Limits...
Differential dynamics of alpha 5 integrin, paxillin, and alpha-actinin during formation and disassembly of adhesions in migrating cells.

Laukaitis CM, Webb DJ, Donais K, Horwitz AF - J. Cell Biol. (2001)

Bottom Line: All localized with their endogenous counterparts and did not perturb migration when expressed at moderate levels. alpha 5-GFP also rescued the adhesive defects in CHO B2 cells, which are alpha 5 integrin deficient.As cells migrated, alpha 5 vesicles also moved from a perinuclear region to the base of the lamellipodium.The alpha 5 vesicles colocalized with transferrin receptor and FM 4-64 dye.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801, USA.

ABSTRACT
To investigate the mechanisms by which adhesions form and disperse in migrating cells, we expressed alpha 5 integrin, alpha-actinin, and paxillin as green fluorescent protein (GFP) fusions. All localized with their endogenous counterparts and did not perturb migration when expressed at moderate levels. alpha 5-GFP also rescued the adhesive defects in CHO B2 cells, which are alpha 5 integrin deficient. In ruffling cells, alpha 5-GFP and alpha-actinin--GFP localized prominently at the leading edge in membrane protrusions. Of the three GFP fusion proteins that we examined, paxillin was the first component to appear visibly organized in protrusive regions of the cell. When a new protrusion formed, the paxillin appeared to remodel from older to newer adhesions at the leading edge. alpha-Actinin subsequently entered adhesions, which translocated toward the cell center, and inhibited paxillin turnover. The new adhesions formed from small foci of alpha-actinin--GFP and paxillin-GFP, which grew in size. Subsequently, alpha 5 integrin entered the adhesions to form visible complexes, which served to stabilize the adhesions. alpha 5-GFP also resided in endocytic vesicles that emanated from the leading edge of protrusions. Integrin vesicles at the cell rear moved toward the cell body. As cells migrated, alpha 5 vesicles also moved from a perinuclear region to the base of the lamellipodium. The alpha 5 vesicles colocalized with transferrin receptor and FM 4-64 dye. After adhesions broke down in the rear, alpha 5-GFP was found in fibrous structures behind the cell, whereas alpha-actinin--GFP and paxillin-GFP moved up the lateral edge of retracting cells as organized structures and then dissipated.

Show MeSH

Related in: MedlinePlus

α5 integrin remains in fibrous structures left behind migrating cells. (a) When α5-GFP–expressing CHO cells were plated on Fn, fluorescent fibers containing the integrin remained behind the migrating cells on the substratum. In cells cotransfected with α5-YFP (b and d) and α-actinin-CFP (c) or paxillin-CFP (e), α5 integrin was found in fibers, whereas neither α-actinin nor paxillin was observed in these structures. Bar, 25 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2150721&req=5

Figure 9: α5 integrin remains in fibrous structures left behind migrating cells. (a) When α5-GFP–expressing CHO cells were plated on Fn, fluorescent fibers containing the integrin remained behind the migrating cells on the substratum. In cells cotransfected with α5-YFP (b and d) and α-actinin-CFP (c) or paxillin-CFP (e), α5 integrin was found in fibers, whereas neither α-actinin nor paxillin was observed in these structures. Bar, 25 μm.

Mentions: As adhesions break down, a fraction of the integrins can be left behind the cell in tracks (Regen and Horwitz 1992; Palecek et al. 1996, Palecek et al. 1998); however, the fate of the cytoskeletal proteins is not known. Thus, we examined the fate of adhesive components at the cell rear as adhesions broke down. When α5-GFP integrin-expressing CHO B2 and CHO K1 cells were plated on 2.5–5 μg/ml Fn, we observed fluorescent fibers extending behind retracting cell regions (Fig. 9 a). A network of “retraction” fibers greater than a cell length was also observed when α5-GFP–expressing WI38 cells were plated on 0.5–2 μg/ml Fn. In some cases, the fibers were left behind as the cells migrated. The length of these fibers was variable. These fibrous structures were membranous, since similar structures were seen behind cells transfected with a palmitoylated membrane-bound GFP (Moriyoshi et al. 1996), but FM 4-64 was not observed in fibers. When CHO K1 cells expressing similar levels of soluble GFP were plated under the same conditions, “retraction” fibers were not observed, indicating that the cytoplasmic volume of these structures is small. Paxillin-CFP and α-actinin–CFP do not localize with α5-YFP in these fibrous networks (Fig. 9, b–e). This suggests that the adhesion is breaking down closer to the integrin than to the actin.


