Visualization of Vesicle Transport Along and Between Distinct Pathways in Neurites of Living Cells GERHARD J. SCHU ¨ TZ, 1 * MARKUS AXMANN, 1 SUSANNE FREUDENTHALER, 1 HANSGEORG SCHINDLER, 1 KOSTYA KANDROR, 2 JOHN C. RODER, 3 AND ANDREAS JEROMIN 4 1 Institute for Biophysics, University of Linz, A-4040 Linz, Austria 2 Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02218 3 Mt. Sinai Hospital, SRLI, Toronto, Ontario, M5G1X5 Canada 4 Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030 KEY WORDS fluorescence microscopy; vesicle tracking; 3D imaging; microtubule; motor mol- ecules; vesicle sorting ABSTRACT Trafficking of secretory vesicles along neurites of PC12 cells was visualized by 2D and 3D real-time imaging using fluorescence microscopy. Vesicle motion along distinct pathways was directly seen. From an overlay of individual pathways, the underlying cytoskeletal filament could be imaged at a subwavelength resolution. Continuous vesicle transport was interrupted by periods of diffusive motion with concomitant pathway changes. Statistical analysis shows that such interruptions were distributed stochastically along the filament, indicating a limited processivity of motor proteins also in a cellular context. Periods of diffusive motion facilitated the interaction with actively transported vesicles. Frequent associations and dissociations of vesicles have been ob- served consistently, pointing to a functional relevance of vesicle cotransport. Microsc. Res. Tech. 63: 159 –167, 2004. © 2004 Wiley-Liss, Inc. INTRODUCTION Continuous supply of nutrients in cells is mediated via the transport of vesicles containing cargo, such as proteins and lipids, along distinct pathways, predomi- nantly microtubules (Sheetz, 1999; Lippincott- Schwartz et al., 2000). In neuronal cells the transport machinery is thought to be optimized for fast delivery of vesicles from the cell body along neurites to sites of growth and communication, the growth cone and syn- apses (Allen et al., 1982; Brady et al., 1982; Kuznetsov et al., 1992; Lin and Scheller, 2000). Neurites contain microtubules, intermediate filaments, and actin fila- ments as the three major classes of cytoskeletal com- ponents (Kobayashi and Mundel, 1998). Transport re- lies on directed movement mediated by the two major types of microtubule-associated motor proteins, kinesin and dynein (Hirokawa, 1998). In neuronal cells the growth of microtubules is polarized, with the growing end directed to the processes, and with kinesin and dynein being responsible for anterograde and retro- grade transport, respectively (Goldstein and Yang, 2000). Detailed information about motor protein mo- tion along microtubules has been obtained in vitro in isolated systems (Vale and Milligan, 2000), yielding processive movements of individual kinesin or dynein molecules over distances of 1.5 m before dissociat- ing from the microtubule (Block et al., 1990). Much less corresponding mechanistic information on transport velocity and processivity is available within living cells. There is currently no clear picture of how pathway changes occur, an important issue in view of the many pathway changes required for the long distance trav- eling from cell body to growth cone (Thorn et al., 2000). Indeed, the limited length of cytoskeletal filaments (Yu and Baas, 1994) and the existence of obstacles along the pathway renders changes of the transport route inevitable. It has been suggested that the observed low processivity (Thorn et al., 2000) may have evolved ei- ther to circumvent these obstacles or to achieve an optimum balance between vectorial transport and ran- dom motion (Taylor and Borisy, 2000). In addition, a recent study reported a more complex transport behav- ior of vesicles, with preassembly or en route assembly of larger vesicle associates, called prototerminals (Ah- mari et al., 2000). Here, we applied real-time 2D and 3D imaging (Schutz et al., 2000b) to resolve mechanistic details during transport of individual motor-driven vesicles. Secretory vesicles were imaged in stable transfected, differentiated neuroendocrine (PC12) cells. These cells share many features with neurons: they undergo calci- um-dependent exocytosis (Meldolesi et al., 1984) and have been used to study the transport of secretory granules in real time (Lang et al., 1997; Lochner et al., 1998; Abney et al., 1999). VAMP-2 was fused to en- hanced green fluorescent protein (VAMP-eGFP) and used as a tool to monitor its trafficking. Like nontagged VAMP-2, VAMP-eGFP labeled a variety of vesicles of different shapes and sizes (Fig. 1), in agreement with earlier results by Papini et al. (1995). The movement of individual vesicles along two- and three-dimensional *Correspondence to: Gerhard J. Schu ¨ tz, Institute for Biophysics, University of Linz, Altenbergerstr. 69, A-4040 Linz, Austria. E-mail: gerhard.schuetz@jku.at Received 26 June 2003; accepted in revised form 6 November 2003 Contract grant sponsor: Austrian Federal Ministry of Science and Transport; Contract grant numbers: GZ 200.025/3-Pr/4/1998; GZ 200.027/3-Pr/2a/1998; Contract grant sponsor: Austrian Research Funds; Contract grant numbers: P12803-MED, P15053. DOI 10.1002/jemt.20016 Published online in Wiley InterScience (www.interscience.wiley.com). MICROSCOPY RESEARCH AND TECHNIQUE 63:159 –167 (2004) © 2004 WILEY-LISS, INC.