PLC-gamma1 regulates fibronectin assembly and cell aggregation

Abstract

Phospholipase C-gamma1 (PLC-gamma1) mediates cell adhesion and migration through an undefined mechanism. Here, we examine the role of PLC-gamma1 in cell-matrix adhesion in a hanging drop assay of cell aggregation. Plcg1 Null (-/-) mouse embryonic fibroblasts formed aggregates that were larger and significantly more resistant to dissociation than cells in which PLC-gamma1 is re-expressed (Null+ cells). Aggregate formation could be disrupted by inhibition of fibronectin interaction with integrins, indicating that fibronectin assembly may mediate aggregate formation. Fibronectin assembly was mediated by integrin alpha5beta1 in both cell lines, while assays measuring fibronectin assembly revealed increased assembly in the Null cells. Null and Null+ cells exhibited equivalent fibronectin mRNA levels and equivalent levels of fibronectin protein in pulse-labeling experiments. However, levels of secreted fibronectin in the conditioned medium were increased in Null cells. The data implicates a negative regulatory role for PLC-gamma1 in cell aggregation by controlling the secretion of fibronectin into the media and its assembly into fibrils.

Figure 1

Assessment of cell aggregation by Null and Null + cells: A) Cells were detached and 5 × 10⁵ cells were re-suspended in cell culture medium. 30 μ1 drops were added to the lid of a 24 well dish and hanging drops were cultured overnight. The next morning, drops were photographed (left panels), pipetted 20 times to disperse, and photographed again (middle panels) using a Leica conventional inverted microscope. All pictures were taken at the same magification. Pictures are representative images of multiple hanging drops. Cellular aggregates were stained for fibronectin and then visualized using a Zeiss confocal microscope (right panels). B) Quantification of aggregate area. Aggregate area was measured from four experiments as described in Materials and Methods. Each dot represents one aggregate. C) Quantification of the number of singlet’s and doublets in photographs before and after pipetting. Values are the average of five different hanging drops. Error bars indicate standard deviation.

Figure 2

Treatment of Hanging Drops with 70 kDa fragment: Null and Null + hanging drops were treated as in Figure 1 except for the addition of 1000 μg/ml, 500 μg/ml, or 100 ug/ml 70 kDa fibronectin fragment.

Figure 3

Assessment of fibronectin assembly in Null and Null + cells: Null and Null + cells were metabolically labeled with ³⁵S methionine for the times indicated. Parts A, B, and C: Top panels - phosphorimager image. Arrows indicate 220 kDa fibronectin; Bottom panels-densitometric analysis. Values are relative to Null cells at 24 hours. Gray bars represent Null cells and white bars represent Null + cells. A) DOC-insoluble fraction; B) DOC-soluble fraction; C) Conditioned media; D) Total cell lysate.

Figure 4

Fibronectin assembly is mediated by Integrin α5β1 in Null and Null + cells: A) Exogenous assembly assay; Cells were plated on 10ug/ml fibronectin or 5 ug/ml vitronectin in fibronectin free medium prior to the addition of biotinylated fibronectin. Cells were incubated overnight followed by collection of lysates and separation into DOC-soluble and insoluble fractions. Left panels: DOC-insoluble fractions. Biotinylated fibronectin is detected by blotting with IR Dye 680 Streptavidin. Vimentin serves as a loading control. Right Panels: DOC-soluble fraction: Actin serves as a loading control. B) Densitometric analysis of DOC insoluble fraction in panel A. Gray bars represent Null cells. White bars represent Null + cells. C) Hanging drop assay: Cells were treated as in figures 1 and 2 except for treatment with 100 μg/ml inhibitory antibodies against integrins β1 (HA 2/5), β3 (2C9.G2) or a combination of both.

Figure 5

Comparison of fibronectin mRNA and protein levels: A) QRT-PCR of RNA extracted from Null and Null + cells. B) Null and Null + cells were plated and cultured overnight. Cell lystates were harvested in RIPA buffer and conditioned medium (CM) was collected. Equal amounts of protein were resolved by SDS-PAGE followed by detection with rabbit FN and mouse actin antibodies. C) Cells were pulsed for 10 minutes with ³⁵S methionine followed by lysis and Immunoprecipitation for fibronectin. Abbreviations FN Fibronectin; TCL total cell lysate D) Null and Null + were pulsed for ³⁵S methionine followed by a two hour chase. The levels of radioactivity were determined using scintillation counter and normalized to cell lysate protein levels.