Paul Fitzgerald, PhD

Dr. Fitzgerald is Professor and Chair of the Department of Cell Biology and Human Anatomy at the University of California-Davis. His research uses genetic engineering approaches to create mice that lack beaded filament proteins. He has discovered that the lenses of these animals develop and differentiate normally, achieving the high degree of structural order that characterizes the lens, but that they are unable to maintain that order with age. Thus, the structural order seen in the lens is inherently unstable, and the beaded filament serves to confer resistance to the age-dependent loss of structure. He now studies the structure of the beaded filament in an effort to determine how it accomplishes its mission.

Recent Publications

2018

Myo/Nog cells are present in the ciliary processes, on the zonule of Zinn and posterior capsule of the lens following cataract surgery.

Exp Eye Res. 2018 Mar 17;:

Authors: Gerhart J, Withers C, Gerhart C, Werner L, Mamalis N, Bravo-Nuevo A, Scheinfeld V, FitzGerald P, Getts R, George-Weinstein M

Myo/Nog cells are present in the ciliary processes, on the zonule of Zinn and posterior capsule of the lens following cataract surgery.

Exp Eye Res. 2018 Mar 17;:

Authors: Gerhart J, Withers C, Gerhart C, Werner L, Mamalis N, Bravo-Nuevo A, Scheinfeld V, FitzGerald P, Getts R, George-Weinstein M

Abstract
Myo/Nog cells, named for their expression of MyoD and noggin, enter the eye during early stages of embryonic development. Their release of noggin is critical for normal morphogenesis of the lens and retina. Myo/Nog cells are also present in adult eyes. Single nucleated skeletal muscle cells designated as myofibroblasts arise from Myo/Nog cells in cultures of lens tissue. In this report we document the presence of Myo/Nog cells in the lens, ciliary body and on the zonule of Zinn in mice, rabbits and humans. Myo/Nog cells were rare in all three structures. Their prevalence increased in the lens and ciliary body of rabbits 24 h following cataract surgery. Rabbits developed posterior capsule opacification (PCO) within one month of surgery. The number of Myo/Nog cells continued to be elevated in the lens and ciliary body. Myo/Nog cells containing alpha smooth muscle actin and striated muscle myosin were present on the posterior capsule and overlaid deformations in the capsule. Myo/Nog cells also were present on the zonule fibers and external surface of the posterior capsule. These findings suggest that Myo/Nog contribute to PCO and may use the zonule fibers to migrate between the ciliary processes and lens.

PMID: 29559302 [PubMed - as supplied by publisher]

Myo/Nog cells are present in the ciliary processes, on the zonule of Zinn and posterior capsule of the lens following cataract surgery.

Exp Eye Res. 2018 Mar 17;:

Authors: Gerhart J, Withers C, Gerhart C, Werner L, Mamalis N, Bravo-Nuevo A, Scheinfeld V, FitzGerald P, Getts R, George-Weinstein M

Deletion of GLUT1 in mouse lens epithelium leads to cataract formation.

Exp Eye Res. 2018 Mar 28;:

Authors: Swarup A, Bell BA, Du J, Han JYS, Soto J, Abel ED, Bravo-Nuevo A, FitzGerald PG, Peachey NS, Philp NJ

Deletion of GLUT1 in mouse lens epithelium leads to cataract formation.

Exp Eye Res. 2018 Mar 28;:

Authors: Swarup A, Bell BA, Du J, Han JYS, Soto J, Abel ED, Bravo-Nuevo A, FitzGerald PG, Peachey NS, Philp NJ

