What is Ena/VASP doing anyway?
. | YES . | . | |
|---|---|---|---|
| 1. Ena/VASP enhances protrusion and propulsion speed | VASP targeted to leading edge = lamellipodia protrusion speed increases (Bear et al., 2000, 2002). | ||
| Results confirmed by CALIa against EGFP (Vitriol et al., 2007). | |||
| FAT1 knockdown to reduce VASP at leading edge = kymograph leading edge smoother (Moeller et al., 2004). | |||
| Listeria (Loisel et al., 1999). | |||
| Beads (Samarin et al., 2003; Plastino et al., 2004b). | |||
| Soft beads (Trichet et al., 2007). | |||
| 2. Ena/VASP inhibits formation of actin branches by the Arp2/3 complex | In solution: fluorescence microscopy, phalloidin-stabilized (Skoble et al., 2001) (conflicting study: [Boujemaa-Paterski et al., 2001], see text). | ||
| In cells, electron microscopy of the leading edge (Bear et al., 2002). | |||
| Comets on beads, Arp2/3 to actin ratio (Samarin et al., 2003). | |||
| Comets on beads, electron microscopy (Plastino et al., 2004b). | |||
| NO | YES | ||
| 3. Ena/VASP nucleates actin polymerization | By pyrene assayb, high (physiological) salt concentration (Barzik et al., 2005). | By pyrene assayb, low salt concentration (Hüttelmaier et al., 1999; Schirenbeck et al., 2006). | |
| Listeria mutants that do not recruit the Arp2/3 complex do not accumulate actin (Skoble et al., 2000). | On beads coated with the ActA domain that binds VASP (Fradelizi et al., 2001; Plastino et al., 2004a). | ||
| On mitochondria that target Ena/VASP proteins via the poly-proline repeats of ActA (Bear et al., 2000). | In conjunction with zyxin, observed by targeting zyxin to mitochondria in cells (Fradelizi et al., 2001). | ||
| 4. Ena/VASP enhances barbed end elongation | By pyrene assayb using F-actin seeds (Bear et al., 2002). | By pyrene assayb using monomeric actin (Skoble et al., 2001). | |
| By pyrene assayb using F-actin seeds (Barzik et al., 2005). | |||
| By pyrene assayb with actin NPFs free in solution (Samarin et al., 2003). | By pyrene assayb with actin NPFs immobilized on beads (Samarin et al., 2003). | ||
| By measuring actin incorporation into comet tails on moving beads (Plastino et al., 2004b). | |||
| 5. Ena/VASP protects filament barbed ends from capping | |||
| –Anti-capping | By pyrene assayb (Samarin et al., 2003). | By pyrene assayb (Bear et al., 2002; Barzik et al., 2005). | |
| –Uncapping | By pyrene assayb (Schirenbeck et al., 2006). | ||
| Lack of capture of capped barbed ends by beads coated with VASP (Bear et al., 2002)c. | |||
. | YES . | . | |
|---|---|---|---|
| 1. Ena/VASP enhances protrusion and propulsion speed | VASP targeted to leading edge = lamellipodia protrusion speed increases (Bear et al., 2000, 2002). | ||
| Results confirmed by CALIa against EGFP (Vitriol et al., 2007). | |||
| FAT1 knockdown to reduce VASP at leading edge = kymograph leading edge smoother (Moeller et al., 2004). | |||
| Listeria (Loisel et al., 1999). | |||
| Beads (Samarin et al., 2003; Plastino et al., 2004b). | |||
| Soft beads (Trichet et al., 2007). | |||
| 2. Ena/VASP inhibits formation of actin branches by the Arp2/3 complex | In solution: fluorescence microscopy, phalloidin-stabilized (Skoble et al., 2001) (conflicting study: [Boujemaa-Paterski et al., 2001], see text). | ||
| In cells, electron microscopy of the leading edge (Bear et al., 2002). | |||
| Comets on beads, Arp2/3 to actin ratio (Samarin et al., 2003). | |||
| Comets on beads, electron microscopy (Plastino et al., 2004b). | |||
| NO | YES | ||
| 3. Ena/VASP nucleates actin polymerization | By pyrene assayb, high (physiological) salt concentration (Barzik et al., 2005). | By pyrene assayb, low salt concentration (Hüttelmaier et al., 1999; Schirenbeck et al., 2006). | |
| Listeria mutants that do not recruit the Arp2/3 complex do not accumulate actin (Skoble et al., 2000). | On beads coated with the ActA domain that binds VASP (Fradelizi et al., 2001; Plastino et al., 2004a). | ||
| On mitochondria that target Ena/VASP proteins via the poly-proline repeats of ActA (Bear et al., 2000). | In conjunction with zyxin, observed by targeting zyxin to mitochondria in cells (Fradelizi et al., 2001). | ||
| 4. Ena/VASP enhances barbed end elongation | By pyrene assayb using F-actin seeds (Bear et al., 2002). | By pyrene assayb using monomeric actin (Skoble et al., 2001). | |
| By pyrene assayb using F-actin seeds (Barzik et al., 2005). | |||
| By pyrene assayb with actin NPFs free in solution (Samarin et al., 2003). | By pyrene assayb with actin NPFs immobilized on beads (Samarin et al., 2003). | ||
| By measuring actin incorporation into comet tails on moving beads (Plastino et al., 2004b). | |||
| 5. Ena/VASP protects filament barbed ends from capping | |||
| –Anti-capping | By pyrene assayb (Samarin et al., 2003). | By pyrene assayb (Bear et al., 2002; Barzik et al., 2005). | |
| –Uncapping | By pyrene assayb (Schirenbeck et al., 2006). | ||
| Lack of capture of capped barbed ends by beads coated with VASP (Bear et al., 2002)c. | |||
Chromophore-assisted laser inactivation.
The pyrene assay is a method of monitoring the change in the amount of F-actin in a solution by following the fluorescence of pyrenyl-actin, which increases when pyrenyl-actin molecules are incorporated into the filament.
In a similar study, Samarin et al. (2003) observed capture of capped barbed ends by beads coated with VASP. However, these authors concluded that this activity was due to VASP's F-actin binding activity and not to uncapping activity.