Viscoelastic properties of epithelial cells

Epithelial cells form tight barriers that line both the outer and inner surfaces of organs and cavities and therefore face diverse environmental challenges. The response to these challenges relies on the cells’ dynamic viscoelastic properties, playing a pivotal role in many biological processes such as adhesion, growth, differentiation, and motility. Therefore, the cells usually adapt their viscoelastic properties to mirror the environment that determines their fate and vitality. Albeit not a high-throughput method, atomic force microscopy is still among the dominating methods to study the mechanical properties of adherent cells since it offers a broad range of forces from Piconewtons to Micronewtons at biologically significant time scales. Here, some recent work of deformation studies on epithelial cells is reviewed with a focus on viscoelastic models suitable to describe force cycle measurements congruent with the architecture of the actin cytoskeleton. The prominent role of the cortex in the cell’s response to external forces is discussed also in the context of isolated cortex extracts on porous surfaces.

P–451 Improving neovascularization and follicle viability in cryopreserved bovine ovarian tissue transplants

Study question Does cryopreservation and transplantation of bovine ovarian medulla-containing cortex tissue improve the viability and vascularization of the graft? Summary answer: Transplantation of bovine ovarian cortex containing medulla has a positive effect on follicular viability and neovascularization of the graft compared to cortex transplantation alone. What is known already For female fertility protection, cryopreservation and retransplantation of ovarian tissue is a widely used method. During cryopreservation, ovarian tissue is exposed to mechanical and hypoxic stress resulting in follicular loss. Moreover, after retransplantation tissue vitality and follicle survival is limited due to ischemia. As follicular viability is of major importance for fertility and hormonal activity, the main focus is on improving vitality and viability of the grafts. In current protocols, ovarian medulla is discarded and merely cortex tissue is preserved. However, medulla tissue predominantly contains blood vessels, thereby obtaining high potential for revascularization processes and thereby supporting tissue vitality. Study design, size, duration This experimental laboratory work was performed during a period of ten months. The rapidly vascularized chorioallantoic-membrane (CAM) of fertilized chicken eggs was used as model system to investigate neovascularization, follicle survival and tissue vitality of different bovine ovarian grafts. In four independent experimental rows four different tissue types (isolated cortex, thick medulla-containing cortex (8 x 10 x 3 mm), thin medulla-containing cortex (5 x 10 x 3 mm) and sole medulla tissue were compared. Participants/materials, setting, methods Out of four bovine ovaries preserved from the slaughterhouse, in total 117 samples of the four different tissue types were primed and cryopreserved by the common slow-freezing protocol. After thawing, grafts were transplanted on separate CAMs at day four of fertilized eggs. After four days of incubation, blood vessels growing towards the grafts were counted. Subsequently, grafts were harvested, digested with collagenase and stained with Neutral Red® to determine the total amount of vital follicles. Main results and the role of chance To investigate the neovascularization, all graft-supplying blood vessels were determined and distinguished between small and thick vessels. Compared to sole cortex, there were more small vessels in the medulla-containing grafts (9,72 vs. 8,65). Especially thin medulla- containing cortex pieces exhibited the highest number of small vessels (9,90). Also in isolated medulla tissue an increased amount of small vessels was observed (9,79). However, the average number of big vessels was not significantly different in all four test groups (Cortex: 2,12; thin medulla-containing cortex: 1,69; thick medulla-containing cortex: 1,5; medulla: 2). The total number of all vessels differed from 10,76 (sole cortex) to 11,75 (medulla-containing grafts), indicating a support of neoangiogenesis by medulla tissue. To further examine whether medulla tissue also alters the amount of vital follicles, Neutral Red® stained vital follicles were determined in all different sample groups. Indeed, in medulla-containing cortex samples was an augmented average number of vital follicles (342,4) compared to sole cortex tissue (256,11). Most vital follicles were detectable in the thick medulla-containing cortex tissue (346,61), closely followed by the thin medulla-containing cortex grafts (338,19). As expected, there was just a rare amount of vital follicles in sole medulla grafts (8,13). Limitations, reasons for caution As the ovarian reserve in cattle is very individual, the prepared ovaries are different in their follicle amount. These individual differences may influence the number of counted follicles. Furthermore, the CAM model is only a short term experimental approach to investigate neovascularization and follicle survival. Wider implications of the findings: According to our results, transplantation of human medulla-containing cortex appears promising. Keeping medulla tissue on the graft seems to improve both follicle viability and revascularization. Our findings need to be proven with human tissue, but might change the preparation of human ovarian tissue for fertility preservation in future. Trial registration number Not applicable

Transient coating of γ-Fe2O3 nanoparticles with glutamate for its delivery to and removal from brain nerve terminals

