Vesicle mobility studied in cultured astrocytes Maja Potokar, Marko Kreft, Tina Pangrs ˇic ˇ, Robert Zorec * Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Medical Faculty, University of Ljubljana, Zaloska 4, SI-1000 Ljubljana, Slovenia Celica Biomedical Sciences Center, Stegne 21c, SI-1000 Ljubljana, Slovenia Received 2 February 2005 Abstract Astrocytes release many neuroactive substances, which are stored in membrane bound vesicles and may play a role in synapse modulation and in the coupling between neuronal activity and the local blood flow. However, the mobility of these vesicles in astro- cytes has not been studied yet. We here used a fluorescently tagged proatrial natriuretic peptide to label single vesicles and dynamic microscopy to monitor their mobility. To track and analyze labeled vesicles, we employed a computer software. We found two modes of vesicle mobility, directional and non-directional. The mobility of non-directional vesicles is likely determined mainly by free diffusion. Only directional vesicles displayed a straight-line motion. The relationship of mean square displacement with time in directional vesicles resembled a quadratic function, indicating that in addition to free diffusion other mechanisms may contribute to vesicle movements in astrocytes, the biophysical properties of which are similar to those of neurons. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Glia; Astrocyte; Tracking; Vesicles; Mobility; Atrial natriuretic peptide In the past decade, astrocytes became a subject of in- tense research. The traditional concept of astrocytes being multifunctional housekeeping cells of neurons, built up in the 20th century, has largely been reconsid- ered. Besides maintaining the optimal environment for functioning of neurons [1], evidence emerged that astro- cytes may be involved in the regulation of synaptogene- sis [2], in direct modulation of synaptic strength in the CNS [3,4], and that they may have an important role in the regulation of brain microcirculation [5,6]. Astrocytes synthesize, store, and release many neuro- active compounds, including neurotransmitters [7], neu- rotrophins, eicosanoids, and neuropeptides [8]. Neurotransmitters released from neurons have been found to evoke an increase in [Ca 2+ ] i in astrocytes [3,9–11]. Moreover, it appears that intracellular calcium signaling may have an important role in the release of certain compounds from astrocytes, such as glutamate [4], atrial natriuretic peptide (ANP) [12], and prostaglan- dins [6]. The mechanism of glutamate and ANP release may involve exocytosis [12,13,15], since these chemical messengers are found in membrane bound organelles and the membrane of which must fuse with the plasma membrane for the cargo to be released into the extracel- lular space. It has been shown that astrocytes possess several components of SNARE molecules [15,16]; key elements for vesicular exocytosis [17]. However, much less is known about the delivery and transport of vesicles to the plasma membrane of astro- cytes to the site of exocytosis. Here we studied the char- acteristics of vesicle mobility in primary cultured rat astrocytes with the DNA construct encoding proANP fused with the emerald green fluorescent protein (pro- ANP-Emd) to label single vesicles, as shown previously [12,18], and used dynamic microscopy to analyze vesicle movements. By employing particle-tracking software we 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.02.030 * Corresponding author. E-mail address: Robert.Zorec@mf.uni-lj.si (R. Zorec). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 329 (2005) 678–683 BBRC