently analyzed by transmission electron microscopy (Loussert Fonta and Humbel 2015). Among the various mono- and polychromatic staining protocols, relatively few do not require prior removal of the resin and osmium (Horobin 1983). Recently, Morikawa et al. (2018) reported the use of an alkaline alcoholic solution of azure B and basic fuchsin as a polychromatic stain for semithin sections of double aldehyde-fixed and epoxy-embedded tissues. Manskikh and Sheval (2020) report now a simple, one-step polychromatic staining protocol using an aqueous alcoholic mixture of neutral red and fast green FCF, which is based on the histological stain introduced by Twort (1924). The adapted Twort’s staining protocol was performed on semithin sections (0.25–0.5 μm thin) of glutaraldehyde- and osmium-fixed and epoxy resin-embedded tissues, and does not require a pretreatment to remove the epoxy resin and osmium. When directly compared with methylene blue-stained semithin sections, the neutral red and fast green FCF provided very high contrast due to the polychromatic staining of the different tissue elements (Fig. 1). The various cellular structures could be clearly distinguished since they were stained at greatly different intensities by neutral red. Of note, collagen and elastic fibers were solely stained by fast green FCF. The authors emphasize two aspects crucial for the proper functioning of the polychromatic stain: optimal osmium post-fixation and carefully chosen incubation time. As shown by the authors, the requirement for osmium fixation provided the means for subsequent analysis by transmission electron microscopy, which may be an important aspect in surgical pathology.
Travel stories on a CD
The translocation of plasma membrane proteins and receptors to the cell nucleus to activate transcription factors is a well-known occurrence in multiple signaling cascades (see Dubovy et al. 2018 for a recent example). In this light, the hyaluron-binding surface receptor CD44 has also been shown to exhibit nuclear translocation via two different mechanisms. In the first, intact surface CD44 is proteolytically cleaved and processed to yield a surface ecto-domain and an intracellular domain fragment which then undergoes nuclear translocation (Nagano and Saya 2004). In the second mechanism, intact whole surface CD44 has been postulated to be internalized into the cytoplasm, followed by nuclear translocation of the unprocessed protein (Lee et al. 2009; Janiszewska et al. 2010). Thorne et al. (2020) have now performed experiments on several cultured cell lines aimed at determining whether they can provide supporting evidence for the nuclear translocation of intact surface CD44. They used multiple antibodies recognizing various domains of the CD44 protein in conjunction with siRNA gene editing techniques, western blotting, subcellular fractionation, and semi-quantitative high-resolution confocal microscopy. The results of these detailed experiments failed to find support for the translocation of intact CD44 protein from the cell surface to the nucleus. Rather, clear support for nuclear transport of the cleaved intracellular domain fragment of CD44 was provided. Moreover, the confocal microscopy imaging experiments combined with co-localization and line-scan intensity analyses demonstrated that nuclear localization of intact surface CD44 protein could be detected when a low-resolution 20 × objective lens (0.75 NA) was used versus a high-resolution 63 × (1.4 NA) objective. The authors caution that limitations in experimental imaging techniques may lead to erroneous conclusions concerning immunofluorescence localization of cellular molecules.
Waste removal in a not so rough neighborhood
Cells have developed multiple well-choreographed mechanisms, all falling under the general term “autophagy”, for removing unwanted cytoplasmic debris and organelles during normal cellular physiology, development and disease (Deng et al. 2018; Mizushima 2018; Offei et al. 2018). The materials for disposal may be selectively targeted to a unique internally formed cytoplasmic structure referred to as the “phagophore” via a variety of autophagy receptors. The origin and formation of the phagophore itself have been the subject of intense investigation, with various possibilities posited depending upon cell type and conditions examined. Damaged and misfolded insoluble cytoplasmic protein aggregates are removed from the cell by a special form of selective autophagy called “aggrephagy” (Metcalf et al. 2012). In earlier work, Roth and colleagues showed that the protein EDEM1, a component of the endoplasmic reticulum-associated protein quality control (ERAD), and RER-associated fibrinogen Aα-γ upon exit from the rough endoplasmic reticulum (RER) form insoluble cytoplasmic aggregates which undergo subsequent autophagy (Zuber et al. 2007; LeFourn et al. 2013). This same group has now extended these earlier studies using multifluorescence confocal microscopy combined with serial section immunoelectron microscopy of fed HepG2 cells to determine which cytoplasmic organelle is involved in providing the membrane component of the phagophores utilized in selective autophagy of insoluble protein aggregates (Park et al. 2020). Their careful high-resolution imaging investigation revealed that in fed HepG2 cells, EDEM1 and fibrinogen Aα-γ insoluble cytoplasmic protein aggregates were engulfed by smooth, ribosome-free subdomains of the RER. Importantly, using fed HepG2 cells, the authors were able to demonstrate that these “smooth” RER domains were structurally different from the RER-formed “omegasomes”, thought to provide the membrane component of phagophores in a starvation-induced autophagy model (Lamb et al. 2013). Their results add further structural proof to the contribution of the RER as the origin of the phagophore membrane in selective autophagy.
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Taatjes, D.J., Roth, J. In focus in HCB. Histochem Cell Biol 153, 71–75 (2020). https://doi.org/10.1007/s00418-020-01843-3