Abstract
The primary energy substrate of the lens is glucose and uptake of glucose from the aqueous humor is dependent on glucose transporters. GLUT1, the facilitated glucose transporter encoded by Slc2a1 is expressed in the epithelium of bovine, human and rat lenses. In the current study, we examined the expression of GLUT1 in the mouse lens and determined its role in maintaining lens transparency by studying effects of postnatal deletion of Slc2a1. In situ hybridization and immunofluorescence labeling were used to determine the expression and subcellular distribution of GLUT1 in the lens. Slc2a1 was knocked out of the lens epithelium by crossing transgenic mice expressing Cre recombinase under control of the GFAP promoter with Slc2a1loxP/loxP mice to generate Slc2a1loxP/loxP;GFAP-Cre+/0 (LensΔGlut1) mice. LensΔGlut1 mice developed visible lens opacities by around 3 months of age, which corresponded temporally with the total loss of detectable GLUT1 expression in the lens. Spectral domain optical coherence tomography (SD-OCT) imaging was used to monitor the formation of cataracts over time. SD-OCT imaging revealed that small nuclear cataracts were first apparent in the lenses of LensΔGlut1 mice beginning at about 2.7 months of age. Longitudinal SD-OCT imaging of LensΔGlut1 mice revealed disruption of mature secondary fiber cells after 3 months of age. Histological sections of eyes from LensΔGlut1 mice confirmed the disruption of the secondary fiber cells. The structural changes were most pronounced in fiber cells that had lost their organelles. In contrast, the histology of the lens epithelium in these mice appeared normal. Lactate and ATP were measured in lenses from LensΔGlut1 and control mice at 2 and 3 months of age. At 2 months of age, when GLUT1 was still detectable in the lens epithelium, albeit at low levels, the amount of lactate and ATP were not significantly different from controls. However, in lenses isolated from 3-month-old LensΔGlut1 mice, when GLUT1 was no longer detectable, levels of lactate and ATP were 50% lower than controls. Our findings demonstrate that in vivo, the transparency of mature lens fiber cells was dependent on glycolysis for ATP and the loss of GLUT1 transporters led to cataract formation. In contrast, lens epithelium and cortical fiber cells have mitochondria and could utilize other substrates to support their anabolic and catabolic needs.

PMID: 29604281 [PubMed - as supplied by publisher]

Deletion of GLUT1 in mouse lens epithelium leads to cataract formation.

Exp Eye Res. 2018 Mar 28;:

Authors: Swarup A, Bell BA, Du J, Han JYS, Soto J, Abel ED, Bravo-Nuevo A, FitzGerald PG, Peachey NS, Philp NJ

2016

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Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells.

Invest Ophthalmol Vis Sci. 2016 Aug 1;57(10):4084-99

Authors: Cheng C, Nowak RB, Biswas SK, Lo WK, FitzGerald PG, Fowler VM

Related Articles

Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells.

Invest Ophthalmol Vis Sci. 2016 Aug 1;57(10):4084-99

Authors: Cheng C, Nowak RB, Biswas SK, Lo WK, FitzGerald PG, Fowler VM

Abstract
PURPOSE: To elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end-capping protein.
METHODS: We investigated F-actin and F-actin-binding protein localization in interdigitations of Tmod1+/+ and Tmod1-/- single mature lens fibers.
RESULTS: F-actin-rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1-/- mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, β2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1-/- mature fibers, β2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1-/- mature fibers.
CONCLUSIONS: These results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a β2-spectrin-actin network stabilized by Tmod1. α-Actinin-crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin-associated proteins required for the formation of paddles between lens fibers.

PMID: 27537257 [PubMed - in process]

Related Articles

Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells.

Invest Ophthalmol Vis Sci. 2016 Aug 1;57(10):4084-99

Authors: Cheng C, Nowak RB, Biswas SK, Lo WK, FitzGerald PG, Fowler VM

Related Articles

Expression of the type VI intermediate filament proteins CP49 and filensin in the mouse lens epithelium.

Mol Vis. 2016;22:970-89

Authors: FitzGerald P, Sun N, Shibata B, Hess JF

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Expression of the type VI intermediate filament proteins CP49 and filensin in the mouse lens epithelium.

