Gelsolin expression increases β1 -integrin affinity and L1210 cell adhesion

Abstract

Integrins are functionally regulated by "inside-out" signaling, in that stimulus-induced signaling pathways act on the intracellular integrin tail to regulate the activity of the receptor on the outside. Both a change in conformation (affinity) and clustering (avidity/valency) of the receptors occurs, but the mechanisms that regulate inside out signaling are not completely understood. Previously, we identified gelsolin in a proteomics screen to identify proteins involved in inside-out control of integrins using the lymphocytic leukemia cell line L1210. Furthermore, we showed that gelsolin was involved in affinity regulation of β1 -integrins in the leukemic cell line U937. Here, we examined how gelsolin regulates β1 -integrin affinity in the leukemia cell line L1210. We show that gelsolin is mainly expressed at the cell membrane and is present near β1 -integrins. The role for actin polymerization in integrin affinity regulation was examined using the actin modulating agent jasplakinolide, which decreased β1 -integrin affinity. Similarly, knock-down of gelsolin in L1210 cells also decreased β1 -integrin affinity and cell adhesion to collagen. These data suggest that increased actin polymerization through gelsolin regulates β1 -integrin affinity and cell adhesion.

Keywords: attachment; cell membrane; cytoskeleton; gelsolin; migration.

Figure 1. Increased active β₁-integrin expression on L1210-A cells

(A) L1210-S and L1210-A cells adhesion to collagen. Expression of (B) CD29 and (C) binding of 9EG7 to L1210-S and L1210-A cell by flow cytometry. (D) L1210-S or L1210-A cells were stained with Calcein-AM and mixed with unstained L1210-A or L1210-S, respectively. The mixed cell populations were stained with CD29 or 9EG7 and DAPI as nuclear stain and imaged by confocal microscopy. Fluorescence intensity quantification of (E) CD29 expression (n=15) and (F) binding of 9EG7 (n=15) to L1210-S and L1210-A cells.

Figure 2. Gelsolin, total β₁- and the high affinity β₁-integrins are expressed at the cell membrane of L1210 cells

(A) L1210-S and L1210-A cells were stained with anti-gelsolin (green) and DAPI (blue) as nuclear stain and imaged by confocal microscopy. (B) L1210-S and L1210-A cells were stained with CD29 (red), 9EG7 (red), anti-gelsolin (green) and DAPI (blue) as nuclear stain and imaged by confocal microscopy. Overlays of CD29 or 9EG7 with gelsolin are depicted.

Figure 3. Actin dynamics affect L1210 β₁-integrin affinity regulation

L1210-S and L1210-were treated with (A) latrunculin B or (B) jasplakinolide and stained for total β₁-integrin (CD29) and active β₁-integrins (9EG7) by flow cytometry. One-way ANOVA with Dunnett’s multiple comparison test was used to perform statistical analysis (* = P<0.05, *** = P<0.001).

Figure 4. Gelsolin knock down decreases cell adhesion

Relative mRNA expression of (A) gelsolin and (B) L-plastin in L1210-S, L1210-A and L1210-A geslolin shRNA knock-down cells. (C) Gelsolin protein expression in L1210-S, L1210-A and L1210-A geslolin shRNA knockdown cells were analyzed by westernblot analysis with anti-gelsolin and anti-β-tubulin as loading control. The western blots shown are representative for five experiments. Mean values are represented ± SEM (n=5). One-way ANOVA with Dunnett’s multiple comparison test was used to perform statistical analysis (* = P<0.05, ** = P<0.01, *** = P<0.001). (D) L1210-A or L1210-A B12 gelsolin shRNA knock-down cells were stained with Calcein-AM and mixed with unstained L1210-A or L1210-A B12 gelsolin shRNA knock-down cells, respectively. The mixed cell populations were stained with CD29 or 9EG7 and DAPI as nuclear stain and imaged by confocal microscopy. Fluorescence intensity quantification of (E) CD29 expression (n=25) and (F) binding of 9EG7 (n=19) on L1210-A or L1210-A B12 gelsolin shRNA knock-down cells. (G) Expression of CD29 and binding of 9EG7 on L1210-S, L1210-A and L1210-A gelsolin shRNA knock-down cells were analyzed by flow cytometry.

Figure 5. Over expression of gelsolin increases β₁-integrin activity on L1210-S cells

(A) L1210-S cells transfected with pEGFP-C1 or pEGFP-C1-mGelsolin were stained with 9EG7 and DAPI as nuclear stain and imaged by confocal microscopy. Fluorescence intensity quantification of (B) L1210-S transfected with pEGFP-C1-mGelsolin (n=4) or (C) L1210-S transfected with pEGFP-C1 (n=4).