Limits...
EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells.

van Rheenen J, Song X, van Roosmalen W, Cammer M, Chen X, Desmarais V, Yip SC, Backer JM, Eddy RJ, Condeelis JS - J. Cell Biol. (2007)

Bottom Line: Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP(2) in living cells.We show that EGF induces a rapid loss of PIP(2) through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin.Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA. jvanrhee@aecom.yu.edu

ABSTRACT
Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP(2); however, this is mainly based on in vitro-binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP(2) in living cells. We show that EGF induces a rapid loss of PIP(2) through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.

Show MeSH

Related in: MedlinePlus

Compartments involved in the cofilin activity cycle. Cofilin molecules cycle through three compartments in the cell; the cytosol, the actin, and the PM compartments. Cofilin at the PM compartment initially translocates to the F-actin compartment upon EGF mediated PIP2 reduction. Cofilin binds and severs actin filaments resulting in filaments with free barbed ends, and cofilin–G-actin complex. The cofilin–G-actin complex cannot bind actin or PM, and therefore diffuses to the cytosol compartment. In the cytosol compartment, cofilin is phosphorylated by LIM-kinase, resulting in the release of cofilin from the cofilin–G-actin complex. Upon cofilin dephosphorylation by SSH, cofilin can reenter the PM or F-actin compartment, starting a new cycle. The cycling of cofilin through the three compartments increases free barbed ends, resulting in newly polymerized actin filaments. The ARP2/3 complex prefers to bind to these newly formed actin filaments, which amplifies the cofilin-induced actin polymerization, resulting in protrusion formation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140025&req=5

fig7: Compartments involved in the cofilin activity cycle. Cofilin molecules cycle through three compartments in the cell; the cytosol, the actin, and the PM compartments. Cofilin at the PM compartment initially translocates to the F-actin compartment upon EGF mediated PIP2 reduction. Cofilin binds and severs actin filaments resulting in filaments with free barbed ends, and cofilin–G-actin complex. The cofilin–G-actin complex cannot bind actin or PM, and therefore diffuses to the cytosol compartment. In the cytosol compartment, cofilin is phosphorylated by LIM-kinase, resulting in the release of cofilin from the cofilin–G-actin complex. Upon cofilin dephosphorylation by SSH, cofilin can reenter the PM or F-actin compartment, starting a new cycle. The cycling of cofilin through the three compartments increases free barbed ends, resulting in newly polymerized actin filaments. The ARP2/3 complex prefers to bind to these newly formed actin filaments, which amplifies the cofilin-induced actin polymerization, resulting in protrusion formation.

Mentions: In this manuscript, we tested the hypothesis that a pool of cofilin is associated with and inhibited by PIP2 in the PM, and that it can be released and activated upon PLC-mediated PIP2 hydrolysis. Using biochemical, FRET, and FLIP experiments, we identified three cofilin compartments in the cell—the cytosolic, PM-associated, and F-actin–bound compartments—the latter two at the periphery of the cell. FLIP experiments showed that cofilin can rapidly cycle between these compartments and that upon EGF stimulation, cofilin molecules at the PM compartment translocate to the F-actin compartment. These observations suggest the activity cycle shown in Fig. 7. In unstimulated cells, cofilin at the cell periphery is mainly localized in the PM compartment. Upon EGF-induced PIP2 reduction, this pool is released from the PM and translocates to the nearby F-actin compartment. The increase in the number of cofilin molecules that bind to actin filaments leads to actin severing, which in turn leads to an increase in free barbed ends and cofilin–G-actin products. The cofilin–G-actin product is not able to bind to F-actin or the PM, and therefore diffuses to the cytosol compartment. Cofilin is released from the cofilin–G-actin complex by phosphorylation in order to reenter the PM or F-actin compartments. The result of releasing cofilin from the PM is an increase in the number of barbed ends in the F-actin compartment followed by the elongation of filaments by polymerization from the barbed ends of the severed filaments. The newly polymerized actin filaments are preferred by the ARP2/3 complex for binding (Ichetovkin et al., 2002). Each ARP2/3 complex nucleates a new daughter branch from the side of the cofilin-generated mother filament, resulting in a large amplification of actin polymerization resulting from cofilin activity. This actin polymerization is the driving force for the formation of membrane protrusions.


EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells.

van Rheenen J, Song X, van Roosmalen W, Cammer M, Chen X, Desmarais V, Yip SC, Backer JM, Eddy RJ, Condeelis JS - J. Cell Biol. (2007)

Compartments involved in the cofilin activity cycle. Cofilin molecules cycle through three compartments in the cell; the cytosol, the actin, and the PM compartments. Cofilin at the PM compartment initially translocates to the F-actin compartment upon EGF mediated PIP2 reduction. Cofilin binds and severs actin filaments resulting in filaments with free barbed ends, and cofilin–G-actin complex. The cofilin–G-actin complex cannot bind actin or PM, and therefore diffuses to the cytosol compartment. In the cytosol compartment, cofilin is phosphorylated by LIM-kinase, resulting in the release of cofilin from the cofilin–G-actin complex. Upon cofilin dephosphorylation by SSH, cofilin can reenter the PM or F-actin compartment, starting a new cycle. The cycling of cofilin through the three compartments increases free barbed ends, resulting in newly polymerized actin filaments. The ARP2/3 complex prefers to bind to these newly formed actin filaments, which amplifies the cofilin-induced actin polymerization, resulting in protrusion formation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140025&req=5

fig7: Compartments involved in the cofilin activity cycle. Cofilin molecules cycle through three compartments in the cell; the cytosol, the actin, and the PM compartments. Cofilin at the PM compartment initially translocates to the F-actin compartment upon EGF mediated PIP2 reduction. Cofilin binds and severs actin filaments resulting in filaments with free barbed ends, and cofilin–G-actin complex. The cofilin–G-actin complex cannot bind actin or PM, and therefore diffuses to the cytosol compartment. In the cytosol compartment, cofilin is phosphorylated by LIM-kinase, resulting in the release of cofilin from the cofilin–G-actin complex. Upon cofilin dephosphorylation by SSH, cofilin can reenter the PM or F-actin compartment, starting a new cycle. The cycling of cofilin through the three compartments increases free barbed ends, resulting in newly polymerized actin filaments. The ARP2/3 complex prefers to bind to these newly formed actin filaments, which amplifies the cofilin-induced actin polymerization, resulting in protrusion formation.
Mentions: In this manuscript, we tested the hypothesis that a pool of cofilin is associated with and inhibited by PIP2 in the PM, and that it can be released and activated upon PLC-mediated PIP2 hydrolysis. Using biochemical, FRET, and FLIP experiments, we identified three cofilin compartments in the cell—the cytosolic, PM-associated, and F-actin–bound compartments—the latter two at the periphery of the cell. FLIP experiments showed that cofilin can rapidly cycle between these compartments and that upon EGF stimulation, cofilin molecules at the PM compartment translocate to the F-actin compartment. These observations suggest the activity cycle shown in Fig. 7. In unstimulated cells, cofilin at the cell periphery is mainly localized in the PM compartment. Upon EGF-induced PIP2 reduction, this pool is released from the PM and translocates to the nearby F-actin compartment. The increase in the number of cofilin molecules that bind to actin filaments leads to actin severing, which in turn leads to an increase in free barbed ends and cofilin–G-actin products. The cofilin–G-actin product is not able to bind to F-actin or the PM, and therefore diffuses to the cytosol compartment. Cofilin is released from the cofilin–G-actin complex by phosphorylation in order to reenter the PM or F-actin compartments. The result of releasing cofilin from the PM is an increase in the number of barbed ends in the F-actin compartment followed by the elongation of filaments by polymerization from the barbed ends of the severed filaments. The newly polymerized actin filaments are preferred by the ARP2/3 complex for binding (Ichetovkin et al., 2002). Each ARP2/3 complex nucleates a new daughter branch from the side of the cofilin-generated mother filament, resulting in a large amplification of actin polymerization resulting from cofilin activity. This actin polymerization is the driving force for the formation of membrane protrusions.

Bottom Line: Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP(2) in living cells.We show that EGF induces a rapid loss of PIP(2) through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin.Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA. jvanrhee@aecom.yu.edu

ABSTRACT
Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP(2); however, this is mainly based on in vitro-binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP(2) in living cells. We show that EGF induces a rapid loss of PIP(2) through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.

Show MeSH
Related in: MedlinePlus