Mol Vis. 2016;22:970-89

Authors: FitzGerald P, Sun N, Shibata B, Hess JF

Abstract
PURPOSE: The differentiated lens fiber cell assembles a filamentous cytoskeletal structure referred to as the beaded filament (BF). The BF requires CP49 (bfsp2) and filensin (bfsp1) for assembly, both of which are highly divergent members of the large intermediate filament (IF) family of proteins. Thus far, these two proteins have been reported only in the differentiated lens fiber cell. For this reason, both proteins have been considered robust markers of fiber cell differentiation. We report here that both proteins are also expressed in the mouse lens epithelium, but only after 5 weeks of age.
METHODS: Localization of CP49 was achieved with immunocytochemical probing of wild-type, CP49 knockout, filensin knockout, and vimentin knockout mice, in sections and in the explanted lens epithelium, at the light microscope and electron microscope levels. The relationship between CP49 and other cytoskeletal elements was probed using fluorescent phalloidin, as well as with antibodies to vimentin, GFAP, and α-tubulin. The relationship between CP49 and the aggresome was probed with antibodies to γ-tubulin, ubiquitin, and HDAC6.
RESULTS: CP49 and filensin were expressed in the mouse lens epithelium, but only after 5 weeks of age. At the light microscope level, these two proteins colocalize to a large tubular structure, approximately 7 × 1 μm, which was typically present at one to two copies per cell. This structure is found in the anterior and anterolateral lens epithelium, including the zone where mitosis occurs. The structure becomes smaller and largely undetectable closer to the equator where the cell exits the cell cycle and commits to fiber cell differentiation. This structure bears some resemblance to the aggresome and is reactive with antibodies to HDAC6, a marker for the aggresome. However, the structure does not colocalize with antibodies to γ-tubulin or ubiquitin, also markers for the aggresome. The structure also colocalizes with actin but appears to largely exclude vimentin and α-tubulin. In the CP49 and filensin knockouts, this structure is absent, confirming the identity of CP49 and filensin in this structure, and suggesting a requirement for the physiologic coassembly of CP49 and filensin.
CONCLUSIONS: CP49 and filensin have been considered robust markers for mouse lens fiber cell differentiation. The data reported here, however, document both proteins in the mouse lens epithelium, but only after 5 weeks of age, when lens epithelial growth and mitotic activity have slowed. Because of this, CP49 and filensin must be considered markers of differentiation for both fiber cells and the lens epithelium in the mouse. In addition, to our knowledge, no other protein has been shown to emerge so late in the development of the mouse lens epithelium, suggesting that lens epithelial differentiation may continue well into post-natal life. If this structure is related to the aggresome, it is a rare, or perhaps unique example of a large, stable aggresome in wild-type tissue.

PMID: 27559293 [PubMed - in process]

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Expression of the type VI intermediate filament proteins CP49 and filensin in the mouse lens epithelium.

Mol Vis. 2016;22:970-89

Authors: FitzGerald P, Sun N, Shibata B, Hess JF

2015

Role of Aquaporin 0 in lens biomechanics.

Biochem Biophys Res Commun. 2015 May 7;

Authors: Sindhu Kumari S, Gupta N, Shiels A, FitzGerald PG, Menon AG, Mathias RT, Varadaraj K

Role of Aquaporin 0 in lens biomechanics.

Biochem Biophys Res Commun. 2015 May 7;

Authors: Sindhu Kumari S, Gupta N, Shiels A, FitzGerald PG, Menon AG, Mathias RT, Varadaraj K

