Recombinant Antibodies Against Subcellular Fractions Used to Track Endogenous Golgi Protein Dynamics in Vivo

Abstract

Generation of specific antibodies against enriched subcellular fractions is a powerful strategy to identify and characterize cellular components. We show that recombinant antibodies can be selected in vitro by phage display against complex subcellular fractions, namely microtubule-binding proteins and Golgi stacks. This technique has allowed us to overcome many limitations of the classical animal-based approach and generate cell biology-compliant antibodies. In addition, we show that intracellular expression of GFP-tagged recombinant antibodies can reveal the dynamics of endogenous proteins in vivo. Endogenous Giantin is very static and outlines the Golgi in living cells. It accumulates neither onto Golgi-derived tubules upon Brefeldin A treatment before Golgi disappearance, nor onto de novo formed Golgi mini-stacks upon microtubule depolymerization, and remains instead on the 'old' pericentriolar Golgi. This suggests that, in contrast to other Golgi matrix proteins, endogenous Giantin is very stably associated with the Golgi and does not efficiently recycle to the ER. Altogether, we show that the antibody phage display technique represents an efficient alternative to rapidly generate versatile antibodies that represent new tools to study protein function.

The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis

“…siRNA duplexes for silencing Nup133, Nup153, and Giantin were described previously Nizak et al, 2003;Walther et al, 2003a). The siRNA duplex used for silencing Tpr was provided by Ulf Nherbass (Institut Pasteur, Paris, France).…”

“…The others antibodies used were affinity-purified rabbit polyclonal antibodies directed against human Nup133 and Nup107 (Belgareh et al, 2001), Nup96/p87 serum affinitypurified by using recombinant Nup96/p87 (residues 1291-1482 of Nup196) immobilized on nitrocellulose (Fontoura et al, 1999), affinity-purified antiNup96 (directed against a synthetic peptide corresponding to aa 1743-1763 of Nup196) (Hase and Cordes, 2003), affinity-purified anti-Sec13 (Tang et al, 1997) and Sec31 (Tang et al, 2000), anti-Tpr (Kuznetsov et al, 2002), or anti-myc epitope (Euromedex, Souffelweyersheim, France); monoclonal antibodies anti-myc (clone 9E10; Sigma-Aldrich, St. Louis, MO), anti-GFP (clone 7.1 and 13.1; Roche Diagnostics, Indianapolis, IN), anti-SC-35 (Sigma-Aldrich), anti-cyclin B1 (Santa Cruz Biotechnology), anti-mitosin (BD Biosciences, San Jose, CA), and mAb414 (Babco, Richmond, CA); the autoimmune CREST serum was obtained from J.C. Courvalin (Institut J. Monod, Paris, France). To detect Giantin, the TA10 scFv (Nizak et al, 2003), obtained from F. Perez (Institut Curie, Paris, France), was used at 1/100 and mixed with a goat anti-myc antibody (Santa Cruz Biotechnology). Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) or Molecular Probes (Eugene, OR).…”

Utilisation desintrabodies: de l’étude des protéines intracellulaires à l’immunisation thérapeutique

“…Par ailleurs, nous avons montré que les intrabodies non bloquants peuvent représenter des outils uniques pour suivre la dynamique des protéines endogènes sans que l'on ait recours à la surexpression de protéines sauvages ou mutées. La dynamique d'une très grosse protéine de l'appareil de Golgi, la giantine, a ainsi pu être étudiée grâce à un scFv anti-giantine étiqueté avec la GFP (green fluorescent protein) et exprimé in vivo [10]. Différents intrabodies fluorescents ont ainsi …”

‘Shotgun immunological’ approach for analysis of a complex subcellular system

“…Selection of single-chain fragment variable (scFv) from bacteriophage-displayed libraries (8,(13)(14)(15) provides a complimentary means of isolating panels of mAbs recognising components of macromolecular structures. Although scFv antibodies are generally of lower binding affinity than their natural counterparts, since they consist of one heavy and one light antibody fragment rather than a pair, their selection is simpler, faster and less labour-intensive.…”

“…Although scFv antibodies are generally of lower binding affinity than their natural counterparts, since they consist of one heavy and one light antibody fragment rather than a pair, their selection is simpler, faster and less labour-intensive. Since no host animal is immunised, scFv mAbs can be generated against toxic and 'self' antigens (13)(14)(15). By-passing immunisation drastically decreases quantities of antigen required for antibody selection.…”