Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.

Echinococcus multilocularis: the impact of ionizing radiation on metacestodes

Alveolar echinococcosis is caused by the metacestode stage of the fox tapeworm Echinococcus multilocularis. Current chemotherapeutical options for the treatment of echinococcosis are not satisfactory, and novel drugs and/or other potential means of therapy are needed. E. multilocularis metacestodes are characterized by almost potentially unlimited growth, and also display other features of cancerous tumours. In this study, we exposed metacestodes that were generated in vitro to 50-100 Gy ionizing irradiation, and subsequently investigated the short-term (10-12 days post-treatment) and long-term (14 weeks post-treatment) effects. We found, that in the short-term, no release of alkaline phosphatase (EmAP) activity as a measure for potentially induced damage and loss of viability could be detected, and that the protein expression pattern and protease activities in vesicle fluids and medium supernatants did not alter dramatically following irradiation. However, irradiation was associated with distinct morphological and ultrastructural alterations in the tissue of metacestodes, affecting most notably cell-cell contacts, mitochondrial shape, glycogen-storage cells and lipid droplet formation. These could be detected already at 10 days following treatment and remained as such also in the long-term. In addition, as determined after 14 weeks of culture, irradiation affected the proliferation and the growth of E. multilocularis metacestodes. Thus, we demonstrate that radiotherapy does not have a clear-cut parasitocidal effect, but can lead to metabolic impairment of E. multilocularis metacestodes, as reflected by the distinct morphological and structural alterations induced by irradiation treatment.

MiniCASCC - A battery driven fog collector for ecosystem research

We developed a small version of the Caltech active strand cloud water collector (CASCC) for biogeochemical investigations in ecological applications. The device is battery powered and thus allows operation at locations where mains power is not available. The collector is designed for sampling periods of up to one week, depending on fog frequency. Our new device is equipped with standard sensors for air temperature, relative humidity, wind, and horizontal visibility for fog detection with a low-cost optical sensor. In mountain areas and during times when clouds are thin the installation of the visibility sensor became a key issue, which limits the potential to estimate liquid water content of the sampled fog. Field tests with 5 devices at three different sites in the Swiss Alps (Niesen) and the Jura Mountains (Lägeren, Switzerland) during two extended summer seasons in 2006 and 2007 showed that in almost all cases it was possible to obtain sample volumes which were large enough for the examination of basic inorganic chemistry of the collected cloud water. Collection rates varied typically from 12 to 30 mL h− 1. The fog droplet cutoff diameter is ≈ 6 μm, which is low enough to include all droplet sizes that are relevant for the liquid water content of typical fog types in the collected samples. From theoretical assumptions of the collection efficiency and theoretical droplet spectra it is possible to estimate the liquid water content of the sampled fog or cloud. Our new fog collector can be constructed and operated at relatively low costs. In combination with chemical and isotopic analyses of the sampled water, this allows to quantify nutrient and pollutant fluxes as is typically needed in ecosystem biogeochemistry studies.

Dynamics of red blood cells in microporous membranes

We have performed microfluidic experiments with erythrocytes passing through a network of microchannels of 20–25 μm width and 5 μm of height. Red blood cells (RBCs) were flowing in countercurrent directions through microchannels connected by μm pores. Thereby, we have observed interesting flow dynamics. All pores were blocked by erythrocytes. Some erythrocytes have passed through pores, depending on the channel size and cell elasticity. Many RBCs split into two or more smaller parts. Two types of splits were observed. In one type, the lipid bilayer and spectrin network were cut at the same time. In the second type, the lipid bilayer reconnected, but the part of spectrin network stayed outside the cell forming a rope like structure, which could eventually break. The microporous membrane results in multiple breakups of the cells, which can have various clinical implications, e.g., glomerulus hematuria and anemia of patients undergoing dialysis. The cell breakup procedure is similar to the one observed in the droplet breakage of viscoelastic liquids in confinement.

