Biomedical subjects

A M Hutcheson

Publications and source records attributed to A M Hutcheson.

13 recordsLinked to original sources

PubMed2004

Human keratin 8 mutations that disturb filament assembly observed in inflammatory bowel disease patients.

We have identified miss-sense mutations in keratin 8 in a subset of patients with inflammatory bowel disease (Crohn disease and ulcerative colitis). Inflammatory bowel diseases are a group of disorders that are polygenic in origin and involve intestinal epithelial breakdown. We investigated the possibility that these keratin mutations might contribute to the course of the disease by adversely affecting the keratin filament network that provides mechanical support to cells in epithelia. The mutations (Gly62 to Cys, Ile63 to Val and Lys464 to Asn) all lie outside the major mutation hotspots associated with severe disease in epidermal keratins, but using a combination of in vitro and cell culture assays we show that they all have detrimental effects on K8/K18 filament assembly in vitro and in cultured cells. The G62C mutation also gives rise to homodimer formation on oxidative stress to cultured intestinal epithelial cells, and homodimers are known to be polymerization incompetent. Impaired keratin assembly resulting from the K8 mutations found in some inflammatory bowel disease patients would be predicted to affect the maintenance and re-establishment of mechanical resilience in vivo, as required during keratin cytoskeleton remodeling in cell division and differentiation, which may lead to epithelial fragility in the gut. Simple epithelial keratins may thus be considered as candidates for genes contributing to a risk of inflammatory bowel disease.

Actin Cytoskeleton↗

PubMed2000

Up-regulation of novel intermediate filament proteins in primary fiber cells: an indicator of all vertebrate lens fiber differentiation?

The early embryonic development and expression patterns of the eye lens specific cytoskeletal proteins, CP49 and CP95, were determined for the chick and were found to be similar in both human and mouse. These proteins, as well as their homologs in other species, are obligate polymerization partners which form unique filamentous structures termed "beaded filaments." CP49 and CP95 appeared as protein products after 3 days of embryonic development in the chick during the elongation of primary fiber cells. Although limited data were obtained for human embryos at these early developmental timepoints, they were consistent with the interpretation that the up-regulation of these lens specific proteins began only after the initiation of lens vesicle closure. In situ hybridization with the mouse lens confirmed that message levels for beaded filament proteins were greatly elevated in differentiating primary fiber cells. Nuclease protection assays established that mRNA levels for CP49 remained relatively constant while CP95 mRNA levels increased once the process of secondary fiber formation was under way. Although present in relatively low abundance, the mRNA for a unique splice variant of CP49, CP49(INS), was also detected early in embryonic development and into adulthood. Peptide-specific antibodies directed against unique predicted sequences were able to confirm the protein expression of CP49(INS) in both embryonic and adult chick lens cells. These data present the first detailed study of the expression of CP49 and CP95 during early lens development. They suggest that the up-regulated expression of CP49 and CP95 could serve as pan-specific markers for all vertebrate lens fiber development.

Animals↗

PubMed1999

The cardiomyopathy and lens cataract mutation in alphaB-crystallin alters its protein structure, chaperone activity, and interaction with intermediate filaments in vitro.

Desmin-related myopathy and cataract are both caused by the R120G mutation in alphaB-crystallin. Desmin-related myopathy is one of several diseases characterized by the coaggregation of intermediate filaments with alphaB-crystallin, and it identifies intermediate filaments as important physiological substrates for alphaB-crystallin. Using recombinant human alphaB-crystallin, the effects of the disease-causing mutation R120G upon the structure and the chaperone activities of alphaB-crystallin are reported. The secondary, tertiary, and quaternary structural features of alphaB-crystallin are all altered by the mutation as deduced by near- and far-UV circular dichroism spectroscopy, size exclusion chromatography, and chymotryptic digestion assays. The R120G alphaB-crystallin is also less stable than wild type alphaB-crystallin to heat-induced denaturation. These structural changes coincide with a significant reduction in the in vitro chaperone activity of the mutant alphaB-crystallin protein, as assessed by temperature-induced protein aggregation assays. The mutation also significantly altered the interaction of alphaB-crystallin with intermediate filaments. It abolished the ability of alphaB-crystallin to prevent those filament-filament interactions required to induce gel formation while increasing alphaB-crystallin binding to assembled intermediate filaments. These activities are closely correlated to the observed disease pathologies characterized by filament aggregation accompanied by alphaB-crystallin binding. These studies provide important insight into the mechanism of alphaB-crystallin-induced aggregation of intermediate filaments that causes disease.

Cardiomyopathies↗

PubMed1999

The eye lens cytoskeleton.

