MOLECULAR ANALYSIS OF OCULAR LENS BEADED FILAMENTS

Project: Research project

Project Details

Description

We are proposing to investigate an ocular lens cytoskeletal structure known
as the beaded filament. This structure, and the two proteins thus far
associated with it, have not been demonstrated in either the lens
epithelium, or in other tissues. Thus the beaded filament appears to be a
structure unique to the differentiating lens fiber cell. For this reason
we feel that the beaded filament must be of seminal importance to normal
lens biology. We are proposing to use subcellular fractionation to purify
the beaded filament, and implicate, by co-purification, any proteins which
might be associated with this structure. Monoclonal antibodies to
candidate proteins will be used to confirm the presence of specific
proteins in the beaded filament. We propose to establish conditions in
which the beaded filament can be reconstituted, initially from disrupted
whole filaments, and subsequently from purified individual constituent
proteins. Using either the reconstitution system developed herein, or a
cell-free translation system programmed with lens mRNA, filament assembly
will be specifically disrupted, using F(ab) fragments of monoclonal
antibodies, cyanogen bromide fragments of beaded filament proteins,
synthetic peptides derived from the primary sequence, or oligonucleotides
synthesized from primary sequence information. In so doing we hope to
pinpoint protein domains involved in beaded filament protein-protein
interactions. We intend to establish the primary amino acid sequence for
the proteins which comprise the beaded filament, by isolating the cDNA for
these proteins from existing cDNA libraries. We plan to utilize this
sequence information to relate the proteins to existing gene families, and
for subsequent investigation of specific binding domains within beaded
filament proteins. Subsequent inquiry will be made into the possibility of
utilizing a differentiating lens explant for further in vitro studies of
beaded filament transcription and translation. In vitro synthesis of
beaded filaments in a cell-free translation system will be employed to
study filament protein synthesis and assembly. Organ culture of whole
lenses, as well as in vitro synthesis will be used to study the
phosphorylation of the beaded filament proteins, the regulation of this
phosphorylation, and the effects of phosphorylation on filament assembly
and disassembly. We believe that such basic knowledge of the system which
generates and maintains cellular shape and tissue level organization in the
lens is fundamental to understanding how the lens achieves and maintains
clarity, and permits us to study the possible role of the cellular
cytoskeleton in the etiology of cataract.
StatusFinished
Effective start/end date8/1/907/31/12

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $375,257.00
  • National Institutes of Health: $369,579.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $380,578.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $467,776.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $391,905.00
  • National Institutes of Health: $432,833.00
  • National Institutes of Health: $396,022.00
  • National Institutes of Health: $385,800.00
  • National Institutes of Health
  • National Institutes of Health: $469,784.00
  • National Institutes of Health: $387,336.00
  • National Institutes of Health: $458,946.00

ASJC

  • Medicine(all)

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