Differential dynamics of alpha 5 integrin, paxillin, and alpha-actinin during formation and disassembly of adhesions in migrating cells.

Laukaitis CM, Webb DJ, Donais K, Horwitz AF - J. Cell Biol. (2001)

α5 integrin remains in fibrous structures left behind migrating cells. (a) When α5-GFP–expressing CHO cells were plated on Fn, fluorescent fibers containing the integrin remained behind the migrating cells on the substratum. In cells cotransfected with α5-YFP (b and d) and α-actinin-CFP (c) or paxillin-CFP (e), α5 integrin was found in fibers, whereas neither α-actinin nor paxillin was observed in these structures. Bar, 25 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2150721&req=5

Figure 9: α5 integrin remains in fibrous structures left behind migrating cells. (a) When α5-GFP–expressing CHO cells were plated on Fn, fluorescent fibers containing the integrin remained behind the migrating cells on the substratum. In cells cotransfected with α5-YFP (b and d) and α-actinin-CFP (c) or paxillin-CFP (e), α5 integrin was found in fibers, whereas neither α-actinin nor paxillin was observed in these structures. Bar, 25 μm.
Mentions: As adhesions break down, a fraction of the integrins can be left behind the cell in tracks (Regen and Horwitz 1992; Palecek et al. 1996, Palecek et al. 1998); however, the fate of the cytoskeletal proteins is not known. Thus, we examined the fate of adhesive components at the cell rear as adhesions broke down. When α5-GFP integrin-expressing CHO B2 and CHO K1 cells were plated on 2.5–5 μg/ml Fn, we observed fluorescent fibers extending behind retracting cell regions (Fig. 9 a). A network of “retraction” fibers greater than a cell length was also observed when α5-GFP–expressing WI38 cells were plated on 0.5–2 μg/ml Fn. In some cases, the fibers were left behind as the cells migrated. The length of these fibers was variable. These fibrous structures were membranous, since similar structures were seen behind cells transfected with a palmitoylated membrane-bound GFP (Moriyoshi et al. 1996), but FM 4-64 was not observed in fibers. When CHO K1 cells expressing similar levels of soluble GFP were plated under the same conditions, “retraction” fibers were not observed, indicating that the cytoplasmic volume of these structures is small. Paxillin-CFP and α-actinin–CFP do not localize with α5-YFP in these fibrous networks (Fig. 9, b–e). This suggests that the adhesion is breaking down closer to the integrin than to the actin.

Bottom Line: All localized with their endogenous counterparts and did not perturb migration when expressed at moderate levels. alpha 5-GFP also rescued the adhesive defects in CHO B2 cells, which are alpha 5 integrin deficient.As cells migrated, alpha 5 vesicles also moved from a perinuclear region to the base of the lamellipodium.The alpha 5 vesicles colocalized with transferrin receptor and FM 4-64 dye.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801, USA.

ABSTRACT
To investigate the mechanisms by which adhesions form and disperse in migrating cells, we expressed alpha 5 integrin, alpha-actinin, and paxillin as green fluorescent protein (GFP) fusions. All localized with their endogenous counterparts and did not perturb migration when expressed at moderate levels. alpha 5-GFP also rescued the adhesive defects in CHO B2 cells, which are alpha 5 integrin deficient. In ruffling cells, alpha 5-GFP and alpha-actinin--GFP localized prominently at the leading edge in membrane protrusions. Of the three GFP fusion proteins that we examined, paxillin was the first component to appear visibly organized in protrusive regions of the cell. When a new protrusion formed, the paxillin appeared to remodel from older to newer adhesions at the leading edge. alpha-Actinin subsequently entered adhesions, which translocated toward the cell center, and inhibited paxillin turnover. The new adhesions formed from small foci of alpha-actinin--GFP and paxillin-GFP, which grew in size. Subsequently, alpha 5 integrin entered the adhesions to form visible complexes, which served to stabilize the adhesions. alpha 5-GFP also resided in endocytic vesicles that emanated from the leading edge of protrusions. Integrin vesicles at the cell rear moved toward the cell body. As cells migrated, alpha 5 vesicles also moved from a perinuclear region to the base of the lamellipodium. The alpha 5 vesicles colocalized with transferrin receptor and FM 4-64 dye. After adhesions broke down in the rear, alpha 5-GFP was found in fibrous structures behind the cell, whereas alpha-actinin--GFP and paxillin-GFP moved up the lateral edge of retracting cells as organized structures and then dissipated.

Show MeSH
Related in: MedlinePlus