Astrocytes, glial cells which outnumber neurons, communicate with neurons and represent integrators in the brain function. We studied the secretory activity of astrocytes by electrophysiological and optical methods and vesicle mobility with confocal microscopy. Our findings were communicated to the researches who work in familiar fileds with the purpose to advance science in the field of research.
F.18 Transfer of new know-how to direct users (seminars, fora, conferences)
COBISS.SI-ID: 28760793The 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.33 Slovenian patent
COBISS.SI-ID: 29542617Rab4 and Rab5 GTPases are key players in the regulation of endocytosis. Although their role has been studied intensively in the past, it is still unclear how they regulate vesicle mobility. In particular, in astrocytes, the most abundant glial cells in the brain, vesicles have been shown to exhibit nondirectional and directional mobility, which can be intermittent, but the underlying switching mechanisms are not known. By using quantitative imaging, we studied the dynamics of single vesicle movements in astrocytes in realtime, by transfecting them with different GDP and GTPlocked mutants of Rab4 and Rab5. Along with the localization of Rab4 and Rab5 on early and late endocytic compartments, we measured the apparent vesicle size by monitoring the area of fluorescent puncta and determined the patterns of vesicle mobility in the presence of wild type and Rab mutants. Dominantnegative and dominantpositive mutants, Rab4 S22N, Rab5 S34N and Rab4 Q67L and Rab5 Q79L, induced an increase in the apparent vesicle size, especially Rab5 mutants. These mutants also significantly reduced vesicle mobility in terms of vesicle track length, maximal displacement and speed. In addition, significant reductions in the fraction of vesicles exhibiting directional mobility were observed in cells expressing Rab4 S22N, Rab4 Q67L, Rab5 S34N and Rab5 Q79L. Our data indicate that changes in the GDPGTP switch apparently not only affect fusion events in endocytosis and recycling, as already proposed, but also affect the molecular interactions determining directional vesicle mobility, likely involving motor proteins and the cytoskeleton.
F.18 Transfer of new know-how to direct users (seminars, fora, conferences)
COBISS.SI-ID: 29708249In astrocytes, as in other eukaryotic cells, vesicles have key cellular functions including constitutive housekeeping of the plasma membrane structure and celltocell communication. On the one hand, vesicle traffic is associated with cell surface morphology exhibiting distinct glial microdomains. These determine the signaling potential and metabolic support for neighboring cells. On the other hand, vesicles are used in astrocytes for the release of vesicleladen chemical messengers. This lecture will address the properties of membranebound vesicles that store gliotransmitters (glutamate, adenosine 5′triphosphate (ATP), peptides), other recycling vesicles, and endocytotic vesicles that are involved in the traffic of plasma membrane receptors such as the class II major histocompatibility molecules (MHCII) and membrane transporters (aquaporin 4 (AQP4) and excitatory amino acid transporter 2 (EAAT2)). Vesicle dynamics greatly depends on intermediate filaments therefore one has to consider that altered vesicle dynamics may be associated with the diseases such as amyotrophic lateral sclerosis, multiple sclerosis, autistic disorders, Alzheimer’s disease, trauma, edema, and states in which astrocytes contribute to neuroinflammation. In multiple sclerosis, for example, fingolimod, a recently introduced drug, apparently also affects vesicle traffic and gliotransmitter release from astrocytes, indicating that this process may well be used as a new physiologic target for the development of new therapies.
B.04 Guest lecture
COBISS.SI-ID: 30886617In immunemediated diseases of the central nervous system (CNS), astrocytes exposed to interferonγ (IFNγ) express major histocompatibility complex (MHC) class II molecules and antigens on their surface. MHC class II molecules are thought to be delivered to the cell surface by membranebound vesicles. However, the characteristics and dynamics of this vesicular traffic are unclear, particularly in reactive astrocytes, which overexpress intermediate filament (IF) proteins that may affect trafficking. The aim of this study was to determine the mobility of MHC class II vesicles in wildtype (WT) astrocytes and in astrocytes devoid of IFs. The identity of MHC class II compartments in WT and IFdeficient astrocytes 48 h after IFNγ activation was determined immunocytochemically by using confocal microscopy. Timelapse confocal imaging and Alexa Fluor546dextran labeling of late endosomes/lysosomes in IFNγ treated cells was used to characterize the motion of MHC class II vesicles. Confocal imaging of primary cultures of WT and IFdeficient astrocytes revealed IFNγ induced MHC class II expression in late endosomes/lysosomes, which were specifically labeled with Alexa Fluor546conjugated dextran. Live imaging revealed faster mobility of dextranpositive vesicles in IFNγtreated versus the untreated astrocytes. Vesicle mobility was reduced in IFNγtreated IFdeficient astrocytes in comparison with WT astrocytes. Thus, the IFNγ induced increase in the mobility of MHC class II compartments is IFdependent. Since reactivity of astrocytes is a hallmark of many CNS pathologies, it is likely that the upregulation of IFs under such conditions allows a faster and therefore a more efficient delivery of MHC class II molecules to the cell surface.
B.06 Other
COBISS.SI-ID: 30887129