ARVO 2014 Annual Meeting Abstracts 233 The fiber cell

ARVO 2014 Annual Meeting Abstracts
233 The fiber cell environment
Monday, May 05, 2014 11:00 AM–12:45 PM
S 330EF Paper Session
Program #/Board # Range: 1681–1687
Organizing Section: Lens
Program Number: 1681
Presentation Time: 11:00 AM–11:15 AM
RNA-sequencing analysis of Tdrd7 mutant lens identifies new
differentially regulated targets
Carrie E. Barnum1, Shaili Patel1, David A. Scheiblin1, Shawn
Polson2, Shinichiro Chuma3, David C. Beebe4, Salil A. Lachke1,
2 1
. Department of Biological Sciences, Univeristy of Delaware,
Newark, DE; 2Center for Bioinfomatics and Computational Biology,
University of Delaware, Newark, DE; 3Institute for Frontier Medical
Sciences, Kyoto University, Kyoto University, Kyoto, Japan;
4
Department of Ophthalmology and Visual Sciences, Washington
University, St. Louis, MO.
Purpose: We recently demonstrated that deficiency of the RNA
granule component gene TDRD7 causes cataracts in human, mouse
and chicken. Tdrd-family proteins are known to function in posttranscriptional control of gene expression in spermatogenesis.
Two different Tdrd7 null mouse mutants have been generated
(one was identified in an ENU-screen and the other is a targeted
germline knockout (KO) mutant), and both exhibit severe cataracts.
To investigate the mechanism of Tdrd7 function in the lens, we
performed detailed phenotypic and molecular characterization of
Tdrd7 KO mouse mutants.
Methods: Phenotypic analysis was performed by light microscopy,
scanning electron microscopy (SEM), confocal microscopy and
laser capture microdissection. Molecular analysis was performed
by running separate RNA-sequencing (RNA-seq) experiments for
isolated “large” (mRNA) and “small” (miRNA, piRNA, snoRNA,
snRNA) RNA fractions to identify differentially regulated transcripts.
Results: In the first three weeks of life, Tdrd7 KO lenses appear
normal in light microscopy until abruptly exhibiting lens defects
by postnatal day (P) 22. However, SEM analysis demonstrates that
fiber cell defects are discernable in Tdrd7 KO lens at P18, indicating
an earlier onset of the phenotype. Large RNA-seq of Tdrd7 KO
P4 lens confirmed Hsbp1 (Hsp27) downregulation and identified
several new differentially regulated mRNA targets, including other
Hsp family members. Small RNA-seq identified piwi-interacting
RNAs (piRNAs) to be expressed in the lens, some of which exhibited
differential regulation in Tdrd7 KO lens. This is intriguing because
piRNAs were previously considered to be restricted to germ cells,
where they are regulated by Tdrd-proteins. Additionally, several
snoRNAs, snRNAs and miRNAs were found to be differentially
regulated in Tdrd7 KO lenses, suggesting a critical function of Tdrd7
in regulating non-protein coding RNAs in the lens.
Conclusions: We used RNA-seq to investigate Tdrd7 KO lens
and have identified many new differentially regulated transcripts.
Significantly, we have identified piRNAs in the lens and demonstrate
that in addition to affecting mRNA profiles, Tdrd7 nullizygosity
affects expression of select snoRNA, snRNA, miRNA and piRNA.
These data suggest that Tdrd7 plays distinct functions, regulating
both mRNAs and non-protein-coding RNAs, to mediate posttranscriptional control of gene expression in lens fiber cells.
Commercial Relationships: Carrie E. Barnum, None; Shaili Patel,
None; David A. Scheiblin, None; Shawn Polson, None; Shinichiro
Chuma, None; David C. Beebe, None; Salil A. Lachke, None
Support: NIH/NEI R01 EY021505, UDRF, Inc., Knights Templar
Eye Foundation, Inc.
Program Number: 1682
Presentation Time: 11:15 AM–11:30 AM
Quantitative Proteomic Analysis of Lipid-Raft Domains from
Lens Fiber Cells
Kevin L. Schey, Zhen Wang. Biochemistry, Vanderbilt University,
Nashville, TN.
Purpose: Lipid rafts are domains within cell membranes that
are rich in cholesterol and sphingolipids and serve to concentrate
specific protein activities. Lens fiber cell membranes contain high
concentrations of sphingomyelin and cholesterol. Lipid rafts could
play critical roles in regulating protein function and in maintaining
lens transparency. The purpose of this study is to characterize lens
proteins that are localized in raft domains and in non-raft membranes
using quantitative proteomic methods.
Methods: Bovine lenses were decapsulated and dissected into cortex
and nucleus regions. The water-insoluble fraction from each region
was divided into two samples and one was treated with methyl-βcyclodextrin to deplete cholesterol and disrupt rafts. Samples were
incubated with detergent (1% Brij 98, 35 mM octyl-glucoside, 600
mM NaCl) at 4 oC for 30 min. and subjected to sucrose density
gradient centrifugation. Proteins from low density to high density
were isolated and precipitated using chloroform/methanol, digested
by trypsin, and analyzed by LC-MS/MS. Raft fractions were
identified based on the enrichment of lipid raft markers. Using
the iTRAQ quantitative proteomics method, raft proteins were
distinguished from non-raft membrane proteins based on abundance
changes upon cholesterol-depletion.
Results: Lipid raft fractions were identified as fractions recovered
from the interface of the 5% and 35% sucrose layers since raft
markers such as flotillin, erlin and prohibitin, were enriched in these
fractions and moved to higher density fractions after cholesterol
depletion. Additional proteins identified as highly enriched in the raft
fractions included caveolins, AQP5, Lim2, and Voltage-dependent
calcium channel subunit alpha-2/delta-1. Some proteins were
detected in both raft and non-raft fractions including AQP0, neural
cell adhesion molecule, Voltage-dependent anion-selective channel
proteins, cytoskeleton-associated protein 4, syntaxin, as well as lipidanchored peripheral membrane proteins such as brain acid soluble
protein 1, paralemmin, ras-related C3 botulinum toxin substrate 1,
and protein kinase C.
Conclusions: Quantitative proteomic analysis revealed components
of the lens fiber cell lipid raft-like, detergent resistant membranes.
These data provide clues to protein sorting into distinct membrane
microdomains and possibly how protein function is altered depending
on the lipid environment.
Commercial Relationships: Kevin L. Schey, None; Zhen Wang,
None
Support: NIH Grant EY13462
Program Number: 1683
Presentation Time: 11:30 AM–11:45 AM
Ankyrin-B Haploinsufficient Lenses Uncover the Importance of
Membrane Subdomain Organization for Fiber Cell Hexagonal
Packing and Mechanical Properties
Vasanth Rao1, Rupalatha Maddala1, Mark D. Walters2, Vann Bennett3.
1
Ophthal & Pharmacology, Duke University, Durham, NC; 2Pratt
School of Engineering, Duke Uuiversity, Durham, NC; 3Department
of Cell Biology, Duke UNiversity School of Medicine, Durham, NC.
Purpose: Fiber cell hexagonal symmetry, membrane organization
and tensile properties are considered to be critical for lens
architecture, transparency and deformability. The molecular
determinants of membrane organization that govern these lens
characteristics, however, are not well defined. In this study, we
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
examined the 3-D organization of various membrane and membraneassociated proteins in normal and Ankyrin-B deficient (AnkB-/+)
mouse lenses in the context of fiber cell geometry and tensile
properties.
Methods: Paraffin-embedded equatorial lens sections derived
from the wild type (WT) and AnkB-/+ mice were evaluated for the
distribution profile of Ank-B & G, β-spectrin, β-actin, periaxin,
Connexin-50, Aquaporin-0, NrCAM, ponsin, β-dystroglycan
and filensin using a Zeiss 780 inverted confocal microscope in
conjunction with Volocity 3D construction software. Changes in lens
stiffness were evaluated using a Micro-strain analyzer.
Results: In WT lenses AnkB & G are distributed in distinct
subdomains throughout the fiber cell membrane including the
long and short arms. High resolution confocal images using 3D
reconstruction reveal a discrete distribution pattern of β-actin,
ponsin and periaxin, which localize predominantly to the tricellular
junctions of fiber cells. β-spectrin and NrCAM exhibit intense
staining throughout the short arms relative to their distribution within
the long arms. In contrast, β-dystroglycan, connexin-50 and filensin
cluster into large domains at the center of long arms of fiber cells.
Aquaporin-0 is distributed throughout the fiber cell. The AnkB-/+
mice show a slight increase in lens weight but remain clear. However,
these lenses reveal disruption of hexagonal geometry of fiber
cells. Additionally, the membrane organization, protein levels and
expression of above described proteins along with L-type calcium
channels and myosin II phosphorylation were significantly disrupted
in the AnkB-/+ mouse lenses. These lenses show a significant
decrease in stiffness compared to WT lenses.
Conclusions: Taken together, this study reveals that Ankyrin-B-based
membrane subdomain organization determines mechanical properties
and hexagonal cell packing in the lens.
Commercial Relationships: Vasanth Rao, None; Rupalatha
Maddala, None; Mark D. Walters, None; Vann Bennett, None
Support: NIH Grant: EY012201and EY18590
Program Number: 1684
Presentation Time: 11:45 AM–12:00 PM
Aquaporin-0 Targets Interlocking Protrusions to Control
Integrity and Transparency of the Mouse Lens
Woo-Kuen Lo1, Sondip K. Biswas1, Lawrence Brako1, Alan Shiels2,
Sumin Gu3, Jean X. Jiang3. 1Neurobiology, Morehouse School
of Medicine, Atlanta, GA; 2Ophthalmology and Visual Sciences,
Washington University School of Medicine, St. Louis, MO;
3
Biochemistry, University of Texas Health Science Center, San
Antonio, TX.
Purpose: Lens fiber cell membranes contain aquaporin-0 (AQP0)
which constitutes approximately 50% of total integral fiber-cell
membrane proteins and plays a dual function as a water channel
protein and an adhesion molecule. Fiber cell membranes also develop
an elaborate interlocking system that is required for maintaining
the structural order, stability and lens transparency. Here we use
an AQP0-deficient mouse model to investigate an unconventional
adhesion role of AQP0 in maintaining a normal structure of
interlocking protrusions in the lens.
Methods: The loss of AQP0 in lens fibers of AQP0-/- mice was
verified by a purified AQP0 polyclonal antibody using Western
blotting and immunofluorescence analyses. Changes in membrane
surface structures of lens fibers of wild-type and AQP0-/- mice at 3
to 12 weeks old were examined with scanning electron microscopy.
Preferential distribution of AQP0 in fiber cell membranes in wildtype controls was analyzed with confocal immunofluorescence and
immunogold labeling using freeze-fracture TEM.
Results: Interlocking protrusions in young differentiating fiber
cells developed normally but showed minor abnormalities at
approximately 50 μm deep from the surface in the absence of
AQP0 in all ages studied. Strikingly, interlocking protrusions in
maturing fiber cells specifically underwent uncontrolled elongation,
deformation and fragmentation while the cells still possessed fairly
normal configurations in the early process. These changes eventually
resulted in fiber-cell separation, breakdown and cataract formation in
the lens core. Immunolabeling at the light and electron microscopic
levels demonstrated that AQP0 was particularly enriched in
interlocking protrusions in wild-type lenses.
Conclusions: This study suggests that AQP0 exerts its primary
adhesion or suppression role specifically to maintain the normal
structure of interlocking protrusions that is critical to the integrity and
transparency of the lens.
Commercial Relationships: Woo-Kuen Lo, None; Sondip K.
Biswas, None; Lawrence Brako, None; Alan Shiels, None; Sumin
Gu, None; Jean X. Jiang, None
Support: NIH/NEI Grant EY05314 to W.K.L., EY012284 to A.S;
EY012085 to J.X.J.
Program Number: 1685
Presentation Time: 12:00 PM–12:15 PM
Extracellular loop positive charges of lens aquaporin 0 play a
significant role in cell-to-cell adhesion
Kulandaiappan Varadaraj1, 2, Sindhu Kumari1. 1Physiology and
Biophysics, State University of New York, Stony Brook, NY; 2SUNY
Eye Institute, New York, NY.
Purpose: Investigate the role of extracellular loops of Aquaporin 0
(AQP0) on cell-to-cell adhesion (CTCA) function.
Methods: Extracellular loops A and C of mouse AQP0 were
substituted with those of AQP1 through polymerase chain reaction
using specific oligonucleotide primers. We have also replaced the
positively charged residues in loop A of AQP0 with structurally
compatible neutral residue to create R33Q and H40Q mutants. Water
permeability (Pw) and CTCA properties of the chimeric and mutant
AQP0 were studied by expressing them in Xenopus oocytes through
cRNA injection and by transfecting into adhesion-deficient mouse
fibroblast L-cells, as appropriate. Pw was studied using the shrinking
and swelling assay. CTCA was tested using a method devised by our
laboratory. Intact (wild type) AQP0 and E-cadherin served as positive
controls while AQP1 served as a negative control for CTCA studies.
Results: AQP0-AQP1-loop A chimera trafficked to the plasma
membrane like the intact AQP0 while AQP0-AQP1-loop C chimera
did not. Pw of AQP0-AQP1-loop A was 45.3 ± 5.8 μm/s while that of
intact AQP0 was 46.4 ± 3.9 μm/s; Pw of control oocytes injected with
distilled water was 10.9 ± 2.5 μm/s. CTCA assays showed that the
adhesion property of AQP0-AQP1-loop A chimera was significantly
reduced (24%; P< 0.001) compared to that of intact AQP0. Mutants
AQP0-R33Q and AQP0-H40Q trafficked and localized at the plasma
membrane like the intact AQP0. Functional studies conducted in
Xenopus oocytes showed no significant difference (P>0.05) in Pw of
AQP0-R33Q (45.7 ± 6.9 μm/s) and AQP0-H40Q (44.7 ± 7.2 μm/s)
compared to that of intact AQP0. However, the CTCA property of
AQP0-R33Q (~22%) and AQP0-H40Q (19%) was significantly
reduced (P< 0.001) in comparison to that of intact AQP0.
Conclusions: The data suggest that extracellular loop A substitution
or point mutation of AQP0 might not have caused significant
alterations in protein folding since there was no obstruction in
protein trafficking or Pw but extracellular loop C substitution
inhibited protein trafficking suggesting alteration/s in protein folding.
Reduction in the CTCA observed for AQP0-AQP1-loop A chimera
as well as AQP0-R33Q and AQP0-H40Q mutants suggest that the
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
conserved positive charges in the Loop A may be critical for the
CTCA function of AQP0.
Commercial Relationships: Kulandaiappan Varadaraj, None;
Sindhu Kumari, None
Support: EY20506
Program Number: 1686
Presentation Time: 12:15 PM–12:30 PM
A Role for Microtubules in Lens Fiber Cell Elongation and Lens
Morphogenesis
Caitlin Logan1, Liping Zhang1, A S. Menko1, 2. 1Pathology Anatomy
and Cell Biology, Thomas Jefferson University, Philadelphia, PA;
2
Wills Vision Research Center at Jefferson, Philadelphia, PA.
Purpose: Tissue development and regeneration involve highordered morphogenetic processes that are governed by elements of
the cytoskeleton in conjunction with cell adhesion molecules. Such
processes are particularly important in the lens whose structure
dictates its function. Microtubules have many roles in the cell, among
them as determinants of directional migration and as the highways
for vesicle transport. Here we investigated the possible role of
microtubules and their interactions with N-cadherin in providing
directionality to fiber cell elongation.
