Microinjection of intermediate filament proteins into living cells with and without preexisting intermediate filament network

Human cells grown in monolayer culture were microinjected with intermediate filament subunit proteins. In fibroblasts with a preexisting vimentin network, injected porcine glial fibrillary acidic protein (GFAP) co-localized with the vimentin network within 24 hours. Phosphorylated GFAP variants were found to become dephosphorylated concomitantly with their incorporation into filamentous structures. After microinjection of either porcine GFAP or murine vimentin into human carcinoma cells lacking cytoplasmic intermediate filaments, we observed that different types of filament networks developed. Whereas vimentin was incorporated into short filaments immediately after injection, GFAP was found to aggregate into rodlike structures. This may indicate a differential filament forming ability of these intermediate filament proteins in vivo.     

Disruption of the keratin filament network during epithelial cell division

The behaviour of keratin filaments during cell division was examined in a wide range of epithelial lines from several species. Almost half of them show keratin disruption as described previously: by immunofluorescence, filaments are replaced during mitosis by a 'speckled' pattern of discrete cytoplasmic dots. In the electron microscope these ' speckles ' are seen as granules around the cell periphery, just below the actin cortical mesh, with no detectable 10 nm filament structure inside them and no keratin filament bundles in the rest of the cytoplasm. A time course of the filament reorganization was constructed from double immunofluorescence data; filaments are disrupted in prophase, and the filament network is intact again by cytokinesis. The phenomenon is restricted to cells rich in keratin filaments, such as keratinocytes; it is unrelated to the co-existence of vimentin in many of these cells, and vimentin is generally maintained as filaments while the keratin is restructured. Some resistance to the effect may be conferred by an extended cycle time. Filament reorganization takes place within minutes, so that a reversible mechanism seems more likely than one involving de novo protein synthesis, at this metabolically quiet stage of the cell cycle.     

Fluorogenic substrate turnover in single living cells

Synopsis Intracellular diffusion properties and enzyme activities in single living cells can be analysed by means of fluorogenic substrates that diffuse into the cells where they are converted into a fluorescent product by an enzymic reaction. The reaction-kinetic analysis of this process as a system of consecutive reactions provides information on the diffusion of the substrate into the cells, on intracellular enzyme activities and on the efflux of the fluorescent product. Separation of diffusion and enzyme-mediated processes is obtained by inducing specific changes of the cellular membrane using gramicidin D. A model for the functional interpretation of the experimental findings is proposed. Application of this method as a viability test for freshly prepared and frozen platelets is discussed.Paper given at the Royal Microscopical Society's European Histochemistry Meeting at Nottingham in September 1975.     

Molecular packing in the feather keratin filament

Avian feathers have a filament-matrix texture and X-ray diffraction studies show that the filament has a helical structure with four repeating units per turn. Each repeating unit consists of a pair of twisted beta-sheets related by a perpendicular diad, and the twist in the sheets is of opposite hand to that of the helix. Each sheet is believed to comprise a 32-residue segment of the feather keratin molecule, which contains around 100 residues, the remainder constituting the matrix. In the present contribution, the sequence of emu feather is mapped to the low-resolution model derived earlier from X-ray studies. This shows that the inner surface of the "beta-sandwich" is densely populated by hydrophobic residues and that the charged residues and cysteine residues lie on the outer surface. In addition, the inner residues in the repeating unit mesh neatly together in layers oriented perpendicular to the filament axis. Amino acid sequences from a range of avian and reptilian keratins were collected and a 32-residue segment corresponding to the filament framework could be identified in every case, supporting the notion that there is a common plan for the filament framework in all of these materials. The hairpin turns in the beta-sheet were also identified and shown to be unusually rich in proline residues and also of variable composition. Two variants of the mapping were found which have complimentary conformations of the hairpin turns and these are illustrated and discussed. Since feather keratin yields a fiber rather than a crystalline X-ray pattern refinement of the model is restricted to trial-and-error methods and the assumptions made in its derivation are critically examined and some possible modifications discussed.     

