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Biomedical subjects

P A Coulombe

Publications and source records attributed to P A Coulombe.

At least 19 recordsLinked to original sources
PubMed2001

The nonhelical tail domain of keratin 14 promotes filament bundling and enhances the mechanical properties of keratin intermediate filaments in vitro.

Keratin filaments arise from the copolymerization of type I and II sequences, and form a pancytoplasmic network that provides vital mechanical support to epithelial cells. Keratins 5 and 14 are expressed as a pair in basal cells of stratified epithelia, where they occur as bundled arrays of filaments. In vitro, bundles of K5-K14 filaments can be induced in the absence of cross-linkers, and exhibit enhanced resistance to mechanical strain. This property is not exhibited by copolymers of K5 and tailless K14, in which the nonhelical tail domain has been removed, or copolymers of K5 and K19, a type I keratin featuring a short tail domain. The purified K14 tail domain binds keratin filaments in vitro with specificity (kD approximately 2 microM). When transiently expressed in cultured cells, the K14 tail domain associates with endogenous keratin filaments. Utilization of the K14 tail domain as a bait in a yeast two-hybrid screen pulls out type I keratin sequences from a skin cDNA library. These data suggest that the tail domain of K14 contributes to the ability of K5-K14 filaments to self-organize into large bundles showing enhanced mechanical resilience in vitro.

Animals↗
PubMed2001

A reporter transgene based on a human keratin 6 gene promoter is specifically expressed in the periderm of mouse embryos.

We report the developmental regulation of a lacZ reporter transgene fused to the promoter region of the human keratin 6a gene. In mouse embryos, the transgene is expressed in the periderm (the outermost layer of embryonic epidermis), as are the endogenous keratin 6 alpha and beta genes. A subset of periderm cells, localized to temporary epithelial fusions, is known to contain keratin 6 protein, and we find that these cells also harbor LacZ enzymatic activity.

Animals↗
PubMed2001

A 'hot-spot' mutation alters the mechanical properties of keratin filament networks.

Keratins 5 and 14 polymerize to form the intermediate filament network in the progenitor basal cells of many stratified epithelia including epidermis, where it provides crucial mechanical support. Inherited mutations in K5 or K14 result in epidermolysis bullosa simplex (EBS), a skin-fragility disorder. The impact that such mutations exert on the intrinsic mechanical properties of K5/K14 filaments is unknown. Here we show, by using differential interference contrast microscopy, that a 'hot-spot' mutation in K14 greatly reduces the ability of reconstituted mutant filaments to bundle under crosslinking conditions. Rheological assays measure similar small-deformation mechanical responses for crosslinked solutions of wild-type and mutant keratins. The mutation, however, markedly reduces the resilience of crosslinked networks against large deformations. Single-particle tracking, which probes the local organization of filament networks, shows that the mutant polymer exhibits highly heterogeneous structures compared to those of wild-type filaments. Our results indicate that the fragility of epithelial cells expressing mutant keratin may result from an impaired ability of keratin polymers to be crosslinked into a functional network.

Biophysical Phenomena↗
PubMed2001

Increased levels of keratin 16 alter epithelialization potential of mouse skin keratinocytes in vivo and ex vivo.

The process of wound repair in adult skin is complex, involving dermal contraction and epithelial migration to repair the lesion and restore the skin's barrier properties. At the wound edge, keratinocytes undergo many changes that engender an epithelialization behavior. The type II keratin 6 and type I keratins 16 and 17 are induced well before cell migration begins, but the role of these proteins is not understood. Forced expression of human K16 in skin epithelia of transgenic mice has been shown to cause dose-dependent skin lesions concomitant with alterations in keratin filament organization and in cell adhesion. Here we show, with the use of a quantitative assay, that these transgenic mice show a delay in the closure of full-thickness skin wounds in situ compared with wild-type and low-expressing K16 transgenic mice. We adapted and validated an ex vivo skin explant culture system to better assess epithelialization in a wound-like environment. Transgenic K16 explants exhibit a significant reduction of keratinocyte outgrowth in this setting. This delay is transgene dose-dependent, and is more severe when K16 is expressed in mitotic compared with post-mitotic keratinocytes. Various lines of evidence suggest that the mechanism(s) involved is complex and not strictly cell autonomous. These findings have important implications for the function of K16 in vivo.

