Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling.

Organs are anatomically compartmentalised to cater for specialised functions. In the small intestine (SI), regionalisation enables sequential processing of food and nutrient absorption. While several studies indicate the critical importance of non-epithelial cells during development and homeostasis, the extent to which these cells contribute to regionalisation during morphogenesis remains unexplored. Here, we identify a mesenchymal-epithelial crosstalk that shapes the developing SI during late morphogenesis. We find that subepithelial mesenchymal cells are characterised by gradients of factors supporting Wnt signalling and stimulate epithelial growth in vitro. Such a gradient impacts epithelial gene expression and regional villus formation along the anterior-posterior axis of the SI. Notably, we further provide evidence that Wnt signalling directly regulates epithelial expression of Sonic Hedgehog (SHH), which, in turn, acts on mesenchymal cells to drive villi formation. Taken together our results uncover a mechanistic link between Wnt and Hedgehog signalling across different cellular compartments that is central for anterior-posterior regionalisation and correct formation of the SI.

Drinking and Water Handling in the Medaka Intestine: A Possible Role of Claudin-15 in Paracellular Absorption?

When euryhaline fish move between fresh water (FW) and seawater (SW), the intestine undergoes functional changes to handle imbibed SW. In Japanese medaka, the potential transcellular aquaporin-mediated conduits for water are paradoxically downregulated during SW acclimation, suggesting paracellular transport to be of principal importance in hyperosmotic conditions. In mammals, intestinal claudin-15 (CLDN15) forms paracellular channels for small cations and water, which may participate in water transport. Since two cldn15 paralogs, cldn15a and cldn15b, have previously been identified in medaka, we examined the salinity effects on their mRNA expression and immunolocalization in the intestine. In addition, we analyzed the drinking rate and intestinal water handling by adding non-absorbable radiotracers, 51-Cr-EDTA or 99-Tc-DTPA, to the water. The drinking rate was >2-fold higher in SW than FW-acclimated fish, and radiotracer experiments showed anterior accumulation in FW and posterior buildup in SW intestines. Salinity had no effect on expression of cldn15a, while cldn15b was approximately 100-fold higher in FW than SW. Despite differences in transcript dynamics, Cldn15a and Cldn15b proteins were both similarly localized in the apical tight junctions of enterocytes, co-localizing with occludin and with no apparent difference in localization and abundance between FW and SW. The stability of the Cldn15 protein suggests a physiological role in water transport in the medaka intestine.

Accelerated redevelopment of vocal skills is preceded by lasting reorganization of the song motor circuitry.

Complex motor skills take considerable time and practice to learn. Without continued practice the level of skill performance quickly degrades, posing a problem for the timely utilization of skilled motor behaviors. Here we quantified the recurring development of vocal motor skills and the accompanying changes in synaptic connectivity in the brain of a songbird, while manipulating skill performance by consecutively administrating and withdrawing testosterone. We demonstrate that a songbird with prior singing experience can significantly accelerate the re-acquisition of vocal performance. We further demonstrate that an increase in vocal performance is accompanied by a pronounced synaptic pruning in the forebrain vocal motor area HVC, a reduction that is not reversed when birds stop singing. These results provide evidence that lasting synaptic changes in the motor circuitry are associated with the savings of motor skills, enabling a rapid recovery of motor performance under environmental time constraints.

Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo.

RESOLFT fluorescence nanoscopy can nowadays image details far beyond the diffraction limit. However, signal to noise ratio (SNR) and temporal resolution are still a concern, especially deep inside living cells and organisms. In this work, we developed a non-deterministic scanning approach based on a real-time feedback system which speeds up the acquisition up to 6-fold and decreases the light dose by 70-90% for in vivo imaging. Also, we extended the information content of the images by acquiring the complete temporal evolution of the fluorescence generated by reversible switchable fluorescent proteins. This generates a series of images with different spatial resolution and SNR, from conventional to RESOLFT images, which combined through a multi-image deconvolution algorithm further enhances the effective resolution. We reported nanoscale imaging of organelles up to 35 Hz and actin dynamics during an invasion process at a depth of 20-30 µm inside a living Caenorhabditis elegans worm.

Forces drive basement membrane invasion in Caenorhabditis elegans.

During invasion, cells breach basement membrane (BM) barriers with actin-rich protrusions. It remains unclear, however, whether actin polymerization applies pushing forces to help break through BM, or whether actin filaments play a passive role as scaffolding for targeting invasive machinery. Here, using the developmental event of anchor cell (AC) invasion in Caenorhabditis elegans, we observe that the AC deforms the BM and underlying tissue just before invasion, exerting forces in the tens of nanonewtons range. Deformation is driven by actin polymerization nucleated by the Arp2/3 complex and its activators, whereas formins and cross-linkers are dispensable. Delays in invasion upon actin regulator loss are not caused by defects in AC polarity, trafficking, or secretion, as appropriate markers are correctly localized in the AC even when actin is reduced and invasion is disrupted. Overall force production emerges from this study as one of the main tools that invading cells use to promote BM disruption in C. elegans.

Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy.