During lens cell differentiation there are a number of very characteristic morphological changes that occur. These include a 50- to 100-fold increase in cell length as the equatorial lens epithelial cells differentiate into fibre cells and the loss of the cellular organelles such as mitochondria, nuclei, Golgi apparatus and endoplasmic reticulum. Coincident with these changes are dramatic alterations in the organisation of the lens fibre cell cytoskeleton and in particular the lens-specific intermediate filament network comprising CP49 and filensin. Cell shape and cell polarisation as well as tissue integrity are all processes that depend upon the cytoskeleton and are therefore important to the lens. The unique aspects of the lenticular cytoskeleton are the subject of this review.

Animals↗

PubMed1999

Intermediate filament interactions can be altered by HSP27 and alphaB-crystallin.

HSP27 and alphaB-crystallin are both members of the small heat shock protein family. alphaB-crystalllin has been proposed to modulate intermediate filaments and recently a mutation in alphaB-crystallin has been identified as the genetic basis of desmin related myopathy. This disease is characterised in its pathology by aggregates of intermediate filaments associated with alphaB-crystallin. Here we report that HSP27 like alphaB-crystallin is associated with glial fibrillary acidic protein and vimentin intermediate filament networks in unstressed U373MG astrocytoma cells. HSP27 is also associated with keratin filaments in MCF7 cells, indicating that this association is not restricted to a particular intermediate filament type. The association of sHSPs with both the soluble and filamentous intermediate filament fractions of U373 cells was demonstrated biochemically. Heat shock or drug treatments induced a co-collapse of intermediate filaments and associated small heat shock proteins. These data show that the presence of HSP27 or alphaB-crystallin could not prevent filament collapse and suggest that the purpose of this association is more than just filament binding. Indeed, in U373MG cells the intermediate filament association with small heat shock proteins is similar to that observed for another protein chaperone, HSC70. In order to discern the effect of different chaperone classes on intermediate filament network formation and maintenance, several in vitro assays were assessed. Of these, falling ball viscometry revealed a specific activity of small heat shock proteins compared to HSC70 that was apparently inactive in this assay. Intermediate filaments form a gel in the absence of small heat shock proteins. In contrast, inclusion of alphaB-crystallin or HSP27 prevented gel formation but not filament assembly. The transient transfection of GFAP into MCF7 cells was used to show that the induction of a completely separate network of intermediate filaments resulted in the specific association of the endogenous HSP27 with these new GFAP filaments. These data lead us to propose that one of the major functions of the association of small heat shock proteins with intermediate filaments is to help manage the interactions that occur between filaments in their cellular networks. This is achieved by protecting filaments against those non-covalent interactions that result when they come into very close proximity as seen from the viscosity experiments and which have the potential to induce intermediate filament aggregation as seen in some disease pathologies.

Cell Compartmentation↗

PubMed1998

cDNA cloning, expression, and assembly characteristics of mouse keratin 16.

There has been speculation as to the existence of the mouse equivalent of human type I keratin 16 (K16). The function of this keratin is particularly intriguing because, in normal epidermis, it is usually confined to hair follicles and only becomes expressed in the suprabasal intrafollicular regions when the epidermis is traumatized. Previous studies suggested that K16 is highly expressed in the skin of mice carrying a truncated K10 gene. We therefore used the skin of heterozygous and homozygous mice to create a cDNA library, and we report here the successful cloning and sequencing of mouse K16. Recent in vitro studies suggested that filaments formed by human K16 are shorter than those formed by other type I keratins. One hypothesis put forward was that a proline residue in the 1B subdomain of the helical domain was responsible. The data presented here demonstrate that this proline is not conserved between mouse and human, casting doubt on the proposed function of this proline residue in filament assembly. In vitro assembly studies showed that mouse K16 produced long filaments in vitro. Also, in contrast to previous observations, transfection studies of PtK2 cells showed that mouse K16 (without the proline) and also human K16 (with the proline) can incorporate into the endogenous K8/K18 network without detrimental effect. In addition, K16 from both species can form filaments de novo when transfected with human K5 into immortalized human lens epithelial cells, which do not express keratins. These results suggest that reduced assembly capabilities due to unusual sequence characteristics in helix 1B are not the key to the unique function of K16. Rather, these data implicate the tail domain of K16 as the more likely protein domain that determines the unique functions.

Amino Acid Sequence↗

PubMed1996

The intermediate filament cytoskeleton of the lens: an ever changing network through development and differentiation. A minireview.

In the eye lens, intermediate filament proteins form two morphologically distinct polymers, 10-nm intermediate-sized filaments and beaded filaments. Coincidently, the beaded filament polymer and the proteins filensin and CP49 are specific to lens fibre cells and are therefore excellent markers for fibre cell differentiation. In the adult lens, filensin and CP49 are maintained throughout all stages of lens fibre cell differentiation whilst vimentin is apparently lost at a specific stage from the deeper cortical fibres. The expression of CP49 and filensin is coincident with the presence of beaded filaments suggesting these proteins are filament components. In association with alpha-crystallin, CP49 and filensin form beaded filaments in vitro. During fibre cell differentiation, filensin and CP49 are post-translationally modified. In the case of filensin, proteolysis results in two functionally distinct fragment sets, one derived from the alpha-helical rod domain and the other from the C-terminal tail domain of filensin. It is proposed that both filensin and CP49 are critically involved in organising the cytoplasmic and plasma membrane domains of the fibre cell and therefore essential to the optical properties of the lens.