Bovine coccygeal intervertebral discs contain multipotent Tie2+ cells which can differentiate into osteogenic and adipogenic lineages

Question: The intervertebral disc (IVD) has a limited regenerative potential and low back pain represents a leading cause of disability [1]. IVD repair strategies require an appropriate cell source that is able to regenerate the damaged tissue such as progenitor stem cells. Recently, progenitor cells that are positive for the angiopoietin re- ceptor (Tie2) in the nucleus pulposus were identified [2]. Here we isolated primary cells from bovine IVD and sorted bovine nucleus pulposus progenitor cells (NPPC) for the marker Tie2. Furthermorewe tested whether Tie2 expressing cells can differentiate into os- teogenic and adipogenic lineages in vitro. Methods: NP cells were obtained from 1 year old bovine tails by sequential digestion with pronase for 1 h and collagenase over- night. Sorted Tie2- and Tie2+ cells were cultured in osteogenic and adipogenic medium for 3 weeks. The formed cell layers from both subpopulations were stained for calcium deposition and fat droplets. Colony forming units were prepared for both cell sus- pensions in methylcellulose-based medium and formed colonies ([10 cells) were analyzed macroscopically after 8 days. Results: After 3 weeks of culture, sorted Tie2+ cells were able to differentiate into osteocytes and adipocytes as characterized by cal- cium deposition and fat droplet formation. By contrast, Tie2- cells generated a weak staining for calcium and no fat droplets were ob- tained (Fig. 1). Sorted Tie2- and Tie2+ subpopulations of cells both formed colonies, however with different morphologies. The colonies formed from Tie2+ cells were spheroid in shape whereas those from Tie2- cells were spread and fibroblastic. Conclusion: Our data showed that Tie2+ cells of the nucleus pul- posus cells are progenitor-like cells that are able to differentiate into osteogenic and adipogenic lineages. Sorting of NPPC for Tie2 may represent a promising strategy with the potential to be used in the clinics for treatment of intervertebral disc damage. References 1. Freemont AJ (2009) The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain. Rheumatology (Oxford) 48:5–10 2. Sakai D, Nakamura Y, Nakai T et al (2012) Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat Commun 3:1264 Acknowledgments: This project was funded by two projects of the Swiss National Science Foundation grant number #IZK0Z3_154384 and #310030_153411.

Angiopoietin-1 receptor Tie2 distinguishes multipotent differentiation capability in bovine coccygeal nucleus pulposus cells.

BACKGROUND The intervertebral disc (IVD) has limited self-healing potential and disc repair strategies require an appropriate cell source such as progenitor cells that could regenerate the damaged cells and tissues. The objective of this study was to identify nucleus pulposus-derived progenitor cells (NPPC) and examine their potential in regenerative medicine in vitro. METHODS Nucleus pulposus cells (NPC) were obtained from 1-year-old bovine coccygeal discs by enzymatic digestion and were sorted for the angiopoietin-1 receptor Tie2. The obtained Tie2- and Tie2+ fractions of cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. Colony-forming units were prepared from both cell populations and the colonies formed were analyzed and quantified after 8 days of culture. In order to improve the preservation of the Tie2+ phenotype of NPPC in monolayer cultures, we tested a selection of growth factors known to have stimulating effects, cocultured NPPC with IVD tissue, and exposed them to hypoxic conditions (2 % O2). RESULTS After 3 weeks of differentiation culture, only the NPC that were positive for Tie2 were able to differentiate into osteocytes, adipocytes, and chondrocytes as characterized by calcium deposition (p < 0.0001), fat droplet formation (p < 0.0001), and glycosaminoglycan content (p = 0.0095 vs. Tie2- NPC), respectively. Sorted Tie2- and Tie2+ subpopulations of cells both formed colonies; however, the colonies formed from Tie2+ cells were spheroid in shape, whereas those from Tie2- cells were spread and fibroblastic. In addition, Tie2+ cells formed more colonies in 3D culture (p = 0.011) than Tie2- cells. During expansion, a fast decline in the fraction of Tie2+ cells was observed (p < 0.0001), which was partially reversed by low oxygen concentration (p = 0.0068) and supplementation of the culture with fibroblast growth factor 2 (FGF2) (p < 0.0001). CONCLUSIONS Our results showed that the bovine nucleus pulposus contains NPPC that are Tie2+. These cells fulfilled formally progenitor criteria that were maintained in subsequent monolayer culture for up to 7 days by addition of FGF2 or hypoxic conditions. We propose that the nucleus pulposus represents a niche of precursor cells for regeneration of the IVD.