Expression and allocation of proteins of the exo-endocytotic machinery in U373 glioma cells: similarities to long-term cultured astrocytes.

Abstract

1. Cultured astrocytes cells release a variety of low and high molecular weight messenger substances and express proteins of the exocytotic pathway including synaptic SNARE proteins. For analyzing the molecular mechanisms of astrocytic messenger release, permanent cell lines with astrocytic properties would provide useful tools. 2. We analyzed the potential of the human malignant astrocytoma-derived cell line U373 MG to express proteins involved in regulated exo- and endocytosis. An immunoblot analysis identified the astrocyte marker glial fibrillary acidic protein, microtubule-associated protein 2, the v-SNAREs VAMP I, VAMP II, and cellubrevin and the t-SNAREs syntaxin I, SNAP-23, and SNAP-25. 3. The cells also express the secretory granule protein secretogranin II. Although secretogranin II immunofluorescence reveals larger fluorescence spots, the majority of the SNARE proteins is associated with smaller organelles. The immunofluorescence is distributed throughout the cytoplasm and accumulates at processes and the growing edges of cells. 4. The organellar association of SNARE proteins was confirmed by heterologous expression of recombinant fusion proteins. Following subcellular fractionation organelles of lower buoyant density carried the majority of VAMP 11. Secretogranin II was associated with organelles of high buoyant density containing a small contribution of VAMP II. 5. The results suggest that U373 MG cells have in common a considerable number of properties with long-term cultured astrocytes rather than with cultured oligodendrocytes or neurons. They contain two types of organelles that can be physically separated and may be employed in the differential release of messengers.