Abstract
Maintenance of proper biomechanics of the eye lens is important for its structural integrity and for the process of accommodation to focus near and far objects. Several studies have shown specialized cytoskeletal systems such as the beaded filament (BF) and spectrin-actin networks contribute to mammalian lens biomechanics; mutations or deletion in these proteins alters lens biomechanics. Aquaporin 0 (AQP0), which constitutes ∼45% of the total membrane proteins of lens fiber cells, has been shown to function as a water channel and a structural cell-to-cell adhesion (CTCA) protein. Our recent ex vivo study on AQP0 knockout (AQP0 KO) mouse lenses showed the CTCA function of AQP0 could be crucial for establishing the refractive index gradient. However, biomechanical studies on the role of AQP0 are lacking. The present investigation used wild type (WT), AQP5 KO (AQP5(-/-)), AQP0 KO (heterozygous KO: AQP0(+/-); homozygous KO: AQP0(-/-); all in C57BL/6J) and WT-FVB/N mouse lenses to learn more about the role of fiber cell AQPs in lens biomechanics. Electron microscopic images exhibited decreases in lens fiber cell compaction and increases in extracellular space due to deletion of even one copy of AQP0. Biomechanical assay revealed that loss of one or both copies of AQP0 caused a significant reduction in compressive load-bearing capacity of the lenses compared to WT lenses. Conversely, loss of AQP5 did not alter the lens load-bearing ability. Compressive load-bearing at the suture area of AQP0(+/-) lenses showed easy separation while WT lens remained intact. These data from KO mouse lenses in conjunction with previous studies on lens-specific BF proteins (CP49 and filensin) suggest that AQP0 and BF proteins could act co-operatively in establishing normal lens biomechanics. We hypothesize that AQP0, with its prolific expression at the fiber cell membrane, could provide anchorage for cytoskeletal structures like BFs and together they help to confer fiber cell shape, architecture and integrity. To our knowledge, this is the first report identifying the involvement of an aquaporin in lens biomechanics. Since accommodation is required in human lenses for proper focusing, alteration in the adhesion and/or water channel functions of AQP0 could contribute to presbyopia.

PMID: 25960294 [PubMed - as supplied by publisher]

Role of Aquaporin 0 in lens biomechanics.

Biochem Biophys Res Commun. 2015 May 7;

Authors: Sindhu Kumari S, Gupta N, Shiels A, FitzGerald PG, Menon AG, Mathias RT, Varadaraj K

Related Articles

An alternative means of retaining ocular structure and improving immunoreactivity for light microscopy studies.

Mol Vis. 2015;21:428-42

Authors: Sun N, Shibata B, Hess JF, FitzGerald PG

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An alternative means of retaining ocular structure and improving immunoreactivity for light microscopy studies.

Mol Vis. 2015;21:428-42

Authors: Sun N, Shibata B, Hess JF, FitzGerald PG

Abstract
PURPOSE: Several properties of ocular tissue make fixation for light microscopy problematic. Because the eye is spherical, immersion fixation necessarily results in a temporal gradient of fixation, with surfaces fixing more rapidly and thoroughly than interior structures. The problem is compounded by the fact that the layers of the eye wall are compositionally quite different, resulting in different degrees of fixation-induced shrinkage and distortion. Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment. This gradient problem is most acute for the lens, where the density of proteins can delay fixation of the central lens for days, and where the fixation gradient parallels the age gradient of lens cells, which complicates data interpretation. Our goal was to identify a simple method for minimizing some of the problems arising from immersion fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques.
METHODS: A simple and inexpensive derivative of the freeze-substitution approach was developed and compared to fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed.
RESULTS: An inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, as is compatibility with in situ hybridization, lectin labeling, and the Click-iT chemistry approach to labeling the thymidine analog EdU. On the negative side, this approach dramatically reduced intrinsic GFP fluorescence.
CONCLUSIONS: A simple, cost-effective derivative of the freeze substitution process is described that is of particular value in the study of rodent or other small eyes, where fixation gradients, globe buckling, retinal detachment, differential shrinkage, autofluorescence, and tissue immunoreactivity have been problematic.

PMID: 25991907 [PubMed - in process]

Related Articles

An alternative means of retaining ocular structure and improving immunoreactivity for light microscopy studies.

Mol Vis. 2015;21:428-42

Authors: Sun N, Shibata B, Hess JF, FitzGerald PG

2014

Related Articles

Rapid light-induced activation of retinal microglia in mice lacking Arrestin-1.

Vision Res. 2014 Sep;102:71-9

Authors: Levine ES, Zam A, Zhang P, Pechko A, Wang X, FitzGerald P, Pugh EN, Zawadzki RJ, Burns ME

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Rapid light-induced activation of retinal microglia in mice lacking Arrestin-1.

