Extended applications of electric cell-substrate impedance sensing for assessment of the structure-function of Alpha2Beta1 integrin

Electric cell-substrate impedance sensing (ECIS) was applied to assess the structure-function of alpha2beta1 integrin, receptor for collagen and laminin. On collagen-coated gold electrodes, expression of this integrin on human rhabdomyosarcoma (RD) cells (RDX2C2) yielded a five-fold increase in resistance when compared with mock transfected RD (RDpF) cells (34.5 +- 5.2 versus 6.5 +- 0.8 OMEGA/cell). An intermediate level of 16 +- 2 OMEGA/cell was measured upon expression of an alpha2beta1 mutant that lacked the alpha2 cytoplasmic domain (RDX2CO). On laminin, the resistance measured for RDX2C2 cells was also higher but only twice that of RDpF cells at 71 +- 4 and 37 +- 4 OMEGA/cell, respectively. In comparison, RDX2CO cells (38 +- 4 OMEGA/cell), exhibiting no enhanced adhesive function, yielded a similar result to that of RDpF cells. On fibronectin, RDX2C2 and RDpF cells, exhibiting comparable levels of adhesion, were similar in resistance measurements at 85 +- 5 and 89 +- 7 OMEGA/cell, respectively. It has been shown that deletion of alpha2 cytoplasmic domain results in dysregulated recruitment of the alpha2beta1 mutant to focal adhesion complexes that mediate binding of fibronectin. RDX2CO cells on fibronectin, exhibiting reduced adhesive function, was associated with noticeably lower resistance (60 +- 4 OMEGA/cell). Monitoring electroporation of the RD plasma membrane also indirectly validated cell attachment as reflected by the resistance measured. Results from this study demonstrated the potential of ECIS for study of the structure-function of beta1 integrin adhesion receptors.NRC publication: Ye

Extended applications of electric cell-substrate impedance sensing for assessment of the structure-function of Alpha2Beta1 integrin

Electric cell-substrate impedance sensing (ECIS) was applied to assess the structure-function of alpha2beta1 integrin, receptor for collagen and laminin. On collagen-coated gold electrodes, expression of this integrin on human rhabdomyosarcoma (RD) cells (RDX2C2) yielded a five-fold increase in resistance when compared with mock transfected RD (RDpF) cells (34.5 +- 5.2 versus 6.5 +- 0.8 OMEGA/cell). An intermediate level of 16 +- 2 OMEGA/cell was measured upon expression of an alpha2beta1 mutant that lacked the alpha2 cytoplasmic domain (RDX2CO). On laminin, the resistance measured for RDX2C2 cells was also higher but only twice that of RDpF cells at 71 +- 4 and 37 +- 4 OMEGA/cell, respectively. In comparison, RDX2CO cells (38 +- 4 OMEGA/cell), exhibiting no enhanced adhesive function, yielded a similar result to that of RDpF cells. On fibronectin, RDX2C2 and RDpF cells, exhibiting comparable levels of adhesion, were similar in resistance measurements at 85 +- 5 and 89 +- 7 OMEGA/cell, respectively. It has been shown that deletion of alpha2 cytoplasmic domain results in dysregulated recruitment of the alpha2beta1 mutant to focal adhesion complexes that mediate binding of fibronectin. RDX2CO cells on fibronectin, exhibiting reduced adhesive function, was associated with noticeably lower resistance (60 +- 4 OMEGA/cell). Monitoring electroporation of the RD plasma membrane also indirectly validated cell attachment as reflected by the resistance measured. Results from this study demonstrated the potential of ECIS for study of the structure-function of beta1 integrin adhesion receptors.NRC publication: Ye

Mycobacterium smegmatis expressing a chimeric protein MPT64-proteolipid protein (PLP) 139-151 reorganizes the PLP-specific T cell repertoire favoring a CD8-mediated response and induces a relapsing experimental autoimmune encephalomyelitis.

We infected SJL mice with a recombinant Mycobacterium smegmatis expressing a chimeric protein containing the self-epitope of proteolipid protein 139\u2013151 (p139) fused to MPT64, a secreted protein of Mycobacterium tuberculosis (rMSp139). Infected mice developed a relapsing experimental autoimmune encephalomyelitis (EAE), showing a prevailing demyelination of the CNS, and disease severity was significantly lower in comparison with the one that follows immunization with p139. rMSp139 was not detected in lymph node or spleen in the course of clinical disease development or in the CNS during relapse. Infection with rMSp139 modified the p139-specific T cell repertoire, recruiting the spontaneous p139-specific repertoire and activating CD4+ T cells carrying the BV4 semiprivate rearrangement. T cells carrying the public BV10 rearrangement that are consistently found in the CNS during flares of disease were not activated by infection with rMSp139 because lymph node APCs infected with rMSp139 selectively fail to present the epitope for which BV10 cells are specific. Simultaneously, rMSp139 expanded p139-specific CD8+ cells more efficiently than immunization with peptide in adjuvant. SJL mice vaccinated against the CDR3 sequence of the BV10 public rearrangement reduced usage of the BV10 cells and displayed reduced symptoms during bouts of EAE. Thus, transient peripheral infection with a CNS-cross\u2013reactive nonpathogenic Mycobacterium induces a relapsing EAE that continues long after clearance of the infectious agent. The composition of the self-reactive repertoire activated determines severity and histology of the resulting disease