Ultrastructural localization of aa-crystallin to the bovine Lens fiber cell cytoskeleton

Paul G FitzGerald, Dcbra Graham

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

Monoclonal antibodies to the bovine aA-crystallin were developed and used to probe the relationship between aA-crystallin and the bovine lens fiber cell Plasma Membrane-Cytoskeleton Complex (PMCC). Superficial bovine lens cortex was washed by repeated homogenization/centrifugation to remove "soluble protein." The resulting Plasma Membrane-Cytoskeleton Complex was covalently immobilized to inert resin, and extensively buffer washed. SDS PAGE and immunoblot analysis of both the covalently immobilized PMCC and of the sequentially-generated subcellular fractions shows that most of the lens alpha crystallin is "soluble" and readily extracted with physiologic buffers. However, this data also shows that 1) Non-alpha crystallins are progressively and quantitatively extracted from the PMCC with buffer, 2) An irreducible level of non-covalently bound alpha crystallin is achieved which cannot be readily extracted from the PMCC, even with 2 M urea, 1% NP40 or 0.4M KC1. Electron microscope level immunocytochemistry was performed on both the covalently immobilized PMCC, as well as on buffer-extracted thick frozen sections, using monoclonal antibodies to the αA-crystallin. The results show a very heavy labelling of both intermediate filaments and beaded filaments, but little or no labelling of fiber cell membranes. The data presented argues that a subtraction of the total αA-crystallin is strongly associated with the fiber cell cytoskeleton complex, and constitutes a quantitatively major component of the lens cytoskeleton fraction.

Original languageEnglish (US)
Pages (from-to)417-436
Number of pages20
JournalCurrent Eye Research
Volume10
Issue number5
DOIs
StatePublished - 1991

Fingerprint

Crystallins
Cytoskeleton
Lenses
Cell Membrane
Buffers
alpha-Crystallins
Monoclonal Antibodies
Subcellular Fractions
Intermediate Filaments
Frozen Sections
Centrifugation
Urea
Polyacrylamide Gel Electrophoresis
Immunohistochemistry
Electrons

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Ultrastructural localization of aa-crystallin to the bovine Lens fiber cell cytoskeleton. / FitzGerald, Paul G; Graham, Dcbra.

In: Current Eye Research, Vol. 10, No. 5, 1991, p. 417-436.

Research output: Contribution to journalArticle

@article{20b421ac34bd4722b8e20a7bb64969ea,
title = "Ultrastructural localization of aa-crystallin to the bovine Lens fiber cell cytoskeleton",
abstract = "Monoclonal antibodies to the bovine aA-crystallin were developed and used to probe the relationship between aA-crystallin and the bovine lens fiber cell Plasma Membrane-Cytoskeleton Complex (PMCC). Superficial bovine lens cortex was washed by repeated homogenization/centrifugation to remove {"}soluble protein.{"} The resulting Plasma Membrane-Cytoskeleton Complex was covalently immobilized to inert resin, and extensively buffer washed. SDS PAGE and immunoblot analysis of both the covalently immobilized PMCC and of the sequentially-generated subcellular fractions shows that most of the lens alpha crystallin is {"}soluble{"} and readily extracted with physiologic buffers. However, this data also shows that 1) Non-alpha crystallins are progressively and quantitatively extracted from the PMCC with buffer, 2) An irreducible level of non-covalently bound alpha crystallin is achieved which cannot be readily extracted from the PMCC, even with 2 M urea, 1{\%} NP40 or 0.4M KC1. Electron microscope level immunocytochemistry was performed on both the covalently immobilized PMCC, as well as on buffer-extracted thick frozen sections, using monoclonal antibodies to the αA-crystallin. The results show a very heavy labelling of both intermediate filaments and beaded filaments, but little or no labelling of fiber cell membranes. The data presented argues that a subtraction of the total αA-crystallin is strongly associated with the fiber cell cytoskeleton complex, and constitutes a quantitatively major component of the lens cytoskeleton fraction.",
author = "FitzGerald, {Paul G} and Dcbra Graham",
year = "1991",
doi = "10.3109/02713689109001750",
language = "English (US)",
volume = "10",
pages = "417--436",
journal = "Current Eye Research",
issn = "0271-3683",
publisher = "Informa Healthcare",
number = "5",

}

TY - JOUR

T1 - Ultrastructural localization of aa-crystallin to the bovine Lens fiber cell cytoskeleton

AU - FitzGerald, Paul G

AU - Graham, Dcbra

PY - 1991

Y1 - 1991

N2 - Monoclonal antibodies to the bovine aA-crystallin were developed and used to probe the relationship between aA-crystallin and the bovine lens fiber cell Plasma Membrane-Cytoskeleton Complex (PMCC). Superficial bovine lens cortex was washed by repeated homogenization/centrifugation to remove "soluble protein." The resulting Plasma Membrane-Cytoskeleton Complex was covalently immobilized to inert resin, and extensively buffer washed. SDS PAGE and immunoblot analysis of both the covalently immobilized PMCC and of the sequentially-generated subcellular fractions shows that most of the lens alpha crystallin is "soluble" and readily extracted with physiologic buffers. However, this data also shows that 1) Non-alpha crystallins are progressively and quantitatively extracted from the PMCC with buffer, 2) An irreducible level of non-covalently bound alpha crystallin is achieved which cannot be readily extracted from the PMCC, even with 2 M urea, 1% NP40 or 0.4M KC1. Electron microscope level immunocytochemistry was performed on both the covalently immobilized PMCC, as well as on buffer-extracted thick frozen sections, using monoclonal antibodies to the αA-crystallin. The results show a very heavy labelling of both intermediate filaments and beaded filaments, but little or no labelling of fiber cell membranes. The data presented argues that a subtraction of the total αA-crystallin is strongly associated with the fiber cell cytoskeleton complex, and constitutes a quantitatively major component of the lens cytoskeleton fraction.

AB - Monoclonal antibodies to the bovine aA-crystallin were developed and used to probe the relationship between aA-crystallin and the bovine lens fiber cell Plasma Membrane-Cytoskeleton Complex (PMCC). Superficial bovine lens cortex was washed by repeated homogenization/centrifugation to remove "soluble protein." The resulting Plasma Membrane-Cytoskeleton Complex was covalently immobilized to inert resin, and extensively buffer washed. SDS PAGE and immunoblot analysis of both the covalently immobilized PMCC and of the sequentially-generated subcellular fractions shows that most of the lens alpha crystallin is "soluble" and readily extracted with physiologic buffers. However, this data also shows that 1) Non-alpha crystallins are progressively and quantitatively extracted from the PMCC with buffer, 2) An irreducible level of non-covalently bound alpha crystallin is achieved which cannot be readily extracted from the PMCC, even with 2 M urea, 1% NP40 or 0.4M KC1. Electron microscope level immunocytochemistry was performed on both the covalently immobilized PMCC, as well as on buffer-extracted thick frozen sections, using monoclonal antibodies to the αA-crystallin. The results show a very heavy labelling of both intermediate filaments and beaded filaments, but little or no labelling of fiber cell membranes. The data presented argues that a subtraction of the total αA-crystallin is strongly associated with the fiber cell cytoskeleton complex, and constitutes a quantitatively major component of the lens cytoskeleton fraction.

UR - http://www.scopus.com/inward/record.url?scp=0025914055&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025914055&partnerID=8YFLogxK

U2 - 10.3109/02713689109001750

DO - 10.3109/02713689109001750

M3 - Article

VL - 10

SP - 417

EP - 436

JO - Current Eye Research

JF - Current Eye Research

SN - 0271-3683

IS - 5

ER -