Mark E. Ireland

A cytoskeletal protein unique to lens fiber cell differentiation

A chick lens urea-soluble polypeptide of estimated mol. wt. 49 000 daltons is unique to fiber cell differentiation and is a component of the beaded-chain filaments of the chick cytoskeleton. Antigenically related proteins are also present in the human and bovine lens. There is no similarity between this protein and actin as determined by immunological analysis and two-dimensional gel electrophoresis.

The cytoskeleton of chick lens cells.

Abstract The cytoskeleton of the chick lens cells was studied by morphological and immunocytochemical methods. Actin, microtubules and intermediate (10 nm) filaments were identified in the epithelial cells. The cortical fiber cells contained intermediate filaments and dispersed chains of protein. In the nuclear fiber cells, the intermediate filaments were absent, while the protein chains were aggregated. While the size of the chain backbone corresponds to actin, the conclusive demonstration that it is actin remains to be shown.

Thermal response in murine L929 cells lacking αB-crystallin expression and αB-crystallin expressing L929 transfectants

We investigated the role of αB-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10−1 isosurvival was 1.7. Expression of αB-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected αB-crystallin on thermoresistance and thermotolerance. Cells stably transfected with αB-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive αB-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of αB-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected αB-crystallin can contribute to increased thermoresistance.

Lens actin: purification and localization.

Actin was purified from the chick lens using DEAE-52 column chromatography followed by hydroxylapatite chromatography. The antibody produced against the purified actin cross-reacted specifically with lens actin from other species in addition to smooth and skeletal muscle actin and labelled the stress bundles of cultured fibroblasts. Actin was localized, using immunological methods, primarily to the plasma membrane of the epithelial and fiber cells of the chick and human lens. Actin filaments were also identified by HMM S-1 labeling in bovine cortical fiber cells. Using this procedure, the actin filaments were found throughout the fiber cell but were mainly concentrated near the plasma membrane and in cell processes. They formed a population distinct from the beaded filaments. The initial DEAE-52 column chromatography was also useful in the initial purification of lens fiber cell intermediate filament protein and two species of beta-crystallins.

Evidence for a calcium activated protease specific for lens intermediate filaments

The calcium mediated loss of intermediate filament protein from lens cytoskeletal preparations was examined by soft laser scanning densitometry of polyacrylamide gels. The time course of proteolysis by the lens Ca++ activated proteinase and inhibition by EGTA or PMSF and leupeptin were also determined. Proteolytic breakdown products were identified on electroblots with specific antiserum to vimentin.

Identification of native actin filaments in chick lens fiber cells

Abstract Native actin filaments were identified in chick lens fiber cells by decoration with the HMM S-1 fraction of chicken skeletal muscle. These filaments are morphologically distinct from the beaded filaments and intermediate filaments.

AGE CHANGES IN THE SKELETON OF THE HUMAN LENS

The cytoskeleton of the human lens (newborn, 24 year‐old and 80 year‐old) was studied by morphological methods. Actin and intermediate (10 nm) filaments were identified in the epithelial cells of all the lenses. In the newborn lens intermediate filaments and chains of protein were found in cortical and nuclear fiber cells. Many intermediate filaments and protein chains in the elongated form were observed in the superficial cortical fiber cells of the 24 year‐old lens. However, in the deep cortical cells the number of intermediate filaments decreased and the protein chains became more coiled and aggregated. In the nuclear region the protein chains were markedly aggregated and intermediate filaments were absent. The 80 year‐old fiber cells contained only protein aggregates in the fiber cells. While the size of the chain backbone corresponds to actin, the conclusive demonstration that it is actin remains to be shown.

Up-regulation of novel intermediate filament proteins in primary fiber cells: an indicator of all vertebrate lens fiber differentiation?

