Assembly of Epithelial Cell Fibrillins

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Assembly of Epithelial Cell Fibrillins Nevena Karaman-Jurukovska, Marcia Simon, Lynn Y. Sakai Journal of Investigative Dermatology Volume 117, Issue 6, Pages 1612-1620 (December 2001) DOI: 10.1046/j.0022-202x.2001.01588.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Fibrillin microfibrils insert into the lamina densa in skin. (A) Large bundles of microfibrils (arrow) traverse the papillary dermis and terminate at the epithelial lamina densa. The lamina densa is often seen separated from the epithelial cell at the specific site where larger microfibril bundles intersect the basal lamina (*). Scale bar: 2 µm. (B) No obvious continuation of microfibrils within the lamina densa is imaged at high magnification. MoAb 69, whose binding induces a periodicity to microfibrils, was used to positively identify fibrillin as a component of this small bundle of microfibrils. Scale bar: 250 nm. Journal of Investigative Dermatology 2001 117, 1612-1620DOI: (10.1046/j.0022-202x.2001.01588.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Fibrillin is localized to the surface of sections cut from lightly fixed neonate skin embedded in LR White media. (A) In unstained sections, MoAb 26 is seen to delineate the basal lamina separating the epithelium (Ep) from the underlying dermis. (B) Revealed at higher magnification in stained sections, MoAb 26 localizes fibrillin to the lamina densa. (C) A similar pattern of fibrillin localization within the lamina densa is revealed by MoAb 69. Scale bars: 250 nm. Journal of Investigative Dermatology 2001 117, 1612-1620DOI: (10.1046/j.0022-202x.2001.01588.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunofluorescent localization of fibrillin-1 in cell cultures. Cultures were grown for 4 d and then labeled with a fibrillin-1-specific monoclonal antibody (MoAb 201). (A) Normal skin fibroblasts. (B) MG63. (C) WISH. (D) HaCaT. Scale bar: 50 µm. Journal of Investigative Dermatology 2001 117, 1612-1620DOI: (10.1046/j.0022-202x.2001.01588.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 HaCaT cultures synthesize a lamina-densa-like matrix. (A) Confluent HaCaT cultures prepared for electron microscopy revealed a fibrous dense mat directly opposed to the culture substrate beneath cells. Scale bar: 250 nm. (B) This lamina-densa-like material was immunolabeled with antifibrillin antibodies (pAb 9543). Journal of Investigative Dermatology 2001 117, 1612-1620DOI: (10.1046/j.0022-202x.2001.01588.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Fibrillin-1 production by cultured cells. (A) Proteins in conditioned medium (M) and cell layers (L) from different cell lines were separated on 5% polyacrylamide gels and analyzed by immunoblotting with fibrillin-1-specific monoclonal antibody (MoAb 15), which recognizes both nonreduced and reduced (+ DTT) molecules. The migration of molecular weight markers is indicated on the left in kDa. (B) Proteins in the conditioned media of equal numbers of WISH and normal skin fibroblasts (NSF) were separated on a 4.5% polyacrylamide gel and immunoblotted using different antibodies. Blots such as this one were quantitated using ImageQuant. Journal of Investigative Dermatology 2001 117, 1612-1620DOI: (10.1046/j.0022-202x.2001.01588.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Fibrillin-1 and fibrillin-2 secretion by immortalized and normal keratinocytes. Protein extracts were made from conditioned medium (M) and cell layers (CL) of immortalized keratinocytes (HaCaT) or NHK grown with 3T3 cells in a serum-containing medium (A, B) or grown without 3T3 cells in KGM (C). The proteins were resolved on 3%-5% gradient gels and analyzed by immunoblotting with monoclonal antibodies to fibrillin-1 (MoAb 15, A) or fibrillin-2 (MoAb 48, B, C). Journal of Investigative Dermatology 2001 117, 1612-1620DOI: (10.1046/j.0022-202x.2001.01588.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Fibrillin derived from human WISH epithelial cells is assembled in cocultures with mouse fibroblasts. (A) Mouse fibroblast cultures are labeled with a fibrillin-1 antiserum (pAb 9543). (B) Fibrillin-1-specific MoAb 201 does not recognize mouse fibrillin-1 in fibroblast cultures. (C) Mouse fibroblast cultures are not labeled with fibrillin-2-specific MoAb 48. (D) WISH cells are stained in punctate patterns with MoAb 201, but no fibrils are observed. (E) Cocultures of human WISH cells and mouse fibroblasts labeled with MoAb 201 demonstrate that human fibrillin-1 is now assembled into fibrils. (F) WISH cells labeled with MoAb 48 show no observable fibrillin-2 fibrils. (G) Labeling of a WISH-mouse fibroblast coculture with MoAb 48 reveals fibrillin-2-containing fibrils. Scale bar: 50 µm. Journal of Investigative Dermatology 2001 117, 1612-1620DOI: (10.1046/j.0022-202x.2001.01588.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Fibrillin-1 from WISH conditioned medium is assembled by mouse fibroblasts. (A) Mouse fibroblasts are labeled with pAb 9543. (B) WISH cells incubated in mouse fibroblast conditioned medium are not induced to assemble fibrils. Punctate staining with MoAb 201 is still obtained. (C) Assembly of fibrillin fibrils by mouse fibroblasts is not inhibited by incubation in WISH conditioned medium. Staining by pAb 9543 is unchanged. (D) Mouse fibroblasts incubated in WISH conditioned medium can incorporate human fibrillin-1 into fibrils. Fibrillar patterns are obtained with MoAb 201. Scale bar: 50 µm. Journal of Investigative Dermatology 2001 117, 1612-1620DOI: (10.1046/j.0022-202x.2001.01588.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Fibrillin-1 forms fibrils in NHK cells cultured in the presence of irradiated mouse 3T3 fibroblasts. (A) NHK cells cultured in serum-free conditions were passaged, grown in chamber slides with irradiated 3T3 cells in serum-containing conditions for 4 d, and stained with MoAb 201. Fibrillin-1 fibrils corresponding to fibroblast outlines were brightly stained. (B) PBS control, instead of primary antibody, showed no background staining. Scale bar: 50 µm. Journal of Investigative Dermatology 2001 117, 1612-1620DOI: (10.1046/j.0022-202x.2001.01588.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions