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Periplakin Interactions with Lens Intermediate and Beaded Filaments
Abstract
The lens assembles two systems of intermediated filaments-vimentin intermediate filament (IF) and highly divergent, lens-specific beaded filament (BF)-sequentially as epithelial cells differentiate into fiber cells. The goal of this study was to identify linker proteins that integrate the different lens IF into the biology of the lens fiber cells.
Antibodies to periplakin were used in coimmunoprecipitation studies to identify proteins that complex with BF and IF in detergent extracts of mouse lens. GST-periplakin fusion proteins were used to confirm coimmunoprecipitation
Yeast two-hybrid analysis was used to establish direct linkage between periplakin and BF/IF proteins and to narrow down binding domains. Immunocytochemistry was used to establish spatial and temporal coexpression of periplakin and BF/IF. results. Periplakin is found complexed to BF and IF in the lens. The COOH terminus of periplakin was shown to have a strong affinity for the CP49 rod 2 domain but not its head or rod 1 domains. Low-level affinity was seen between the filensin rod domain and periplakin. Periplakin localization in lens overlapped with BF and IF.
Despite divergence in primary sequence, predicted secondary structure, and filament structure, CP49 has conserved the capacity to bind a common IF linker protein, periplakin, and shares that binding capacity with the other major lens IF protein, vimentin. This suggests that mutations in periplakin have the potential to emulate the cataract seen in lenses with defective BF proteins.
... 36 Periplakin, an IF-associated protein (IFAP), has been shown to bind both IFs and beaded filaments, directly interacting with vimentin and phakinin. 37 Periplakin and periaxin are also part of the EPPD (ezrin, periaxin, periplakin, desmoyokin) junction complex in lens. 38 Although their roles are not well understood, both periaxin and periplakin could anchor IFs to the plasma membrane through protein-protein interactions. ...
... AQP0 has been demonstrated to interact with beaded filaments, but AQP0 did not pull down with periplakin-BF complexes in previous studies, suggesting there may be another protein anchor connecting periplakin and BF to the plasma membrane. 37 Brain acid soluble protein 1 has been detected in lens but little is known about its function in this tissue. 39 In the brain, the growth-associated protein BASP1 is localized to the plasma membrane at the tip of elongating axons. ...
Purpose:
To quantify protein changes in the morphologically distinct remodeling zone (RZ) and adjacent regions of the human lens outer cortex using spatially directed quantitative proteomics.
Methods:
Lightly fixed human lens sections were deparaffinized and membranes labeled with fluorescent wheat germ agglutinin (WGA-TRITC). Morphology directed laser capture microdissection (LCM) was used to isolate tissue from four distinct regions of human lens outer cortex: differentiating zone (DF), RZ, transition zone (TZ), and inner cortex (IC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the plasma membrane fraction from three lenses (21-, 22-, and 27-year) revealed changes in major cytoskeletal proteins including vimentin, filensin, and phakinin. Peptides from proteins of interest were quantified using multiple reaction monitoring (MRM) mass spectrometry and isotopically-labeled internal peptide standards.
Results:
Results revealed an intermediate filament switch from vimentin to beaded filament proteins filensin and phakinin that occurred at the RZ. Several other cytoskeletal proteins showed significant changes between regions, while most crystallins remained unchanged. Targeted proteomics provided accurate, absolute quantification of these proteins and confirmed vimentin, periplakin, and periaxin decrease from the DF to the IC, while filensin, phakinin, and brain acid soluble protein 1 (BASP1) increase significantly at the RZ.
Conclusions:
Mass spectrometry-compatible fixation and morphology directed laser capture enabled proteomic analysis of narrow regions in the human lens outer cortex. Results reveal dramatic cytoskeletal protein changes associated with the RZ, suggesting that one role of these proteins is in membrane deformation and/or the establishment of ball and socket joints in the human RZ.
... Substantial accumulations of CP49 and filensin filaments in fiber cells under low temperatures might increase light scattering to lead to the formation of cold cataract, suggesting the involvement of cytoskeleton and other related proteins such as alpha-crystallin. Several proteins, including aquaporin 0 and periplakin, have been reported to be potential linkers between intermediate filaments and plasma membrane or cytoskeleton structures [18][19][20][21]. The distribution of lens intermediate filament proteins filensin and CP49 is highly regulated in different regions of inner fiber cells [10,22]; posttranslational modifications of filensin and CP49 proteins also occur in the transition zone, which is similar to the location in zone II ( Figure 5A) of inner fibers [23]. ...
Purpose:
To investigate the molecular and cellular mechanisms of cataract induced by cold temperatures in young lenses of wild-type C57BL/6J (B6), wild-type 129SvJae (129), and filensin knockout (KO) mice. To determine how lens intermediate filament proteins, filensin (BFSP1) and CP49 (BFSP2), are involved in the formation of cold cataract.
Methods:
The formation of cold cataract was examined in enucleated lenses at different temperatures and was imaged under a dissecting microscope. Lens vibratome sections were prepared, immunostained with different antibodies and fluorescent probes, and then imaged with a laser confocal microscope to evaluate the protein distribution and the membrane and cytoskeleton structures in the lens fibers.
Results:
Postnatal day 14 (P14) wild-type B6 lenses showed cataracts dependent on cold temperatures in interior fibers about 420-875 µm (zone III) and 245-875 µm (zone II and zone III) from the lens surface, under 25 °C and 4 °C, respectively. In contrast, wild-type 129 (with CP49 gene deletion) and filensin KO (on the B6 background) lenses did not have cold cataracts at 25 °C but displayed a reduced cold cataract, especially in zone III, at 4 °C. Immunofluorescent staining data revealed that CP49 and filensin proteins were uniformly distributed in fiber cell cytosols without cold cataracts but accumulated or aggregated in the cell boundaries of the fibers where cold cataracts appeared.
Conclusions:
CP49 and filensin are important components for the formation of cold cataract in young B6 mouse lenses. Accumulated or aggregated CP49 and filensin beaded intermediate filaments in fiber cell boundaries might directly or indirectly contribute to the light scattering of cold cataract. Cold cataract in zone II is independent of beaded intermediate filaments. CP49 and filensin intermediate filaments and other lens proteins probably form distinct high molecular organizations to regulate lens transparency in interior fibers.
... Interestingly, the small C-terminal linker domain is sufficient for specific interactions with intermediate filament (IF) proteins K8, K18, and vimentin (Kazerounian et al., 2002). The linker domain served as a hub for the unique interactions of PPL with lens fiber cell-specific IF proteins CP49 and filensin (Yoon and FitzGerald, 2009). Although PPL dependent K8 reorganization was demonstrated to be critical for maintenance of epithelial integrity in migrating cells, its downregulation perturbed keratin re-arrangement and cable formation resulting in impaired wound closure (Long et al., 2006). ...
The architecture of the cytoskeleton and its remodeling are tightly regulated by dynamic reorganization of keratin-rich intermediate filaments. Plakin family proteins associate with the network of intermediate filaments (IFs) and affect its reorganization during migration, differentiation, and response to stress. The smallest plakin, periplakin (PPL), interacts specifically with intermediate filament proteins K8, K18, and vimentin via its C-terminal linker domain. Here, we show that periplakin is SUMOylated at a conserved lysine in its linker domain (K1646) preferentially by small ubiquitin-like modifier 1 (SUMO1). Our data indicate that PPL SUMOylation is essential for the proper reorganization of the keratin IF network. Stresses perturbing intermediate-filament and cytoskeletal architecture induce hyper--SUMOylation of periplakin. Okadaic acid induced hyperphosphorylation-dependent collapse of the keratin IF network results in a similar hyper-SUMOylation of PPL. Strikingly, exogenous overexpression of a non-SUMOylatable periplakin mutant (K1646R) induced aberrant bundling and loose network interconnections of the keratin filaments. Time-lapse imaging of cells expressing the K1646R mutant showed the enhanced sensitivity of keratin filament collapse upon okadaic acid treatment. Our data identify an important regulatory role for periplakin SUMOylation in dynamic reorganization and stability of keratin IFs.
... The expression of mutant forms of both proteins is correlated with the development of cataracts, implicating a role for these proteins in lens cell and tissue organization. In support of this, in fiber cells, both vimentin and beaded filaments terminate at junctional complexes established by N-cadherin, cadherin-11, Pg, periplakin, and plectin (Leonard et al. 2008;Yoon and FitzGerald 2009). Intriguingly, these cadherin-IF complexes congregate along the short edge of the hexagonal cells, whereas, along the long edge, beaded filaments feed into desmoyokin (AHNAK)/ periplakin-based adhesions (Straub et al. 2003). ...
