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SignificanceThe plant plasma membrane acts as the front line for cellular perception of the environment. As such, signaling and transport proteins which perceive or transport environmental signals, developmental cues, and nutrients are located within it. A number of studies have revealed that proteins located within the plasma membrane do not simply freely diffuse within its plane. Rather, proteins are localized in nanodomains. In addition to the plasma membrane, plant cells also have an extracellular matrix, the cell wall. Here we have shown that the cell wall has a role in regulating the dynamics and size of plasma-membrane nanodomains for proteins involved in morphogenesis (PIN3) and pathogen perception (FLS2). Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.

The organization of chromatin into higher-order structures and its condensation process represent one of the key challenges in structural biology. This is important for elucidating several disease states. To address this long-standing problem, development of advanced imaging methods has played an essential role in providing understanding into mitotic chromosome structure and compaction. Amongst these are two fast evolving fluorescence imaging technologies, specifically fluorescence lifetime imaging (FLIM) and super-resolution microscopy (SRM). FLIM in particular has been lacking in the application of chromosome research while SRM has been successfully applied although not widely. Both these techniques are capable of providing fluorescence imaging with nanometer information. SRM or “nanoscopy” is capable of generating images of DNA with less than 50 nm resolution while FLIM when coupled with energy transfer may provide less than 20 nm information. Here, we discuss the advantages and limitations of both methods followed by their contribution to mitotic chromosome studies. Furthermore, we highlight the future prospects of how advancements in new technologies can contribute in the field of chromosome science.

A Correction to this paper has been published: https://doi.org/10.1007/s10577-021-09662-5

Gap junctions provide a route for small molecules to pass directly between cells. Toxic species may spread through junctions into ‘bystander’ cells, which may be exploited in chemotherapy and radiotherapy. However, this may be prevented by junction closure, and therefore an understanding of the dose-dependency of inhibition of communication and bystander effects is important. Low-energy ionising radiation (ultrasoft X-rays) provides a tool for the study of bystander effects because the area of exposure may be carefully controlled, and thus target cells may be clearly defined. Loss of gap junction-mediated intercellular communication between irradiated cells was dose-dependent, indicating that closure of junctions is proportional to dose. Closure was associated with hyperphosphorylation of connexin43. Inhibition of communication occurred in bystander cells but was not proportional to dose. Inhibition of communication at higher radiation doses may restrict the spread of inhibitory factors, thus protecting bystander cells. The reduction in communication that takes place in bystander cells was dependent on cells being in physical contact, and not on the release of signalling factors into the medium.

Characteristic aluminum K (${\\rm Al}_{{\\rm K}}$) (energy of 1.5 keV) and copper L (${\\rm Cu}_{{\\rm L}}$) (energy of ∼0.96 keV) ultrasoft X rays have been used to investigate the effectiveness of the numerous low-energy secondary electrons produced by low-linear energy transfer (LET) ionizing radiation. Cellular inactivation and induction and rejoining of DNA double-strand breaks (DSBs) in Chinese hamster V79-4 cells irradiated as monolayers with these ultrasoft X radiations have been studied under aerobic and anaerobic conditions. The mean cell thickness, determined by confocal laser scanning fluorescence microscopy, was used to calculate the mean dose to the nucleus of the irradiated cells. Relative to 60 Co γ rays, the relative biological effectiveness (RBE) for cellular inactivation at 10% survival is 1.7 ± 0.1 and 2.3 ± 0.3 for ${\\rm Al}_{{\\rm K}}$ and ${\\rm Cu}_{{\\rm L}}$ ultrasoft X rays, respectively. The RBE values for induction of DSBs of 2.5 ± 0.2 and 3.0 ± 0.3 for ${\\rm Al}_{{\\rm K}}$ and ${\\rm Cu}_{{\\rm L}}$ X rays, respectively, were determined after irradiation at 277 K using the technique of pulsed-field gel electrophoresis. Induction of DSBs is linearly dependent on dose. Oxygen enhancement ratios of 1.9 and 2.1 for cellular inactivation and DSB induction, respectively, were obtained with ${\\rm Al}_{{\\rm K}}$ X rays. These values are less than those for 60 Co γ radiation. The repair kinetics for rejoining of DSBs after a dose of 15 Gy is similar for both X-ray energies and 60 Co γ rays with a first half-life of 18-22 ± 5 min. From these studies, it is suggested that induction of DSBs by low-LET radiations such as 60 Co γ rays reflects clustered damage produced predominantly by low-energy electron \"track ends,\" which represent about 30% of the total dose.