Methods: Co-immunoprecipitation analysis was performed on
chick embryo lenses microdissected into four distinct zones of
differentiation to analyze association of tubulin/acetylated tubulin
with N-cadherin. The role of tubulin in fiber cell elongation
was examined by treating E10 lenses in organ culture with the
microtubule depolymerizing drug nocodazole. Treated lenses were
microdissected as above and immunoblotted for tubulin expression/
acetylation. Lens cryosections were labeled for α-tubulin, acetylated
tubulin, N-cadherin, and/or F-actin.
Results: N-Cadherin interacts with tubulin primarily in the cortical
fiber zone, where lens fiber cells elongate. No association was
detected between N-cadherin and the acetylated form of tubulin.
Disassembly of microtubules with nocodazole affected fiber cell
elongation and directionality. High doses of nocodazole also
affected interactions between fiber cells and epithelial cells along the
epithelial-fiber interface (EFI). These effects were accompanied by
changes in levels of F-actin and increased localization of N-cadherin
along the EFI. These results provide the first demonstration of a role
for microtubules in lens fiber cell elongation, in lens morphogenesis
and in the maintenance of lens tissue integrity.
Conclusions: Microtubules have an important role in the
determination of proper lens morphogenesis.
Commercial Relationships: Caitlin Logan, None; Liping Zhang,
None; A S. Menko, None
Support: R01 EY014258
Methods: Transgenic expression of histone 2B (H2B)-GFP and
DNA-staining dyes were used to monitor the distribution and
disassembly of fiber cell nuclei and chromatin behavior in live lenses
by 3-dimensional confocal laser microscopic imaging. Western
blotting and immunostaining were performed to elucidate the
molecular and cellular changes.
Results: Distinct nuclei distribution was observed along the anterior
and posterior equatorial panel during fiber cell differentiation and
elongation. Chromatins in fiber cell nuclei displayed sequential
changes during fiber cell maturation. All inner fiber cells lost their
nuclei at about 150 μm distance from the lens surface. However,
denucleation of fiber cells often occurred long before reaching the
nuclei-free zone. During denucleation, H2B-GFP proteins were
diffused along both anterior and posterior directions and then were
evenly distributed with cell boundaries of inner fibers. Altered
distribution of fiber cell nuclei and aberrant distribution and/or
aggregation of the H2B-GFP proteins were detected in different
cataractous lenses. In addition, aberrant aggregation of H2B-GFP
proteins was also detected in newly formed cortical mature fiber cells
in aged wild-type lenses.
Conclusions: This work reveals that inner fiber cell denucleation
occurs at around 100-150 μm distance from the lens surface rather
than an old assumption that fiber cell denucleation proceeds only
in several cell layers before the nuclei-free zone. Aging impairs
fiber cells denucleation by blocking the disassembly of nuclear
proteins and induces the aggregation of nuclear proteins such as
H2B. Different types of cataracts caused by various gene mutations
are associated with uniquely altered distribution and/or disassembly
of fiber cell nuclei. Coordinated functions of intercellular gap
junction communication, intermediate filaments and alpha-crystallin
chaperons precisely regulate the distribution and disassembly of fiber
cell nuclei during cell maturation.
Commercial Relationships: Xiaohua Gong, None; Catherine
Cheng, None; Wiktor Stopka, None; Jing Zeng, None; Chun-hong
Xia, None
Support: Supported by grants EY013849 from the National Eye
Institute
Program Number: 1687
Presentation Time: 12:30 PM–12:45 PM
Lens fiber cell denucleation and cataractogenesis
Xiaohua Gong1, 2, Catherine Cheng1, Wiktor Stopka2, Jing Zeng1,
Chun-hong Xia1. 1Vision Sci School of Optometry, University of
California, Berkeley, Berkeley, CA; 2UC Berkeley/UCSF Joint
Graduate Program in Bioengineering, University of California,
Berkeley, Berkeley, CA.
Purpose: To investigate the mechanisms that control nuclei
distribution, chromatin behavior or denucleation process during lens
fiber cell differentiation and maturation in normal and cataractous
mice caused by various mutations, such as alpha-crystallin gene
mutations, intermediate filament protein CP49 gene deletion and
connexin gene knockouts. Moreover, to evaluate how aging affects
fiber cell differentiation and maturation.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].

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