Animals↗
PubMed2000

Introducing a null mutation in the mouse K6alpha and K6beta genes reveals their essential structural role in the oral mucosa.

Mammalian genomes feature multiple genes encoding highly related keratin 6 (K6) isoforms. These type II keratins show a complex regulation with constitutive and inducible components in several stratified epithelia, including the oral mucosa and skin. Two functional genes, K6alpha and K6beta, exist in a head-to-tail tandem array in mouse genomes. We inactivated these two genes simultaneously via targeting and homologous recombination. K6 null mice are viable and initially indistinguishable from their littermates. Starting at two to three days after birth, they show a growth delay associated with reduced milk intake and the presence of white plaques in the posterior region of dorsal tongue and upper palate. These regions are subjected to greater mechanical stress during suckling. Morphological analyses implicate the filiform papillae as being particularly sensitive to trauma in K6alpha/K6beta null mice, and establish the complete absence of keratin filaments in their anterior compartment. All null mice die about a week after birth. These studies demonstrate an essential structural role for K6 isoforms in the oral mucosa, and implicate filiform papillae as being the major stress bearing structures in dorsal tongue epithelium.

Animals↗
PubMed2000

The 'ins' and 'outs' of intermediate filament organization.

A major function shared by several types of cytoplasmic intermediate filaments (IFs) is to stabilize cellular architecture against the mechanical forces it is subjected to. As for other fibrous cytoskeletal arrays, a crucial determinant of this function is the spatial organization of IFs in the cytoplasm. However, very few crossbridging proteins are specific for IFs - most IF-associated proteins known to exert a structural role act to tether IFs to other major cytoskeletal elements, such as F-actin, microtubules or adhesion complexes. In addition, IFs are endowed with the ability to participate in their own organization. This intriguing property is probably connected to the unusual degree of sequence diversity and sequence-specific regulation that characterize IF genes and their proteins. This dependence upon a combination of extrinsic and intrinsic determinants contributes to distinguish IFs from other fibrous cytoskeletal polymers and is key to their function.

Eukaryotic Cells↗
PubMed2000

Keratin 17 expression in the hard epithelial context of the hair and nail, and its relevance for the pachyonychia congenita phenotype.

The hard-keratin-containing portion of the murine hair shaft displays a positive immunoreactivity with an antibody against the soft epithelial keratin, K17. The K17-expressing cell population is located in the medulla compartment of the hair. Consistent with this observation, K17-containing cells also occur in the presumptive medulla precursor cells located in the hair follicle matrix. Western blot analysis of hair extracts prepared from a number of mouse strains confirms this observation and suggests that K17 expression in the hair shaft is a general trait in this species. The expression of K17 in human hair extracts is restricted to eyebrow and facial hair samples. These are the major sites for the occurrence of the pili torti (twisted hair) phenotype in the type 2 (Jackson-Lawler) form of pachyonychia congenita, previously shown to arise from inherited K17 mutations. Given that all forms of pachyonychia congenita show an involvement of the nail, we compared the expression of the two other genes mutated in pachyonychia congenita diseases, K6 and K16, with that of K17 in human nail. All three keratins are abundantly expressed within the nail bed epithelium, whereas K17 protein is expressed in the nail matrix, which contains the epithelial cell precursors for the nail plate. Our data suggest a role for K17 in the formation and maintenance of various skin appendages and directly support the concept that pachyonychia congenita is a disease of the nail bed.

Animals↗
PubMed2000

Forced expression of keratin 16 alters the adhesion, differentiation, and migration of mouse skin keratinocytes.