Vision Res. 2014 Sep;102:71-9

Authors: Levine ES, Zam A, Zhang P, Pechko A, Wang X, FitzGerald P, Pugh EN, Zawadzki RJ, Burns ME

Abstract
Microglia dynamically prune synaptic contacts during development, and digest waste that accumulates in degeneration and aging. In many neurodegenerative diseases, microglial activation and phagocytosis gradually increase over months or years, with poorly defined initial triggering events. Here, we describe rapid retinal microglial activation in response to physiological light levels in a mouse model of photoreceptor degeneration that arises from defective rhodopsin deactivation and prolonged signaling. Activation, migration and proliferation of microglia proceeded along a well-defined time course apparent within 12h of light onset. Retinal imaging in vivo with optical coherence tomography revealed dramatic increases in light-scattering from photoreceptors prior to the outer nuclear layer thinning classically used as a measure of retinal neurodegeneration. This model is valuable for mechanistic studies of microglial activation in a well-defined and optically accessible neural circuit, and for the development of novel methods for detecting early signs of pending neurodegeneration in vivo.

PMID: 25091460 [PubMed - in process]

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Rapid light-induced activation of retinal microglia in mice lacking Arrestin-1.

Vision Res. 2014 Sep;102:71-9

Authors: Levine ES, Zam A, Zhang P, Pechko A, Wang X, FitzGerald P, Pugh EN, Zawadzki RJ, Burns ME

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Loss of Sip1 leads to migration defects and retention of ectodermal markers during lens development.

Mech Dev. 2014 Feb;131:86-110

Authors: Manthey AL, Lachke SA, FitzGerald PG, Mason RW, Scheiblin DA, McDonald JH, Duncan MK

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Loss of Sip1 leads to migration defects and retention of ectodermal markers during lens development.

Mech Dev. 2014 Feb;131:86-110

Authors: Manthey AL, Lachke SA, FitzGerald PG, Mason RW, Scheiblin DA, McDonald JH, Duncan MK

Abstract
SIP1 encodes a DNA-binding transcription factor that regulates multiple developmental processes, as highlighted by the pleiotropic defects observed in Mowat-Wilson syndrome, which results from mutations in this gene. Further, in adults, dysregulated SIP1 expression has been implicated in both cancer and fibrotic diseases, where it functionally links TGFβ signaling to the loss of epithelial cell characteristics and gene expression. In the ocular lens, an epithelial tissue important for vision, Sip1 is co-expressed with epithelial markers, such as E-cadherin, and is required for the complete separation of the lens vesicle from the head ectoderm during early ocular morphogenesis. However, the function of Sip1 after early lens morphogenesis is still unknown. Here, we conditionally deleted Sip1 from the developing mouse lens shortly after lens vesicle closure, leading to defects in coordinated fiber cell tip migration, defective suture formation, and cataract. Interestingly, RNA-Sequencing analysis on Sip1 knockout lenses identified 190 differentially expressed genes, all of which are distinct from previously described Sip1 target genes. Furthermore, 34% of the genes with increased expression in the Sip1 knockout lenses are normally downregulated as the lens transitions from the lens vesicle to early lens, while 49% of the genes with decreased expression in the Sip1 knockout lenses are normally upregulated during early lens development. Overall, these data imply that Sip1 plays a major role in reprogramming the lens vesicle away from a surface ectoderm cell fate towards that necessary for the development of a transparent lens and demonstrate that Sip1 regulates distinctly different sets of genes in different cellular contexts.

PMID: 24161570 [PubMed - indexed for MEDLINE]

Related Articles

Loss of Sip1 leads to migration defects and retention of ectodermal markers during lens development.

Mech Dev. 2014 Feb;131:86-110

Authors: Manthey AL, Lachke SA, FitzGerald PG, Mason RW, Scheiblin DA, McDonald JH, Duncan MK

2013

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Electron paramagnetic resonance analysis of the vimentin tail domain reveals points of order in a largely disordered region and conformational adaptation upon filament assembly.

