Abstract
Binding of anti-Fx1A to Heymann nephritis antigens (HA) on rat glomerular epithelial cells (GECs) in culture leads to capping and disappearance of antigens from the cell surface. This process may contribute to the formation of glomerular subepithelial immune deposits in vivo. The authors differentially extracted GECs to determine whether HA redistribution is mediated by cytoskeletal components. Observations were made by phase-contrast and immunofluorescence microscopy on primary and passaged GECs in monolayer culture and spectrofluorimetry on GECs in suspension. GEC-bound sheep anti-Fx1A IgG was detected by fluoresceinated anti-sheep IgG. Microfilaments were identified by rhodamine-phalloidin staining of F-actin. After cross-linking HA on GECs by anti-fx1A IgG at 0 C, GECs remained polygonal in shape and had diffuse granular IgG staining of their plasma membranes. Treatment of GECs at 0 C with hypotonic buffer containing 0.5% Triton X-100 produced microfilament-rich cytoskeletons that retained the shape of unextracted GECs. Further incubation with DNase I at 37 C removed microfilaments (mean fluorescence declined by 90%) and resulted in the rounding of cytoskeletons. After Triton X-100 treatment, 85% of initial GEC-bound anti-Fx1A IgG remained, but only 29% remained after DNase I. In contrast to intact IgG, detergent-extraction resulted in the complete loss of GEC-bound anti-Fx1A Fab'. Anti-Fx1A IgG did not bind to GECs pretreated with Triton X-100. Thus, cross-linking of HA by anti-Fx1A converts HA from a detergent-soluble, membrane-associated form to an insoluble, cytoskeleton-bound form. Attachment of cross-linked HA to the cytoskeleton is mediated by microfilaments.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 373-384 |
| Number of pages | 12 |
| Journal | American Journal of Pathology |
| Volume | 129 |
| Issue number | 2 |
| State | Published - 1987 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Pathology and Forensic Medicine
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Anti-fx1A induces association of Heymann nephritis antigens with microfilaments of cultured glomerular visceral epithelial cells. / Cybulsky, A. V.; Quigg, R. J.; Badalamenti, John; Salant, D. J.
In: American Journal of Pathology, Vol. 129, No. 2, 1987, p. 373-384.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Anti-fx1A induces association of Heymann nephritis antigens with microfilaments of cultured glomerular visceral epithelial cells
AU - Cybulsky, A. V.
AU - Quigg, R. J.
AU - Badalamenti, John
AU - Salant, D. J.
PY - 1987
Y1 - 1987
N2 - Binding of anti-Fx1A to Heymann nephritis antigens (HA) on rat glomerular epithelial cells (GECs) in culture leads to capping and disappearance of antigens from the cell surface. This process may contribute to the formation of glomerular subepithelial immune deposits in vivo. The authors differentially extracted GECs to determine whether HA redistribution is mediated by cytoskeletal components. Observations were made by phase-contrast and immunofluorescence microscopy on primary and passaged GECs in monolayer culture and spectrofluorimetry on GECs in suspension. GEC-bound sheep anti-Fx1A IgG was detected by fluoresceinated anti-sheep IgG. Microfilaments were identified by rhodamine-phalloidin staining of F-actin. After cross-linking HA on GECs by anti-fx1A IgG at 0 C, GECs remained polygonal in shape and had diffuse granular IgG staining of their plasma membranes. Treatment of GECs at 0 C with hypotonic buffer containing 0.5% Triton X-100 produced microfilament-rich cytoskeletons that retained the shape of unextracted GECs. Further incubation with DNase I at 37 C removed microfilaments (mean fluorescence declined by 90%) and resulted in the rounding of cytoskeletons. After Triton X-100 treatment, 85% of initial GEC-bound anti-Fx1A IgG remained, but only 29% remained after DNase I. In contrast to intact IgG, detergent-extraction resulted in the complete loss of GEC-bound anti-Fx1A Fab'. Anti-Fx1A IgG did not bind to GECs pretreated with Triton X-100. Thus, cross-linking of HA by anti-Fx1A converts HA from a detergent-soluble, membrane-associated form to an insoluble, cytoskeleton-bound form. Attachment of cross-linked HA to the cytoskeleton is mediated by microfilaments.
AB - Binding of anti-Fx1A to Heymann nephritis antigens (HA) on rat glomerular epithelial cells (GECs) in culture leads to capping and disappearance of antigens from the cell surface. This process may contribute to the formation of glomerular subepithelial immune deposits in vivo. The authors differentially extracted GECs to determine whether HA redistribution is mediated by cytoskeletal components. Observations were made by phase-contrast and immunofluorescence microscopy on primary and passaged GECs in monolayer culture and spectrofluorimetry on GECs in suspension. GEC-bound sheep anti-Fx1A IgG was detected by fluoresceinated anti-sheep IgG. Microfilaments were identified by rhodamine-phalloidin staining of F-actin. After cross-linking HA on GECs by anti-fx1A IgG at 0 C, GECs remained polygonal in shape and had diffuse granular IgG staining of their plasma membranes. Treatment of GECs at 0 C with hypotonic buffer containing 0.5% Triton X-100 produced microfilament-rich cytoskeletons that retained the shape of unextracted GECs. Further incubation with DNase I at 37 C removed microfilaments (mean fluorescence declined by 90%) and resulted in the rounding of cytoskeletons. After Triton X-100 treatment, 85% of initial GEC-bound anti-Fx1A IgG remained, but only 29% remained after DNase I. In contrast to intact IgG, detergent-extraction resulted in the complete loss of GEC-bound anti-Fx1A Fab'. Anti-Fx1A IgG did not bind to GECs pretreated with Triton X-100. Thus, cross-linking of HA by anti-Fx1A converts HA from a detergent-soluble, membrane-associated form to an insoluble, cytoskeleton-bound form. Attachment of cross-linked HA to the cytoskeleton is mediated by microfilaments.
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M3 - Article
VL - 129
SP - 373
EP - 384
JO - American Journal of Pathology
JF - American Journal of Pathology
SN - 0002-9440
IS - 2
ER -