Avariety of intermediate filament (IF) types show intricate association with plasma membrane proteins, including receptors and adhesion molecules. The molecular basis of linkage of IFs to desmosomes at sites of cell-cell interaction and hemidesmosomes at sites of cell-matrix adhesion has been elucidated and involves IF-associated proteins. However, IFs also interact with focal adhesions and cell-surface molecules, including dystroglycan. Through such membrane interactions, it is well accepted that IFs play important roles in the establishment and maintenance of tissue integrity. However, by organizing cell-surface complexes, IFs likely regulate, albeit indirectly, signaling pathways that are key to tissue homeostasis and repair. © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.
... The following methodology was utilized in this study. Eyes were removed from 8 to 9 week C57 mice, frozen in dry ice-cooled propane, placed in 97% methanol and 3% glacial acetic acid at À80 for 48 h before freeze substitution, immersed in 100% ethanol, embedded in paraffin and sectioned at 4 mm (Yoon and FitzGerald, 2009). Sections of the adult mouse eye were examined for the expression of G8 protein and Noggin mRNA or MyoD mRNA as described previously (Gerhart et al., 2001(Gerhart et al., , 2000(Gerhart et al., , 2006. ...
Myo/Nog cells are essential for eye development in the chick embryo and respond to injury in adult tissues. These cells express mRNA for the skeletal muscle specific transcription factor MyoD, the bone morphogenetic protein (BMP) inhibitor Noggin and the cell surface protein recognized by the G8 monoclonal antibody (mAb). In this study, we determined that Myo/Nog cells are present in low numbers in the retina of the mouse eye. G8-positive Myo/Nog cells were distinguished from neuronal, Muller and microglial cells that were identified with antibodies to calretinin, Chx10, glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1, respectively. In the neonatal retina, the number of Myo/Nog cells increased in parallel with cell death induced by transient exposure to hyperoxia. In this model of retinopathy of prematurity, depletion of Myo/Nog cells by intravitreal injection of the G8 mAb and complement increased cell death. These findings demonstrate that Myo/Nog cells are a distinct population of cells, not previously described in the retina, which increases in response to retinal damage and mitigate hypoxia-induced cell death.
... Antibodies: To assess the impact of freeze substitution on the preservation of immunoreactivity, six different polyclonal antibody preparations were used to compare the freeze substitution routines to formalin immersion fixation. Antibodies included: affinity-purified rabbit anti-vimentin (vim) [5], affinity-purified rabbit anti-periplakin (Ppl) [6], affinitypurified anti-CP49 (CP49) [5], rabbit anti p57 Kip 2 (p57 Kip2; Abcam Cat# ab4058, gift from Nadean Brown, UC Davis, Davis, CA), rabbit anti-gamma-tubulin (gift of Ken Beck, UC Davis), and chicken anti-plectin prepared in-house from recombinant plectin fragment (plectin). Sections were de-paraffinized, blocked with tris-buffered-saline with 0.1% Tween-20 (TBS-T), and 5% powdered milk. ...
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.
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.
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.
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.
... Since the subretinal MG/MΦ are localized precisely where artifactual retinal detachments occur during regular specimen preparation for retinal sections, we used a quickfreezing of eyeballs followed by a freeze-substitution technique. This method has been shown to be effective in 1) preventing artifactual retinal detachment, 2) minimizing ice crystal formation, 3) preserving cellular morphology, and 4) preserving antigenicity [30][31][32][33][34]. ...
Background
Microglia/macrophages (MG/M¿) are found in the subretinal space in both mice and humans. Our goal was to study the spatial and temporal distribution, the phenotype, and gene expression of subretinal MG/M¿ in mice with normal retinas and compare them to mice with known retinal pathology.Methods
We studied C57BL/6 mice with (C57BL/6N), or without (C57BL/6J) the rd8 mutation in the Crb1 gene (which, in the presence of yet unidentified permissive/modifying genes, leads to a retinal degeneration), and documented their fundus appearance and the change with aging. Immunostaining of retinal pigment epithelium (RPE) flat mounts was done for 1) Ionized calcium binding adaptor (Iba)-1, 2) Fc¿III/II Receptor (CD16/CD32, abbreviated as CD16), and 3) Macrophage mannose receptor (MMR). Reverse-transcription quantitative PCR (RT-qPCR) was done for genes involved in oxidative stress, complement activation and inflammation.ResultsThe number of yellow fundus spots correlated highly with subretinal Iba-1+ cells. The total number of subretinal MG/M¿ increased with age in the rd8 mutant mice, but not in the wild-type (WT) mice. There was a centripetal shift in the distribution of the subretinal MG/M¿ with age. Old rd8 mutant mice had a greater number of CD16+ MG/M¿. CD16+ cells had morphological signs of activation, and this was most prominent in old rd8 mutant mice (P <1x10¿8 versus old WT mice). Subretinal MG/M¿ in rd8 mutant mice also expressed iNOS and MHC-II, and had ultrastructural signs of activation. Finally, rd8 mutant mouse RPE/ MG/M¿ RNA isolates showed an upregulation of Ccl2, CFB, C3, NF-kß, CD200R and TNF-alpha. The retinas of rd8 mutant mice showed upregulation of HO-1, C1q, C4, and Nrf-2.Conclusions
When compared to C57BL/6J mice, C57BL/6N mice demonstrate increased accumulation of subretinal MG/M¿, displaying phenotypical, morphological, and gene-expression characteristics consistent with a pro-inflammatory shift. These changes become more prominent with aging and are likely due to the combination of the rd8 mutation and yet unidentified permissive/modulatory genes in the C57BL/6N mice. In contrast, aging leads to a scavenging phenotype in the C57BL/6J subretinal microglia/macrophages.
... Retinas were fixed by freeze substitution as described in Yoon and FitzGerald, 2009. Briefly, methanol-acetic acid fixative (97% methanol and 3% acetic acid) and propane were chilled to dry ice temperature in advance. ...
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 (OCT) 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.
... Thus, PPL has EVPL-independent functions in other tissues, including the liver. Indeed, many in vitro studies have revealed that PPL may modulate the intracellular cytoskeletal network through interaction with a number of molecules other than EVPL, including vimentin, keratin 8 (K8), beaded filament structural component 2 (also known as cytoskeletal protein 49 kDa), filensin, plectin, and collagen type XVII (also known as bullous pemphigoid antigen 2) [14,15,18,19]. PPL has also been shown to inhibit intracellular signal transduction through interaction with family A (rhodopsin-like) G-protein-coupled receptors [20][21][22]. ...
Periplakin (PPL) is a rod-shaped cytolinker protein thought to connect cellular adhesion junctional complexes to cytoskeletal filaments. PPL serves as a structural component of the cornified envelope in the skin and interacts with various types of proteins in cultured cells; its level decreases dramatically during tumorigenic progression in human epithelial tissues. Despite these intriguing observations, the physiological roles of PPL, especially in non-cutaneous tissues, are still largely unknown. Because we observed a marked fluctuation of PPL expression in mouse liver in association with the bile acid receptor farnesoid X receptor (FXR) and cholestasis, we sought to characterize the role of PPL in the liver and determine its contributions to the etiology and pathogenesis of cholestasis.
Time- and context-dependent expression of PPL in various mouse models of hepatic and renal disorders were examined by immunohistochemistry, western blotting, and quantitative real-time polymerase chain reactions.
The hepatic expression of PPL was significantly decreased in Fxr-/- mice. In contrast, the expression was dramatically increased during cholestasis, with massive PPL accumulation observed at the boundaries of hepatocytes in wild-type mice. Interestingly, the hepatic accumulation of PPL resulting from cholestasis was reversible. In addition, similar accumulation of PPL at cellular boundaries was found in epithelial cells around renal tubules upon ureteral obstruction.
PPL may be involved in the temporal accommodation to fluid stasis in different tissues. Further examination of the roles for PPL may lead to the discovery of a novel mechanism for cellular protection by cytolinkers that is applicable to many tissues and in many contexts.
Abstract Background Methylation at cytosines (mCG) is a well-known regulator of gene expression, but its requirements for cellular differentiation have yet to be fully elucidated. A well-studied cellular differentiation model system is the eye lens, consisting of a single anterior layer of epithelial cells that migrate laterally and differentiate into a core of fiber cells. Here, we explore the genome-wide relationships between mCG methylation, chromatin accessibility and gene expression during differentiation of eye lens epithelial cells into fiber cells. Results Whole genome bisulfite sequencing identified 7621 genomic loci exhibiting significant differences in mCG levels between lens epithelial and fiber cells. Changes in mCG levels were inversely correlated with the differentiation state-specific expression of 1285 genes preferentially expressed in either lens fiber or lens epithelial cells (Pearson correlation r = − 0.37, p
In order to study the mechanisms involved in the development of posterior capsule opacification (PCO) we compared in vivo developed PCO with PCO formed in tissue culture with focus on the periphery of the lens capsule to evaluate lens regeneration potential. We studied 3 human tissue groups: Cultured lens capsules after mock cataract surgery (n = 6, 30 days), lens capsules from donors that had previously undergone cataract surgery (IOL capsules) (n = 12) and intact lenses (n = 6). All samples were stained with Vimentin, alpha Smooth Muscle Actin, Picro Sirius Red (for collagen) and Paired box protein (Pax6). We found that cultured capsules and less developed IOL capsules consisted mainly of monolayers of mesenchymal cells, while more developed IOL capsules, contained lens epithelial cells (LECs), globular cells and lens fiber cells. Many IOL capsule samples expressed collagen I and III in areas where cells were in contact with the IOL. Pax6 had a similar dispersed distribution in less developed IOL capsules and cultured capsules, while more developed IOL capsules and intact lenses, concentrated Pax6 in LECs at the equatorial lens bow. The similarities between cultured capsules and less developed IOL capsules indicate that our in vitro developed PCO is comparable to early in vivo developed PCO. The similar morphology of more developed IOL capsules and intact lenses seems to indicate an attempt at lens regeneration.