Animals↗

PubMed1995

In vitro studies on the assembly properties of the lens proteins CP49, CP115: coassembly with alpha-crystallin but not with vimentin.

A rapid one-step purification procedure for CP49, an intermediate filament protein found in the lens, is described using reverse-phase HPLC. This protein is one of the major intermediate filament proteins of the lens fibre cells and is found in both the water insoluble fraction (WIF) and the water soluble fraction (WSF) of the lens. In order to better understand the physiological role of CP49 in lens transparency we have purified CP49 from both compartments and compared the in vitro assembly characteristics of both by electron microscopy and sedimentation assays. Our studies showed that CP49, when mixed with another lens intermediate filament protein, CP115, forms 10 nm intermediate filaments. Vimentin, another intermediate filament protein found in the lens, was unable to coassemble with CP115, thus demonstrating the specificity of the interaction of CP49 with CP115. CP49 isolated from either the WIF or the WSF formed 10-nm filaments with CP115 and indicated that CP49 from both these lens cell compartments had similar in vitro assembly characteristics. This also suggested that the post-translational modifications observed for CP49 from the different compartments was of little apparent consequence to filament formation. The inability to reconstitute beaded filaments from CP49 and CP115 suggested that other lens proteins may be needed in the reconstitution assay before these lens specific cytoskeletal elements could be repolymerised from their purified protein components. CP49 and CP115 were therefore assembled in the presence of alpha-crystallins and a beaded filament structure was observed as has been seen with type III intermediate filament proteins assembled with alpha-crystallins.

Animals↗

PubMed1995

Vimentin and CP49/filensin form distinct networks in the lens which are independently modulated during lens fibre cell differentiation.

The cells of the eye lens contain the type III intermediate filament protein vimentin, as well as two other intermediate filament proteins, CP49 and filensin. These two proteins appear to be unique to the differentiated lens fibre cell. Immunoblotting and confocal microscopy were used to describe changes which occur in these three intermediate filament proteins and the networks they form during fibre cell differentiation and maturation. The vimentin network was present in both epithelial cells and some fibre cells. Fibre cells were vimentin positive up to a specific point 2-3 mm in from the lens capsule where the vimentin signal was drastically reduced. The CP49/filensin network was not present in the undifferentiated epithelial cells but emerged in the differentiating fibre cells. This latter network exhibited a principally plasma membrane localization in younger fibre cells but became more cytoplasmic in older fibre cells. This change also occurred at a distinct point in fibre cell differentiation, much earlier than the observed loss of the vimentin network. The subcellular changes in the distributions of these cytoskeletal networks were correlated to the loss of the fibre cell nucleus, another feature of fibre cell differentiation. No correlation was found to changes in the vimentin network but nuclear loss did coincide with changes in the CP49/filensin network. Concomitant with nuclear pyknosis, there were also changes in the nuclear lamina as well as infringement of the nuclear compartment by CP49, as shown by confocal microscopy. This study demonstrates vimentin and the CP49/filensin network to be independent in the lens but both networks undergo dramatic changes in subcellular distribution during the differentiation/maturation of the fibre cell. Only changes in the CP49/filensin network can be correlated to nuclear loss. Thus in the lens, unlike mammalian erythropoiesis which is also characterized by nuclear loss, the vimentin network does not appear linked to nuclear retention.

Animals↗

PubMed1995

Filensin is proteolytically processed during lens fiber cell differentiation by multiple independent pathways.

Filensin is a lens-specific intermediate filament protein, expressed in the lens fiber cells but not the lens epithelium. Using antibodies to filensin and the other lens intermediate filament proteins, vimentin and CP49, the codistribution of filensin with CP49 and independence of this network from the vimentin network was confirmed. Monoclonal and polyclonal antibodies to peptides and specific subdomains of filensin were used to follow changes in the subcellular distribution of filensin during bovine lens fiber cell differentiation. Filensin is shown to be extensively processed during lens fiber cell differentiation to give protein fragments derived from distinct protein domains, one corresponding to the N-terminal non-alpha-helical/and rod domain and the other to the C-terminal non-alpha-helical tail domain. Immunoblotting analysis using anti-filensin peptide polyclonal antibodies suggested that the two fragment sets arose separately. Residues 331 to 430 in filensin have been identified as an important region in the processing pathway(s). Our results clarify previous confusion in the literature regarding the processing of filensin which arose because of the similar relative electrophoretic mobilities by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the different fragment sets. The predicted secondary structure characteristics of the different domains of filensin suggests different functions for the two fragment sets to give filensin a dual role in the lens. This suggestion is supported by the subtly different subcellular distributions in the peripheral and mature fiber cells of the two filensin fragment sets.

