Technical Reference #1777

1777.

Rapid neurite outgrowth in neurosecretory cells and neurons in sustained by the exocytosis of a cytoplasmic organelle; the enlargeosome Gabriella Racchetti; Anna Lorusso; Carsten Schulte; Daniela Gavello; Valentina Carabelli; Rosalba D'Alessandro; Jacopo Meldolesi, National Institute of Neuroscience-Italy, Journal of Cell Science, 123(1777), (2010)
Link To Paper

Keywords:
Rac1; Nerve-cell differentiation; Neuronal primary cultures; PC12 cells; SH-SY5Y cells; Vamp4

Materials & Methods:
Sources: wt-PC12 and PC12-27 clones SH-SY5Y cells anti-Ahnak monoclonal and anti-VAMP4 antibodies and the siRNAs for VAMP4 VAMP7 VAMP8 and scrambled were as in Cocucci et al. (Cocucci et al. 2008); PC12-27/trk108 as in Leoni et al. (Leoni et al.1999); HeLa cells as in Borgonovo et al. (Borgonovo et al. 2002); ionomycin Fura-2 and BAPTA-AM as in Cocucci et al. (Cocucci et al. 2004). The flagged constitutively active (G12V) and dominant-negative (T17N) chicken Rac1 and the HA-ARF6 constructs were gifts of Ivan DeCurtis (San Raffaele Institute Milan Italy) and Michel Lazdunski (Institut de Pharmacologie Moléculaire et Cellulaire Sophia Antipolis France). Murine anti-Rac1 was from BD Bioscience San José CA; anti-b3-tubulin from Covance Berkeley CA; anti-NeuN and antineurofilaments from Millipore Billerica MA; anti-GFAP from Dako Glostrup Denmark; Y27632 from Calbiochem La Jolla CA; NGF from Alomone Jerusalem Israel; W56 peptide from Tocris Ellisville MO; latrunculin A and jasplakinolide from Invitrogen Eugene OR; anti-MAP2 NSC23766 actinomycin brefeldin A cyclohexamide nocodazol hispidin LY294002 PD98059 and rottlerin from Sigma- Aldrich Italy Milan Italy.

Microscopic Technique
Inverted Microscopy

Cell Type(s)
PC12-27 and SH-SY5Y cells