Envoplakin and periplakin are the two smallest plakin family cytoskeletal linker proteins that connect intermediate filaments to cellular junctions and other membrane locations. These two plakins have a structural role in the assembly of the cornified envelope (CE), the terminal stage of epidermal differentiation. Analysis of gene-targeted mice lacking both these plakins and the third initial CE scaffold protein, involucrin, demonstrate the importance of the structural integrity of CE for a proper epidermal barrier function. It has emerged that periplakin, which also has a wider tissue distribution than envoplakin, has additional, independent roles. Periplakin participates in the cytoskeletal organization also in other tissues and interacts with a wide range of membrane-associated proteins such as kazrin and butyrophilin BTN3A1. This review covers methods used to understand periplakin and envoplakin functions in cell culture models, including siRNA ablation of periplakin expression and the use of tagged protein domain constructs to study localization and interactions. In addition, assays that can be used to analyze CEs and epidermal barrier function in gene-targeted mice are described and discussed.
To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina.
Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC.
Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh(-/-) eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh(-/-) mice. Greatly reduced Cfh protein immunohistological signals in the Cfh(-/-) eyes also supported the specificity of the Cfh protein distribution results.
Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC.
Transparency of the ocular lens depends on symmetric packing and membrane organization of highly elongated hexagonal fiber cells. These cells possess an extensive, well-ordered cortical cytoskeleton to maintain cell shape and to anchor membrane components. Periaxin (Prx), a PDZ domain protein involved in myelin sheath stabilization, is also a component of adhaerens plaques in lens fiber cells. Here we show that Prx is expressed in lens fibers and exhibits maturation dependent redistribution, clustering discretely at the tricellular junctions in mature fiber cells. Prx exists in a macromolecular complex with proteins involved in membrane organization including ankyrin-B, spectrin, NrCAM, filensin, ezrin and desmoyokin. Importantly, Prx knockout mouse lenses were found to be softer and more easily deformed than normal lenses, revealing disruptions in fiber cell hexagonal packing, membrane skeleton and membrane stability. These observations suggest a key role for Prx in maturation, packing, and membrane organization of lens fiber cells. Hence, there may be functional parallels between the roles of Prx in membrane stabilization of the myelin sheath and the lens fiber cell.
The Arabidopsis thaliana genome encodes about 386 proteins with coiled-coil domains of at least 50 amino acids in length. In mammalian systems, many coiled-coil proteins are part of various cytoskeletal networks including intermediate filament protein, actin-binding proteins and MAP (microtubule-associated proteins). Immunological evidence suggests that some of these cytoskeletal proteins, such as lamins, keratins and tropomyosins, may be conserved in Arabidopsis. However, coiled-coil proteins are of low complexity, and thus, traditional sequence comparison algorithms, such as BLAST may not detect homologies. Here, we use the PROPSEARCH algorithm to detect putative coiled-coil cytoskeletal protein homologues in Arabidopsis. This approach reveals putative intermediate filament protein homologues of filensin, lamin and keratin; putative actin-binding homologues of ERM (ezrin/radixin/moesin), periplakin, utrophin, tropomyosin and paramyosin, and putative MAP homologues of restin/CLIP-170 (cytoplasmic linker protein-170). We suggest that the AtFPP (Arabiopsis thaliana filament-like plant protein) and AtMAP70 (Arabidopsis microtubule-associated protein 70) families of coiled-coil proteins may, in fact, be related to lamins and function as intermediate filament proteins.
Intermediate filaments (IFs) are a key component of the cytoskeleton in virtually all vertebrate cells, including those of the lens of the eye. IFs help integrate individual cells into their respective tissues. This Review focuses on the lens-specific IF proteins beaded filament structural proteins 1 and 2 (BFSP1 and BFSP2) and their role in lens physiology and disease. Evidence generated in studies in both mice and humans suggests a critical role for these proteins and their filamentous polymers in establishing the optical properties of the eye lens and in maintaining its transparency. For instance, mutations in both BFSP1 and BFSP2 cause cataract in humans. We also explore the potential role of BFSP1 and BFSP2 in aging processes in the lens.
A null mutation was introduced into the mouse desmin gene by homologous recombination. The desmin knockout mice (Des −/−)
develop normally and are fertile. However, defects were observed after birth in skeletal, smooth, and cardiac muscles (Li,
Z., E. Colucci-Guyon, M. Pincon-Raymond, M. Mericskay, S. Pournin, D. Paulin, and C. Babinet. 1996. Dev. Biol. 175:362–366; Milner, D.J., G. Weitzer, D. Tran, A. Bradley, and Y. Capetanaki. 1996. J. Cell Biol. 134:1255– 1270). In the present study we have carried out a detailed analysis of somitogenesis, muscle formation, maturation,
degeneration, and regeneration in Des −/− mice. Our results demonstrate that all early stages of muscle differentiation
and cell fusion occur normally. However, after birth, modifications were observed essentially in weight-bearing muscles such
as the soleus or continually used muscles such as the diaphragm and the heart. In the absence of desmin, mice were weaker
and fatigued more easily. The lack of desmin renders these fibers more susceptible to damage during contraction. We observed
a process of degeneration of myofibers, accompanied by macrophage infiltration, and followed by a process of regeneration.
These cycles of degeneration and regeneration resulted in a relative increase in slow myosin heavy chain (MHC) and decrease
in fast MHC. Interestingly, this second wave of myofibrillogenesis during regeneration was often aberrant and showed signs
of disorganization. Subsarcolemmal accumulation of mitochondria were also observed in these muscles. The lack of desmin was
not compensated by an upregulation of vimentin in these mice either during development or regeneration. Absence of desmin
filaments within the sarcomere does not interfere with primary muscle formation or regeneration. However, myofibrillogenesis
in regenerating fibers is often abortive, indicating that desmin may be implicated in this repair process. The results presented
here show that desmin is essential to maintain the structural integrity of highly solicited skeletal muscle.
Specific interactions between desmoplakins I and II (DP I and II) and other desmosomal or cytoskeletal molecules have been difficult to determine in part because of the complexity and insolubility of the desmosome and its constituents. We have used a molecular genetic approach to investigate the role that DP I and II may play in the association of the desmosomal plaque with cytoplasmic intermediate filaments (IF). A series of mammalian expression vectors encoding specific predicted domains of DP I were transiently expressed in cultured cells that form (COS-7) and do not form (NIH-3T3) desmosomes. Sequence encoding a small antigenic peptide was added to the 3' end of each mutant DP cDNA to facilitate immunolocalization of mutant DP protein. Light and electron microscopical observations revealed that DP polypeptides including the 90-kD carboxy-terminal globular domain of DP I specifically colocalized with and ultimately resulted in the complete disruption of IF in both cell lines. This effect was specific for IF as microtubule and microfilament networks were unaltered. This effect was also specific for the carboxyl terminus of DP, as the expression of the 95-kD rod domain of DP I did not visibly alter IF networks. Immunogold localization of COS-7 cells transfected with constructs including the carboxyl terminus of DP demonstrated an accumulation of mutant protein in perinuclear aggregates within which IF subunits were sequestered. These results suggest a role for the DP carboxyl terminus in the attachment of IF to the desmosome in either a direct or indirect manner.
Desmoplakins (DPs) I and II are two major related proteins located in the innermost portion of the desmosomal plaque where it is thought they may play a role in attaching intermediate filaments (IF) to the cell surface. We have isolated and sequenced human cDNA clones encoding two major DP domains and a portion of a third. These clones can be divided into two classes that we believe to represent DPI and DPII cDNAs; our evidence suggests that the DPII message is derived at least in part from the processing of a larger transcript encoded by a single gene. Computer-aided analysis of the DPI-predicted amino acid sequence indicates that the central domain, which contains the heptad repeat characteristic of many alpha-fibrous proteins, will participate in the formation of a coiled coil dimer approximately 130 nm in length. The periodicity of acidic and basic residues in the rod suggests that DPI will aggregate with itself or similar molecules into higher order filamentous structures. The carboxyl terminus contains three regions with significant homology, each of which comprises almost five repeats of a 38-residue motif. It is likely that these regions each fold into a compact globular conformation stabilized by intrachain ionic interactions. Comparison of the predicted amino acid sequence of a cDNA encoding a portion of the 230-kDa bullous pemphigoid antigen (Stanley, J. R., Tanaka, T., Mueller, S., Klaus-Kovtun, V., and Roop, D. (1988) J. Clin. Invest. 82, 1864-1870) with DP revealed the presence of a 38-residue repeat with striking similarity to that of the DPs. Significantly, the periodicity in acidic and basic residues of these domains is the same as that found in the 1B rod domain of IF proteins. This suggests the possibility that the DPs might interact with IF via their common periodicity of charged residues.