Protein Sci. 2013 Jan;22(1):47-55

Authors: Hess JF, Budamagunta MS, Aziz A, FitzGerald PG, Voss JC

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Electron paramagnetic resonance analysis of the vimentin tail domain reveals points of order in a largely disordered region and conformational adaptation upon filament assembly.

Protein Sci. 2013 Jan;22(1):47-55

Authors: Hess JF, Budamagunta MS, Aziz A, FitzGerald PG, Voss JC

Abstract
Very little data have been reported that describe the structure of the tail domain of any cytoplasmic intermediate filament (IF) protein. We report here the results of studies using site directed spin labeling and electron paramagnetic resonance (SDSL-EPR) to explore the structure and dynamics of the tail domain of human vimentin in tetramers (protofilaments) and filaments. The data demonstrate that in contrast to the vimentin head and rod domains, the tail domains are not closely apposed in protofilaments. However, upon assembly into intact IFs, several sites, including positions 445, 446, 451, and 452, the conserved "beta-site," become closely apposed, indicating dynamic changes in tail domain structure that accompany filament elongation. No evidence is seen for coiled-coil structure within the region studied, in either protofilaments or assembled filaments. EPR analysis also establishes that more than half of the tail domain is very flexible in both the assembly intermediate and the intact IF. However, by positioning the spin label at distinct sites, EPR is able to identify both the rod proximal region and sites flanking the beta-site motif as rigid locations within the tail. The rod proximal region is well assembled at the tetramer stage with only slight changes occurring during filament elongation. In contrast, at the beta site, the polypeptide backbone transitions from flexible in the assembly intermediate to much more rigid in the intact IF. These data support a model in which the distal tail domain structure undergoes significant conformational change during filament elongation and final assembly.

PMID: 23109052 [PubMed - indexed for MEDLINE]

Related Articles

Electron paramagnetic resonance analysis of the vimentin tail domain reveals points of order in a largely disordered region and conformational adaptation upon filament assembly.

Protein Sci. 2013 Jan;22(1):47-55

Authors: Hess JF, Budamagunta MS, Aziz A, FitzGerald PG, Voss JC

Related Articles

Carbon turnover in the water-soluble protein of the adult human lens.

Mol Vis. 2013;19:463-75

Authors: Stewart DN, Lango J, Nambiar KP, Falso MJ, FitzGerald PG, Rocke DM, Hammock BD, Buchholz BA

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Carbon turnover in the water-soluble protein of the adult human lens.

Mol Vis. 2013;19:463-75

Authors: Stewart DN, Lango J, Nambiar KP, Falso MJ, FitzGerald PG, Rocke DM, Hammock BD, Buchholz BA

Abstract
PURPOSE: Human eye lenses contain cells that persist from embryonic development. These unique, highly specialized fiber cells located at the core (nucleus) of the lens undergo pseudo-apoptosis to become devoid of cell nuclei and most organelles. Ostensibly lacking in protein transcriptional capabilities, it is currently believed that these nuclear fiber cells owe their extreme longevity to the perseverance of highly stable and densely packed crystallin proteins. Maintaining the structural and functional integrity of lenticular proteins is necessary to sustain cellular transparency and proper vision, yet the means by which the lens actually copes with a lifetime of oxidative stress, seemingly without any capacity for protein turnover and repair, is not completely understood. Although many years of research have been predicated upon the assumption that there is no protein turnover or renewal in nuclear fiber cells, we investigated whether or not different protein fractions possess protein of different ages by using the (14)C bomb pulse.
METHODS: Adult human lenses were concentrically dissected by gently removing the cell layers in water or shaving to the nucleus with a curved micrometer-controlled blade. The cells were lysed, and the proteins were separated into water-soluble and water-insoluble fractions. The small molecules were removed using 3 kDa spin filters. The (14)C/C was measured in paired protein fractions by accelerator mass spectrometry, and an average age for the material within the sample was assigned using the (14)C bomb pulse.
RESULTS: The water-insoluble fractions possessed (14)C/C ratios consistent with the age of the cells. In all cases, the water-soluble fractions contained carbon that was younger than the paired water-insoluble fraction.
CONCLUSIONS: As the first direct evidence of carbon turnover in protein from adult human nuclear fiber cells, this discovery supports the emerging view of the lens nucleus as a dynamic system capable of maintaining homeostasis in part due to intricate protein transport mechanisms and possibly protein repair. This finding implies that the lens plays an active role in the aversion of age-related nuclear (ARN) cataract.

