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. 2002 Jan;13(1):1-11.
doi: 10.1091/mbc.01-10-0481.

Function of the tetraspanin CD151-alpha6beta1 integrin complex during cellular morphogenesis

Xin A Zhang  1 , Alexander R Kazarov, Xiuwei Yang, Alexa L Bontrager, Christopher S Stipp, Martin E Hemler
Affiliations

Function of the tetraspanin CD151-alpha6beta1 integrin complex during cellular morphogenesis

Xin A Zhang et al. Mol Biol Cell. 2002 Jan.

Abstract

Upon plating on basement membrane Matrigel, NIH3T3 cells formed an anastomosing network of cord-like structures, inhibitable by anti-alpha6beta1 integrin antibodies. For NIH3T3 cells transfected with human CD151 protein, the formation of a cord-like network was also inhibitable by anti-CD151 antibodies. Furthermore, CD151 and alpha6beta1 were physically associated within NIH3T3 cells. On removal of the short 8-amino acid C-terminal CD151 tail (by deletion or exchange), exogenous CD151 exerted a dominant negative effect, as it almost completely suppressed alpha6beta1-dependent cell network formation and NIH3T3 cell spreading on laminin-1 (an alpha6beta1 ligand). Importantly, mutant CD151 retained alpha6beta1 association and did not alter alpha6beta1-mediated cell adhesion to Matrigel. In conclusion, the CD151-alpha6beta1 integrin complex acts as a functional unit that markedly influences cellular morphogenesis, with the CD151 tail being of particular importance in determining the "outside-in" functions of alpha6beta1-integrin that follow ligand engagement. Also, antibodies to alpha6beta1 and CD151 inhibited formation of endothelial cell cord-like networks, thus pointing to possible relevance of CD151-alpha6beta1 complexes during angiogenesis.