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteinswith divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles inmorphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.

The high‐resolution spatial induction of ultraviolet (UV) photoproducts in mammalian cellular DNA is a goal of many scientists who study UV damage and repair. Here we describe how UV photoproducts can be induced in cellular DNA within nanometre dimensions by near‐diffraction‐limited 750 nm infrared laser radiation. The use of multiphoton excitation to induce highly localized DNA damage in an individual cell nucleus or mitochondrion will provide much greater resolution for studies of DNA repair dynamics and intracellular localization as well as intracellular signalling processes and cell–cell communication. The technique offers an advantage over the masking method for localized irradiation of cells, as the laser radiation can specifically target a single cell and subnuclear structures such as nucleoli, nuclear membranes or any structure that can be labelled and visualized by a fluorescent tag. It also increases the time resolution with which migration of DNA repair proteins to damage sites can be monitored. We define the characteristics of localized DNA damage induction by near‐infrared radiation and suggest how it may be used for new biological investigations.

The application of near infrared (NIR)-light to living systems has been suggested as a potential method to enhance tissue repair, decrease inflammation, and possibly mitigate cancer therapy-associated side effects. In this study, we examined the effect of exposing three cell lines: breast cancer (MCF7), non-cancer breast cells (MCF10A), and lung fibroblasts (IMR-90), to 734 nm NIR-light for 20 minutes per day for six days, and measuring changes in cellular senescence. Positive senescent populations were induced using doxorubicin. Flow cytometry was used to assess relative levels of senescence together with mitochondria-related variables. Exposure to NIR-light significantly increased the level of senescence in MCF7 cells (13.5%; P<0.01), with no observable effects on MCF10A or IMR-90 cell lines. NIR-induced senescence was associated with significant changes in mitochondria homeostasis, including raised ROS level (36.0%; P<0.05) and mitochondrial membrane potential (14.9%; P<0.05), with no changes in mitochondrial Ca2+. These results suggest that NIR-light exposure can significantly arrest the proliferation of breast cancer cells via inducing senescence, while leaving non-cancerous cell lines unaffected.

We report on the first experimental characterization of a laser-wakefield accelerator able to deliver, in a single pulse, doses in excess of \\unit[1]{Gy} on timescales of the order of a hundred femtoseconds, reaching unprecedented average dose-rates up to \\unit[10\\(^{13}\\)]{Gy/s}. The irradiator is demonstrated to deliver doses tuneable up to \\unit[2.2]{Gy} in a cm\\(^2\\) area and with a high degree of longitudinal and transverse uniformity in a single irradiation. In this regime, proof-of-principle irradiation of patient-derived glioblastoma stem-like cells and human skin fibroblast cells show indications of a differential cellular response, when compared to reference irradiations at conventional dose-rates. These include a statistically significant increase in relative biological effectiveness (\\(1.40\\pm0.08\\) at 50\\% survival for both cell lines) and a significant reduction of the relative radioresistance of tumour cells. Data analysis provides preliminary indications that these effects might not be fully explained by induced oxygen depletion in the cells but may be instead linked to a higher complexity of the damages triggered by the ultra-high density of ionising tracks of femtosecond-scale radiation pulses. These results demonstrate an integrated platform for systematic radiobiological studies at unprecedented beam durations and dose-rates, a unique infrastructure for translational research in radiobiology at the femtosecond scale.

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport and cellular signalling. Recent models suggest that for these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signalling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using sub-diffraction-limited Airyscan confocal microscopy. In addition, single particle tracking reveals that these proteins move at different rates within the membrane. We show that the actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by affecting single particle dynamics of two proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for differentially regulating protein cluster size and dynamics thereby contributing to the formation and functionality of membrane nanodomains.