Two types of exocytosis are known, full fusion and transient fusion, first type is dominant form in melanotrophs, which are therefore appropriate cell type for measurements of bulk exocytosis. Sfingolipid sphingosine recruits protein VAMP2 for SNARE complex formation, which is important for membrane fusion. We discovered that sphingosine and its structure homologue fingolimod increased exocytosis, if added extracellularly. We also studied exocytosis in lactotrophs, where transient fusion is dominant mode of exocytosis. We measure exocytosis by monitoring discrete steps in membrane capacitance, which is proportional to the changes in surface area of plasma membrane. In the presence of sphingosine the frequency of transient and full-fusion events increased. Vesi cles with larger diameters proceeded to full fusion, while smaller vesicles remained entrapped in transient exocytosis. Labeling with fluorescent antibodies recognizing prolactin (PRL) showed that the average diameter of secreted, but undegradated vesicle content was larger than diameter of unsecreted vesicles. PRL- and VAMP2-antibodies revealed that the intensity of VAMP2 signal was independent of PRL vesicle diameter. However, the density of VAMP2 signals was reduced in larger vesicles. We propose that sphingosine-mediated facilitation of regulated exocytosis is not only related to the number of SNARE complexes per vesicle but also depends on the vesicle size, which may determine the transition between transient and full-fusion exocytosis.
D.09 Tutoring for postgraduate students
COBISS.SI-ID: 784247Small monomeric GTPases function as molecular switches and are important for the regulation of the vesicular traffic.The role of RAB proteins in the vesicular traffic of vesicles in astrocytes, which are the most abundant glial cells in the mammalian CNS, is poorly understood. We have studied their role by transfecting astrocytes with the plasmids encoding RAB4A and RAB5A wild type, the dominant negative and the dominant positive forms. We proceeded by testing whether astrocytes express GDI1 or GDI2 proteins and their potential role in teh vesicular mobility. Our results show that RAB4A and RAB5A are present on the sub-cellular structures of the early and late endocytic pathway. Astrocytes expressing the dominant-negative or the dominant positive forms of RAB4A/5A exhibit enlarged vesicles, which is most evident in cells expressing dominant positive forms.The mutants also affect vesicle mobility, which is significantly decreased. We have proven that astrocytes express GDI1 and GDI2. Inhibition of the expression of these two proteins decreases vesicle mobility.
D.09 Tutoring for postgraduate students
COBISS.SI-ID: 262313984Astrocytes have an important role in modulating signal transmission in synapses between neurons, because they release gliotransmitters. Regulated exocytosis represents one of the mechanisms of the release of gliotransmitters and it consists of vesicle fusion with the plasma membrane. Fusion pore formation enables diffusion of gliotransmitters into extracellular space. Subsequently, the fusion pore closes or the vesicle merges with the plasma membrane. In the first case the surface area of the membrane increases transiently and in the second it increases for longer period of time. Direct measurements of plasma membrane fluctuations due to regulated exocytosis in astrocytes has not been performed yet. Surface area alternations can be monitored by applying the high resolution patch clamp technique. Cell-attached mode of membrane capacitance measurements enables monitoring of reversible and irreversible discrete steps in capacitance (parameter linearly dependant on the surface area) and subsequent evaluation of exocytosis types (transient or full fusion). Results showed that transient fusion dominates in astrocytes. It occurs in more than 90% of the cases. The average vesicle capacitance amplitudes are 0,41 ± 0,06 fF (vesicle diameter of 100 nm) and have the fusion pores opened in average for 91 ± 6 ms. Stimulation with adenosine triphosphate (ATP) influences the physiology of astrocytes. Results revealed that stimulation with ATP (1 mM) increases the frequency of transient events with average amplitudes of 0,41 ± 0,01 fF. Additionally, it induces secretion of a second population with average amplitudes of vesicle capacitance of 1,50 ± 0,03 fF, representing vesicles with diameters of around 300 nm and prolonged dwell-time (0,16 ± 0,01 before stimulation vs. 0,27 ± 0,01 s after stimulation). After the stimulation with ATP the fusion pore expands, which enables stimulated release of gliotransmitters.
D.10 Educational activities
COBISS.SI-ID: 35338245The invention relates to a method of screening for a compound useful in the treatment of a disease selected from neurodegenerative diseases and neuro-inflammatory diseases, said method comprising providing a test cell; staining at least one organelle of said test cell; contacting said test cell with a test compound; and recording the path of said stained organelle in said test cell. Suitable compounds are identified from a comparison of the recorded path with a suitable reference.
F.06 Development of a new product
COBISS.SI-ID: 29542617Super resolution microscopy is the cutting edge technology with the resolution that is at list twice better than the resolution of conventional far field microscopy. One of the key instuments of super resolution microscopy is STED technology based instrument, which is now located also in Slovenia. It represents the most advanced technology in the field of optical microscopy, comprised of most advanced optics, mechanics and electronics. It enables observation of living objects at resolution between 35 and 40 nm with the potential to visualize objects beyond these limits. The conference with a workshop of the demonstration of the super-resolution microscopy is dedicated to the introduction of this technological novelty.
B.01 Organiser of a scientific meeting
COBISS.SI-ID: 2814543