Aggregation of wool keratin intermediate filament proteins

The wool keratin intermediate filament proteins were isolated as their S-carboxymethyl derivatives (S-carboxymethylkerateine A, SCMKA) and purified by gel filtration to remove residual non-helical protein of low molecular weight. The alpha-helix content of purified SCMKA was approximately 62% in agreement with that predicted for the alpha-helical coiled-coil segments from the amino acid sequences of the subunits. In aqueous buffer at pH 11 or in n-propanol (20% v/v) at pH 9.2 very large aggregates are dissociated and SCMKA exists largely as a mixture of the dimer (two-chain coiled-coil of Mr approximately 103,000) and the tetramer. The protein species are not in rapidly reversible equilibrium as judged from gel filtration and sedimentation equilibrium. It is probable that species with a range of association constants are present. The equilibrium is shifted towards the dimer with change of pH from 9.2 to 11 or by the addition of 20% (v/v) n-propanol. The tetrameric proteolytic digestion product which is derived from the 1B segment of the alpha-helical rod section of the keratin molecule dissociates in a similar way to intact SCMKA with increase of pH and in the presence of n-propanol. This indicates the importance of this region of the rod domain in the initial stages of the assembly of the filament. Electrostatic and hydrophobic interactions are implicated in the association of the two-chain coiled-coil to the tetramer both in intact SCMKA and the 1B segment tetramer. The results are discussed in relation to the intact dimeric and tetrameric complexes obtained from other intermediate filament types.     

Spatiotemporal analysis of flow-induced intermediate filament displacement in living endothelial cells

The distribution of hemodynamic shear stress throughout the arterial tree is transduced by the endothelium into local cellular responses that regulate vasoactivity, vessel wall remodeling, and atherogenesis. Although the exact mechanisms of mechanotransduction remain unknown, the endothelial cytoskeleton has been implicated in transmitting extracellular force to cytoplasmic sites of signal generation via connections to the lumenal, intercellular, and basal surfaces. Direct observation of intermediate filament (IF) displacement in cells expressing green fluorescent protein-vimentin has suggested that cytoskeletal mechanics are rapidly altered by the onset of fluid shear stress. Here, restored images from time-lapse optical sectioning fluorescence microscopy were analyzed as a four-dimensional intensity distribution function that represented IF positions. A displacement index, related to the product moment correlation coefficient as a function of time and subcellular spatial location, demonstrated patterns of IF displacement within endothelial cells in a confluent monolayer. Flow onset induced a significant increase in IF displacement above the nucleus compared with that measured near the coverslip surface, and displacement downstream from the nucleus was larger than in upstream areas. Furthermore, coordinated displacement of IF near the edges of adjacent cells suggested the existence of mechanical continuity between cells. Thus, quantitative analysis of the spatiotemporal patterns of flow-induced IF displacement suggests redistribution of intracellular force in response to alterations in hemodynamic shear stress acting at the lumenal surface.     

Elucidating the early stages of keratin filament assembly

Because of extraordinarily tight coiled-coil associations of type I and type II keratins, the composition and structure of keratin subunits has been difficult to determine. We report here the use of novel genetic and biochemical methods to explore the early stages of keratin filament assembly. Using bacterially expressed humans K5 and K14, we show that remarkably, these keratins behave as 1:1 complexes even in 9 M urea and in the presence of a reducing agent. Gel filtration chromatography and chemical cross-linking were used to identify heterodimers and heterotetramers as the most stable building blocks of keratin filament assembly. EM suggested that the dimer consists of a coiled-coil of K5 and K14 aligned in register and in parallel fashion, and the tetramer consists of two dimers in antiparallel fashion, without polarity. In 4 M urea, both end-to-end and lateral packing of tetramers occurred, leading to a variety of larger heteromeric complexes. The coexistence of multiple, higher-ordered associations under strongly denaturing conditions suggests that there may not be a serial sequence of events leading to the assembly of keratin intermediate filaments, but rather a number of associations may take place in parallel.     