In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution-binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.
To establish the fate of the Golgi apparatus and the endoplasmic reticulum (ER) during lens fiber differentiation.
Organelles were visualized by confocal or electron microscopy. For fluorescence microscopy, organelles were labeled with fluorescent probes or antibodies raised against organelle-resident proteins. The cytoplasmic volume was reconstructed from optical sections using volume rendering techniques.
The Golgi apparatus was located apically in epithelial cells. In the annular pad, Golgi elements were transformed into ribbon-like structures running parallel to the long axes of the cells. Toward the lens equator, the Golgi apparatus fragmented. In the lens fibers, the Golgi apparatus was detected only in the superficial cells. The ER was present as vesicular or tubular elements in both epithelial and cortical fiber cells, and ER probes co-labeled the nuclear membrane and revealed that the ER and nuclei disappeared coincidentally in the deep cortex. Using a lipophilic dye and volume rendering, the relationships between organelles could be evaluated in three dimensions.
The Golgi apparatus was not a prominent organelle in differentiating lens fibers. In contrast, the ER was more abundant and extended to the edge of the organelle-free region, where it was degraded along with the nuclei and mitochondria.
The cDNA coding for calf filensin, a membrane-associated protein of the lens fiber cells, has been cloned and sequenced. The predicted 755-amino acid-long open reading frame shows primary and secondary structure similarity to intermediate filament (IF) proteins. Filensin can be divided into an NH2-terminal domain (head) of 38 amino acids, a middle domain (rod) of 279 amino acids, and a COOH-terminal domain (tail) of 438 amino acids. The head domain contains a di-arginine/aromatic amino acid motif which is also found in the head domains of various intermediate filament proteins and includes a potential protein kinase A phosphorylation site. By multiple alignment to all known IF protein sequences, the filensin rod, which is the shortest among IF proteins, can be subdivided into three subdomains (coils 1a, 1b, and 2). A 29 amino acid truncation in the coil 2 region accounts for the smaller size of this domain. The filensin tail contains 6 1/2 tandem repeats which match analogous motifs of mammalian neurofilament M and H proteins. We suggest that filensin is a novel IF protein which does not conform to any of the previously described classes. Purified filensin fails to form regular filaments in vitro (Merdes, A., M. Brunkener, H. Horstmann, and S. D. Georgatos. 1991. J. Cell Biol. 115:397-410), probably due to the missing segment in the coil 2 region. Participation of filensin in a filamentous network in vivo may be facilitated by an assembly partner.
The fiber cell of the vertebrate ocular lens assembles a cytoskeletal structure, the beaded filament, which contains two proteins
unique to the fiber cell: CP49 (phakinin) and CP115/CP95 (filensin). We report here the complete primary sequence and gene
structure for human CP49. These data show that CP49 is a member of the intermediate filament family, but highly unusual in
several regards. 1) CP49 primary sequence does not permit unambiguous assignment to any existing class of intermediate filament
protein, but exhibits a gene structure that is identical to the Type I cytokeratins. 2) CP49 essentially lacks one of the
three major domains that characterize all intermediate filament proteins, the carboxyl-terminal tail domain. 3) CP49 shows
substitutions at 3 of 4 residues in the otherwise highly conserved intermediate filament protein motif LNDR. Notably, this
divergence includes an Arg to Cys substitution that has only been observed in the mutant human cytokeratin K14, a mutation
shown to cause the skin blistering seen in the genetic disorder Dowling-Meara epidermolysis bullosa simplex.
Plectin is a widely expressed high molecular weight protein that is involved in cytoskeleton-membrane attachment in epithelial cells, muscle, and other tissues. The human autosomal recessive disorder epidermolysis bullosa with muscular dystrophy (MD-EBS) shows epidermal blister formation at the level of the hemidesmosome and is associated with a myopathy of unknown etiology. Here, plectin was found to be absent in skin and cultured keratinocytes from an MD-EBS patient by immunofluorescence and immunoprecipitation, suggesting that plectin is a candidate gene/protein system for MD-EBS mutation. The 14800-bp human plectin cDNA was cloned and sequenced. The predicted 518-kD polypeptide has homology to the actin-binding domain of the dystrophin family at the amino terminus, a central rod domain, and homology to the intermediate filament-associated protein desmoplakin at the carboxyl terminus. The corresponding human gene (PLEC1), consisting of 33 exons spanning >26 kb of genomic DNA was cloned, sequenced, and mapped to chromosomal band 8q24. Homozygosity by descent was observed in the consanguineous MD-EBS family with intragenic plectin polymorphisms. Direct sequencing of PCR-amplified plectin cDNA from the patient's keratinocytes revealed a homozygous 8-bp deletion in exon 32 causing a frameshift and a premature termination codon 42 bp downstream. The clinically unaffected parents of the proband were found to be heterozygous carriers of the mutation. These results establish the molecular basis of MD-EBS in this family and clearly demonstrate the important structural role for plectin in cytoskeleton-membrane adherence in both skin and muscle.
By immunogold labeling, we demonstrate that "millipede-like" structures seen previously in mammalian cell cytoskeletons after removal of actin by treatment with gelsolin are composed of the cores of vimentin IFs with sidearms containing plectin. These plectin sidearms connect IFs to microtubules, the actin-based cytoskeleton and possibly membrane components. Plectin binding to microtubules was significantly increased in cells from transgenic mice lacking IFs and was reversed by microinjection of exogenous vimentin. These results suggest the existence of a pool of plectin which preferentially associates with IFs but may also be competed for by microtubules. The association of IFs with microtubules did not show a preference for Glu-tubulin. Nor did it depend upon the presence of MAP4 since plectin links were retained after specific immunodepletion of MAP4. The association of IFs with stress fibers survived actin depletion by gelsolin suggesting that myosin II minifilaments or components closely associated with them may play a role as plectin targets. Our results provide direct structural evidence for the hypothesis that plectin cross-links elements of the cytoskeleton thus leading to integration of the cytoplasm.
To determine the morphologic and biochemical events preceding the breakdown of fiber cell nuclei in the primate lens.
Monkey lens slices were labeled with fluorescent probes and optically sectioned using a confocal microscope. The distribution of nuclear histones was visualized by immunofluorescence. DNA and cellular membranes were imaged simultaneously by staining with SYTO 17 and 3,3'-dihexyloxacarbocyanine iodide, respectively. The condition of fiber cell DNA during differentiation was determined by an in situ DNA fragmentation assay. The assay was adapted to allow the detection of DNA fragments with 3'-OH or 3'-PO4 termini.
Monkey lens fiber nuclei passed through distinct stages before disintegrating. In the outer cell layers, the nuclei were large, smooth, and oval-shaped with prominent nucleoli. Deeper in the lens, they had a flattened profile with whorls of membranous material and nucleic acid accumulated at one end. At this point, histone immunofluorescence was reduced and the nucleoli had a characteristic, spoked appearance. At the border of the organelle-free zone, the intracellular membranes (including the nuclear envelope) disappeared, and particulate material was released from the nuclei into the cytoplasm. This material was stained by SYTO-17 and the DNA fragmentation assay, indicating that it contained fragmented DNA with 3'-OH termini.
The denucleation process in the primate lens differs from that described recently in the embryonic chicken lens. In particular, the extrusion of nuclear material and persistence of DNA-rich particles in the fiber cytoplasm are novel features. One similarity between the denucleation process in these species is the appearance of 3'-OH ends in the DNA after the loss of the nuclear membrane.
Desmoplakin (DP), plakoglobin (PG), and plakophilin 1 (PP1) are desmosomal components lacking a transmembrane domain, thus making them candidate linker proteins for connecting intermediate filaments and desmosomes. Using deletion and site-directed mutagenesis, we show that remarkably, removal of approximately 1% of DP's sequence obliterates its ability to associate with desmosomes. Conversely, when linked to a foreign protein, as few as 86 NH2-terminal DP residues are sufficient to target to desmosomes efficiently. In in vitro overlay assays, the DP head specifically associates with itself and with desmocollin 1a (Dsc1a). In similar overlay assays, PP1 binds to DP and Dsc1a, and to a lesser extent, desmoglein 1 (Dsg1), while PG binds to Dsg1 and more weakly to Dsc1a and DP. Interestingly, like DP, PG and PP1 associate with epidermal keratins, although PG is considerably weaker in its ability to do so. As judged by overlay assays, the amino terminal head domain of type II keratins appears to have a special importance in establishing these connections. Taken together, our findings provide new insights into the complexities of the links between desmosomes and intermediate filaments (IFs). Our results suggest a model whereby at desmosome sites within dividing epidermal cells, DP and PG anchor to desmosomal cadherins and to each other, forming an ordered array of nontransmembrane proteins that then bind to keratin IFs. As epidermal cells differentiate, PP1 is added as a molecular reinforcement to the plaque, enhancing anchorage to IFs and accounting at least partially for the increase in numbers and stability of desmosomes in suprabasal cells.