Injury to the skin results in an induction of keratins K6, K16, and K17 concomitant with activation of keratinocytes for reepithelialization. Forced expression of human K16 in skin epithelia of transgenic mice causes a phenotype that mimics several aspects of keratinocyte activation. Two types of transgenic keratinocytes, with forced expression of either human K16 or a K16-C14 chimeric cDNA, were analyzed in primary culture to assess the impact of K16 expression at a cellular level. High K16-C14-expressing and low K16-expressing transgenic keratinocytes behave similar to wild type in all aspects tested. In contrast, high K16-expressing transgenic keratinocytes show alterations in plating efficiency and calcium-induced differentiation, but proliferate normally. Migration of keratinocytes is reduced in K16 transgenic skin explants compared with controls. Finally, a subset of high K16-expressing transgenic keratinocytes develops major changes in the organization of keratin filaments in a time- and calcium concentration-dependent manner. These changes coincide with alterations in keratin content while the steady-state levels of K16 protein remain stable. We conclude that forced expression of K16 in progenitor skin keratinocytes directly impacts properties such as adhesion, differentiation, and migration, and that these effects depend upon determinants contained within its carboxy terminus.

Amino Acid Sequence↗
PubMed2000

Dose-dependent linkage, assembly inhibition and disassembly of vimentin and cytokeratin 5/14 filaments through plectin's intermediate filament-binding domain.

Plectin, the largest and most versatile member of the cytolinker/plakin family of proteins characterized to date, has a tripartite structure comprising a central 200 nm-long (&agr;)-helical rod domain flanked by large globular domains. The C-terminal domain comprises a short tail region preceded by six highly conserved repeats (each 28-39 kDa), one of which (repeat 5) contains plectin's intermediate filament (IF)-binding site. We used recombinant and native proteins to assess the effects of plectin repeat 5-binding to IF proteins of different types. Quantitative Eu(3+)-based overlay assays showed that plectin's repeat 5 domain bound to type III IF proteins (vimentin) with preference over type I and II cytokeratins 5 and 14. The ability of both types of IF proteins to self-assemble into filaments in vitro was impaired by plectin's repeat 5 domain in a concentration-dependent manner, as revealed by negative staining and rotary shadowing electron microscopy. This effect was much more pronounced in the case of vimentin compared to cytokeratins 5/14. Preassembled filaments of both types became more and more crosslinked upon incubation with increasing concentrations of plectin repeat 5. However, at high proportions of plectin to IF proteins, disassembly of filaments occurred. Again, vimentin filaments proved considerably more sensitive towards disassembly than those composed of cytokeratins 5 and 14. In general, IFs formed from recombinant proteins were found to be slightly more responsive towards plectin influences than their native counterparts. A dose-dependent plectin-inflicted collapse and putative disruption of IFs was also observed in vivo after ectopic expression of vimentin and plectin's repeat 5 domain in cotransfected vimentin-deficient SW13 (vim(-)) cells. Our results suggest an involvement of plectin not only in crosslinking and stabilization of cytoskeletal IF networks, but also in regulation of their dynamics.

Animals↗
PubMed1999

The functional diversity of epidermal keratins revealed by the partial rescue of the keratin 14 null phenotype by keratin 16.

The type I epidermal keratins K14 and K16 are remarkably similar at the primary sequence level. While a structural function has been clearly defined for K14, we have proposed that a function of K16 may be to play a role in the process of keratinocyte activation that occurs after acute injury to stratified epithelia. To compare directly the functions of the two keratins we have targeted the expression of the human K16 cDNA to the progenitor basal layer of the epidermis of K14 null mice. Mice null for K14 blister extensively and die approximately 2 d after birth (Lloyd, C., Q.C. Yu, J. Cheng, K. Turksen, L. Degenstein, E. Hutton, and E. Fuchs. 1995. J. Cell Biol. 129:1329-1344). The skin of mice expressing K16 in the absence of K14 developed normally without evidence of blistering. However, as the mice aged they featured extensive alopecia, chronic epidermal ulcers in areas of frequent physical contact, and alterations in other stratified epithelia. Mice expressing a control K16-C14 cDNA also rescue the blistering phenotype of the K14 null mice with only a small percentage exhibiting minor alopecia. While K16 is capable of rescuing the blistering, phenotypic complementation in the resulting skin is incomplete due to the multiple age dependent anomalies. Despite their high sequence similarity, K16 and K14 are not functionally equivalent in the epidermis and other stratified epithelia and it is primarily the carboxy-terminal approximately 105 amino acids of K16 that define these differences.

