Mateja Gabrijel

Intermediate filaments attenuate stimulation-dependent mobility of endosomes/lysosomes in astrocytes.

Intermediate filament (IF) proteins upregulation is a hallmark of astrocyte activation and reactive gliosis, but its pathophysiological implications remain incompletely understood. A recently reported association between IFs and directional mobility of peptidergic vesicles allows us to hypothesize that IFs affect vesicle dynamics and exocytosis‐mediated astrocyte communication with neighboring cells. Here, we ask whether the trafficking of recycling vesicles (i.e., those fused to and then retrieved from the plasma membrane) and endosomes/lysosomes depends on IFs. Recycling vesicles were labeled by antibodies against vesicle glutamate transporter 1 (VGLUT1) and atrial natriuretic peptide (ANP), respectively, and by lysotracker, which labels endosomes/lysosomes. Quantitative fluorescence microscopy was used to monitor the mobility of labeled vesicles in astrocytes, derived from either wild‐type (WT) mice or mice deficient in glial fibrillary acidic protein and vimentin (GFAP−/−Vim−/−), the latter lacking astrocyte IFs. Stimulation with ionomycin or ATP enhanced the mobility of VGLUT1‐positive vesicles and reduced the mobility of ANP‐positive vesicles in WT astrocytes. In GFAP−/−Vim−/− astrocytes, both vesicle types responded to stimulation, but the relative increase in mobility of VGLUT1‐positive vesicles was more prominent compared with nonstimulated cells, whereas the stimulation‐dependent attenuation of ANP‐positive vesicles mobility was reduced compared with nonstimulated cells. The mobility of endosomes/lysosomes decreased following stimulation in WT astrocytes. However, in GFAP−/−Vim−/− astrocytes, a small increase in the mobility of endosomes/lysosomes was observed. These findings show that astrocyte IFs differentially affect the stimulation‐dependent mobility of vesicles. We propose that upregulation of IFs in pathologic states may alter the function of astrocytes by deregulating vesicle trafficking.

IFN-γ-induced increase in the mobility of MHC class II compartments in astrocytes depends on intermediate filaments

BackgroundIn immune-mediated diseases of the central nervous system, astrocytes exposed to interferon-γ (IFN-γ) can 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 membrane-bound 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 wild-type (WT) astrocytes and in astrocytes devoid of IFs.MethodsThe identity of MHC class II compartments in WT and IF-deficient astrocytes 48 h after IFN-γ activation was determined immunocytochemically by using confocal microscopy. Time-lapse confocal imaging and Alexa Fluor546-dextran labeling of late endosomes/lysosomes in IFN-γ treated cells was used to characterize the motion of MHC class II vesicles. The mobility of vesicles was analyzed using ParticleTR software.ResultsConfocal imaging of primary cultures of WT and IF-deficient astrocytes revealed IFN-γ induced MHC class II expression in late endosomes/lysosomes, which were specifically labeled with Alexa Fluor546-conjugated dextran. Live imaging revealed faster movement of dextran-positive vesicles in IFN-γ-treated than in untreated astrocytes. Vesicle mobility was lower in IFN-γ-treated IF-deficient astrocytes than in WT astrocytes. Thus, the IFN-γ-induced increase in the mobility of MHC class II compartments is IF-dependent.ConclusionsSince reactivity of astrocytes is a hallmark of many CNS pathologies, it is likely that the up-regulation of IFs under such conditions allows a faster and therefore a more efficient delivery of MHC class II molecules to the cell surface. In vivo, such regulatory mechanisms may enable antigen-presenting reactive astrocytes to respond rapidly and in a controlled manner to CNS inflammation.

Astrocytic Vesicle Mobility in Health and Disease

Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5′-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

Fused Late Endocytic Compartments and Immunostimulatory Capacity of Dendritic–Tumor Cell Hybridomas

Late endocytic compartments, containing MHC class II molecules in antigen presenting cells, fuse to each other in order to deliver antigens to these molecules. We have shown previously that fusion of late endocytic compartments takes place also in hybridomas. Therefore, we investigate here whether the level of fused late endocytic compartments affects the immunostimulatory capacity of hybridomas obtained by the electrofusion of dendritic and tumor cells. The level of fused late endocytic compartments in a single hybridoma cell was assessed and samples of electrofused cells were then cocultured with autologous T cells, resulting in the priming of naïve T cells. To test the immunostimulatory capacity of hybridoma cells, T-cell-induced cytotoxicity of tumor cells was assayed. The results demonstrate that in vitro cytotoxic T cell responses are enhanced if a higher percentage of fused late endocytic compartments is present in the cell population of electrofused hybridoma cells.

Chapter 12 Exocytosis: The Pulsing Fusion Pore

Abstract The elaborate intracellular membrane system of eukaryotic cells participates in vesicle trafficking and represents an important basis exploited in cell-to-cell signaling. Communication between cells involves the release of neurotransmitters, hormones and other chemical messengers that are stored in secretory vesicles and granules. A key event in the release of these primary messengers is exocytosis, consisting of fusion between the vesicle and the plasma membrane. This leads to the formation of a fusion pore through which a diffusional continuum between the vesicle lumen and the extracellular space is established. In the past, in vitro studies of biological membrane fusion considered this an almost impossible process, because large pressures had to be delivered to counteract the electrostatic repulsion owing to negatively charged membrane surfaces. It is only a decade or so that the omnipresent fusion between biological membranes started to be understood in greater detail. Since the SNARE hypothesis was proposed about a decade ago, several proteins have been identified to play a role in exocytosis, and attempts to define minimal molecular machinery for regulated exocytosis have been considered. However, several studies provided evidence for multiple modes of exocytosis, and that exocytosis may not necessarily lead to the release of vesicle cargo. The aim of this chapter is to review the results obtained on pituitary cells, specialized to release a number of important hormones and to highlight that there are multiple mechanisms of exocytosis present in the same cell. Moreover, the goal is to address elementary properties of exocytosis, consisting of the interaction between a single vesicle and the plasma membrane. These studies indicate that the long-thought concept of membrane fusion as an irreversible process will have to be changed. Here we discuss an unusually regular reversible opening of the fusion pore termed “the pulsing pore”.