Envoplakin and periplakin are two plakins that are precursors of the epidermal cornified envelope. We studied their distribution and interactions by transfection of primary human keratinocytes and other cells. Full-length periplakin localized to desmosomes, the interdesmosomal plasma membrane and intermediate filaments. Full length envoplakin also localized to desmosomes, but mainly accumulated in nuclear and cytoplasmic aggregates with associated intermediate filaments. The envoplakin rod domain was required for aggregation and the periplakin rod domain was necessary and sufficient to redistribute envoplakin to desmosomes and the cytoskeleton, confirming earlier predictions that the proteins can heterodimerize. The linker domain of each protein was required for intermediate filament association. Like the NH(2) terminus of desmoplakin, that of periplakin localized to desmosomes; however, in addition, the periplakin NH(2) terminus accumulated at cell surface microvilli in association with cortical actin. Endogenous periplakin was redistributed from microvilli when keratinocytes were treated with the actin disrupting drug Latrunculin B. We propose that whereas envoplakin and periplakin can localize independently to desmosomes, the distribution of envoplakin at the interdesmosomal plasma membrane depends on heterodimerization with periplakin and that the NH(2) terminus of periplakin therefore plays a key role in forming the scaffold on which the cornified envelope is assembled.
Periplakin is a component of desmosomes and the epidermal cornified envelope. Its N-terminal domain interacts with the plasma membrane; it heterodimerises with envoplakin via its rod domain; and its C-terminus interacts with intermediate filaments. Periplakin has the shortest C-terminus of the plakin family, comprising only the linker domain found in all conventional plakins. By transient transfection of COS7 cells and primary human epidermal keratinocytes with deletion mutants of the periplakin C-terminus we mapped sequences required for intermediate filament interaction to two regions of the linker motif that are most highly conserved amongst the plakins. The results were confirmed by overlay assays of the binding of in vitro translated periplakin constructs to keratins and vimentin. We found that envoplakin and periplakin could still associate with each other when parts of their rod domains were deleted and, surprisingly, that removal of the entire rod domain did not completely inhibit their interaction. Co-transfection of constructs containing the C-termini of envoplakin and periplakin suggested that the periplakin C-terminus may stabilise the interaction of the envoplakin C-terminus with intermediate filaments. We conclude that the periplakin C-terminus plays an important role in linking periplakin and envoplakin to intermediate filaments.
To deduce the function of the lens-specific cytoskeletal structure, the beaded filament, by blocking expression of the fiber cell-specific beaded filament protein CP49.
The first exon of the mouse CP49 gene was deleted by using targeted genomic deletion techniques. Gene deletion was assessed through Southern blot analysis and PCR. Translation and protein expression were characterized by Northern and Western blot analysis of both CP49 and its assembly partner filensin. The architecture of knockout lenses was compared with that of wild-type lenses at the histologic level by light microscopy. Lens clarity was assessed in situ by direct ophthalmic examination and slit lamp microscopy.
Transcription and translation of CP49 were successfully negated in knockout animals. Lenses homozygous for the CP49 deletion showed no obvious changes in lens architecture at the light microscope level. Filensin levels were sharply reduced, although filensin mRNA levels appeared unchanged. Direct examination of lenses showed no obvious loss of lens clarity, but slit lamp examination revealed the emergence of opacification in even the youngest animals. The opacification worsened with age.
The absence of CP49 causes a subtle loss of optical clarity in the ocular lens, a loss that worsens with age. However, CP49 is not essential for the assumption or maintenance of overall fiber cell shape or long-range order of fiber cells. CP49 appears to regulate the protein levels of its assembly partner filensin, suggesting a mechanism for the regulation of beaded filament protein stoichiometry.
The anucleate prismoid fiber cells of the eye lens are densely packed to form a tissue in which the plasma membranes and their associated cytoplasmic coat form a single giant cell-cell adhesive complex, the cortex adhaerens. Using biochemical and immunoprecipitation methods in various species (cow, pig, rat), in combination with immunolocalization microscopy, we have identified two different major kinds of cortical complex. In one, the transmembrane glycoproteins N-cadherin and cadherin-11 [which also occur in heterotypic ('mixed') complexes] are associated with alpha- and beta-catenin, plakoglobin (proportions variable among species), p120ctn and vinculin. The other complex contains ezrin, periplakin, periaxin and desmoyokin (and so is called the EPPD complex), usually together with moesin, spectrin(s) and plectin. In sections through lens fiber tissue, the short sides of the lens fiber hexagons appear to be enriched in the cadherin-based complexes, whereas the EPPD complexes also occur on the long sides. Moreover, high resolution double-label fluorescence microscopy has revealed, on the short sides, a finer, almost regular mosaicism of blocks comprising the cadherin-based, catenin-containing complexes, alternating with patches formed by the EPPD complexes. The latter, a new type of junctional plaque ensemble of proteins hitherto known only from certain other cell types, must be added to the list of major lens cortex proteins. We here discuss its possible functional importance for the maintenance of lens structure and functions, notably clear and sharp vision.
Envoplakin and periplakin are two plakins that are precursors of the epidermal cornified envelope. We studied their distribution and interactions by transfection of primary human keratinocytes and other cells. Full-length periplakin localized to desmosomes, the interdesmosomal plasma membrane and intermediate filaments. Full length envoplakin also localized to desmosomes, but mainly accumulated in nuclear and cytoplasmic aggregates with associated intermediate filaments. The envoplakin rod domain was required for aggregation and the periplakin rod domain was necessary and sufficient to redistribute envoplakin to desmosomes and the cytoskeleton, confirming earlier predictions that the proteins can heterodimerize. The linker domain of each protein was required for intermediate filament association. Like the NH2 terminus of desmoplakin, that of periplakin localized to desmosomes; however, in addition, the periplakin NH2 terminus accumulated at cell surface microvilli in association with cortical actin. Endogenous periplakin was redistributed from microvilli when keratinocytes were treated with the actin disrupting drug Latrunculin B. We propose that whereas envoplakin and periplakin can localize independently to desmosomes, the distribution of envoplakin at the interdesmosomal plasma membrane depends on heterodimerization with periplakin and that the NH2 terminus of periplakin therefore plays a key role in forming the scaffold on which the cornified envelope is assembled.
A Monte-Carlo method is used to simulate the re-entrant corner growth of an fcc (111) crystal surface on which emerged the outcrop of a twin lamella of type A, composed of parallel stacking faults bounded by partial dislocations. The results are compared with those of twin lamellae of type B, stacking fault, and screw dislocation mechanisms. It is found that the activity of the re-entrant corner mechanism induced by the A-type lamella is smaller than that of the B-type twin lamella mechanism, and that it is larger than that of the stacking fault mechanism. However, it is less than that of the screw dislocation mechanism at low supersaturation.
A portion of the intracellular domain of Type XVII collagen, used as a bait in a yeast two-hybrid screen of an epidermal keratinocyte cDNA library, identified overlapping cDNA clones that showed a high degree of homology to envoplakin and other members of the plakin family of intermediate filament connector molecules. Subsequent cloning allowed identification of contiguous cDNA sequences with an open reading frame of 5268 bp encoding a putative polypeptide of 1756 amino acids with a computed molecular mass of 204.7 kDa. Northern analysis using these cDNA clones revealed a prominent band of approximately 6.5 kb in keratinocytes, which was barely detectable in fibroblasts. Multiple tissue RNA analysis showed that this protein is highly expressed in tissues with a prominent component of epithelial cells. This novel member of the plakin family was designated periplakin. The human gene (PPL) was mapped to the interval between D16S510 and D16S509 by radiation hybrid mapping, corresponding to chromosomal band 16p13. Murine ESTs having 97.2% amino acid identity to the human sequence were identified. Interspecific backcross mapping was used to place the murine periplakin gene (Ppl) 0.53 cM distal to marker D16mit32 on the proximal part of murine chromosome 16, close to the locus of mahoganoid (md), a mouse hair mutant. Mapping of this gene in human and mouse will allow evaluation of periplakin as a candidate locus for disorders of epithelial fragility, with or without other phenotypes.
