Localization of VE-cadherin in plasmalemmal cholesterol rich microdomains and the effects of cholesterol depletion on VE-cadherin mediated cell-cell adhesion.

VE-cadherin is the predominant adhesion molecule in vascular endothelial cells being responsible for maintenance of the endothelial barrier function by forming adhesive contacts (adherens junctions) to neighbouring cells. We found by use of single molecule fluorescence microscopy that VE-cadherin is localised in preformed clusters when not inside adherens junctions. These clusters depend on the integrity of the actin cytoskeleton and are localised in cholesterol rich microdomains of mature endothelial cells as found by membrane fractionation. The ability to form and maintain VE-cadherin based junctions was probed using the laser tweezer technique, and we found that cholesterol depletion has dramatical effects on VE-cadherin mediated adhesion. While a 30% reduction of the cholesterol-level results in an increase of adhesion, excessive cholesterol depletion by about 60% leads to an almost complete loss of VE-cadherin function. Nevertheless, the cadherin concentration in the membrane and the single molecule kinetic parameters of the cadherin are not changed. Our results suggest that the actin cytoskeleton, junction-associated proteins and protein-lipid assemblies in cholesterol-rich micro-domains mutually stabilise each other to form functional adhesion contacts.

Extraplastidial cytidinediphosphate diacylglycerol synthase activity is required for vegetative development in Arabidopsis thaliana.

Cytidinediphosphate diacylglycerol synthase (CDS) catalyzes the activation of phosphatidic acid to cytidinediphosphate (CDP)-diacylglycerol, a central intermediate in glycerolipid biosynthesis in prokaryotic and eukaryotic organisms. Cytidinediphosphate-diacylglycerol is the precursor to phosphatidylinositol, phosphatidylglycerol (PG) and cardiolipin of eukaryotic phospholipids that are essential for various cellular functions. Isoforms of CDS are located in plastids, mitochondria and the endomembrane system of plants and are encoded by five genes in Arabidopsis. Two genes have previously been shown to code for the plastidial isoforms which are indispensable for the biosynthesis of plastidial PG, and thus biogenesis and function of thylakoid membranes. Here we have focused on the extraplastidial CDS isoforms, encoded by CDS1 and CDS2 which are constitutively expressed contrary to CDS3. We provide evidence that these closely related CDS genes code for membrane proteins located in the endoplasmic reticulum and possess very similar enzymatic properties. Development and analysis of Arabidopsis mutants lacking either one or both CDS1 and CDS2 genes clearly shows that these two genes have redundant functions. As reflected in the seedling lethal phenotype of the cds1cds2 double mutant, plant cells require at least one catalytically active microsomal CDS isoform for cell division and expansion. According to the altered glycerolipid composition of the double mutant in comparison with wild-type seedlings, it is likely that the drastic decrease in the level of phosphatidylinositol and the increase in phosphatidic acid cause defects in cell division and expansion.

Two closely related genes of Arabidopsis encode plastidial cytidinediphosphate diacylglycerol synthases essential for photoautotrophic growth.

Cytidinediphosphate diacylglycerol synthase (CDS) catalyzes the formation of cytidinediphosphate diacylglycerol, an essential precursor of anionic phosphoglycerolipids like phosphatidylglycerol or -inositol. In plant cells, CDS isozymes are located in plastids, mitochondria, and microsomes. Here, we show that these isozymes are encoded by five genes in Arabidopsis (Arabidopsis thaliana). Alternative translation initiation or alternative splicing of CDS2 and CDS4 transcripts can result in up to 10 isoforms. Most of the cDNAs encoding the various plant isoforms were functionally expressed in yeast and rescued the nonviable phenotype of the mutant strain lacking CDS activity. The closely related genes CDS4 and CDS5 were found to encode plastidial isozymes with similar catalytic properties. Inactivation of both genes was required to obtain Arabidopsis mutant lines with a visible phenotype, suggesting that the genes have redundant functions. Analysis of these Arabidopsis mutants provided further independent evidence for the importance of plastidial phosphatidylglycerol for structure and function of thylakoid membranes and, hence, for photoautotrophic growth.

The function of 7D-cadherins: a mathematical model predicts physiological importance for water transport through simple epithelia.

