Limits...
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.

Show MeSH

Related in: MedlinePlus

Phase diagram for actin-fascin-arp2/3 complex.Condition is: 7 µM G-actin, Arp2/3 complex and fascin concentration was changed according to the graph. Condition for images is: 3 nM, 200 nM and 3 µM fascin; 6.25 nM, 12.5 nM and 6.25 nM Arp2/3 complex (a, b and c, respectively). Experimental points demonstrate different type of structures formed; these are represented by squares - entangled network, triangles - stars and circles - asters. Phase changes from asters to stars (blue line) and from stars to entangled bundle network (red line). Lines of phase separation are plotted as averages of two points of different structures. For low concentration of Arp2/3 complex phase transition is more abrupt for the same change in fascin concentration. No transition to entangled network was visible for high concentrations of Arp2/3 complex.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2538570&req=5

pone-0003297-g003: Phase diagram for actin-fascin-arp2/3 complex.Condition is: 7 µM G-actin, Arp2/3 complex and fascin concentration was changed according to the graph. Condition for images is: 3 nM, 200 nM and 3 µM fascin; 6.25 nM, 12.5 nM and 6.25 nM Arp2/3 complex (a, b and c, respectively). Experimental points demonstrate different type of structures formed; these are represented by squares - entangled network, triangles - stars and circles - asters. Phase changes from asters to stars (blue line) and from stars to entangled bundle network (red line). Lines of phase separation are plotted as averages of two points of different structures. For low concentration of Arp2/3 complex phase transition is more abrupt for the same change in fascin concentration. No transition to entangled network was visible for high concentrations of Arp2/3 complex.

Mentions: In Fig. 3 we present a phase diagram of the system, depicting the regions of existence of the three types of experimentally observed structures, as a function of the initial [Arp2/3] and [fascin]. The data shown here corresponds to experiments conducted at [G-actin] = 7 µM; a similar qualitative behavior was observed for the other concentrations of actin we have tested, ranging between 1 µM and 5 µM (data not shown). Careful inspection shows that below 3 nM of fascin only aster-like structures form (circles), regardless of [Arp2/3]. Increasing [fascin], for a given amount of Arp2/3 complex, induces structural transitions into stars (inverted triangles). The amount of fascin required to mediate this transition increases nonlinearly with [Arp2/3], saturating above 40 nM Arp2/3, (the blue curve marks the approximate boundary between the two regions). While only few nano-molars of fascin are sufficient to induce the transition from asters to stars, the subsequent transition to ‘networks’ (squares) requires much larger amounts of fascin (several µM; the red curve marks the approximate boundary between the two regions). We note that a transition to ‘networks’ is observed only for sufficiently low [Arp2/3] (<25 nM), within this region, the amount of fascin required for the nucleation of bundles increases monotonically with Arp2/3 content.


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)

Phase diagram for actin-fascin-arp2/3 complex.Condition is: 7 µM G-actin, Arp2/3 complex and fascin concentration was changed according to the graph. Condition for images is: 3 nM, 200 nM and 3 µM fascin; 6.25 nM, 12.5 nM and 6.25 nM Arp2/3 complex (a, b and c, respectively). Experimental points demonstrate different type of structures formed; these are represented by squares - entangled network, triangles - stars and circles - asters. Phase changes from asters to stars (blue line) and from stars to entangled bundle network (red line). Lines of phase separation are plotted as averages of two points of different structures. For low concentration of Arp2/3 complex phase transition is more abrupt for the same change in fascin concentration. No transition to entangled network was visible for high concentrations of Arp2/3 complex.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003297-g003: Phase diagram for actin-fascin-arp2/3 complex.Condition is: 7 µM G-actin, Arp2/3 complex and fascin concentration was changed according to the graph. Condition for images is: 3 nM, 200 nM and 3 µM fascin; 6.25 nM, 12.5 nM and 6.25 nM Arp2/3 complex (a, b and c, respectively). Experimental points demonstrate different type of structures formed; these are represented by squares - entangled network, triangles - stars and circles - asters. Phase changes from asters to stars (blue line) and from stars to entangled bundle network (red line). Lines of phase separation are plotted as averages of two points of different structures. For low concentration of Arp2/3 complex phase transition is more abrupt for the same change in fascin concentration. No transition to entangled network was visible for high concentrations of Arp2/3 complex.
Mentions: In Fig. 3 we present a phase diagram of the system, depicting the regions of existence of the three types of experimentally observed structures, as a function of the initial [Arp2/3] and [fascin]. The data shown here corresponds to experiments conducted at [G-actin] = 7 µM; a similar qualitative behavior was observed for the other concentrations of actin we have tested, ranging between 1 µM and 5 µM (data not shown). Careful inspection shows that below 3 nM of fascin only aster-like structures form (circles), regardless of [Arp2/3]. Increasing [fascin], for a given amount of Arp2/3 complex, induces structural transitions into stars (inverted triangles). The amount of fascin required to mediate this transition increases nonlinearly with [Arp2/3], saturating above 40 nM Arp2/3, (the blue curve marks the approximate boundary between the two regions). While only few nano-molars of fascin are sufficient to induce the transition from asters to stars, the subsequent transition to ‘networks’ (squares) requires much larger amounts of fascin (several µM; the red curve marks the approximate boundary between the two regions). We note that a transition to ‘networks’ is observed only for sufficiently low [Arp2/3] (<25 nM), within this region, the amount of fascin required for the nucleation of bundles increases monotonically with Arp2/3 content.

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