Celica Biomedical Center

Ljubljana, Slovenia

Celica Biomedical Center

Ljubljana, Slovenia
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Jorgacevski J.,University of Ljubljana | Jorgacevski J.,Celica Biomedical Center | Kreft M.,University of Ljubljana | Kreft M.,Celica Biomedical Center | And 4 more authors.
Cell Calcium | Year: 2012

Regulated exocytosis, which involves fusion of secretory vesicles with the plasma membrane, is an important mode of communication between cells. In this process, signalling molecules that are stored in secretory vesicles are released into the extracellular space. During the initial stage of fusion, the interior of the vesicle is connected to the exterior of the cell with a narrow, channel-like structure: the fusion pore. It was long believed that the fusion pore is a short-lived intermediate state leading irreversibly to fusion pore dilation. However, recent results show that the diameter of the fusion pore can fluctuate, suggesting that the fusion pore is a subject of stabilization. A possible mechanism is addressed in this article, involving the local anisotropicity of membrane constituents that can stabilize the fusion pore. The molecular nature of such a stable fusion pore to predict how interacting molecules (proteins and/or lipids) mediate changes that affect the stability of the fusion pore and exocytosis is also considered. The fusion pore likely attains stability via multiple mechanisms, which include the shape of the lipid and protein membrane constituents and the interactions between them. © 2012 Elsevier Ltd.

Rituper B.,University of Ljubljana | Flasker A.,University of Ljubljana | Gucek A.,University of Ljubljana | Chowdhury H.H.,University of Ljubljana | And 4 more authors.
Cell Calcium | Year: 2012

Since the 1970s, much effort was been expended researching mechanisms of regulated exocytosis. Early work focused mainly on the role of proteins. Most notably the discovery of SNARE proteins in the 1980s and the zippering hypothesis brought us much closer to understanding the complex interactions in membrane fusion between vesicle and plasma membranes, a pivotal component of regulated exocytosis. However, most likely due to the predictions of the Singer-Nicholson fluid mosaic membrane model, the lipid components of the exocytotic machinery remained largely overlooked. Lipids were considered passive constituents of cellular membranes, not contributing much, if anything, to the process of exocytosis and membrane fusion. Since the 1990s, this so-called proteocentric view has been gradually giving way to the new perspective best described with the term proteolipidic. Many lipids were found to be of great importance in the regulation of exocytosis. Here we highlight the role of cholesterol. Furthermore, by using high-resolution cell-attached membrane capacitance measurements, we have monitored unitary exocytotic events in cholesterol-depleted membranes. We show that the frequency of these events is attenuated, providing evidence at the single vesicle level that cholesterol directly influences the merger of the vesicle and the plasma membranes. © 2012 Elsevier Ltd.

Gucek A.,University of Ljubljana | Vardjan N.,University of Ljubljana | Vardjan N.,Celica Biomedical Center | Zorec R.,University of Ljubljana | And 2 more authors.
Neurochemical Research | Year: 2012

Astrocytes, a type of glial cells in the brain, are eukaryotic cells, and a hallmark of these are subcellular organelles, such as secretory vesicles. In neurons vesicles play a key role in signaling. Upon a stimulus-an increase in cytosolic concentration of free Ca2+ ([Ca2+] i)-the membrane of vesicle fuses with the presynaptic plasma membrane, allowing the exit of neurotransmitters into the extracellular space and their diffusion to the postsynaptic receptors. For decades it was thought that such vesiclebased mechanisms of gliotransmitter release were not present in astrocytes. However, in the last 30 years experimental evidence showed that astrocytes are endowed with mechanisms for vesicle- and non-vesicle-βased gliotransmitter release mechanisms. The aim of this review is to focus on exocytosis, which may play a role in gliotransmission and also in other forms of cell-to-cell communication, such as the delivery of transporters, ion channels and antigen presenting molecules to the cell surface. © Springer Science+Business Media, LLC 2012.