BACKGROUND: 7D-cadherins like LI-cadherin are cell adhesion molecules and represent exceptional members of the cadherin superfamily. Although LI-cadherin was shown to act as a functional Ca2+-dependent adhesion molecule, linking neighboring cells together, and to be dysregulated in a variety of diseases, the physiological role is still enigmatic. Interestingly 7D-cadherins occur only in the lateral plasma membranes of cells from epithelia of water transporting tissues like the gut, the liver or the kidney. Furthermore LI-cadherin was shown to exhibit a highly cooperative Ca2+-dependency of the binding activity. Thus it is tempting to assume that LI-cadherin regulates the water transport through the epithelium in a passive fashion by changing its binding activity in dependence on the extracellular Ca2+. RESULTS: We developed a simple mathematical model describing the epithelial lining of a lumen with a content of variable osmolarity covering an interstitium of constant osmolarity. The width of the lateral intercellular cleft was found to influence the water transport significantly. In the case of hypertonic luminal content a narrow cleft is necessary to further increase concentration of the luminal content. If the cleft is too wide, the water flux will change direction and water is transported into the lumen. Electron microscopic images show that in fact areas of the gut can be found where the lateral intercellular cleft is narrow throughout the lateral cell border whereas in other areas the lateral intercellular cleft is widened. CONCLUSIONS: Our simple model clearly predicts that changes of the width of the lateral intercellular cleft can regulate the direction and efficiency of water transport through a simple epithelium. In a narrow cleft the cells can increase the concentration of osmotic active substances easily by active transport whereas if the cleft is wide, friction is reduced but the cells can hardly build up high osmotic gradients. It is now tempting to speculate that 7D-cadherins, owing to their location and their Ca2+-dependence, will adapt their binding activity and thereby the width of the lateral intercellular cleft automatically as the Ca2+-concentration is coupled to the overall electrolyte concentration in the lateral intercellular cleft. This could provide a way to regulate the water resorption in a passive manner adapting to different osmotic conditions.

A simple and cheap aerosol penetrometer for filter testing using an electronic cigarette.

Background: During the coronavirus disease 2019 (COVID-19) pandemic face masks grew in importance as their use by the general population was recommended by health officials in order to minimize the risk of infection and prevent further spread of the virus. To ensure health protection of medical personnel and other system relevant staff, it is of considerable interest to quickly test if a certain lot of filtering facepiece masks meets the requirements or if the penetration changes under different conditions. As certified penetrometers are rather expensive and were difficult to obtain during the COVID-19 pandemic, we describe two quite simple and cheap methods to quickly test the filter penetration based on an electronic cigarette. Methods: The first method uses a precision scale, the second method uses a light scattering detector to measure the filter penetration. To make sure these two methods yield reliable results, both were tested with freshly cut filter samples covering the range of approx. 2 % to 60 % filter penetration and compared to the results of a certified penetrometer. Results: The comparison of the two methods with the certified penetrometer showed a good correlation and therefore allow a quick and rather reliable estimation of the penetration. Conclusions: Several examples about the use of faulty masks and the resulting health risks show that simple, fast, cheap and broadly available methods for filter characterization might be useful in these days.

A novel device for elimination of cancer cells from blood specimens.

Circulating tumor cells (CTCs) are derivatives of solid cancerous lesions that detach from the tumor mass and enter the blood circulation. CTCs are considered to be the precursors of metastasis in several cancer types. They are present in the blood of cancer patients as single cells or clusters, with the latter being associated with a higher metastatic potential. Methods to eliminate CTCs from the bloodstream are currently lacking. Here, we took advantage of the lower shear stress-resistance of cancer cells compared to blood cells, and developed a device that can eliminate cancer cells without blood damage. The device consists of an axial pump and a coupled rotating throttle, controllable to prevent local blood flow impairment, yet maintaining a constant shear performance. When processing cancer cells through our device, we observe cancer cell-cluster disruption and viability reduction of single cancer cells, without noticeable effects on human blood cells. When injecting cancer cell-containing samples into tumor-free recipient mice, processed samples fail to generate metastasis. Together, our data show that a selective disruption of cancer cells is possible while preserving blood cells, paving the way towards the development of novel, implantable tools for CTC disruption and metastasis prevention.

Modeling of Zinc Dynamics in the Synaptic Cleft: Implications for Cadherin Mediated Adhesion and Synaptic Plasticity.

While the numerous influences of synaptically released zinc on synaptic efficiency during long-term potentiation have been discussed by many authors already, we focused on the possible effect of zinc on cadherins and therefore its contribution to morphological changes in the context of synaptic plasticity. The difficulty with gaining insights into the dynamics of zinc-cadherin interaction is the inability to directly observe it on a suitable timescale. Therefore our approach was to establish an analytical model of the zinc diffusion dynamics in the synaptic cleft and experimentally validate, if the theoretical concentrations at the periphery of the synaptic cleft are sufficient to significantly modulate cadherin-mediated adhesion. Our results emphasize, that synaptically released zinc might have a strong accelerating effect on the morphological changes involved in long-term synaptic plasticity. The approach presented here might also prove useful for investigations on other synaptically released trace metals.

Water transport through the intestinal epithelial barrier under different osmotic conditions is dependent on LI-cadherin trans-interaction.