Bacterial plasmids containing cDNA sequences specific for keratins were constructed from mRNA of cultured human epidermal cells. Two separate classes of cloned cDNAs were identified by positive hybrid selection: one class removed from total human epidermal mRNA a fraction that was translated into 56 and 58 kilodalton (kd) keratins, and the other class selected mRNAs that translated into a mixture of 50 kd and 46 kd keratins. When probes specific for these two keratin classes were hybridized with human DNA digested with a restriction endonuclease that does not cleave within the probe, two distinct patterns of about ten fragments each were observed. Most of the hybridizing genomic fragments corresponded to complete cDNA sequences, and it is estimated that each of the two classes is encoded by about 10 genes. When the probes were hybridized with DNA from different species, all vertebrates were found to contain discrete sequences homologous to both human keratin probes. Within each vertebrate species, the two probes always hybridized with approximately equal intensities to nonoverlapping sets of genomic sequences, suggesting a coordinate evolution between the two subfamilies of keratin genes. This finding has important functional implications for keratin filament assembly.
Cultured human epidermal cells and human stratum corneum (callus) contain a number of keratins of different molecular size, but the size distribution is not the same in the two cases. To characterize these keratins in more detail, we compared them by amino acid analysis, immunological reactivity and one-dimensional peptide mapping (Cleveland et al., 1977). No differences in amino acid compositon could be detected among keratins of stratum corneum differing in molecular size by as much as 50%, suggesting that some repeating structure may be present in these molecules. Examination of polypeptide fragments produced by partial enzymatic hydrolysis showed strong similarities among all the keratins of stratum corneum and of cultured epidermal cells, even extending to the keratins of rodents; but the keratins of similar size, whether of stratum corneum or cultured cells, were more closely related than keratins of different size. This conclusion was supported by studies of the immunological reactivity of the keratins. How the epidermal cell generates a family of keratins is a problem of considerable interest. The differences in size and structure between the keratins of stratum corneum and cultured epidermal cells suggest that the epidermal cell can modify the expression its keratin genes.
Cytologic details of the lens cells of the human and monkey have been studied with specific emphasis on their maturation process. Lens cells maintain the basal attachment until the nuclei are eliminated. After the denucleation, the cells develop specific joining apparatus. The mature lens cells, which occupy the major part of the lens, are held to each other firmly by knob and socket joints in the superficial cortex and by fine ridges in the deep lens.Cells in all maturation stages are present within the lens. Except for the decreased number of the elongating cells at the bow zone in older age, there is no specific structural difference between lenses of different age groups.
Desmoplakins (DPs) I and II are two major related proteins located in the desmosomal plaque where they have been proposed to play a role in attaching intermediate filaments (IF) to the inner cell surface. The predicted amino acid sequence of DP was obtained by analysis of overlapping cDNA clones. Computer‐aided analysis suggests that DPI will form a dumbbell‐shaped homodimer, with a central α ‐helical coiled coil rod domain of 132 nm and two globular end domains. The DPII molecule is missing 599 residues from the central domain, resulting in a rod about one third the length of DPI. The carboxyl terminus comprises three subdomains each containing almost 5 repeats of a 38 residue repeating motif with a periodicity in acidic and basic residues similar to that found in the rod domain of IF proteins. This suggests a possible mechanism by which these proteins might interact. The amino terminus contains groups of heptad repeats that are predicted to form at least two major α ‐helical rich bundles.
A series of c‐ myc ‐tagged mammalian expression vectors encoding specific predicted domains of DPI were transiently expressed in COS‐7 cells. Light and electron microscopical observations revealed that DP polypeptides including the 90 kDa carboxyl terminal globular domain of DPI specifically colocalized with and ultimately resulted in the complete disruption of keratin and vimentin IF. This effect was specific for the carboxyl terminus, as the expression of the 95 kDa rod domain of DPI did not visibly alter IF networks. Immunogold localization of COS‐7 cells transfected with constructs including the carboxyl terminus of DP demonstrated an accumulation of mutant protein within which IF subunits were sequestered. These results suggest a role for the DP carboxyl terminus in the attachment of IF to the desmosomal plaque in either a direct or indirect manner.
During normal differentiation, lens fiber cells lose their nuclei, mitochondria, and other membrane-bound organelles. In the present study, a slice preparation of the embryonic chicken lens was used with laser scanning confocal microscopy to study the spatial and temporal patterns of organelle breakdown during embryonic development. At all stages examined, mitochondria in lens epithelial cells were present in perinuclear clusters. In contrast, early in development, lens fiber cells contained extremely elongated mitochondria (> 100 microns) that were distributed throughout the cytoplasm and oriented along the long axis of the cells. By the 8th day of embryonic development (E8), the mitochondria in the central fiber cells began to fragment. At the same time, the nuclei in these cells became smaller and more spherical. By E10, mitochondrial staining in the central fibers became punctate. Electron microscopy of this region revealed swollen mitochondria with disrupted cristae. By E12, cells in the central region of the lens lacked mitochondria and nuclei. The loss of nuclei and mitochondria from a given cell was coincident and abrupt (2-4 hr), occurring in a previously unsuspected domain situated about 300 microns from the anterior surface of the lens. A cytoskeletal component, actin, persisted in the central cells indicating that organelle degradation represents a selective process and not simply the global degradation of supramolecular structures. Throughout embryonic development, the organelle-free region grew at approximately the same rate as the lens and, by the time of hatching, had expanded to match the diameter of the pupil.(ABSTRACT TRUNCATED AT 250 WORDS)
Lenses were obtained from the eyes of four different classes of Chordates, including Mammalia (rat, mouse, cow, human), Aves (chicken), Amphibia (tiger salamander), and Osteichthyes (steelhead), as well as from one Mollusca (squid). Buffer soluble, urea soluble and urea insoluble fractions were prepared from each, and probed by western blot analysis for the presence of the lens fiber cell 115 and 49 kD beaded filament proteins. Application of both polyclonal and monoclonal antibodies revealed that an immunologic homologue to the bovine fiber cell 115 kD protein is present in all examples of Chordates tested, and that this homologue possessed properties very similar to those of its bovine counterpart. Both monoclonal and polyclonal antibodies revealed an immunologically cross-reactive homologue in squid as well, but suggested that the squid protein had a native molecular weight of closer to 70-80 kD. A monoclonal antibody to the bovine 49 kD beaded filament protein was successful at identifying an immunologic homologue to this protein in mouse, chicken, and tiger salamander. Ultrastructural analysis of rat, human, and fish lenses showed that a beaded filament was present in these lenses, which was indistinguishable from that seen in the bovine lens. In the squid a filamentous, beaded structure was observed, but it differed from that seen in the bovine lens. We conclude from the data presented that the beaded filament, and its constituent proteins, are well-conserved. This data should facilitate the identification of lens cytoskeletal proteins and structure in a wide range of animal models, and establish that probes for these proteins may be of broad applicability.
Two alternative methods for the study of the lens cytoskeleton are described which serve to overcome some of the difficulties imparted by the unique biology of the lens. The first technique involves rapid freezing, thick sectioning, and selective extraction and/or fixation of the lens section. This approach offers several advantages: 1) enhanced visualization of the cytoskeleton, 2) avoidance of fixation gradients, 3) free access for immunocytochemical probes, 4) retention of tissue-wide spatial relationships, with a sharp increase in the resolution of regional analysis, and 5) the capacity for correlative morphological and biochemical comparisons. The second method involves the covalent immobilization of the plasma membrane-cytoskeleton complex (PMCC) to acrylamide beads. This approach permits: 1) avoidance of fixation in the immunocytochemical analysis of lens cytoskeleton and plasma membranes 2) rapid processing of multiple, small-quantity samples for immunocytochemistry/biochemical analysis 3) cleaner and more rapid analysis of cytoskeletal extraction conditions. Both approaches, while particularly suited to the study of the lens PMCC, may also be of value to the study of the PMCC of other tissues, particularly where preservation/analysis of regional relationships is essential.
Electron microscope level immunocytochemistry was used to localize a lens fiber cell-specific protein with an Mr of 115 kd. Affinity-purified polyclonal antibodies were utilized on sections of detergent-extracted, acrylic-embedded lens cortical fiber cells. Monoclonal antibodies were utilized for pre-embeddment labelling of a subcellular fraction of lens fiber cells generated by homogenization, and high-speed centrifugation. The results indicate that the Mr 115 kd antigen is a component of the lens fiber cell cytoskeleton, specifically the beaded filament (BF), a cytoskeletal element thought to be unique to the differentiated lens fiber cell.
Western blot analysis using a monoclonal antibody raised against a lens fiber cell-specific, extrinsic membrane protein reveals several immunologically related bands in fractions derived from bovine lens. Previous work suggests that the parent molecule is the Mr 115 species, and that lower molecular weight bands represent the products of a progressive, step-wise, post-translational degradation. In this report we compare the extent of proteolytic degradation in extracts prepared from the lens cortex and lens nucleus, using both protease-suppressive and protease-permissive isolation protocols. The results suggest that the observed degradation is a result of in vivo post-translational modification of the Mr 115 antigen, and thus represents physiologic aging of this protein. This analysis also suggests that degradation alters the solubility and/or membrane affinity of this antigen, resulting in a progressive shift to the insoluble phase.
