Para-Hydroxycinnamic Acid Mitigates Senescence and Inflammaging in Human Skin Models.

Para-hydroxycinnamic acid (pHCA) is one of the most abundant naturally occurring hydroxycinnamic acids, a class of chemistries known for their antioxidant properties. In this study, we evaluated the impact of pHCA on different parameters of skin aging in in vitro skin models after H2O2 and UV exposure. These parameters include keratinocyte senescence and differentiation, inflammation, and energy metabolism, as well as the underlying molecular mechanisms. Here we demonstrate that pHCA prevents oxidative stress-induced premature senescence of human primary keratinocytes in both 2D and 3D skin models, while improving clonogenicity in 2D. As aging is linked to inflammation, referred to as inflammaging, we analyzed the release of IL-6, IL-8, and PGE2, known to be associated with senescence. All of them were downregulated by pHCA in both normal and oxidative stress conditions. Mechanistically, DNA damage induced by oxidative stress is prevented by pHCA, while pHCA also exerts a positive effect on the mitochondrial and glycolytic functions under stress. Altogether, these results highlight the protective effects of pHCA against inflammaging, and importantly, help to elucidate its potential mechanisms of action.

NET4 and RabG3 link actin to the tonoplast and facilitate cytoskeletal remodelling during stomatal immunity.

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function being downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure involves a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune-triggered osmotic changes and actin cytoskeletal remodelling likely driving compact vacuolar morphologies.

Expansion Microscopy of Plant Cells (PlantExM).

Expansion microscopy (ExM) achieves super-resolution imaging without the need for sophisticated super-resolution microscopy hardware through a combination of physical and optical magnification. Samples are fixed, stained, and embedded in a swellable gel. Following cross-linking of fluorophores to the gel matrix, the components of the sample are digested away and the gel expanded in water. Labeled objects which are too close to be resolved by diffraction-limited microscopy are moved far enough apart that these can now be resolved as individual objects on a standard confocal. Originally developed for animal cells and tissues, ExM for plants requires the additional consideration of cell wall digestion. Super-resolution can be limited in plants due to the size of cells, light scattering of tissues, and variations in refractive index. By removing the components which cause these limitations, ExM opens up the possibility of super-resolution at depth within plant tissues for the first time. Here we describe our method for PlantExM which is optimized for cytoskeleton resolution, which, when also coupled with compatible optical super-resolution technologies, can produce images of the plant cytoskeleton in unprecedented detail.

Light Microscopy Technologies and the Plant Cytoskeleton.

The cytoskeleton is a dynamic and diverse subcellular filament network, and as such microscopy is an essential technology to enable researchers to study and characterize these systems. Microscopy has a long history of observing the plant world not least as the subject where Robert Hooke coined the term "cell" in his publication Micrographia. From early observations of plant morphology to today's advanced super-resolution technologies, light microscopy is the indispensable tool for the plant cell biologist. In this mini review, we will discuss some of the major modalities used to examine the plant cytoskeleton and the theory behind them.

A Potential Role of Keratinocyte-Derived Bilirubin in Human Skin Yellowness and Its Amelioration by Sucrose Laurate/Dilaurate.

Sallow and/or dull skin appearance is greatly attributable to the yellow components of skin tone. Bilirubin is a yellow chromophore known to be made in the liver and/or spleen and is transported throughout the body via the blood stream. Recent publications suggest bilirubin may be synthesized in other cells/organs, including the skin. We found human keratinocytes express the transcripts involved in bilirubin biosynthesis. In parallel, we also found human keratinocytes could indeed synthesize bilirubin in monolayer keratinocytes and in a 3D human skin-equivalent model. The synthesized amount was substantial enough to contribute to skin yellowness. In addition, oxidative stress enhanced bilirubin production. Using UnaG, a protein that forms a fluorescent species upon binding to bilirubin, we also visualized the intracellular expression of bilirubin in keratinocytes. Finally, we screened a compound library and discovered that the sucrose laurate/dilaurate (SDL) combination significantly reduced bilirubin levels, as well as bilirubin-mediated yellowness. In conclusion, bilirubin is indeed synthesized in epidermal keratinocytes and can be upregulated by oxidative stress, which could contribute to chronic or transient yellow skin tone appearance. Application of SDL diminishes bilirubin generation and may be a potential solution to mitigate yellowish and/or dull skin appearance.

NETWORKED2-subfamily proteins regulate the cortical actin cytoskeleton of growing pollen tubes and polarised pollen tube growth.

We have recently characterised NET2A as a pollen-specific actin-binding protein that binds F-actin at the plasma membrane of growing pollen tubes. However, the role of NET2 proteins in pollen development and fertilisation have yet to be elucidated. To further characterise the role of Arabidopsis NET2 proteins in pollen development and fertilisation, we analysed the subcellular localisation of NET2A over the course of pollen grain development and investigated the role of the NET2 family using net2 loss-of-function mutants. We observed NET2A to localise to the F-actin cytoskeleton in developing pollen grains as it underwent striking structural reorganisations at specific stages of development and during germination and pollen tube growth. Furthermore, net2 loss-of-function mutants exhibited striking morphological defects in the early stages of pollen tube growth, arising from frequent changes to pollen tube growth trajectory. We observed defects in the cortical actin cytoskeleton and actin-driven subcellular processes in net2 mutant pollen tubes. We demonstrate that NET2 proteins are essential for normal actin-driven pollen development highlighting an important role for the NET2 family members in regulating pollen tube growth during fertilisation.

Multiple C2 domains and transmembrane region proteins (MCTPs) tether membranes at plasmodesmata.

In eukaryotes, membrane contact sites (MCS) allow direct communication between organelles. Plants have evolved a unique type of MCS, inside intercellular pores, the plasmodesmata, where endoplasmic reticulum (ER)-plasma membrane (PM) contacts coincide with regulation of cell-to-cell signalling. The molecular mechanism and function of membrane tethering within plasmodesmata remain unknown. Here, we show that the multiple C2 domains and transmembrane region protein (MCTP) family, key regulators of cell-to-cell signalling in plants, act as ER-PM tethers specifically at plasmodesmata. We report that MCTPs are plasmodesmata proteins that insert into the ER via their transmembrane region while their C2 domains dock to the PM through interaction with anionic phospholipids. A Atmctp3/Atmctp4 loss of function mutant induces plant developmental defects, impaired plasmodesmata function and composition, while MCTP4 expression in a yeast Δtether mutant partially restores ER-PM tethering. Our data suggest that MCTPs are unique membrane tethers controlling both ER-PM contacts and cell-to-cell signalling.

Pulling force deforms hair follicle root sheath nuclei and surrounding dermal collagen matrix differently at infundibulum, isthmus and suprabulbar regions.

The biomechanical properties of the collagenous dermal matrix are well described but responses to mechanical force by the hair follicles have not been characterised so far. We applied a pulling force on hair follicles to visualise and quantify changes in the keratin-14 and involucrin-positive cell layers of the follicles using nuclear dimensions as an indicator of tissue deformation. Moreover, we used second-harmonic generation imaging to visualise changes in the dermal collagen. We report how the anatomical regions of the follicle respond to the force. Nuclei of the isthmus region were most affected. The nuclei in both K14-positive outer root sheath cells and in involucrin-positive cells were significantly compressed, whereas the response in the infundibulum and suprabulbar regions was more variable. The deformation of the nuclei did not correlate with lamin A/C expression. The changes in the collagenous matrix were distinct at different depths of the dermis as collagen fibrils were compressed closer to each other in the region adjacent to upper suprabulbar follicle and pulled apart near the infundibulum. Thus, the responses to the force are locally defined and the cells in the permanent and cycling parts of the follicle behave differently.

Leaves of isoprene-emitting tobacco plants maintain PSII stability at high temperatures.

At high temperatures, isoprene-emitting plants display a higher photosynthetic rate and a lower nonphotochemical quenching (NPQ) compared with nonemitting plants. The mechanism of this phenomenon, which may be very important under current climate warming, is still elusive. NPQ was dissected into its components, and chlorophyll fluorescence lifetime imaging microscopy (FLIM) was used to analyse the dynamics of excited chlorophyll relaxation in isoprene-emitting and nonemitting plants. Thylakoid membrane stiffness was also measured using atomic force microscope (AFM) to identify a possible mode of action of isoprene in improving photochemical efficiency and photosynthetic stability. We show that, when compared with nonemitters, isoprene-emitting tobacco plants exposed at high temperatures display a reduced increase of the NPQ energy-dependent component (qE) and stable (1) chlorophyll fluorescence lifetime; (2) amplitude of the fluorescence decay components; and (3) thylakoid membrane stiffness. Our study shows for the first time that isoprene maintains PSII stability at high temperatures by preventing the modifications of the surrounding environment, namely providing a more steady and homogeneous distribution of the light-absorbing centres and a stable thylakoid membrane stiffness. Isoprene photoprotects leaves with a mechanism alternative to NPQ, enabling plants to maintain a high photosynthetic rate at rising temperatures.

Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution.

For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.