Aging↗
PubMed1999

Keratin filament suspensions show unique micromechanical properties.

All epithelial cells feature a prominent keratin intermediate filament (IF) network in their cytoplasm. Studies in transgenic mice and in patients with inherited epithelial fragility syndromes showed that a major function of keratin IFs is to provide mechanical support to epithelial cell sheets. Yet the micromechanical properties of keratin IFs themselves remain unknown. We used rheological methods to assess the properties of suspensions of epidermal type I and type II keratin IFs and of vimentin, a type III IF polymer. We find that both types of IFs form gels with properties akin to visco-elastic solids. With increasing deformation they display strain hardening and yield relatively rapidly. Remarkably, both types of gels recover their preshear properties upon cessation of the deformation. Repeated imposition of small deformations gives rise to a progressively stiffer gel for keratin but not vimentin IFs. The visco-elastic moduli of both gels show a weak dependence upon the frequency of the input shear stress and the concentration of the polymer, suggesting that both steric and nonsteric interactions between individual polymers contribute to the observed mechanical properties. In support of this, the length of individual polymers contributes only modestly to the properties of IF gels. Collectively these properties render IFs unique among cytoskeletal polymers and have strong implications for their function in vivo.

Animals↗
PubMed1999

Using transgenic models to study the pathogenesis of keratin-based inherited skin diseases.

In the past decade, the production of transgenic animals whose genome is modified to contain DNA transgenes of interest has significantly contributed to expand our understanding of the molecular etiology and pathobiology of several inherited skin diseases. This technology has led to the discovery that mutations affecting specific keratin genes are responsible for a wide spectrum of inherited bullous diseases, which are collectively characterized by blistering after minor trauma. Type I and type II keratin proteins are restricted to, and very abundant in, epithelial cells, where they occur as a pancytoplasmic network of cytoskeletal filaments. Although it had long been suspected that a primary function of keratin filaments may be to contribute to the physical strength of epithelial sheets, a formal demonstration came from studies of transgenic mouse models and patients suffering from keratin-based blistering diseases. Here we review the basic characteristics of keratin gene and their proteins and relate them to the molecular pathogenesis of relevant inherited skin blistering diseases. A particular emphasis is placed on the role of transgenic mouse models in the past, current, and future studies of these genodermatoses.

Animals↗
PubMed1998

The type I keratin 19 possesses distinct and context-dependent assembly properties.

Keratins (K), the cytoplasmic intermediate filament (IF) proteins of epithelial cells, are encoded by a multigene family and expressed in a tissue- and differentiation-specific manner. In human skin, keratinocytes of the basal layer of epidermis and the outer root sheath of hair follicles express K5 and K14 as their main keratins. A small subpopulation of basal cells exhibiting stem-cell like characteristics express, in addition, K19. At 40 kDa, this keratin is the smallest IF protein due to an exceptionally short carboxyl-terminal domain. We examined the assembly properties of K19 and contrasted them to K14 in vitro and in vivo. Relative to K5-K14, we find that K5-K19 form less stable tetramers that polymerize into shorter and narrower IFs in vitro. When transiently co-expressed in cultured baby hamster kidney cells, the K5 and K19 combination fails to form a filamentous array, whereas the K5-K14 and K8-K19 ones readily do so. Transient expression of K19 in the epithelial cell lines T51B-Ni and A431 results in its integration into the endogenous keratin network with minimal if any perturbation. Collectively, these results indicate that K19 possesses assembly properties that are distinct from those of K14 and suggest that it may impart unique properties to the basal cells expressing it in skin epithelia.

Animals↗
PubMed1998

Onset of keratin 17 expression coincides with the definition of major epithelial lineages during skin development.