Glutamate is the main excitatory neurotransmitter in the central nervous system and excessive extracellular glutamate concentration is a characteristic feature of stroke, brain trauma, and epilepsy. Also, glutamate is a potential tumor growth factor. Using radiolabeled ʟ-[14C]glutamate and magnetic fields, we developed an approach for monitoring the biomolecular coating (biocoating) with glutamate of the surface of maghemite (γ-Fe2O3) nanoparticles. The nanoparticles decreased the initial rate of ʟ-[14C]glutamate uptake, and increased the ambient level of ʟ-[14C]glutamate in isolated cortex nerve terminals (synaptosomes). The nanoparticles exhibit a high capability to adsorb glutamate/ʟ-[14C]glutamate in water. Some components of the incubation medium of nerve terminals, that is, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and NaH2PO4, decreased the ability of γ-Fe2O3 nanoparticles to form a glutamate biocoating by about 50% and 90%, respectively. Only 15% of the amount of glutamate biocoating obtained in water was obtained in blood plasma. Albumin did not prevent the formation of a glutamate biocoating. It was shown that the glutamate biocoating is a temporal dynamic structure at the surface of γ-Fe2O3 nanoparticles. Also, components of the nerve terminal incubation medium and physiological fluids responsible for the desorption of glutamate were identified. Glutamate-coated γ-Fe2O3 nanoparticles can be used for glutamate delivery to the nervous system or for glutamate adsorption (but with lower effectiveness) in stroke, brain trauma, epilepsy, and cancer treatment following by its subsequent removal using a magnetic field. γ-Fe2O3 nanoparticles with transient glutamate biocoating can be useful for multifunctional theranostics.

Polarity of the ascidian egg cortex before fertilization

The unfertilized ascidian egg displays a visible polar organization along its animal-vegetal axis. In particular, the myoplasm, a mitochondria-rich subcortical domain inherited by the blastomeres that differentiate into muscle cells, is mainly situated in the vegetal hemisphere. We show that, in the unfertilized egg, this vegetal domain is enriched in actin and microfilaments and excludes microtubules. This polar distribution of microfilaments and microtubules persists in isolated cortices prepared by shearing eggs attached to a polylysine-coated surface. The isolated cortex is further characterized by an elaborate network of tubules and sheets of endoplasmic reticulum (ER). This cortical ER network is tethered to the plasma membrane at discrete sites, is covered with ribosomes and contains a calsequestrin-like protein. Interestingly, this ER network is distributed in a polar fashion along the animal-vegetal axis of the egg: regions with a dense network consisting mainly of sheets or tightly knit tubes are present in the vegetal hemisphere only, whereas areas characterized by a sparse tubular ER network are uniquely found in the animal hemisphere region. The stability of the polar organization of the cortex was studied by perturbing the distribution of organelles in the egg and depolymerizing microfilaments and microtubules. The polar organization of the cortical ER network persists after treatment of eggs with nocodazole, but is disrupted by treatment with cytochalasin B. In addition, we show that centrifugal forces that displace the cytoplasmic organelles do not alter the appearance and polar organization of the isolated egg cortex. These findings taken together with our previous work suggest that the intrinsic polar distribution of cortical membranous and cytoskeletal components along the animal-vegetal axis of the egg are important for the spatial organization of calcium-dependent events and their developmental consequences.

Secretory organelles of Paramecium cells (trichocysts) are not remarkably acidic compartments.

Acridine orange (AO) trapping in conjunction with fluorescence microscopy was applied to Paramecium cells. Trichocysts were not labeled when analyzed with an image intensification system (as opposed to a lysosomal population). Only with increasing intensity of ultraviolet light (UV) did trichocysts (and to some extent the cytosol) exhibit orange fluorescence, both effects being paralleled by increasing cell damage. Therefore, in comparison with the reported cytosolic pH (6.8), trichocysts cannot be considered as essentially acidic compartments. This is supported by experiments in vitro, using isolated cortex fragments or isolated fractions of membrane-bounded trichocysts (greater than or equal to 90% non-leaky). Again, during UV illumination orange fluorescence was observed even in the absence of ATP and Mg2+. Furthermore, this AO fluorescence and the condensation state of trichocyst contents were not affected by NH3 or by any of the widely differing ion- and H(+)-exchange inhibitors or ionophores tested. Decondensation of trichocyst contents occurred only when Ca2+ ionophore A23187 or X537A was incorporated into trichocyst membranes and when Ca2+ was then added. In this case all trichocysts partially decondensed within their intact membranes. We conclude that AO might be trapped in trichocysts by the abundant acidic secretory components during observation with UV light, rather than by acidic luminal pH.

Demonstration of calcium uptake and release by sea urchin egg cortical endoplasmic reticulum.

The calcium indicator dye fluo-3/AM was loaded into the ER of isolated cortices of unfertilized eggs of the sea urchin Arbacia punctulata. Development of the fluorescent signal took from 8 to 40 min and usually required 1 mM ATP. The signal decreased to a minimum level within 30 s after perfusion with 1 microM InsP3 and increased within 5 min when InsP3 was replaced with 1 mM ATP. Also, the fluorescence signal was lowered rapidly by perfusion with 10 microM A23187 or 10 microM ionomycin. These findings demonstrate that the cortical ER is a site of ATP-dependent calcium sequestration and InsP3-induced calcium release. A light-induced wave of calcium release, traveling between 0.7 and 2.8 microns/s (average speed 1.4 microns/s, N = 8), was sometimes observed during time lapse recordings; it may therefore be possible to use the isolated cortex preparation to investigate the postfertilization calcium wave.