PMID: 23441119 [PubMed - indexed for MEDLINE]

Related Articles

Carbon turnover in the water-soluble protein of the adult human lens.

Mol Vis. 2013;19:463-75

Authors: Stewart DN, Lango J, Nambiar KP, Falso MJ, FitzGerald PG, Rocke DM, Hammock BD, Buchholz BA

2012

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The structure of vimentin linker 1 and rod 1B domains characterized by site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) and X-ray crystallography.

J Biol Chem. 2012 Aug 17;287(34):28349-61

Authors: Aziz A, Hess JF, Budamagunta MS, Voss JC, Kuzin AP, Huang YJ, Xiao R, Montelione GT, FitzGerald PG, Hunt JF

Related Articles

The structure of vimentin linker 1 and rod 1B domains characterized by site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) and X-ray crystallography.

J Biol Chem. 2012 Aug 17;287(34):28349-61

Authors: Aziz A, Hess JF, Budamagunta MS, Voss JC, Kuzin AP, Huang YJ, Xiao R, Montelione GT, FitzGerald PG, Hunt JF

Abstract
Despite the passage of ∼30 years since the complete primary sequence of the intermediate filament (IF) protein vimentin was reported, the structure remains unknown for both an individual protomer and the assembled filament. In this report, we present data describing the structure of vimentin linker 1 (L1) and rod 1B. Electron paramagnetic resonance spectra collected from samples bearing site-directed spin labels demonstrate that L1 is not a flexible segment between coiled-coils (CCs) but instead forms a rigid, tightly packed structure. An x-ray crystal structure of a construct containing L1 and rod 1B shows that it forms a tetramer comprising two equivalent parallel CC dimers that interact with one another in the form of a symmetrical anti-parallel dimer. Remarkably, the parallel CC dimers are themselves asymmetrical, which enables them to tetramerize rather than undergoing higher order oligomerization. This functionally vital asymmetry in the CC structure, encoded in the primary sequence of rod 1B, provides a striking example of evolutionary exploitation of the structural plasticity of proteins. EPR and crystallographic data consistently suggest that a very short region within L1 represents a minor local distortion in what is likely to be a continuous CC from the end of rod 1A through the entirety of rod 1B. The concordance of this structural model with previously published cross-linking and spectral data supports the conclusion that the crystallographic oligomer represents a native biological structure.

PMID: 22740688 [PubMed - indexed for MEDLINE]

Related Articles

The structure of vimentin linker 1 and rod 1B domains characterized by site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) and X-ray crystallography.

J Biol Chem. 2012 Aug 17;287(34):28349-61

Authors: Aziz A, Hess JF, Budamagunta MS, Voss JC, Kuzin AP, Huang YJ, Xiao R, Montelione GT, FitzGerald PG, Hunt JF

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Long-term effects of intravitreal injection of GMP-grade bone-marrow-derived CD34+ cells in NOD-SCID mice with acute ischemia-reperfusion injury.

Invest Ophthalmol Vis Sci. 2012 Feb;53(2):986-94

Authors: Park SS, Caballero S, Bauer G, Shibata B, Roth A, Fitzgerald PG, Forward KI, Zhou P, McGee J, Telander DG, Grant MB, Nolta JA

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Long-term effects of intravitreal injection of GMP-grade bone-marrow-derived CD34+ cells in NOD-SCID mice with acute ischemia-reperfusion injury.