Animals↗

PubMed1994

Identification of two N-terminal non-alpha-helical domain motifs important in the assembly of glial fibrillary acidic protein.

The non-alpha-helical N-terminal domain of intermediate filament proteins plays a key role in filament assembly. Previous studies have identified a nonapeptide motif, SSYRRIFGG, in the non-alpha-helical N-terminal domain of vimentin that is required for assembly. This motif is also found in desmin, peripherin and the type IV intermediate filament proteins. GFAP is the only type III intermediate filament protein in which this motif is not readily identified. This study has identified two motifs in the non-alpha-helical N-terminal domain of mouse GFAP that play important roles in GFAP assembly. One motif is located at the very N terminus and has the consensus sequence, MERRRITS-ARRSY. It has some characteristics in common with the vimentin nonapeptide motif, SSYRRIFGG, including its location in the non-alpha-helical N-terminal domain and a concentration of arginine residues. Unlike the vimentin motif in which even conserved sequence changes affect filament assembly, the GFAP consensus sequence, MERRRITS-ARRSY, can be replaced by a completely unrelated sequence; namely, the heptapeptide, MVRANKR, derived from the lambda cII protein. When fused to GFAP sequences with sequential deletions of the N-terminal domain, the lambda cII heptapeptide was used to help identify a second motif, termed the RP-box, which is located just upstream of the GFAP alpha-helical rod domain. This RP-box affected the efficiency of filament assembly as well as protein-protein interactions in the filament, as shown by sedimentation assays and electron microscopy. These results are supported by previous data, which showed that the dramatic reorganization of GFAP within cells was due to phosphorylation-dephosphorylation of a site located in this RP-box. The results in this study suggest the RP-box motif to be a key modulator in the mechanism of GFAP assembly, and support a role for this motif in both the nucleation and elongation phases of filament assembly. The RP-box motif in GFAP has the consensus sequence, RLSL-RM-PP. Sequences similar to the GFAP RP-box motif are also to be found in vimentin, desmin and peripherin. Like GFAP, these include phosphorylation and proteolysis sites and are adjacent to the start of the central alpha-helical rod domain, suggesting that this motif of general importance to type III intermediate filament protein assembly.

Amino Acid Sequence↗

PubMed1992

The 53kDa polypeptide component of the bovine fibre cell cytoskeleton is derived from the 115kDa beaded filament protein: evidence for a fibre cell specific intermediate filament protein.

The 115kDa protein found enriched in the PMCC (plasma membrane-cytoskeleton complex) fraction of the cortex in bovine lens fibre cells is proteolytically processed to a stable 53kDa product. The 115 kDa protein and the 53kDa polypeptide have been purified by a combination of ion exchange and hydroxyapatite chromatography. Tryptic peptide mapping using reverse phase HPLC and subsequent peptide sequencing confirmed that the 53kDa polypeptide is derived from the 115kDa protein. The 53kDa fragment is also a component of the PMCC as well as being a major component of the urea soluble fraction of lens plasma membranes which have been extracted with buffers containing 1M KC1. The 53kDa polypeptide has escaped identification as a breakdown product of the 115kDa protein because it is not recognised by a commonly used monoclonal antibody, R2D2, specific for the bovine 115kDa protein. This result suggests that proteolysis is important in determining the function(s) of the 115kDa protein, and that part of this function is satisfied by the 53kDa protein core. Both the purified 115kDa protein and the 53kDa polypeptide were unable to form either beaded or intermediate filaments on their own but they were able to form short 10nm rods indicative of an intermediate stage in intermediate filament assembly. Comparison ot the assembly properties of the 53 and 115kDa proteins indicate that there are sequences in the 115kDa protein which inhibit in vitro assembly. This is similar to the situation with neurofilament proteins. We suggest that the 115kDa protein is a lens-specific intermediate filament protein.

Amino Acid Sequence↗

PubMed1975

Handling and staining epoxy resin sections for light microscopy.

Epoxy resin sections 0 1-1.0 mum thick of specimens embedded for electron microscopy, were collected from the ultramicrotome using strips of cover slip, and, after drying on a hot plate, were bulk stained in specially made troughs. After treatment with periodic acid, the sections were stained first in basic fuchsin at 70 degrees C and then in alkaline methylene blue at room temperature. The handling technique allows accurate collection of serial sections without interrupting the sectioning process, and the stain combination is suitable for different specimens in different epoxy resins and is unaffected by storage for more than 1 year.

Epoxy Resins↗