In the intestine water has to be reabsorbed from the chymus across the intestinal epithelium. The osmolarity within the lumen is subjected to high variations meaning that water transport often has to take place against osmotic gradients. It has been hypothesized that LI-cadherin is important in this process by keeping the intercellular cleft narrow facilitating the buildup of an osmotic gradient allowing water reabsorption. LI-cadherin is exceptional among the cadherin superfamily with respect to its localization along the lateral plasma membrane of epithelial cells being excluded from adherens junction. Furthermore it has 7 but not 5 extracellular cadherin repeats (EC1-EC7) and a small cytosolic domain. In this study we identified the peptide VAALD as an inhibitor of LI-cadherin trans-interaction by modeling the structure of LI-cadherin and comparison with the known adhesive interfaces of E-cadherin. This inhibitory peptide was used to measure LI-cadherin dependency of water transport through a monolayer of epithelial CACO2 cells under various osmotic conditions. If LI-cadherin trans-interaction was inhibited by use of the peptide, water transport from the luminal to the basolateral side was impaired and even reversed in the case of hypertonic conditions whereas no effect could be observed at isotonic conditions. These data are in line with a recently published model predicting LI-cadherin to keep the width of the lateral intercellular cleft small. In this narrow cleft a high osmolarity can be achieved due to ion pumps yielding a standing osmotic gradient allowing water absorption from the gut even if the faeces is highly hypertonic.

Adaptive camouflage: what can be learned from the wetting behaviour of the tropical flat bugs Dysodius lunatus and Dysodiusmagnus.

The neotropical flat bug species Dysodius lunatus and Dysodius magnus show a fascinating camouflage principle, as their appearance renders the animal hardly visible on the bark of trees. However, when getting wet due to rain, bark changes its colour and gets darker. In order to keep the camouflage effect, it seems that some Dysodius species benefit from their ability to hold a water film on their cuticle and therefore change their optical properties when also wetted by water. This camouflage behaviour requires the insect to have a hydrophilic surface and passive surface structures which facilitate the liquid spreading. Here we show morphological and chemical characterisations of the surface, especially the cuticular waxes of D. magnus Scanning electron microscopy revealed that the animal is covered with pillar-like microstructures which, in combination with a surprising chemical hydrophilicity of the cuticle waxes, render the bug almost superhydrophilic: water spreads immediately across the surface. We could theoretically model this behaviour assuming the effect of hemi-wicking (a state in which a droplet sits on a rough surface, partwise imbibing the structure around).  Additionally the principle was abstracted and a laser-patterned polymer surface, mimicking the structure and contact angle of Dysodius wax, shows exactly the behaviour of the natural role model - immediate spreading of water and the formation of a thin continuous water film changing optical properties of the surface.

The plant hopper Issus coleoptratus can detoxify phloem sap saponins including the degradation of the terpene core.

Issus coleoptratus is a small plant hopper which mainly feeds on the phloem sap from ivy. Although all parts of ivy are poisonous as the plant contains saponins, especially hederasaponins, I. coleoptratus can cope with the poison. In contrast to other animals like the stick insect Carausius morosus which accumulates saponins in its body, I. coleoptratus can degrade and disintegrate not only the saponins but even the genines, i.e. the triterpene core of the substances. This is perhaps made possible by a specialised midgut and/or the salivary glands. When the glands and the gut are dissected and added to saponins in solution, the saponins, including the genines, are degraded ex vivo.

Mitochondrial Ca(2+) mobilization is a key element in olfactory signaling.

In olfactory sensory neurons (OSNs), cytosolic Ca(2+) controls the gain and sensitivity of olfactory signaling. Important components of the molecular machinery that orchestrates OSN Ca(2+) dynamics have been described, but key details are still missing. Here, we demonstrate a critical physiological role of mitochondrial Ca(2+) mobilization in mouse OSNs. Combining a new mitochondrial Ca(2+) imaging approach with patch-clamp recordings, organelle mobility assays and ultrastructural analyses, our study identifies mitochondria as key determinants of olfactory signaling. We show that mitochondrial Ca(2+) mobilization during sensory stimulation shapes the cytosolic Ca(2+) response profile in OSNs, ensures a broad dynamic response range and maintains sensitivity of the spike generation machinery. When mitochondrial function is impaired, olfactory neurons function as simple stimulus detectors rather than as intensity encoders. Moreover, we describe activity-dependent recruitment of mitochondria to olfactory knobs, a mechanism that provides a context-dependent tool for OSNs to maintain cellular homeostasis and signaling integrity.

Heterotypic trans-interaction of LI- and E-cadherin and their localization in plasmalemmal microdomains.