Monoclonal and polyclonal antibodies have been produced against a lens fiber cell extrinsic membrane protein, with a relative molecular weight of approximately 115 kd. Enzyme Linked Immunosorbent Assays (ELISA) of retina, ciliary body-iris, liver, and skeletal muscle, utilizing these antibodies, suggest that the antigen is unique to the lens. Immunocytochemistry indicates that the antigen is present only in the differentiated fiber cell, and is absent from the lens epithelium. Further, immunocytochemical reactivity is predominantly associated with the fiber cell plasma membrane. However, sequential extraction of fiber cell homogenate, followed by quantitative, competitive ELISA analysis, indicates that most of the antigen is recovered in the neutral buffer extract. ELISA analysis using monoclonal antibodies indicates that an analogous antigen is present in human and rabbit lenses. On the basis of these results we characterize this antigen as a conserved extrinsic membrane protein, which is unique to the differentiated lens fiber cell. The relationship of this antigen to a previously described Mr 95 beaded filament-associated protein is discussed.
A novel, simple and relatively rapid method is described for the isolation of the intermediate-sized filament protein vimentin from eye lens tissue. Chromatofocusing is applied as the sole purification step. The apparent isoelectric point of the protein in 6 M urea and at 22 degrees C is 4.9. Electrophoretic mobility on one- and two-dimensional polyacrylamide gels, solubility in 6 M urea and amino acid composition were used for identification.
A study of the synthesis of the intermediate filament protein, vimentin, is reported here. The following systems were examined: the epithelial cells of the organ-cultured rabbit lens, the epithelial and cortical fiber cells of the organ-cultured adult chicken lens and the epithelial cells of the rabbit lens grown in tissue culture. Vimentin is actively synthesized by all of the above mentioned cells.
Denucleating process of the mouse lens has been studied electron microscopically. The first step appears as a transformation in which the appearance of the nucleus becomes more like that of the cytoplasm. When the appearance of both nuclear and cytoplasmic substances becomes indistinguishable, the nuclear membrane disappears without disturbing the size or other structure of the cell. Extrusion of the nuclear substance or degeneration of the nucleus are not observed during the denucleation. Fragments of nucleolus remain within the denucleated lens for a long period of time. Disturbance of the enucleation process may cause certain cataractous changes of the lens.
Cells of the inner layers of the epidermis contain small keratins (46-58K), whereas the cells of the outer layers contain large keratins (63-67K) in addition to small ones. The changes in keratin composition that take place within each cell during the course of its terminal differentiation result largely from changes in synthesis. Cultured epidermal cells resemble cells of the inner layers of the epidermis in synthesizing only small keratins. The cultured cells possess translatable mRNA only for small keratins, whereas mRNA extracted from whole epidermis can be translated into both large and small keratins. As no synthesis takes place in the outermost layer of the epidermis (stratum corneum), the keratins of this layer must be synthesized earlier, but in some cases they then become smaller: this presumably occurs by post-translational processing of the molecules during the final stages of differentiation. Stratified squamous epithelia of internal organs do not form a typical stratum corneum and do not make the large keratins characteristic of epidermis. Their keratins are also different from those of cultured keratinocytes, implying that they have embarked on an alternate route of terminal keratin synthesis.
Antibody prepared against chick lens vimentin cross-reacts with chick fibroblast vimentin and with vimentin of mammalian, reptilian, amphibian and fish lenses. This protein is localized in the epithelial and cortical fiber cells and is progressively lost from the deeper cortical cells. It is absent from the nuclear cells. Lens vimentin is readily oxidized to form high molecular components.
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.
Two proteins, with molecular weights of 49 (CP49) and 115 kDa (CP115) as judged by SDS PAGE, have been shown by immunocytochemistry to be components of the beaded filament, a cytoskeletal structure thus far demonstrated only in the lens fiber cell. We have used antibodies reactive with CP49 to screen a mouse lens cDNA expression library. An immunoreactive clone with an approximately 1.0 kb insert was identified and purified. DNA sequence analysis shows the presence of an open reading frame that extends from the upstream cloning site for 660 bases. Contained within this reading frame are 2 peptide sequences nearly identical to 2 peptide sequences obtained from purified bovine CP49. Northern analysis revealed that the mRNA for the CP49 is not detectable in mouse brain, muscle, lung, liver, or heart. A search of the Genbank database showed that the partial cDNA sequence for the murine CP49 is unique, but that this partial sequence shows a strong similarity to multiple members of the intermediate filament family, with greatest similarity to type I acidic cytokeratins. The data presented here suggests that the CP49 is related to, and possibly represents a new member of the intermediate filament family. These data, in concert with previously published work, suggests 1) the CP49 and CP115, which have been localized to the beaded filament, are related to the family of IF proteins, and 2) these two proteins comprise a cytoskeletal structure which is structurally distinct from classical 8-11 nm intermediate filaments, thus possibly comprising a structurally novel form of intermediate filament.
Filensin, a 100 kDa, membrane-associated, cytoskeletal protein, is uniquely expressed in the lens fiber cell (Merdes, A., Brunkener, M., Horstmann, H., and Georgatos, S. D. (1991) J. Cell Biol. 115, 397-410). I cloned and sequenced a full-length chicken lens cDNA encoding filensin, also known as CP95 (Ireland, M. and Maisel, H. (1989) Lens and Eye Toxicity Research 6, 623-638). The deduced amino acid sequence of 657 residues contained an internal 280 residue heptad repeat domain with sequence similarities to the rod domain of intermediate filament proteins. The putative filensin rod domain could be divided into three alpha-helical segments (1A, 1B and 2) separated by short, non-helical linkers. The sequence of the amino-terminal end of the filensin rod domain contained the highly conserved intermediate filament segment 1A motif (Conway, J. F. and Parry, D. A. D. (1988) Int. J. Biol. Macromol. 10, 79-98). Allowing conservative amino acid substitutions, the sequence of the carboxy-terminal end of the filensin rod domain was similar to that of the highly conserved intermediate filament rod carboxy terminus. The alpha-helical segments of the shorter filensin rod domain aligned with the corresponding segments of intermediate filament proteins by allowing a gap of four heptad repeats in the amino-terminal half of filensin segment 2. Filensin rod segment 2 contained the characteristic stutter in heptad repeat phasing, nine heptads from the end of the intermediate filament rod. The overall sequence identity between the rod domains of filensin and individual intermediate filament proteins was 20 to 25%, approximately the level of sequence identity observed between intermediate filament proteins of different types. The open reading frame of chicken filensin predicted a 657 amino acid protein with molecular mass of 76 kDa. Embryonic chicken filensin migrated in SDS-PAGE as a triplet of 102, 105 and 109 kDa, while rooster filensin migrated as a 105 and 109 kDa doublet. Antibodies to filensin labeled lens fiber cells but not lens epithelial cells. By immunofluorescence methods filensin was localized to the fiber cell plasma membranes, including the ends of elongated fiber cells.
Typified by rapid degeneration of sensory neurons, dystonia musculorum mice have a defective BPAG1 gene, known to be expressed in epidermis. We report a neuronal splice form, BPAG1n, which localizes to sensory axons. Both isoforms have a coiled-coil rod, followed by a carboxy domain that associates with intermediate filaments. However, the amino terminus of BPAG1n differs from BPAG1e in that it contains a functional actin-binding domain. In transfected cells, BPAG1n coaligns neurofilaments and microfilaments, establishing this as a cytoskeletal protein interconnecting actin and intermediate filament cytoskeletons. In BPAG1 null mice, axonal architecture is markedly perturbed, consistent with a failure to tether neurofilaments to the actin cytoskeleton and underscoring the physiological relevance of this protein.
The ocular lens fiber cell assembles a novel cytoskeletal element, the Beaded Filament, from CP49 and filensin, two proteins expressed only in the differentiated lens fiber cell. We report the primary sequence, secondary structural analysis, gene structure and Yeast Two Hybrid interaction data for human filensin, and develop a consensus model of filensin from the human and previously reported bovine and chicken filensin sequences. This consensus model, combined with gene structure and Yeast Two Hybrid studies establish that filensin is a member of the Intermediate Filament family of proteins. Specifically, filensin exhibits (1) divergence at amino acid sequence motifs otherwise highly conserved among intermediate filament proteins, (2) a loss of 29 amino acids from the central rod domain which is unique among cytoplasmic intermediate filament proteins, (3) an absence of sequence identity with any existing class of intermediate filament protein, (4) a gene structure unique among intermediate filament family, (5) an inability to dimerize with representatives of Type I, II, and III intermediate filament proteins. Thus, at each level of analysis, we find that filensin is similar to the consensus model of intermediate filament proteins, supporting our conclusion that filensin's relatedness to the IF family is not the consequence of convergent evolution. However, filensin also shows unique or extreme distinctions from the consensus intermediate filament protein at each level of analysis, indicating that filensin constitutes a novel class of IF protein. Some of filensin's unique features are incompatible with current models of IF assembly. Analysis of filensin gene structure suggests that the 29 amino acid reduction in the central rod domain was not the result of a single splice site mutation, the mechanism suggested for the transition between nuclear lamins and cytoplasmic intermediate filament proteins.
