Fluorescence-based super-resolution-microscopy strategies for chromatin studies

Super-resolution microscopy (SRM) is a prime tool to study chromatin organisation at near biomolecular resolution in the native cellular environment. With fluorescent labels DNA, chromatin-associated proteins and specific epigenetic states can be identified with high molecular specificity. The aim of this review is to introduce the field of diffraction-unlimited SRM to enable an informed selection of the most suitable SRM method for a specific chromatin-related research question. We will explain both diffraction-unlimited approaches (coordinate-targeted and stochastic-localisation-based) and list their characteristic spatio-temporal resolutions, live-cell compatibility, image-processing, and ability for multi-colour imaging. As the increase in resolution, compared to, e.g. confocal microscopy, leads to a central role of the sample quality, important considerations for sample preparation and concrete examples of labelling strategies applicable to chromatin research are discussed. To illustrate how SRM-based methods can significantly improve our understanding of chromatin functioning, and to serve as an inspiring starting point for future work, we conclude with examples of recent applications of SRM in chromatin research.</p

The patterned assembly and stepwise Vps4-mediated disassembly of composite ESCRT-III polymers drives archaeal cell division

ESCRT-III family proteins form composite polymers that deform and cut membrane tubes in the context of a wide range of cell biological processes across the tree of life. In reconstituted systems, sequential changes in the composition of ESCRT-III polymers induced by the AAA-adenosine triphosphatase Vps4 have been shown to remodel membranes. However, it is not known how composite ESCRT-III polymers are organized and remodeled in space and time in a cellular context. Taking advantage of the relative simplicity of the ESCRT-III-dependent division system in Sulfolobus acidocaldarius, one of the closest experimentally tractable prokaryotic relatives of eukaryotes, we use super-resolution microscopy, electron microscopy, and computational modeling to show how CdvB/CdvB1/CdvB2 proteins form a precisely patterned composite ESCRT-III division ring, which undergoes stepwise Vps4-dependent disassembly and contracts to cut cells into two. These observations lead us to suggest sequential changes in a patterned composite polymer as a general mechanism of ESCRT-III-dependent membrane remodeling. </p

Altered T Cell Memory and Effector Cell Development in Chronic Lymphatic Filarial Infection That Is Independent of Persistent Parasite Antigen

Chronic lymphatic filarial (LF) infection is associated with suppression of parasite-specific T cell responses that persist even following elimination of infection. While several mechanisms have been implicated in mediating this T cell specific downregulation, a role for alterations in the homeostasis of T effector and memory cell populations has not been explored. Using multiparameter flow cytometry, we investigated the role of persistent filarial infection on the maintenance of T cell memory in patients from the filarial-endemic Cook Islands. Compared to filarial-uninfected endemic normals (EN), microfilaria (mf) positive infected patients (Inf) had a reduced CD4 central memory (TCM) compartment. In addition, Inf patients tended to have more effector memory cells (TEM) and fewer effector cells (TEFF) than did ENs giving significantly smaller TEFF ∶ TEM ratios. These contracted TCM and TEFF populations were still evident in patients previously mf+ who had cleared their infection (CLInf). Moreover, the density of IL-7Rα, necessary for T memory cell maintenance (but decreased in T effector cells), was significantly higher on memory cells of Inf and CLInf patients, although there was no evidence for decreased IL-7 or increased soluble IL7-Rα, both possible mechanisms for signaling defects in memory cells. However, effector cells that were present in Inf and CLInf patients had lower percentages of HLA-DR suggesting impaired function. These changes in T cell populations appear to reflect chronicity of infection, as filarial-infected children, despite the presence of active infection, did not show alterations in the frequencies of these T cell phenotypes. These data indicate that filarial-infected patients have contracted TCM compartments and a defect in effector cell development, defects that persist even following clearance of infection. The fact that these global changes in memory and effector cell compartments do not yet occur in infected children makes early treatment of LF even more crucial

Temperature-responsive and biocompatible nanocarriers based on clay nanotubes for controlled anti-cancer drug release

Administration of temperature-responsive drug carriers that release anticancer drugs at high temperatures can benefit hyperthermia therapies because of the synergistic effect of anticancer drug molecules and high temperature on killing the cancer cells. In this study, we design and characterize a new temperature-responsive nanocarrier based on a naturally occurring and biocompatible clay mineral, halloysite nanotubes. Poly(N-isopropylacrylamide) brushes were grown on the surface of halloysite nanotubes using a combination of mussel-inspired dopamine polymerization and surface-initiated atom transfer radical polymerization. The chemical structure of the hybrid materials was investigated using X-ray photoelectron spectroscopy, thermogravimetric analysis and energy-dispersive X-ray spectroscopy. The hybrid material was shown to have a phase transition temperature of about 32 °C, corresponding to a 40 nm thick polymer layer surrounding the nanotubes. Cell studies suggested that grafting of poly(N-isopropylacrylamide) brushes on the polydopamine-modified halloysite nanotubes suppresses the cytotoxicity caused by the polydopamine interlayer and drug release studies on nanotubes loaded with doxorubicin showed that thanks to the poly(N-isopropylacrylamide) brushes a temperature-dependent drug release is observed. Finally, a fluorescent dye molecule was covalently attached to the polymer-grafted nanotubes and stimulated emission depletion nanoscopy was used to confirm the internalization of the nanotubes in HeLa cells