The present innovation represents a platform comprising in vitro cell systems, methods and the standard compound for manipulation and/or monitoring of subcellular organelle traffic, important for the understanding of the fundamental cellular processes as well as for the applications in advanced approaches in medicine, biotechnology, pharmaceutical industry and other related fields. The innovation and novelty of the proposed patent application is the combination (platform) of approaches, consisting of the i) method for monitoring subcellular organelle dynamics to screen for biologically active substances (i.e. constituents of sera, cerebrospinal fluid and other biologically relevant small inorganic and organic substances and mixtures of these), ii) method for the determination of the quality of cells destined to be used in advanced medical therapies and iii) method for assaying and controlling the production of recombinant proteins within eukaryotic cells, in relation to organelle traffic. The invention offers the basis for the development and optimization of different pharmacologically relevant compounds for the treatment/modulation of pathological states, associated with the intercellular communication and hence with subcellular organelle traffic.
F.21 Development of new health/diagnostic methods/procedures
COBISS.SI-ID: 29542617Astrocytes 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 release of hormones and neurotransmitters, mediated by regulated exocytosis, can be modified by regulation of the fusion pore, which is considered to be stable and narrow initially. Eventually, the fusion pore expands fully (full-fusion exocytosis), or reversibly closes (transient exocytosis). In the latter case, the release of vesicle cargo depends on the fusion pore dwell-time and diameter. However, the molecular events mediating transitions between stages of exocytosis are unclear. By using the high-resolution patch-clamp capacitance technique, we studied single vesicles to see if Munc18 protein, interacting with the membrane fusion-mediating SNARE proteins, affects fusion pore properties. We transfected lactotrophs with Munc18-1 mutants to affect the interaction of Munc18-1 with Synt1 (R39C), Rab3A (E466K) and Mints (P242S). Compared with wild-type, Munc18-1 E466K increased the frequency of the fusion event. The latter two mutants increased the fusion pore dwell-time. All the mutants stabilized narrow fusion pores and increased the amplitude of fusion events, likely due to preferential fusion of larger vesicles, since over-expression of Munc18-1 R39C did not affect the average size of vesicles, as determined by STED microscopy. Single molecule atomic force microscopy experiments revealed that wild-type Munc18-1, but not Munc18-1 R39C, abrogates the interaction between Syb2 and Synt1 binary trans complexes. However, neither forms of Munc18-1 affected the interaction of Syb2 with the preformed binary cis Synt1-SNAP25B complexes. This indicates that Munc18-1 favors tethering of Syb2-containing vesicles to the preformed binary cis complex of Synt1-SNAP25B at the plasma membrane. Munc18-1 and ternary SNARE complex tune fusion pores via multiple converging mechanisms involving Munc18-1 interactions with Synt1, Rab3A and Mints.
F.18 Transfer of new know-how to direct users (seminars, fora, conferences)
COBISS.SI-ID: 28841433