The developing chicken embryo lens provides a unique model for examining the relationship between alpha6 integrin expression and cell differentiation, since multiple stages of differentiation are expressed concurrently at one stage of development. We demonstrate that alpha6 integrin is likely to mediate the inductive effects of laminin on lens differentiation as well as to function in a matrix-independent manner along the cell-cell interfaces of the differentiating cortical lens fiber cells. Both alpha6 isoform expression and its linkage to the cytoskeleton were regulated in a differentiation-specific manner. The association of alpha6 integrin with the Triton-insoluble cytoskeleton increased as the lens cells differentiated, reaching its highest levels in the cortical fiber region where the lens fiber cells are formed. In this region of the lens alpha6 integrin was uniquely localized along the cell-cell borders of the differentiating fiber cells, similar to beta1. alpha6beta4, the primary transmembrane protein of hemidesmosomes, is also expressed in the lens, but in the absence of hemidesmosomes. Differential expression of alpha6A and alpha6B isoforms with lens cell differentiation was seen at both the mRNA and the protein levels. RT-PCR studies demonstrated that alpha6B was the predominant isoform expressed both early in development, embryonic day 4, and in the epithelial regions of the day 10 embryonic lens. Isoform switching, with alpha6A now the predominant isoform, occurred in the fiber cell zones. Immunoprecipitation studies showed that alpha6B, which is characteristic of undifferentiated cells, was expressed by the lens epithelial cells but was dramatically reduced in the lens fiber zones. Expression of alpha6B began to drop as the cells initiated their differentiation and then dropped precipitously in the cortical fiber zone. In contrast, expression of the alpha6A isoform remained high until the cells became terminally differentiated. alpha6A was the predominant isoform expressed in the cortical fiber region. The down-regulation of alpha6B relative to alpha6A provides a developmental switch in the process of lens fiber cell differentiation.
To define the remodeling of lens fiber cell intermediate filaments (IF) that occurs with both development and differentiation.
Prenatal and postnatal mice were probed for the IF proteins phakosin, filensin, and vimentin, using light microscope immunocytochemical methodology.
The pattern of vimentin accumulation in elongating fiber cells changed with development. Early in development vimentin first emerged predominantly as focal accumulations in the basal region of both epithelial and primary fiber cells. A light diffuse cytoplasmic staining was also noted. Later in embryonic development, and through maturity, vimentin in fiber cells was predominantly associated with the plasma membrane with no anterior-posterior polarity. Phakosin and filensin were first detected in the very latest stages of primary fiber elongation and continued to accumulate well after cells had completed elongation. Initially, these proteins accumulated in the anterior half of the fiber cells and were cytoplasmic in distribution. After P13, the pattern of initial distribution in differentiating fiber cells changed to a predominantly plasma membrane localization. Neither beaded filament protein showed focal basal accumulations. In mature lenses, all three proteins ultimately disappeared from the nuclear fiber cells.
Beaded filament protein accumulation lags significantly behind both primary and secondary fiber cell elongation, suggesting a functional role subsequent to elongation. The subcellular distribution of vimentin and the beaded filament proteins showed marked differences within the cell, with differentiation, and with development. The differences in time of initial synthesis and in distribution of these IF proteins may bear on hypotheses about the role of IFs in fiber cell elongation and in structural-functional polarity of the fiber cell.
By connecting cytoskeletal elements to each other and to junctional complexes, the plakin family of cytolinkers plays a crucial role in orchestrating cellular development and maintaining tissue integrity. Plakins are built from combinations of interacting domains that bind to microfilaments, microtubules, intermediate filaments, cell-adhesion molecules and members of the armadillo family. Plakins are involved in both inherited and autoimmune diseases that affect the skin, neuronal tissue, and cardiac and skeletal muscle. Here, we describe the members of the plakin family and their interaction partners, and give examples of the cellular defects that result from their dysfunction.
The striate form of palmoplantar keratoderma is a rare autosomal dominant disorder affecting palm and sole skin. Genetic heterogeneity of striate palmoplantar keratoderma has been demonstrated with pathogenic mutations in the desmosomal proteins desmoplakin and desmoglein 1. We have studied a four-generation family of British descent with striate palmoplantar keratoderma. Ultrastructural studies show that intermediate filaments of suprabasal keratinocytes are finer than those of the basal layer. In addition, desmosome numbers are normal, but their inner plaques and midline structures are attenuated. Microsatellite markers were used to screen candidate loci including the epidermal differentiation complex on 1q, the desmoplakin locus on 6p, the type I and II keratin gene clusters on chromosomes 12q and 17q, and the desmosomal cadherin gene cluster on chromosome 18q. Significant genetic linkage to chromosome 12q was observed using marker D12S368, with a maximum two-point lod score of 3.496 at a recombination fraction of 0. Direct sequencing of the keratin 1 gene revealed a frameshift mutation in exon 9 that leads to the partial loss of the glycine loop motif in the V2 domain and the gain of a novel 70 amino acid peptide. Using expression studies we show that the V2 domain is essential for normal function of keratin intermediate filaments.
The serine/threonine kinase protein kinase B (PKB/c-Akt) acts downstream of the lipid kinase phosphoinositide 3-kinase (PI3K) and functions as an essential mediator in many growth-factor-induced cellular responses such as cell cycle regulation, cell survival and transcriptional regulation. PI3K activation generates 3'-phosphorylated phosphatidylinositol lipids (PtdIns3P) and PKB activation requires PtdIns3P-dependent membrane translocation and phosphorylation by upstream kinases. However PKB activation and function is also regulated by interaction with other proteins. Here we show binding of PKB to periplakin, a member of the plakin family of cytolinker proteins. Interaction between PKB and periplakin was mapped to part of the pleckstrin homology (PH) domain of PKB, which is probably not involved in lipid binding, and indeed binding to periplakin did not affect PKB activation. We therefore investigated the possibility that periplakin may act as a scaffold or localization signal for PKB. In cells endogenous periplakin localizes to different cellular compartments, including plasma membrane, intermediate filament structures, the nucleus and mitochondria. Overexpression of the C-terminal part of periplakin, encompassing the PKB binding region, results in predominant intermediate filament localization and little nuclear staining. This also resulted in inhibition of nuclear PKB signalling as indicated by inhibition of PKB-dependent Forkhead transcription factor regulation. These results suggest a possible role for periplakin as a localization signal in PKB-mediated signalling.
Plectin, desmoplakin, and the 230-kDa bullous pemphigoid antigen (BPAG1), members of the plakin family of proteins, are multifunctional cytolinkers, connecting the cytoskeletal structures to the cell adhesion complexes. Envoplakin and periplakin are components of the cornified envelope, but less is known about their role in tissues other than the stratified epithelium. Our tissue-wide survey utilizing RT-PCR revealed that periplakin, like plectin and desmoplakin, has a wide tissue distribution, but envoplakin expression is limited to certain tissues only, and BPAG1 is clearly specific for epidermal keratinocytes. Plectin, desmoplakin and BPAG1 are known to bind to the intermediate filaments through their C-terminal domains. The short C-terminal domain of periplakin is composed only of the linker domain, a region highly homologous between the plakin proteins. Here we demonstrate, through the use of yeast two-hybrid assay, a specific interaction of the periplakin linker domain with keratin 8 and vimentin. Co-expression of each plakin linker domain with keratin 8 revealed that periplakin and BPAG1 linkers co-localize with keratin signals in HaCaT cells, plectin and desmoplakin linkers were detected both in the nucleus and in cytoplasm together with the overexpressed keratin 8, while envoplakin linker localized independently into the nucleus. These results suggest that, in spite of its high homology and structural similarity with envoplakin, periplakin is functionally closer to the well-characterized plakin proteins plectin and desmoplakin, and thus may function tissue-wide as a scaffolding protein in intermediate filament assembly.
In this report, the phenotype associated with the first targeted knockout of the lens specific intermediate filament gene CP49 is described. Several surprising observations have been made. The first was that no cataract was observed despite the fact that the beaded filaments of the lens fibre cells had been disrupted. Light scatter and the lens optical properties had, however, deteriorated in the CP49 knockout lenses compared to litter mate controls. These changes were accompanied by dramatic changes in plasma membrane organisation of the fibre cells as revealed by detailed morphological examinations and providing the second surprising result. The CP49 knockout mouse is therefore an important model to study the functional link between lens transparency, the cytoskeleton and plasma membrane organisation.