The early embryonic development and expression patterns of the eye lens specific cytoskeletal proteins, CP49 and CP95, were determined for the chick and were found to be similar in both human and mouse. These proteins, as well as their homologs in other species, are obligate polymerization partners which form unique filamentous structures termed “beaded filaments.” CP49 and CP95 appeared as protein products after 3 days of embryonic development in the chick during the elongation of primary fiber cells. Although limited data were obtained for human embryos at these early developmental timepoints, they were consistent with the interpretation that the up‐regulation of these lens specific proteins began only after the initiation of lens vesicle closure. In situ hybridization with the mouse lens confirmed that message levels for beaded filament proteins were greatly elevated in differentiating primary fiber cells. Nuclease protection assays established that mRNA levels for CP49 remained relatively constant while CP95 mRNA levels increased once the process of secondary fiber formation was under way. Although present in relatively low abundance, the mRNA for a unique splice variant of CP49, CP49INS, was also detected early in embryonic development and into adulthood. Peptide‐specific antibodies directed against unique predicted sequences were able to confirm the protein expression of CP49INS in both embryonic and adult chick lens cells. These data present the first detailed study of the expression of CP49 and CP95 during early lens development. They suggest that the up‐regulated expression of CP49 and CP95 could serve as pan‐specific markers for all vertebrate lens fiber development. Anat Rec 258:25–33, 2000.

Functional characterization of insulin and IGF-I receptors in chicken lens epithelial and fiber cells

Insulin and insulin-like growth factor I (IGF-I) play a role in lens cell growth and development. The binding of these hormones to their respective receptors with its concomitant signal transduction is an important step in these cellular processes. Hormone binding to adult chicken lens insulin and IGF-I receptors, partially purified from epithelial and fiber cells, was studied to examine this activity in lens. The associated stimulation of receptor-mediated tyrosine kinase by the hormones was also studied. At an insulin concentration of 0.02 nM, specific binding was similar for epithelial and fiber receptor preparations (Epi = 0.23 +/- 0.03 fmol, Fib = 0.19 +/- 0.02 fmol). Displacement studies revealed that there was also no difference between epithelial and fiber receptor preparations in the concentration of insulin necessary for half maximal displacement of specific [125I]-insulin binding (IC50: Epi = 0.32 nM +/- 0.07 nM, Fib = 0.31 nM +/- 0.05 nM). Comparison of IGF-I (0.02 nM) binding to receptor preparations from epithelial and fiber cells demonstrated that specific binding was similar in the two preparations (Epi = 0.50 +/- 0.05 fmol, Fib = 0.42 +/- 0.05 fmol). Also, there was no difference in the concentration of IGF-I necessary for half maximal displacement of specific [125I]-IGF-I binding (IC50 = Epi: 0.27 +/- 0.05 nM, Fib: 0.28 +/- 0.04 nM). The ability of IGF-I to displace bound [125I]-insulin was also examined. The IC50 for IGF-I binding to the insulin receptors isolated from epithelial and fiber cells was 37.4 +/- 2.4 nM, and 35.4 +/- 2.8 nM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The second messenger pathway for germ cell-mediated stimulation of Sertoli cells

Treatment of cultured rat Sertoli cells with FSH or dibutyryl cAMP for 30 min resulted in phosphorylation of the same Sertoli cell proteins. Different Sertoli cell proteins were phosphorylated after calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. A23187 stimulated the phosphorylation of hsp27, while TPA alone had no effect. TPA plus A23187 resulted in phosphorylation of a 14 kDa protein, in addition to hsp27. The effect of TPA plus A23187 was identical to that of germ cells on Sertoli cell protein phosphorylation. FSH-stimulated cAMP production by Sertoli cells was reduced by prior exposure of Sertoli cells to germ cells. The results indicate that germ cells stimulate Sertoli cells by the inositol trisphosphate/diacylglycerol mediated second messenger pathway. The results also suggest that the germ cell-activated pathway interacts within Sertoli cells to modulate Sertoli cell response to FSH.