Cadherins are calcium-dependent adhesion molecules important for tissue morphogenesis and integrity. LI-cadherin and E-cadherin are the two prominent cadherins in intestinal epithelial cells. Whereas LI-cadherin belongs to the subfamily of 7D (seven-domain)-cadherins defined by their seven extracellular cadherin repeats and short intracellular domain, E-cadherin is the prototype of classical cadherins with five extracellular domains and a highly conserved cytoplasmic part that interacts with catenins and thereby modulates the organization of the cytoskeleton. Here, we report a specific heterotypic trans-interaction of LI- with E-cadherin, two cadherins of distinct subfamilies. Using atomic force microscopy and laser tweezer experiments, the trans-interaction of LI- and E-cadherin was characterized on the single-molecule level and on the cellular level, respectively. This heterotypic interaction showed similar binding strength (20-52 pN at 200-4000 nm/s) and lifetime (0.8 s) as the respective homotypic interactions of LI- and E-cadherin. VE-cadherin, another classical cadherin, did not bind to LI-cadherin. In enterocytes, LI-cadherin and E-cadherin are located in different membrane regions. LI-cadherin is distributed along the basolateral membrane, whereas the majority of E-cadherin is concentrated in adherens junctions. This difference in membrane distribution was also reflected in Chinese hamster ovary cells stably expressing either LI- or E-cadherin. We found that LI-cadherin is localized almost exclusively in cholesterol-rich fractions, whereas E-cadherin is excluded from these membrane fractions. Given their different membrane localization in enterocytes, the heterotypic trans-interaction of LI- and E-cadherin might play a role during development of the intestinal epithelium when the cells do not yet have elaborate membrane specializations.

Investigating the locomotion of the sandfish in desert sand using NMR-imaging.

The sandfish (Scincus scincus) is a lizard having the remarkable ability to move through desert sand for significant distances. It is well adapted to living in loose sand by virtue of a combination of morphological and behavioural specializations. We investigated the bodyform of the sandfish using 3D-laserscanning and explored its locomotion in loose desert sand using fast nuclear magnetic resonance (NMR) imaging. The sandfish exhibits an in-plane meandering motion with a frequency of about 3 Hz and an amplitude of about half its body length accompanied by swimming-like (or trotting) movements of its limbs. No torsion of the body was observed, a movement required for a digging-behaviour. Simple calculations based on the Janssen model for granular material related to our findings on bodyform and locomotor behaviour render a local decompaction of the sand surrounding the moving sandfish very likely. Thus the sand locally behaves as a viscous fluid and not as a solid material. In this fluidised sand the sandfish is able to "swim" using its limbs.

Transglutaminase 1 stabilizes beta-actin in endothelial cells correlating with a stabilization of intercellular junctions.

Microvascular endothelial monolayers from mouse myocardium become resistant to various barrier-compromising stimuli correlating with the expression of transglutaminase 1 (TGase1) and its translocation towards cellular junctions. In contrast, endothelial monolayers from mouse lung microvessels do not express TGase1 and remain sensitive to barrier-compromising stimuli corresponding to the known in vivo sensitivity of the lung microvasculature. Using the TGase-substrate 5-(biotinamido)-pentylamine, specific TGase inhibitors and RNAi, one target protein of TGase1 in endothelial cells was found to be beta-actin, suggesting that tissue-specific stabilization of the cortical actin filament network by intracellular TGase1 activity may play a role in controlling barrier properties of endothelial monolayers.

Role of transglutaminase 1 in stabilisation of intercellular junctions of the vascular endothelium.

Microvascular endothelial monolayers from mouse myocardium (MyEnd) cultured for up to 5 days postconfluency became increasingly resistant to various barrier-compromising stimuli such as low extracellular Ca(2+) and treatment with the Ca(2+) ionophore A23187 and with the actin depolymerising compound cytochalasin D. In contrast, microvascular endothelial monolayers from mouse lung microvessels (PulmEnd) remained sensitive to these conditions during the entire culture period which corresponds to the well-known in vivo sensitivity of the lung microvasculature to Ca(2+) depletion and cytochalasin D treatment. One molecular difference between pulmonary and myocardial endothelial cells was found to be transglutaminase 1 (TGase1) which is strongly expressed in myocardial endothelial cells but is absent from pulmonary endothelial cells. Resistance of MyEnd cells to barrier-breaking conditions correlated strongly with translocation of TGase1 to intercellular junctions. Simultaneous inhibition of intracellular and extracellular TGase activity by monodansylcadaverine (MDC) strongly weakened barrier properties of MyEnd monolayers, whereas inhibition of extracellular TGases by the membrane-impermeable active site-directed TGase inhibitor R281 did not reduce barrier properties. Weakening of barrier properties could be also induced in MyEnd cells by downregulation of TGase1 expression using RNAi-based gene silencing. These findings suggest that crosslinking activity of intracellular TGase1 at intercellular junctions may play a role in controlling barrier properties of endothelial monolayers.