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Lattice complex assembled by noncompetitive anti-EGFR antibodies regulates actin cytoskeletal reorganization.

ABSTRACT: Background:Recent evidence of clinical trials highlights that the combination of two noncompetitive anti-EGFR antibodies can benefit patients with several cancers. Previous studies propose that a lattice complex assembled by antibodies and EGFR down-regulates surface EGFR by rapid internalization of the complex. However, there remains a paucity of evidence and understanding on the existence of a lattice complex on cell surface and its cellular processes of internalization. Methods:Herein, we used three dimensions structured illumination microscopy to directly observe the actual morphology of the lattice complex formed on Hela cell membrane after noncompetitive anti-EGFR antibody combinations, and we explored the internalized mechanism of noncompetitive antibody combinations by constructing a PIP2 consumption system. Result:We observed the lattice complex (length?>?1 ?m) on the surface of living cell after preincubation with Cetuximab and H11, but combination of Cetuximab and single domain antibody 7D12 fails to assemble the lattice, these results demonstrates the importance of symmetrical structure of conventional antibody for lattice formation. Interestingly, the lattice complex assembles along with cytoskeletal fibers, and its internalization recruits a large amount of PIP2 and triggers the rearrangement of F-actin. Conclusions:The above data suggests that large-size lattice complex affects membrane fluidity and dynamic reorganization of cytoskeletal, which may be responsible for its rapid internalization. These new insight will aid in current rational combination design of anti-EGFR antibodies.

SUBMITTER: Huang D

PROVIDER: S-EPMC7171787 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Lattice complex assembled by noncompetitive anti-EGFR antibodies regulates actin cytoskeletal reorganization.

Huang Dianshuai D Lu Tianqi T Du Xingyu X Xi Xi X Zhang Xin X Zhang Xitian X Zhang Haoran H Sun Fei F

Cancer cell international 20200421

<h4>Background</h4>Recent evidence of clinical trials highlights that the combination of two noncompetitive anti-EGFR antibodies can benefit patients with several cancers. Previous studies propose that a lattice complex assembled by antibodies and EGFR down-regulates surface EGFR by rapid internalization of the complex. However, there remains a paucity of evidence and understanding on the existence of a lattice complex on cell surface and its cellular processes of internalization.<h4>Methods</h4 ...[more]

PMID: 32336949

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Project description:Noncollagenous domain 1 (NC1)-peptide is a biologically active peptide derived from the C-terminal region of collagen α3(IV) chain, a structural constituent protein at the basement membrane in the rat testis, likely via proteolytic cleavage of matrix metalloproteinase 9. Studies have shown that this NC1 peptide regulates testis function by inducing Sertoli cell blood-testis barrier (BTB) remodeling and is also capable of inducing elongate spermatid exfoliation through its disruptive effects on the organization of actin- and microtubule (MT)-based cytoskeletons at these cell adhesion sites. However, the underlying molecular mechanism remains unknown. NC1 peptide was found to exert its biologic effects through an activation of small GTPase cell division control protein 42 homolog (Cdc42) because cooverexpression of the dominant negative mutant of Cdc42 [namely, Cdc42-T17N (via a single mutation of amino acid residue 17 from the N terminus from Thr to Asn by site-directed mutagenesis, making it constitutively inactive)] and NC1 peptide was able to block the NC1 peptide-induced Sertoli cell tight junction-permeability barrier disruption. Their cooverexpression also blocked the NC1 peptide-induced misdistribution of BTB-associated proteins at the cell-cell interface and also disruptive cytoskeletal organization of F-actin and MTs through changes in spatial expression of the corresponding actin and MT regulatory proteins. Interestingly, NC1 peptide was also found to induce an up-regulation of phosphorylated (p)-ribosomal protein S6 (rpS6) (namely, p-rpS6-S235/S236) and a concomitant down-regulation of p-Akt1/2 (namely, p-Akt1-S473 and p-Akt2-S474), but these changes could not be blocked by overexpression of Cdc42-T17N. More importantly, NC1 peptide-induced Cdc42 activation was effectively blocked by treatment of Sertoli cell epithelium with a p-Akt1/2 activator SC79, which is also capable of blocking NC1 peptide-induced down-regulation of p-Akt1-S473 and p-Akt2/S474, but not p-rpS6-S235/S236 up-regulation. In summary, these findings illustrate that Cdc42 is working downstream of the mammalian target of rapamycin complex 1/rpS6/Akt1/2 signaling pathway to support NC1 peptide-mediated effects on Sertoli cell function in the testis using the rat as an animal model.-Su, W., Cheng, C. Y. Cdc42 is involved in NC1 peptide-regulated BTB dynamics through actin and microtubule cytoskeletal reorganization.

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Dataset Information

Lattice complex assembled by noncompetitive anti-EGFR antibodies regulates actin cytoskeletal reorganization.

Publications

Lattice complex assembled by noncompetitive anti-EGFR antibodies regulates actin cytoskeletal reorganization.

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