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Figures

Figure 1
Figure 1
NIH3T3 cells form cord-like structures when plated on Matrigel. (A) Mock or human C0151-NIH3T3 cell CD151-NIH3T3 cell transfectants were grown in 5% FBS-DMEM on the surface of Matrigel for 8 h and then photographed. Monoclonal antibodies to mouse α6 integrin (GoH3), and human CD151 (1A5) were added at 5 μg/ml at the beginning of the experiment. CD151 was well expressed on the surface of NIH3T3-CD151 cells (∼100 MFI units, see Figure 4). (B) Cells were grown as in A but for 30 h. (C) NIH3T3-CD151-α3 double-transfected cells were grown in 5% FBS-DMEM on the surface of Matrigel for 7 d and then photographed. Monoclonal antibodies to mouse α6 integrin, human α3 integrin (IIF5), human CD151 (5C11), mouse CD9 (KMC9), and mouse CD44 (KM114) were each added at 10 μg/ml at the beginning of the experiment. Levels of transfected α3 (∼55 MFI) were somewhat higher than that of endogenous α6 (∼26 MFI). As seen elsewhere, CD44 (Kawano et al., 2000) and CD9 (unpublished data) are highly expressed on the surface of NIH3T3 cells. Magnification, ×10.
Figure 1
Figure 1
NIH3T3 cells form cord-like structures when plated on Matrigel. (A) Mock or human C0151-NIH3T3 cell CD151-NIH3T3 cell transfectants were grown in 5% FBS-DMEM on the surface of Matrigel for 8 h and then photographed. Monoclonal antibodies to mouse α6 integrin (GoH3), and human CD151 (1A5) were added at 5 μg/ml at the beginning of the experiment. CD151 was well expressed on the surface of NIH3T3-CD151 cells (∼100 MFI units, see Figure 4). (B) Cells were grown as in A but for 30 h. (C) NIH3T3-CD151-α3 double-transfected cells were grown in 5% FBS-DMEM on the surface of Matrigel for 7 d and then photographed. Monoclonal antibodies to mouse α6 integrin, human α3 integrin (IIF5), human CD151 (5C11), mouse CD9 (KMC9), and mouse CD44 (KM114) were each added at 10 μg/ml at the beginning of the experiment. Levels of transfected α3 (∼55 MFI) were somewhat higher than that of endogenous α6 (∼26 MFI). As seen elsewhere, CD44 (Kawano et al., 2000) and CD9 (unpublished data) are highly expressed on the surface of NIH3T3 cells. Magnification, ×10.
Figure 1
Figure 1
NIH3T3 cells form cord-like structures when plated on Matrigel. (A) Mock or human C0151-NIH3T3 cell CD151-NIH3T3 cell transfectants were grown in 5% FBS-DMEM on the surface of Matrigel for 8 h and then photographed. Monoclonal antibodies to mouse α6 integrin (GoH3), and human CD151 (1A5) were added at 5 μg/ml at the beginning of the experiment. CD151 was well expressed on the surface of NIH3T3-CD151 cells (∼100 MFI units, see Figure 4). (B) Cells were grown as in A but for 30 h. (C) NIH3T3-CD151-α3 double-transfected cells were grown in 5% FBS-DMEM on the surface of Matrigel for 7 d and then photographed. Monoclonal antibodies to mouse α6 integrin, human α3 integrin (IIF5), human CD151 (5C11), mouse CD9 (KMC9), and mouse CD44 (KM114) were each added at 10 μg/ml at the beginning of the experiment. Levels of transfected α3 (∼55 MFI) were somewhat higher than that of endogenous α6 (∼26 MFI). As seen elsewhere, CD44 (Kawano et al., 2000) and CD9 (unpublished data) are highly expressed on the surface of NIH3T3 cells. Magnification, ×10.
Figure 2
Figure 2
Association of wild-type and mutant CD151 with α6 integrin. NIH3T3 transfectants were lysed in 1% Brij 99, and then immunoprecipitations were carried out using anti-human CD151 mAb 5C11 (lanes b, d, and f), anti-human A15 mAb A2 M 30.3 (lane h), and anti-mouse α6 integrin mAb GoH3 (lanes a, c, e, and g). Proteins were resolved by nonreducing 10% SDS-PAGE and blotted with anti-α6 integrin polyclonal antibody 6843. The arrow indicates the position of the integrin α6 subunit. Expression levels for wild-type and mutant CD151 are indicated in Figure 4 below.
Figure 3
Figure 3
Role of the CD151 C-terminal cytoplasmic domain during morphogenesis on Matrigel. The indicated transfectants were cultured in 5% FBS-DMEM on the surface of Matrigel for 24 h. Magnification, ×10. Wild-type CD151 and all mutant CD151 proteins were expressed at comparable levels (see Figure 4).
Figure 4
Figure 4
Cell surface expression of wild-type and mutant CD151 in NIH3T3 cells. Stable NIH3T3 transfectants were stained with negative control mAb P3 (dashed line), anti-human A15 mAb B2D (A15-wt transfectants) or anti-human CD151 mAb 5C11 (for all other transfectants, including mock) and analyzed by flow cytometry. Schematic diagrams indicate regions from CD151 (black), A15 (gray), NAG2 (hatched), and green fluorescent protein (oval).
Figure 5
Figure 5
Time lapse formation of reticular structures. (A) NIH3T3-CD151 wild-type cells were grown on Matrigel for 7 h, and photos were obtained from the same randomly chosen field at hourly intervals. (B) The same experiment was carried out using NIH3T3-CD151-c-A15 cells. Bar, 100 μM. (A video supplement to this figure was prepared from images recorded at 5-min intervals, over a period of 13 h).
Figure 6
Figure 6
Comparison of cell spreading for CD151 transfectants on laminin-1 and fibronectin. NIH3T3 transfectants were seeded onto coverslips coated with either laminin-1 or fibronectin. (A) Spread cells were readily defined as cells that had increased their surface contact area by at least two- to threefold, as they began to show a flattened morphology. Each bar represents the mean ± SD from three separate experiments. (B) Photos of spread cells are shown at ×20 magnification. Coverslips were coated with laminin (Lm, 10 μg/ml), fibronectin (Fn, 10 μg/ml), or Matrigel (Mgl, diluted 1/30 from stock solution, according to manufacturer's instructions; BD Labware, Bedford, MA). Cell spreading was carried out in serum-free DMEM at 37°C for 30 min. For cells on laminin-1, PDGF (40 ng/ml) was included to enhance spreading.
Figure 6
Figure 6
Comparison of cell spreading for CD151 transfectants on laminin-1 and fibronectin. NIH3T3 transfectants were seeded onto coverslips coated with either laminin-1 or fibronectin. (A) Spread cells were readily defined as cells that had increased their surface contact area by at least two- to threefold, as they began to show a flattened morphology. Each bar represents the mean ± SD from three separate experiments. (B) Photos of spread cells are shown at ×20 magnification. Coverslips were coated with laminin (Lm, 10 μg/ml), fibronectin (Fn, 10 μg/ml), or Matrigel (Mgl, diluted 1/30 from stock solution, according to manufacturer's instructions; BD Labware, Bedford, MA). Cell spreading was carried out in serum-free DMEM at 37°C for 30 min. For cells on laminin-1, PDGF (40 ng/ml) was included to enhance spreading.
Figure 7
Figure 7
Perturbation of capillary-like structures formed by human endothelial cells. HUVECs were seeded on Matrigel for 24 h, in the presence of monoclonal antibodies (anti-integrin α6, GoH3; anti-CD151, 5C11; anti-CD147, 8G6; anti-integrin αV, P3G8), each at 10 μg/ml. The CD151 and CD147 molecules and the α6 and αV integrins are each very well expressed on HUVECs (Leukocyte Typing VI, 1998). Photos were obtained after 24 h. Magnification, ×10.
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