Prebil M.,University of Ljubljana | Jensen J.,Norwegian School of Sport Sciences | Zorec R.,University of Ljubljana | Zorec R.,Celica Biomedical Center | And 2 more authors.
Archives of Physiology and Biochemistry | Year: 2011

Astrocytes are glial cells, which play a significant role in a number of processes, including the brain energy metabolism. Their anatomical position between blood vessels and neurons make them an interface for effective glucose uptake from blood. After entering astrocytes, glucose can be involved in different metabolic pathways, e.g. in glycogen production. Glycogen in the brain is localized mainly in astrocytes and is an important energy source in hypoxic conditions and normal brain functioning. The portion of glucose metabolized into glycogen molecules in astrocytes is as high as 40%. It is thought that the release of gliotransmitters (such as glutamate, neuroactive peptides and ATP) into the extracellular space by regulated exocytosis supports a significant part of communication between astrocytes and neurons. On the other hand, neurotransmitter action on astrocytes has a significant role in brain energy metabolism. Therefore, understanding the astrocytes energy metabolism may help understanding neuron-astrocyte interactions. © 2011 Informa UK, Ltd.

Kreft M.,University of Ljubljana | Kreft M.,Celica Biomedical Center | Luksic M.,University of Ljubljana | Zorec T.M.,University of Ljubljana | And 3 more authors.
Cellular and Molecular Life Sciences | Year: 2013

Astrocytes interact with neurons and endothelial cells and may mediate exchange of metabolites between capillaries and nerve terminals. In the present study, we investigated intracellular glucose diffusion in purified astrocytes after local glucose uptake. We used a fluorescence resonance energy transfer (FRET)-based nano sensor to monitor the time dependence of the intracellular glucose concentration at specific positions within the cell. We observed a delay in onset and kinetics in regions away from the glucose uptake compared with the region where we locally super-fused astrocytes with the d-glucose-rich solution. We propose a mathematical model of glucose diffusion in astrocytes. The analysis showed that after gradual uptake of glucose, the locally increased intracellular glucose concentration is rapidly spread throughout the cytosol with an apparent diffusion coefficient (D app) of (2.38 ± 0.41) × 10-10 m2 s-1 (at 22-24 C). Considering that the diffusion coefficient of d-glucose in water is D = 6.7 × 10-10 m2 s-1 (at 24 C), D app determined in astrocytes indicates that the cytosolic tortuosity, which hinders glucose molecules, is approximately three times higher than in aqueous solution. We conclude that the value of D app for glucose measured in purified rat astrocytes is consistent with the view that cytosolic diffusion may allow glucose and glucose metabolites to traverse from the endothelial cells at the blood-brain barrier to neurons and neighboring astrocytes. © 2012 Springer Basel.

Vardjan N.,Celica Biomedical Center | Vardjan N.,University of Ljubljana | Jorgacevski J.,Celica Biomedical Center | Jorgacevski J.,University of Ljubljana | And 2 more authors.
Neuroscientist | Year: 2013

Exocytosis is a multistage process involving a merger between the vesicle and the plasma membranes, leading to the formation of a fusion pore, a channel, through which secretions are released from the vesicle to the cell exterior. A stimulus may influence the pore by either dilating it completely (full-fusion exocytosis) or mediating a reversible closure (transient exocytosis). In neurons, these transitions are short-lived and not accessible for experimentation. However, in some neuroendocrine cells, initial fusion pores may reopen several hundred times, indicating their stability. Moreover, these pores are too narrow to pass luminal molecules to the extracellular space, termed release-unproductive. However, on stimulation, their diameter dilates, initiating the release of cargo without de novo fusion pore formation. To explain the stability of the initial narrow fusion pores, anisotropic membrane constituents with non-axisymmetrical shape were proposed to accumulate in the fusion pore membrane. Although the nature of these is unclear, they may consist of lipids and proteins, including SNAREs, which may facilitate and regulate the pre- and post-fusional stages of exocytosis. In the future, a more detailed insight into the molecular control of fusion pore stabilization and regulation will generate a better understanding of fusion pore physiology in health and disease. © The Author(s) 2012.

Rituper B.,University of Ljubljana | Davletov B.,University of Cambridge | Zorec R.,University of Ljubljana | Zorec R.,Celica Biomedical Center
Clinical Lipidology | Year: 2010

Exocytosis is a highly conserved and ubiquitous process of eukaryotic cells responsible for the release of signaling molecules into extracellular space. Exocytosis involves trafficking, docking and eventually fusion of vesicles, carrying various cargo, with the plasma membrane. Until recently, the membrane fusion was considered to be predominantly mediated by proteins such as SNAP receptors, Muncs and Rabs, where lipids only played a passive role. However, newer studies portray lipids differently. Not only do lipids have a significantly more important role in membrane merger as previously believed, they also appear to be critical for regulating the entire process of exocytosis. The purpose of this article is to highlight the importance of specific lipids and lipid-protein interactions in regulated release of neurotransmitters and hormones. © 2010 Future Medicine Ltd.