Invest Ophthalmol Vis Sci. 2012 Feb;53(2):986-94

Authors: Park SS, Caballero S, Bauer G, Shibata B, Roth A, Fitzgerald PG, Forward KI, Zhou P, McGee J, Telander DG, Grant MB, Nolta JA

Abstract
PURPOSE: To determine long-term safety of intravitreal administration of good manufacturing practice (GMP)-grade human bone-marrow-derived CD34(+) cells in NOD-SCID (nonobese diabetic-severe combined immunodeficiency) mice with acute retinal ischemia-reperfusion injury, a model for retinal vasculopathy.
METHOD: Acute ischemia-reperfusion injury was induced in the right eye of adult NOD-SCID mice (n = 23) by transient elevation of intraocular pressure. Seven days later, 12 injured eyes and 5 normal contralateral eyes were injected each intravitreally with 5 × 10(4) CD34(+) cells isolated under GMP conditions from a healthy human donor bone marrow using an immunomagnetic cell isolation system. The remaining 11 injured eyes were not treated and served as controls. Mice were euthanized 1 day, 4 months, and 8 months later. Both eyes were enucleated and examined by immunohistochemical analysis and hematoxylin and eosin staining. Among mice followed for 8 months, electroretinography (ERG) was performed on both eyes before euthanization. All major organs were examined grossly and histologically after serial sectioning.
RESULTS: Immunohistochemical staining 4 months after injection showed detectable CD34(+) cells in the retinal vasculature. ERG at 8 months after CD34(+) cell injection showed signals that were similar in untreated eyes. Histology of the enucleated eyes injected with CD34(+) cells showed no intraocular tumor or abnormal tissue growth after 8 months. Histologic analysis of all major organs showed no abnormal proliferation of human cells.
CONCLUSIONS: Intravitreal administration of GMP-grade human bone-marrow-derived CD34(+) cells appears to be well tolerated long-term in eyes with acute retinal ischemic injury. A clinical trial will start to further explore this therapy.

PMID: 22247454 [PubMed - indexed for MEDLINE]

Related Articles

Long-term effects of intravitreal injection of GMP-grade bone-marrow-derived CD34+ cells in NOD-SCID mice with acute ischemia-reperfusion injury.

Invest Ophthalmol Vis Sci. 2012 Feb;53(2):986-94

Authors: Park SS, Caballero S, Bauer G, Shibata B, Roth A, Fitzgerald PG, Forward KI, Zhou P, McGee J, Telander DG, Grant MB, Nolta JA

2010

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Site-directed spin labeling and electron paramagnetic resonance determination of vimentin head domain structure.

J Biol Chem. 2010 May 14;285(20):15278-85

Authors: Aziz A, Hess JF, Budamagunta MS, Voss JC, Fitzgerald PG

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Site-directed spin labeling and electron paramagnetic resonance determination of vimentin head domain structure.

J Biol Chem. 2010 May 14;285(20):15278-85

Authors: Aziz A, Hess JF, Budamagunta MS, Voss JC, Fitzgerald PG

Abstract
Intermediate filament (IF) proteins have been predicted to have a conserved tripartite domain structure consisting of a largely alpha-helical central rod domain, flanked by head and tail domains. However, crystal structures have not been reported for any IF or IF protein. Although progress has been made in determining central rod domain structure, no structural data have been reported for either the head or tail domains. We used site-directed spin labeling and electron paramagnetic resonance to analyze 45 different spin labeled mutants spanning the head domain of vimentin. The data, combined with results from a previous study, provide strong evidence that the polypeptide backbones of the head domains form a symmetric dimer of closely apposed backbones that fold back onto the rod domain, imparting an asymmetry to the dimer. By following the behavior of spin labels during the process of in vitro assembly, we show that head domain structure is dynamic, changing as a result of filament assembly. Finally, because the vimentin head domain is the major site of the phosphorylation that induces disassembly at mitosis, we studied the effects of phosphorylation on head domain structure and demonstrate that phosphorylation drives specific head domain regions apart. These data provide the first evidence-based model of IF head domain structure.

PMID: 20231271 [PubMed - indexed for MEDLINE]

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Authors: Aziz A, Hess JF, Budamagunta MS, Voss JC, Fitzgerald PG