Fusion Pore Diameter Regulation by Cations Modulating Local Membrane Anisotropy

The fusion pore is an aqueous channel that is formed upon the fusion of the vesicle membrane with the plasma membrane. Once the pore is open, it may close again (transient fusion) or widen completely (full fusion) to permit vesicle cargo discharge. While repetitive transient fusion pore openings of the vesicle with the plasma membrane have been observed in the absence of stimulation, their frequency can be further increased using a cAMP-increasing agent that drives the opening of nonspecific cation channels. Our model hypothesis is that the openings and closings of the fusion pore are driven by changes in the local concentration of cations in the connected vesicle. The proposed mechanism of fusion pore dynamics is considered as follows: when the fusion pore is closed or is extremely narrow, the accumulation of cations in the vesicle (increased cation concentration) likely leads to lipid demixing at the fusion pore. This process may affect local membrane anisotropy, which reduces the spontaneous curvature and thus leads to the opening of the fusion pore. Based on the theory of membrane elasticity, we used a continuum model to explain the rhythmic opening and closing of the fusion pore

Histological Skin Remodeling Following Autologous Fibroblast Application

ABSTRACT The aim of this study was to quantify the effectiveness of intradermal application of autologous fibroblasts on lean tissue structures. The histological sections of the skin were analysed and evaluated for the expansion potential of autologous fibroblasts in the control skin patch area and the nearby pre-treated skin patch into which we had injected expanded autologous fibroblasts nine month earlier. The results show that the pre-injection of fibroblasts into the dermis leads to a long-term rejuvenation of the skin, as evaluated from the histological appearance and from the significantly increased density of fibroblasts in the pre-injected skin vs. controls, from around 60% to over 80%, determined as the percent of lean tissue by a novel image analysis approach. Interestingly, the rate of the in vitro fibroblast expansion from the pre-injected area of the skin was reduced in comparison with the controls, consistent with the view that fibroblasts exhibit a limited cell-division potential and that fibroblasts from the pre-injected skin already experienced expansion nine month earlier prior to the injection into the skin. We conclude that autologous fibroblast application results in a significant long-term augmentation of the lean tissue elements of the skin

Exocytotic release of ATP from cultured astrocytes.

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca(2+)](i) also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca(2+)](i) attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca(2+)-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X(3) receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca(2+). Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared

Erratum to “Fusion Pore Diameter Regulation by Cations Modulating Local Membrane Anisotropy”

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Amnijska membrana kot biološki nosilec, njena priprava in uporaba v regenerativni medicini v Sloveniji

Izhodišča: Amnijska membrana (AM) je notranja stran posteljice, ki obdaja in ščiti zarodek. AM je večplastna struktura, ki je sestavljena iz amnijskih epitelijskih celic, amnijskih mezenhimskih stromalnih celic, bazalne lamine in vezivnega tkiva. Iz njene zgradbe izhajajo tudi lastnosti AM, zaradi katerih se že vrsto let uporablja v terapevtske namene, predvsem v oftalmologiji, saj pospešuje epitelizacijo, deluje kot substrat za celice, zmanjšuje fibrozo in neovaskularizacijo tkiva ter deluje protimikrobno. Zaradi mehanskih lastnosti AM, ki so posledica predvsem molekul zunajceličnega matriksa bazalne lamine in vezivnega tkiva, se AM v zadnjih letih vedno pogosteje uporablja tudi kot biološki nosilec v regenerativni medicini.   Zaključki: Regenerativna medicina je interdisciplinarno področje raziskav in kliničnih aplikacij, ki uporablja načela bioloških in inženirskih znanosti za razvoj živih tkivnih ali organskih nadomestkov. V regenerativni medicini ločimo tri pristope: 1) vsaditev funkcionalnih celic, med drugim tudi matičnih celic, v poškodovano ali okvarjeno tkivo, 2) uporaba različnih sintetičnih materialov ali materialov naravnega izvora, ki pomagajo pri ponovnem oblikovanju poškodovanega ali okvarjenega tkiva in 3) tkivno inženirstvo, tj. uporaba ustreznih nosilcev, ki spodbujajo rast tkivno specifičnih celic in oblikovanje novega tkiva. V prispevku predstavljamo tudi uporabo amnijske membrane kot biološkega nosilca v regenerativni medicini v Sloveniji

cAMP-Mediated stabilization of fusion pores in cultured rat pituitary lactotrophs

Regulated exocytosis mediates the release of hormones and transmitters. The last step of this process is represented by the merger between the vesicle and the plasma membranes, and the formation of a fusion pore. Once formed, the initially stable and narrow fusion pore may reversibly widen (transient exocytosis) or fully open (full-fusion exocytosis). Exocytosis is typically triggered by an elevation in cytosolic calcium activity. However, other second messengers, such as cAMP, have been reported to modulate secretion. The way in which cAMP influences the transitions between different fusion pore states remains unclear. Here, hormone release studies show that prolactin release from isolated rat lactotrophs stimulated by forskolin, an activator of adenylyl cyclases, and by membrane-permeable cAMP analog (dbcAMP), exhibit a biphasic concentration dependency. Although at lower concentrations (2-10 μm forskolin and 2.5-5 mm dbcAMP) these agents stimulate prolactin release, an inhibition is measured at higher concentrations (50 μm forskolin and 10-15 mm dbcAMP). By using high-resolution capacitance (Cm) measurements, we recorded discrete increases in Cm, which represent elementary exocytic events. An elevation of cAMP leaves the frequency of full-fusion events unchanged while increasing the frequency of transient events. These exhibited a wider fusion pore as measured by increased fusion pore conductance and a prolonged fusion pore dwell time. The probability of observing rhythmic reopening of transient fusion pores was elevated by dbcAMP. In conclusion, cAMP-mediated stabilization of wide fusion pores prevents vesicles from proceeding to the full-fusion stage of exocytosis, which hinders vesicle content discharge at high cAMP concentrations

Aspects of astrocyte energy metabolism, amino acid neurotransmitter homoeostasis and metabolic compartmentation

Astrocytes are key players in brain function; they are intimately involved in neuronal signalling processes and their metabolism is tightly coupled to that of neurons. In the present review, we will be concerned with a discussion of aspects of astrocyte metabolism, including energy-generating pathways and amino acid homoeostasis. A discussion of the impact that uptake of neurotransmitter glutamate may have on these pathways is included along with a section on metabolic compartmentation

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives