pCAG-gap-Venus was prepared while described previously (27). regulate axon growth or branching. Moreover, shRNA-mediated knockdown of Rab17 manifestation resulted in a dramatically reduced quantity of dendritic spines, probably because of impaired filopodia formation. These findings possess exposed the 1st molecular link between membrane Kynurenic acid sodium trafficking and dendritogenesis. dendrites and axons, respectively. Considerable dendritic morphogenesis, which includes the generation and elaboration of considerable dendrite arbors followed by retraction and pruning, occurs in the brain to establish appropriate neural circuits (1). During the maturation step of dendritogenesis, filopodia from your dendritic shaft are morphologically and functionally converted into spines, which are tiny bulbous protrusions that receive input through a single synapse with an axon (2). The dendritic morphogenesis is vital to the proper formation of neuronal circuits, because problems in dendrite patterning and/or spine formation often cause severe neurodevelopmental disorders (1, 2). However, the molecular mechanisms by which dendritogenesis and postsynaptic development occur are poorly recognized. Membrane trafficking is definitely a well known biological process that is involved in a wide variety of cellular events, including cell polarization. In neurons, for example, axon-specific molecules (glutamate transporter and synaptotagmin I) (3, 4) and dendrite-specific molecules (AMPA receptors and NMDA receptors) (5, 6) are thought to be targeted to axons and dendrites, respectively, by individual as yet unestablished trafficking mechanisms. Rab small GTPases are conserved membrane trafficking proteins in all eukaryotes, and they Kynurenic acid sodium mediate numerous methods in membrane trafficking, including vesicle budding, vesicle CD14 movement, vesicle docking to specific membranes, and vesicle fusion. The numbers of Rab isoforms vary with the varieties, ranging from 11 in budding yeasts to 60 in mammals, and the development in the Kynurenic acid sodium number of Rab isoforms in higher eukaryotes is likely to be related to specialized membrane trafficking pathways in specialized cell types (7, 8). Some Rab proteins have actually been shown to be specifically localized in axons and to operate axon-specific trafficking events (Rab3 and Rab27 in synaptic vesicle trafficking) (9C11). Involvement of additional Rab isoforms in postsynaptic functions has also been reported (12C16), but they are not dendrite-specific Rab proteins (they are also present and function in axons; observe Refs. 17 and 18), and no Rab protein that specifically functions in dendritic morphogenesis offers ever been reported. In this study, we discovered that Rab17 is the only Rab isoform that is predominantly targeted to the dendrites of mouse hippocampal neurons. Rab17 is definitely localized at dendritic growth cones, shafts, filopodia, and adult spines, but it is mostly absent in axons. We also found that Rab17 mediates dendrite growth and branching but that it does not regulate axon growth or branching. Moreover, knockdown of Rab17 manifestation resulted in a dramatically reduced quantity of spines probably because of impaired filopodia formation. Possible functions of Rab17 in dendritic morphogenesis and postsynaptic development are discussed based on our findings. EXPERIMENTAL Methods Antibodies The following antibodies used in this study were acquired commercially: anti-actin goat polyclonal antibody and anti-c-Myc (9E10) mouse monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-actin mouse monoclonal antibody (ABM, Richmond, Canada); anti-neurofilament-H mouse monoclonal antibody (American Study Products, Belmont, MA); anti-Tau (5E2) mouse monoclonal antibody, anti-NMDAR1 (MAB363) mouse monoclonal antibody, anti-GluR2 (MAB397) mouse monoclonal antibody, and anti-MAP2 chick polyclonal antibody (Millipore Corp., Billerica, MA); anti-calcium/calmodulin-dependent protein kinase II (CaMKII)2 (6G9) mouse monoclonal antibody (Calbiochem, La Jolla, LA); anti-PSD95 (7E3C1B8) mouse monoclonal antibody (Pierce); anti-EEA1 rabbit polyclonal antibody (Cell Signaling Technology, Beverly, MA); anti-EEA1 mouse monoclonal antibody (BD Transduction Laboratories, Lexington, KY); anti-GFP rabbit polyclonal antibody and horseradish peroxidase-conjugated anti-GAPDH (3H12) mouse monoclonal antibody (MBL, Nagoya, Japan); anti-synaptophysin (SVP-38) monoclonal antibody and horseradish peroxidase-conjugated anti-FLAG tag (M2) mouse monoclonal antibody (Sigma-Aldrich); and anti-Rab11 rabbit polyclonal antibody and Alexa 488/594/633-conjugated anti-mouse/rabbit/goat/chick/guinea pig IgG goat antibody (Invitrogen). Anti-synaptotagmin (Syt) I N-terminal rabbit polyclonal antibody was prepared as explained previously (19). Anti-Rab17 rabbit polyclonal antibody and anti-GFP guinea pig polyclonal antibody were raised against GST-Rab17 and GST-EGFP, respectively, and affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad) as explained previously (20). Plasmid Building pEGFP-C1 vectors (Clontech) harboring cDNAs of 41 Kynurenic acid sodium different human being or mouse Rab proteins.