Prebil M.,University of Ljubljana | Chowdhury H.H.,University of Ljubljana | Chowdhury H.H.,Celica Biomedical Center | Zorec R.,University of Ljubljana | And 3 more authors.
Biochemical and Biophysical Research Communications | Year: 2011

Astrocytes which lie between brain capillaries and neuronal terminals are the primary site of glucose uptake and have a key role in coupling synaptic activity to glucose utilization in the central nervous system (CNS). We used a fluorescence resonance energy transfer (FRET) based approach to monitor cytosolic glucose in astrocytes. We determined the effect of increasing extracellular glucose concentrations on FRET ratio as a measure of increased cytosolic glucose in astrocytes. By briefly raising extracellular glucose concentration, astrocytes responded promptly by increased cytosolic glucose levels, which was manifested by decreased time-dependent FRET ratio. The FRET ratio fall-time recorded at low extracellular d-glucose concentration change (from 0 to 0.5. mM) was 53. s, whereas 17. s was recorded by raising extracellular concentration of d-glucose from 0 to 10. mM, which is likely due to facilitated d-glucose entry along the increased d-glucose gradient across the plasmalemma. The relationship between the extracellular glucose concentration and the FRET ratio change is limited to the maximal ratio change, where the d-glucose plasma membrane permeability is balanced by the cytosolic utilization. We measured the effect of extracellular ATP, an important extracellular messenger for astrocyte-to-astrocyte communication, on intracellular glucose concentration. The results show that stimulation of astrocytes with ATP (1. mM) decreases cytosolic glucose concentration with a time constant of ∼145. s. The mechanism of this change is discussed. © 2011 Elsevier Inc.

Singh P.,University of Ljubljana | JorgaAevski J.,University of Ljubljana | JorgaAevski J.,Celica Biomedical Center | Kreft M.,University of Ljubljana | And 10 more authors.
Nature Communications | Year: 2014

Exocytic transmitter release is regulated by the SNARE complex, which contains a vesicular protein, synaptobrevin2 (Sb2). However, Sb2 vesicular arrangement is unclear. Here we use super-resolution fluorescence microscopy to study the prevalence and distribution of endogenous and exogenous Sb2 in single vesicles of astrocytes, the most abundant glial cells in the brain. We tag Sb2 protein at C- and N termini with a pair of fluorophores, which allows us to determine the Sb2 length and geometry. To estimate total number of Sb2 proteins per vesicle and the quantity necessary for the formation of fusion pores, we treat cells with ATP to stimulate Ca 2+ -dependent exocytosis, increase intracellular alkalinity to enhance the fluorescence presentation of yellow-shifted pHluorin (YpH), appended to the vesicle lumen domain of Sb2, and perform photobleaching of YpH fluorophores. Fluorescence intensity analysis reveals that the total number of endogenous Sb2 units or molecules per vesicle is â ‰ 25. © 2014 Macmillan Publishers Limited. All rights reserved.

Rituper B.,Institute of Pathophysiology | Gucek A.,Institute of Pathophysiology | Jorgacevski J.,Institute of Pathophysiology | Jorgacevski J.,Celica Biomedical Center | And 5 more authors.
Nature Protocols | Year: 2013

In order to understand exocytosis and endocytosis, it is necessary to study these processes directly. An elegant way to do this is by measuring plasma membrane capacitance (Cm), a parameter proportional to cell surface area, the fluctuations of which are due to fusion and fission of secretory and other vesicles. Here we describe protocols that enable high-resolution C m measurements in macroscopic and microscopic modes. Macroscopic mode, performed in whole-cell configuration, is used for measuring bulk C m changes in the entire membrane area, and it enables the introduction of exocytosis stimulators or inhibitors into the cytosol through the patch pipette. Microscopic mode, performed in cell-attached configuration, enables measurements of Cm with attofarad resolution and allows characterization of fusion pore properties. Although we usually apply these protocols to primary pituitary cells and astrocytes, they can be adapted and used for other cell types. After initial hardware setup and culture preparation, several Cm measurements can be performed daily. © 2013 Nature America, Inc. All rights reserved.

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