The Tight skin mouse: demonstration of mutant fibrillin-1 production and assembly into abnormal microfibrils

Abstract

Mice carrying the Tight skin (Tsk) mutation harbor a genomic duplication within the fibrillin-1 (Fbn 1) gene that results in a larger than normal in-frame Fbn 1 transcript. In this study, the consequences of the Tsk mutation for fibrillin-containing microfibrils have been examined. Dermal fibroblasts from Tsk/+ mice synthesized and secreted both normal fibrillin (approximately 330 kD) and the mutant oversized Tsk fibrillin-1 (approximately 450 kD) in comparable amounts, and Tsk fibrillin-1 was stably incorporated into cell layers. Immunohistochemical and ultrastructural analyses of normal and Tsk/+ mouse skin highlighted differences in the gross organization and distribution of microfibrillar arrays. Rotary shadowing of high Mr preparations from Tsk/+ skin demonstrated the presence of abundant beaded microfibrils. Some of these had normal morphology and periodicity, but others were distinguished by diffuse interbeads, longer periodicity, and tendency to aggregate. The presence of a structurally abnormal population of microfibrils in Tsk/+ skin was unequivocally demonstrated after calcium chelation and in denaturating conditions. Scanning transmission electron microscopy highlighted the presence of more mass in Tsk/+ skin microfibrils than in normal mice skin microfibrils. These data indicate that Tsk fibrillin-1 polymerizes and becomes incorporated into a discrete population of beaded microfibrils with altered molecular organization.

Figure 1

(A) SDS-PAGE analysis of newly synthesized fibrillin-1 radioimmunoprecipitated from medium of normal and Tsk/+ dermal fibroblast cultures. Newly synthesized, labeled fibrillin-1 was radioimmunoprecipitated from the medium of dermal fibroblasts that had been labeled with ³⁵[S]cys/met for 17 h. Tsk/+ cells synthesized and then secreted both normal (∼330 kD) and Tsk fibrillin-1 (∼450 kD) in comparable amounts. Lanes 1, 3 and 5, medium from normal mice cells; lanes 2, 4, and 6, medium from Tsk/+ mice cells. Lanes 1 and 2, crude medium; lanes 3 and 4, fibrillin-1 immunoprecipitated with PF2 antiserum; lanes 5 and 6, immunoprecipitation controls using normal rabbit serum. (B) Incorporation of the oversized Tsk monomer into extracellular matrix. Normal and Tsk fibroblast cultures were pulsed for 1 h with ³⁵[S]met/ cys and chased in culture medium containing 2% fetal calf serum for 24 h. Medium and extracellular matrix extracts were harvested after this period of time. The medium and extracellular matrix of both cell cultures contains the normal fibrillin (∼330 kD). (small arrow). In addition, medium and extracellular matrix of the Tsk cell strain contain the oversized fibrillin-1 monomer (∼450 kD; large arrow).

Figure 1

(A) SDS-PAGE analysis of newly synthesized fibrillin-1 radioimmunoprecipitated from medium of normal and Tsk/+ dermal fibroblast cultures. Newly synthesized, labeled fibrillin-1 was radioimmunoprecipitated from the medium of dermal fibroblasts that had been labeled with ³⁵[S]cys/met for 17 h. Tsk/+ cells synthesized and then secreted both normal (∼330 kD) and Tsk fibrillin-1 (∼450 kD) in comparable amounts. Lanes 1, 3 and 5, medium from normal mice cells; lanes 2, 4, and 6, medium from Tsk/+ mice cells. Lanes 1 and 2, crude medium; lanes 3 and 4, fibrillin-1 immunoprecipitated with PF2 antiserum; lanes 5 and 6, immunoprecipitation controls using normal rabbit serum. (B) Incorporation of the oversized Tsk monomer into extracellular matrix. Normal and Tsk fibroblast cultures were pulsed for 1 h with ³⁵[S]met/ cys and chased in culture medium containing 2% fetal calf serum for 24 h. Medium and extracellular matrix extracts were harvested after this period of time. The medium and extracellular matrix of both cell cultures contains the normal fibrillin (∼330 kD). (small arrow). In addition, medium and extracellular matrix of the Tsk cell strain contain the oversized fibrillin-1 monomer (∼450 kD; large arrow).

