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Pericellular Brush and Mechanics of Guinea Pig Fibroblast Cells Studied with AFM.

ABSTRACT: The atomic force microscopy (AFM) indentation method combined with the brush model can be used to separate the mechanical response of the cell body from deformation of the pericellular layer surrounding biological cells. Although self-consistency of the brush model to derive the elastic modulus of the cell body has been demonstrated, the model ability to characterize the pericellular layer has not been explicitly verified. Here we demonstrate it by using enzymatic removal of hyaluronic content of the pericellular brush for guinea pig fibroblast cells. The effect of this removal is clearly seen in the AFM force-separation curves associated with the pericellular brush layer. We further extend the brush model for brushes larger than the height of the AFM probe, which seems to be the case for fibroblast cells. In addition, we demonstrate that an extension of the brush model (i.e., double-brush model) is capable of detecting the hierarchical structure of the pericellular brush, which, for example, may consist of the pericellular coat and the membrane corrugation (microridges and microvilli). It allows us to quantitatively segregate the large soft polysaccharide pericellular coat from a relatively rigid and dense membrane corrugation layer. This was verified by comparison of the parameters of the membrane corrugation layer derived from the force curves collected on untreated cells (when this corrugation membrane part is hidden inside the pericellular brush layer) and on treated cells after the enzymatic removal of the pericellular coat part (when the corrugations are exposed to the AFM probe). We conclude that the brush model is capable of not only measuring the mechanics of the cell body but also the parameters of the pericellular brush layer, including quantitative characterization of the pericellular layer structure.

SUBMITTER: Dokukin M

PROVIDER: S-EPMC4944720 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Pericellular Brush and Mechanics of Guinea Pig Fibroblast Cells Studied with AFM.

Dokukin Maxim M Ablaeva Yulija Y Kalaparthi Vivekanand V Seluanov Andrei A Gorbunova Vera V Sokolov Igor I

Biophysical journal 20160701 1

The atomic force microscopy (AFM) indentation method combined with the brush model can be used to separate the mechanical response of the cell body from deformation of the pericellular layer surrounding biological cells. Although self-consistency of the brush model to derive the elastic modulus of the cell body has been demonstrated, the model ability to characterize the pericellular layer has not been explicitly verified. Here we demonstrate it by using enzymatic removal of hyaluronic content o ...[more]

PMID: 27410750

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Project description:Cytomegalovirus is a leading cause of congenital disease and a vaccine is a high priority. The viral gB glycoprotein is essential for infection on all cell types. The guinea pig is the only small animal model for congenital CMV (cCMV), but requires guinea pig cytomegalovirus (GPCMV). Various GPCMV gB vaccine strategies have been investigated but not with a full length protein. Previous GPCMV gB vaccines have failed to fully protect against cCMV, with approximately 50% efficacy. In an effort to define the basis of GPCMV gB based vaccine failure, we evaluated recombinant defective Ad vectors encoding GPCMV gB full length (gBwt), or truncated protein lacking transmembrane domain (gBTMD). Both candidate vaccines evoked high anti-gB titers and neutralized virus infection on fibroblast cells but had varying weaker results on non-fibroblasts (renal epithelial and placental trophoblasts). Non-fibroblast cells are dependent upon the viral pentamer complex (PC) for endocytic pathway cell entry. In contrast, fibroblasts cells that express the viral receptor platelet derived growth factor receptor alpha (PDGFRA) to enable entry by direct cell fusion independent of the PC. Anti-gBwt sera was approximately 2-fold (renal epithelial) to 3-fold (fibroblasts) more effective at neutralizing virus compared to anti-gBTMD sera. Both gB vaccines were weakest against virus neutralization on trophoblasts. Knockout of PDGFRA cell receptor on fibroblast cells (GPKO) rendered virus dependent upon the PC pathway for cell entry and anti-gB GPCMV NA50 was more similar to epithelial cells. In a gBwt vaccine protection study, vaccination of animals significantly reduced, but did not prevent dissemination of wild type GPCMV challenge virus to target organs. Depletion of complement in vivo had limited impact on vaccine efficacy. Overall, a full length gB antigen has the potential to improve neutralizing antibody titer but fails to fully prevent virus dissemination and likely congenital infection.

Dataset Information

Pericellular Brush and Mechanics of Guinea Pig Fibroblast Cells Studied with AFM.

Publications

Pericellular Brush and Mechanics of Guinea Pig Fibroblast Cells Studied with AFM.

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