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First published online June 14, 2004
doi: 10.1242/10.1242/jcs.01285

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Visualizing Ras signalling in real-time

Laboratory of Molecular Signalling, The Babraham Institute, Babraham Research Campus, Babraham, Cambridge, CB2 4AT, UK

View larger version (25K): [in a new window]   Fig. 1. A model for the subcellular localization of mammalian Ras proteins. A protein farnesyl transferase adds a farnesyl group (prenylation) to the cysteine residue of the Ras CAAX motif (A=aliphatic, X=any amino acid), which thereby anchors the protein to the endoplasmic reticulum (ER). Rce1 (Ras and a-factor-converting enzyme) and isoprenylcysteine carboxyl methyltransferase remove the AAX and methylate the farnesylcysteine residue. H-Ras and N-Ras (and possibly K-Ras4A) are palmitoylated on cysteine residues in their hypervariable domains and enter the classical secretory pathway en route to the plasma membrane (PM). Palmitoylation is an unstable modification and might be a means by which the rate of Ras trafficking can be regulated (Hancock, 2003). Studies indicate that the nucleotide status of Ras can also influence the stability of this modification (Baker et al., 2003). K-Ras has a polylysine sequence instead of cysteine residues and is not palmitoylated; instead, it bypasses the Golgi via a non-classical secretory pathway. This might depend on microtubules (Thissen et al., 1997), although there has been no evidence that fluorescently tagged K-Ras4B is delivered along microtubule tracks in live cells.

View larger version (14K): [in a new window]   Fig. 2. Ras activity probes. These reporters are based on the Ras-binding domain (RBD) from Raf-1 fused to a fluorescent protein. Active Ras has been detected by the degree of membrane localization, in some cases with the additional sensitivity of a FRET indicator. Numbering applies to the human Raf-1 primary sequence. CR1 is the N-terminal conserved region of Raf-1 containing the RBD and cysteine-rich domain (CRD). CR2 is a serine- and threonine-rich region containing regulatory phosphorylation sites. CR3 is the catalytic domain of the kinase. Studies that have used various different domains as reporters are indicated and discussed in the text.

View larger version (33K): [in a new window]   Fig. 3. Localization of ectopic H-Ras-GTP in non-starved CHO cells. CHO cells were transfected with GFP-RBD (51-131) in (A) or cotransfected with H-Ras (B-D) and fixed 24 h later. (A) The GFP-RBD is diffusely localized throughout the cytoplasm and is not clearly associated with any intracellular structure or feature at the plasma membrane. Although the GFP-RBD can be occasionally seen in small membrane ruffles, this is probably due to the nonspecific trapping of fluorescent protein since GFP alone can produce a similar localization. Single confocal z-section shown. (B) Co-transfection with H-Ras causes the recruitment of the GFP-RBD to perinuclear structures, and plasma membrane ruffles, in non-starved cells. Single confocal z-section shown. (C) Immunostaining for total Ras expression. (D) Maximum projection overlay of eight equally spaced z-sections from the dorsal to the ventral cell surface showing co-localization of GFP-RBD (green) and H-Ras (red).

View larger version (103K): [in a new window]   Fig. 4. Mitogen-stimulated activation of H-Ras on the PM and Golgi. COS-1 cells were transiently co-transfected with GFP-RBD (51-131) and H-Ras. 24 h after transfection, cells were serum-starved overnight and live-cell imaging was performed at 37°C. Digital images of a single cell were obtained before and after stimulation with EGF, as indicated, with identical acquisition settings. The nucleus (N) is marked. The arrow indicates activated regions of the PM and the arrowhead indicates the position of the Golgi. Figure reproduced with permission from the Nature Publishing Group (http://www.nature.com/ncb/) (Chiu et al., 2002).

View larger version (31K): [in a new window]   Fig. 5. The Raichu-Ras FRET probe. (A) Schematic representation of Raichu-Ras bound to GDP or GTP. (B) EGF activation of Ras. Intensity-modulated display mode (IMD) images of COS-1 cells expressing Raichu-Ras or Raichu-Rap1 and stimulated by EGF. Figure reproduced with permission from the Nature Publishing Group (http://www.nature.com/nature/) (Mochizuki et al., 2001).