Figure 2

Confocal laser scanning analysis of normal and Tsk/+ mice skin. (A) a and c, normal mouse skin; b and d, Tsk/+ mouse skin. In the normal mouse, PF2 antibody stains the papillary microfibrillar apparatus beneath and along the basement membrane region, perivascular microfibrils, and the surrounding microfibrillar matrix around hair follicles. The identical regions in the Tsk/+ mouse are labeled, but in comparison with normal mouse skin, the intensity of the immune signal is substantially increased. Bar, 20 μm. (B) a and c, normal mouse; b and d, Tsk/+ mouse. In the normal mouse, 5507 antibody strongly stains the papillary microfibrillar apparatus beneath the dermal–epidermal junction and the matrix surrounding the hair follicles. The same structures are immunolabeled in Tsk/+ skin, but the intensity is reduced. Bar, 20 μm.

Figure 2

Confocal laser scanning analysis of normal and Tsk/+ mice skin. (A) a and c, normal mouse skin; b and d, Tsk/+ mouse skin. In the normal mouse, PF2 antibody stains the papillary microfibrillar apparatus beneath and along the basement membrane region, perivascular microfibrils, and the surrounding microfibrillar matrix around hair follicles. The identical regions in the Tsk/+ mouse are labeled, but in comparison with normal mouse skin, the intensity of the immune signal is substantially increased. Bar, 20 μm. (B) a and c, normal mouse; b and d, Tsk/+ mouse. In the normal mouse, 5507 antibody strongly stains the papillary microfibrillar apparatus beneath the dermal–epidermal junction and the matrix surrounding the hair follicles. The same structures are immunolabeled in Tsk/+ skin, but the intensity is reduced. Bar, 20 μm.

Figure 3

Ultrastructural study of normal and Tsk/+ mice skin. a, c, and e, normal mouse; b, d, and f, Tsk/+ mouse. a and b are low-power views (5,000×) showing basal keratinocytes, the dermo–epidermal junction as represented by the lamina densa (open arrows), and the upper dermis. In both mice, the dermis is dominated by collagen bundles. Microfibrillar bundles and elastic fibers are indicated by closed arrows. b reveals the presence of larger microfibrillar bundles with abundant microfibrils but little elastin. c–f are high magnification views of elastic fibers and microfibrillar bundles. These appear less well defined in Tsk/+ skin (d and f) than in normal skin (c and e). In particular, the striation pattern of microfibrils (arrowheads) seen with the normal animals (c and e) was less apparent in the Tsk/+ mice skin. Bars: (a and b) 3,000 nm; (c–f) 250 nm.

Figure 4

Rotary shadowing analysis of normal and Tsk/+ skin microfibrils. a and c, normal skin microfibrils; b and d, Tsk/+ skin microfibrils. (a) Normal mice skin microfibrils exhibited well-organized packing and regular diameter. (b) Tsk/+ mice skin microfibrils. Some appeared normal in morphology and periodicity, whereas others appeared as periodic rows of beads with indistinct interbeads and repeat distances longer than normal (arrowheads). (c) Normal mice skin microfibrils after incubation for 10 min in 5 mM EDTA. (d) Tsk/+ mice skin microfibrils after incubation for 10 min in 5 mM EDTA. The apparently normal microfibrils responded to EDTA as control mice microfibrils, but the abnormal microfibrils remained morphologically distinct (arrowheads). Grids were examined using a JEOL 1200EX electron microscope at an accelerating voltage of 100 kV. Bars, 100 nm.

Figure 5

Rotary shadowing analysis of microfibrillar aggregates isolated from Tsk/+ skin. Numerous extensive microfibril clusters were present in the Tsk/+ skin preparations. (a and b) Microfibril clusters after isolation in native, calcium-containing state. (c and d) Microfibril clusters after 10-min incubation in 5 mM EDTA. Grids were examined using a JEOL 1200EX electron microscope at an accelerating voltage of 100 kV. Bars, 100 nm.