Reversibly photoswitchable fluorescent proteins (rsFPs) are gaining popularity as tags for optical nanoscopy because they make it possible to image with lower light doses. However, green rsFPs need violet-blue light for photoswitching, which is potentially phototoxic and highly scattering. We developed new rsFPs based on FusionRed that are reversibly photoswitchable with green-orange light. The rsFusionReds are bright and exhibit rapid photoswitching, thereby enabling nanoscale imaging of living cells.

Role of Gln222 in Photoswitching of Aequorea Fluorescent Proteins: A Twisting and H-Bonding Affair?

Reversibly photoswitchable fluorescent proteins (RSFPs) admirably combine the genetic encoding of fluorescence with the ability to repeatedly toggle between a bright and dark state, adding a new temporal dimension to the fluorescence signal. Accordingly, in recent years RSFPs have paved the way to novel applications in cell imaging that rely on their reversible photoswitching, including many super-resolution techniques such as F-PALM, RESOLFT, and SOFI that provide nanoscale pictures of the living matter. Yet many RSFPs have been engineered by a rational approach only to a limited extent, in the absence of clear structure-property relationships that in most cases make anecdotic the emergence of the photoswitching. We reported [ Bizzarri et al. J. Am Chem Soc. 2010 , 102 , 85 ] how the E222Q replacement is a single photoswitching mutation, since it restores the intrinsic cis-trans photoisomerization properties of the chromophore in otherwise nonswitchable Aequorea proteins of different color and mutation pattern (Q-RSFPs). We here investigate the subtle role of Q222 on the excited-state photophysics of the two simplest Q-RSFPs by a combined experimental and theoretical approach, using their nonswitchable anacestor EGFP as benchmark. Our findings link indissolubly photoswitching and Q222 presence, by a simple yet elegant scenario: largely twisted chromophore structures around the double bond (including hula-twist configurations) are uniquely stabilized by Q222 via H-bonds. Likely, these H-bonds subtly modulate the electronic properties of the chromophore, enabling the conical intersection that connects the excited cis to ground trans chromophore. Thus, Q222 belongs to a restricted family of single mutations that change dramatically the functional phenotype of a protein. The capability to distinguish quantitatively T65S/E222Q EGFP ("WildQ", wQ) from the spectrally identical EGFP by quantitative Optical Lock-In Detection (qOLID) witnesses the relevance of this mutation for cell imaging.

Fluorescent Diarylethene Photoswitches-A Universal Tool for Super-Resolution Microscopy in Nanostructured Materials.

Super-resolution fluorescence microscopy allows for unprecedented in situ visualization of biological structures, but its application to materials science has so far been comparatively limited. One of the main reasons is the lack of powerful dyes that allow for labeling and photoswitching in materials science systems. In this study it is shown that appropriate substitution of diarylethenes bearing a fluorescent closed and dark open form paves the way for imaging nanostructured materials with three of the most popular super-resolution fluorescence microscopy methods that are based on different concepts to achieve imaging beyond the diffraction limit of light. The key to obtain optimal resolution lies in a proper control over the photochemistry of the photoswitches and its adaption to the system to be imaged. It is hoped that the present work will provide researchers with a guide to choose the best photoswitch derivative for super-resolution microscopy in materials science, just like the correct choice of a Swiss Army Knife's tool is essential to fulfill a given task.

Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier.

In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS) to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED) images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that transport their cargo directly through the skin barrier, but mainly burst and fuse with the outer lipid layers of the stratum corneum. It was also found that the flexible liposomes showed a greater delivery of the fluorophore into the stratum corneum, indicating that they functioned as chemical permeability enhancers.

Systematic variation of gel-phase texture in phospholipid membranes.

The tilted gel phase of lipid bilayers can display in-plane orientational texture due to long-range alignment of the molecular director. We explore systematic variations of texture defects in a series of binary phospholipid membranes. Using polarized two-photon fluorescence microscopy, the texture pattern of single domains is revealed. The appearance of a central vortex-type defect in each domain correlates with a particular range of hydrophobic mismatch values h > 1 nm at the domain border while domains with h < 1 nm correlate with uniformly aligned texture. The central vortex defect is characterized by a defect angle, indicating its bend or splay nature. Using image analysis, we measure the defect angle and find that it has primarily bend character for small mismatch values (h ≈ 1 nm) and primarily splay nature for larger values of h. For domains containing a vortex, the domain shape is decoupled from the texture while for uniformly textured domains there is a preferred texture orientation of ≃45° along the domain border. The results establish a foundation for understanding texture phenomena in compositionally complex membranes.

Hydrophobic Mismatch Triggering Texture Defects in Membrane Gel Domains.

The orientational texture of gel-phase lipid bilayers is a phenomenon that can structure membrane domains. Using two-photon polarized fluorescence microscopy and image analysis, we map the lateral variation of the lipid orientation (the texture) in single domains. With this method, we uncover a lipid-induced transition between vortex and uniform textures in binary phospholipid bilayers. By tuning the lipid composition, the hydrophobic mismatch at the domain boundary can be varied systematically as monitored by AFM. Low hydrophobic mismatch correlates with domains having uniform texture, while higher mismatch values correlate with a vortex-type texture. The defect pattern created during early growth persists in larger domains, and a minimal model incorporating the anisotropic line tension and the vortex energy can rationalize this finding. The results suggest that the lipid composition and the domain nucleation process are critical factors that determine the texture pattern of membrane domains.