recombinant heterodimer integrin ecd (α5β1  (R&D Systems)

Journal: The Journal of biological chemistry

Article Title: The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites.

doi: 10.1016/j.jbc.2021.100481

Figure Lengend Snippet: Figure 5. Association of transmembrane protein 2 (TMEM2) with integrins via interactions between the extracellular domains. A and B, targeting of TMEM2 to focal adhesions (FAs) does not require the cyto- plasmic domain of TMEM2. In this experiment, mCherry-mTMEM2 (full length) and mCherry-mTMEM2/Δcyto (Δcyto) cells were analyzed for their in situ hyaluronan (HA) degradation activities. To allow specific analysis of the activity of the full-length mouse TMEM2 and its Δcyto deletion mutant, expression of endogenous human TMEM2 was silenced by siRNA treatment prior to the assay. A, in situ HA degradation assays were performed on substrate immobilized with FA-HA, as described in Experimental procedures section. Note that the pattern of in situ HA degradation is indistinguishable between mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 10 μm. B, immunostaining for vinculin in mCherry-mTMEM2 and mCherry-mTMEM2/Δcyto cells on the FA-HA substrate. Note that the sites of HA degradation colocalize with vinculin-immunoreactive puncta in both mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 2 μm. C–E, TMEM2 associates with integrins via extracellular interactions. C, cell surface–expressed TMEM2 is coimmunoprecipitated with integrin α5β1. mCherry-mTMEM2 cells were treated with the membrane-impermeable crosslinker 3’,3’-dithiobis(sulfosuccinimidyl

Article Snippet: Two micrograms of recombinant heterodimer of integrin ECD (α5β1; R&D Systems: 3230-A5-050; αLβ2; R&D Systems: 3868-AV-050) were applied to the TMEM2–ECD–bound and control unbound resin and incubated in HBSS++ overnight at 4 C. After extensive washing, bound materials were eluted by boiling in SDS-PAGE sample buffer, and eluents were analyzed by SDSPAGE and immunoblotting with rabbit polyclonal antiintegrin α5 (Proteintech; 10569-1-AP), rabbit monoclonal anti-integrin β1 (Abcam; ab52971), rabbit polyclonal antiintegrin β2 (Proteintech; 10544-1-AP), or mouse monoclonal anti-polyhistidine (Sigma; A7058; clone: HIS-1, peroxidase conjugated).

Techniques: In Situ, Activity Assay, Mutagenesis, Expressing, Immunostaining, Membrane

Journal: The Journal of biological chemistry

Article Title: The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites.

doi: 10.1016/j.jbc.2021.100481

Figure Lengend Snippet: Figure 6. A model for the role of transmembrane protein 2 (TMEM2) in integrin-mediated cell adhesion and migration. Our results suggest that TMEM2-dependent degradation of hyaluronan (HA) is critical for cells to form strong cell–matrix adhesion on HA-rich extracellular matrix (ECM). A, high levels of HA in the ECM are inhibitory to the direct engagement of integrins to their ECM ligands. B, in the presence of TMEM2, HA in the ECM is locally removed, which generates a microenvironment that is permissible to the direct integrin–ECM engagement. C, the association between TMEM2 and integrins promotes the FA formation and maturation via further removal of HA in the vicinity of the integrin–ECM engagement. D, this in turn facilitates integrin clustering, integrin-mediated downstream signaling, and cellular responses. See the text for further discussion.

Article Snippet: Two micrograms of recombinant heterodimer of integrin ECD (α5β1; R&D Systems: 3230-A5-050; αLβ2; R&D Systems: 3868-AV-050) were applied to the TMEM2–ECD–bound and control unbound resin and incubated in HBSS++ overnight at 4 C. After extensive washing, bound materials were eluted by boiling in SDS-PAGE sample buffer, and eluents were analyzed by SDSPAGE and immunoblotting with rabbit polyclonal antiintegrin α5 (Proteintech; 10569-1-AP), rabbit monoclonal anti-integrin β1 (Abcam; ab52971), rabbit polyclonal antiintegrin β2 (Proteintech; 10544-1-AP), or mouse monoclonal anti-polyhistidine (Sigma; A7058; clone: HIS-1, peroxidase conjugated).

Techniques: Migration

Journal: The Journal of Biological Chemistry

Article Title: The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites

doi: 10.1016/j.jbc.2021.100481

Figure Lengend Snippet: Association of transmembrane protein 2 (TMEM2) with integrins via interactions between the extracellular domains. A and B , targeting of TMEM2 to focal adhesions (FAs) does not require the cytoplasmic domain of TMEM2. In this experiment, mCherry-mTMEM2 ( full length ) and mCherry-mTMEM2/Δcyto (Δ cyto ) cells were analyzed for their in situ hyaluronan (HA) degradation activities. To allow specific analysis of the activity of the full-length mouse TMEM2 and its Δcyto deletion mutant, expression of endogenous human TMEM2 was silenced by siRNA treatment prior to the assay. A , in situ HA degradation assays were performed on substrate immobilized with FA-HA, as described in section. Note that the pattern of in situ HA degradation is indistinguishable between mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 10 μm. B , immunostaining for vinculin in mCherry-mTMEM2 and mCherry-mTMEM2/Δcyto cells on the FA-HA substrate. Note that the sites of HA degradation colocalize with vinculin-immunoreactive puncta in both mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 2 μm. C – E , TMEM2 associates with integrins via extracellular interactions. C , cell surface–expressed TMEM2 is coimmunoprecipitated with integrin α5β1. mCherry-mTMEM2 cells were treated with the membrane-impermeable crosslinker 3',3'-dithiobis(sulfosuccinimidyl propionate), and the lysates from these cells were immunoprecipitated with anti-mCherry antibody, followed by immunoblotting analysis with antibodies to α5, β1, and mCherry. D , the extracellular domain of TMEM2 directly binds α5β1. Binding between TMEM2 extracellular domain (ECD) and the α5β1 ECD heterodimer was analyzed by a pull-down assay. E , the ECD of TMEM2 directly binds αLβ2 (lymphocyte function-associated antigen-1). Binding between TMEM2 ECD and the αLβ2 ECD heterodimer was analyzed by a pull-down assay.

Article Snippet: Two micrograms of recombinant heterodimer of integrin ECD (α5β1; R&D Systems: 3230-A5-050; αLβ2; R&D Systems: 3868-AV-050) were applied to the TMEM2–ECD–bound and control unbound resin and incubated in HBSS ++ overnight at 4 °C.

Techniques: In Situ, Activity Assay, Mutagenesis, Expressing, Immunostaining, Membrane, Immunoprecipitation, Western Blot, Binding Assay, Pull Down Assay