The type I keratin 17 (K17) shows a peculiar localization in human epithelial appendages including hair follicles, which undergo a growth cycle throughout adult life. Additionally K17 is induced, along with K6 and K16, early after acute injury to human skin. To gain further insights into its potential function(s), we cloned the mouse K17 gene and investigated its expression during skin development. Synthesis of K17 protein first occurs in a subset of epithelial cells within the single-layered, undifferentiated ectoderm of embryonic day 10.5 mouse fetuses. In the ensuing 48 h, K17-expressing cells give rise to placodes, the precursors of ectoderm-derived appendages (hair, glands, and tooth), and to periderm. During early development, there is a spatial correspondence in the distribution of K17 and that of lymphoid-enhancer factor (lef-1), a DNA-bending protein involved in inductive epithelial-mesenchymal interactions. We demonstrate that ectopic lef-1 expression induces K17 protein in the skin of adult transgenic mice. The pattern of K17 gene expression during development has direct implications for the morphogenesis of skin epithelia, and points to the existence of a molecular relationship between development and wound repair.

Age Factors↗
PubMed1998

Functional differences between keratins of stratified and simple epithelia.

Dividing populations of stratified and simple epithelial tissues express keratins 5 and 14, and keratins 8 and 18, respectively. It has been suggested that these keratins form a mechanical framework important to cellular integrity, since their absence gives rise to a blistering skin disorder in neonatal epidermis, and hemorrhaging within the embryonic liver. An unresolved fundamental issue is whether different keratins perform unique functions in epithelia. We now address this question using transgenic technology to express a K16-14 hybrid epidermal keratin transgene and a K18 simple epithelial keratin transgene in the epidermis of mice null for K14. Under conditions where the hybrid epidermal keratin restored a wild-type phenotype to newborn epidermis, K18 partially but not fully rescued. The explanation does not appear to reside in an inability of K18 to form 10-nm filaments with K5, which it does in vitro and in vivo. Rather, it appears that the keratin network formed between K5 and K18 is deficient in withstanding mechanical stress, leading to perturbations in the keratin network in regions of the skin that are subjected either to natural or to mechanically induced trauma. Taken together, these findings suggest that the loss of a type I epidermal keratin cannot be fully compensated by its counterpart of simple epithelial cells, and that in vivo, all keratins are not equivalent.

Animals↗
PubMed1998

The two functional keratin 6 genes of mouse are differentially regulated and evolved independently from their human orthologs.

The type II keratin 6 (K6) features a complex expression pattern, with a constitutive component in a subset of stratified epithelia and an inducible component following injury and other types of acute challenges. Multiple genes encoding highly related K6 isoforms have been described for human and bovine, a unique feature among mammalian keratin genes. Here we report on the cloning and characterization of two functional genes and their cDNAs encoding the K6 isoforms in mouse and two related pseudogenes. A systematic comparison of the mouse and human K6 genes suggests that they evolved independently after these species diverged. The mK6alpha and mK6beta genes are organized in tandem with the same transcriptional orientation in the mouse genome. Similar to the human isoforms, the coding sequences for mK6alpha and mK6beta isoforms show approximately 95% identity. The two mouse K6 genes are differentially regulated at the mRNA level in several stratified epithelia. The mK6alpha isoform mRNA clearly predominates in intact trunk skin of adult mice, where it is restricted to the outer root sheath of hair follicles. Both mRNAs are induced in epidermis and proximal hair follicles as early as 1 h following acute injury or topical application of phorbol esters and subsequently increase to a comparable extent but with different kinetics. These novel findings have important implications for the evolution, regulation, and function of K6 genes in mammalian species.

Amino Acid Sequence↗
PubMed1998

A dysfunctional desmin mutation in a patient with severe generalized myopathy.

Mice lacking desmin produce muscle fibers with Z disks and normal sarcomeric organization. However, the muscles are mechanically fragile and degenerate upon repeated contractions. We report here a human patient with severe generalized myopathy and aberrant intrasarcoplasmic accumulation of desmin intermediate filaments. Muscle tissue from this patient lacks the wild-type desmin allele and has a desmin gene mutation encoding a 7-aa deletion within the coiled-coil segment of the protein. We show that recombinant desmin harboring this deletion cannot form proper desmin intermediate filament networks in cultured cells, nor is it able to assemble into 10-nm filaments in vitro. These findings provide direct evidence that a mutation in desmin can cause human myopathies.

Amino Acid Sequence↗