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Arp2/3 branched actin network mediates filopodia-like bundles formation in vitro.

Ideses Y, Brill-Karniely Y, Haviv L, Ben-Shaul A, Bernheim-Groswasser A - PLoS ONE (2008)

Bottom Line: Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower.Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy.As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed.

View Article: PubMed Central - PubMed

Affiliation: Ben-Gurion University of the Negev, Beer-Sheva, Israel.

ABSTRACT
During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]). Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched) surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed. This process is essentially irreversible on the time scale of actin polymerization. Two structural parameters, L, which is proportional to the length of filament tips at the aster periphery and b, the spacing between their origins, dictate the onset of bundling; both depending on [Arp2/3]. Cells may use a similar mechanism to regulate filopodia formation along the cell leading edge. Such a mechanism may allow cells to have control over the localization of filopodia by recruiting specific proteins that regulate filaments length (e.g., Dia2) to specific sites along lamellipodia.

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Mean star-like bundles' length, LFin, dependence on the [Arp2/3] and [fascin].(a) Conditions are: [G-actin] = 7 µM; [Arp2/3] = 6.25 nM (green dots), 25 nM (blue dots), and 100 nM (red dots). For all three cases, the mean bundles' length, LFin, increases with [fascin]. (b) Conditions are: 7 µM G-actin; 100 nM fascin and variable amounts of Arp2/3 complex. The data shows that the average of bundles length, LFin, decrease with [Arp2/3] monotonically (error bars are ±SD).
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pone-0003297-g005: Mean star-like bundles' length, LFin, dependence on the [Arp2/3] and [fascin].(a) Conditions are: [G-actin] = 7 µM; [Arp2/3] = 6.25 nM (green dots), 25 nM (blue dots), and 100 nM (red dots). For all three cases, the mean bundles' length, LFin, increases with [fascin]. (b) Conditions are: 7 µM G-actin; 100 nM fascin and variable amounts of Arp2/3 complex. The data shows that the average of bundles length, LFin, decrease with [Arp2/3] monotonically (error bars are ±SD).

Mentions: For all the concentrations of Arp2/3 that we have tested, the mean final length of bundles, LFin, was found to increase with [fascin], reaching asymptotically a maximal ([Arp2/3] dependent) value, (Fig. 5a). The system appears more sensitive to fascin addition when [Arp2/3] is lower (as reflected by the steeper slopes in Fig. 5a). Consequently, the amount of fascin required to reach decreases with the decrease in [Arp2/3]. In contrast, for a fixed concentration of fascin, we found that LFin decreases monotonously with [Arp2/3], (as seen for [fascin] = 100 nM in Fig. 5b). The inverse correlation between LFin and [Arp2/3] results from the reduced amount of actin monomers available for bundle growth when [Arp2/3] is increased, as discussed in more detail in the theory section below.


Arp2/3 branched actin network mediates filopodia-like bundles formation in vitro.

Ideses Y, Brill-Karniely Y, Haviv L, Ben-Shaul A, Bernheim-Groswasser A - PLoS ONE (2008)

Mean star-like bundles' length, LFin, dependence on the [Arp2/3] and [fascin].(a) Conditions are: [G-actin] = 7 µM; [Arp2/3] = 6.25 nM (green dots), 25 nM (blue dots), and 100 nM (red dots). For all three cases, the mean bundles' length, LFin, increases with [fascin]. (b) Conditions are: 7 µM G-actin; 100 nM fascin and variable amounts of Arp2/3 complex. The data shows that the average of bundles length, LFin, decrease with [Arp2/3] monotonically (error bars are ±SD).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2538570&req=5

pone-0003297-g005: Mean star-like bundles' length, LFin, dependence on the [Arp2/3] and [fascin].(a) Conditions are: [G-actin] = 7 µM; [Arp2/3] = 6.25 nM (green dots), 25 nM (blue dots), and 100 nM (red dots). For all three cases, the mean bundles' length, LFin, increases with [fascin]. (b) Conditions are: 7 µM G-actin; 100 nM fascin and variable amounts of Arp2/3 complex. The data shows that the average of bundles length, LFin, decrease with [Arp2/3] monotonically (error bars are ±SD).
Mentions: For all the concentrations of Arp2/3 that we have tested, the mean final length of bundles, LFin, was found to increase with [fascin], reaching asymptotically a maximal ([Arp2/3] dependent) value, (Fig. 5a). The system appears more sensitive to fascin addition when [Arp2/3] is lower (as reflected by the steeper slopes in Fig. 5a). Consequently, the amount of fascin required to reach decreases with the decrease in [Arp2/3]. In contrast, for a fixed concentration of fascin, we found that LFin decreases monotonously with [Arp2/3], (as seen for [fascin] = 100 nM in Fig. 5b). The inverse correlation between LFin and [Arp2/3] results from the reduced amount of actin monomers available for bundle growth when [Arp2/3] is increased, as discussed in more detail in the theory section below.

Bottom Line: Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower.Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy.As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed.

View Article: PubMed Central - PubMed

Affiliation: Ben-Gurion University of the Negev, Beer-Sheva, Israel.

ABSTRACT
During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]). Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched) surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed. This process is essentially irreversible on the time scale of actin polymerization. Two structural parameters, L, which is proportional to the length of filament tips at the aster periphery and b, the spacing between their origins, dictate the onset of bundling; both depending on [Arp2/3]. Cells may use a similar mechanism to regulate filopodia formation along the cell leading edge. Such a mechanism may allow cells to have control over the localization of filopodia by recruiting specific proteins that regulate filaments length (e.g., Dia2) to specific sites along lamellipodia.

Show MeSH
Related in: MedlinePlus