Insights into the dynamic properties of keratin intermediate filaments in living epithelial cells

The properties of keratin intermediate filaments (IFs) have been studied after transfection with green fluorescent protein (GFP)-tagged K18 and/or K8 (type I/II IF proteins). GFP-K8 and -K18 become incorporated into tonofibrils, which are comprised of bundles of keratin IFs. These tonofibrils exhibit a remarkably wide range of motile and dynamic activities. Fluorescence recovery after photobleaching (FRAP) analyses show that they recover their fluorescence slowly with a recovery t(1/2) of approximately 100 min. The movements of bleach zones during recovery show that closely spaced tonofibrils (<1 microm apart) often move at different rates and in different directions. Individual tonofibrils frequently change their shapes, and in some cases these changes appear as propagated waveforms along their long axes. In addition, short fibrils, termed keratin squiggles, are seen at the cell periphery where they move mainly towards the cell center. The motile properties of keratin IFs are also compared with those of type III IFs (vimentin) in PtK2 cells. Intriguingly, the dynamic properties of keratin tonofibrils and squiggles are dramatically different from those of vimentin fibrils and squiggles within the same cytoplasmic regions. This suggests that there are different factors regulating the dynamic properties of different types of IFs within the same cytoplasmic regions.     

Vimentin and keratin intermediate filament systems in cultured PtK2 epithelial cells are interrelated.

Certain cultured epithelial cells contain separate vimentin and keratin-type intermediate filament networks. The intracellular injection of monoclonal antibodies directed against either vimentin or keratin filaments into PtK2 cultured epithelial cells specifically disrupted the organization of both filament types. Neither antibody had any effect when injected into cells which, while containing vimentin or keratin filaments, lacked the specific filament type which that antibody recognized. These experiments suggest that keratin and vimentin filament networks are associated in some way with one another.     

Mouse differentiation-specific keratins 1 and 10 require a preexisting keratin scaffold to form a filament network

Keratins 1 (K1) and 10 (K10) are the predominant cytoskeletal intermediate filaments of epidermal cells during transition from the proliferative to the terminal differentiation stage. In situ, formation of the K1/K10 intermediate filament network occurs in the cytoplasm of cells with a preexisting cytoskeleton composed of keratins 5 and 14. To define cytoskeletal interactions permissive for formation of the K1/K10 filamentous network, active copies of mouse K1 and K10 genes were introduced into fibroblasts (NIH 3T3) which do not normally express these proteins. Transient and stable transfectants, as well as heterokaryons produced by fusions with epithelial cells, were evaluated for expression of K1 and K10 proteins and filament formation using specific antibodies. In contrast to keratin pairs K5/K14 and K8/K18, the K1/K10 pair failed to form an extensive keratin filament network on its own, although small isolated dense K1/K10 filament bundles were observed throughout the cytoplasm by EM. K1 and K10 filaments integrated only into the preexisting K5/K14 network upon fusion of the NIH 3T3 (K1/K10) cells with epithelial cells expressing endogenous K5/K14 or with NIH 3T3 cells which were transfected with active copies of the K5 and K14 genes. When combinations of active recombinant gene constructs for keratins 1, 5, 10, and 14 were tested in transient NIH 3T3 transfections, the most intact cytokeratin network observed by immunofluorescence was formed by the K5/K14 pair. The K1/K14 pair was capable of forming a cytoskeletal network, but the network was poorly developed, and usually perinuclear. Transfection of K10 in combination with K5 or K1 resulted in cytoplasmic agglomerates, but not a cytoskeleton. These results suggest that the formation of the suprabasal cytoskeleton in epidermis is dependent on the preexisting basal cell intermediate filament network. Furthermore, restrictions on filament formation appear to be more stringent for K10 than for K1.     

The intermediate filament protein keratin 8 is a novel cytoplasmic substrate for c-Jun N-terminal kinase.

Keratins 8 (K8) and 18 are the primary intermediate filaments of simple epithelia. Phosphorylation of keratins at specific sites affects their organization, assembly dynamics, and their interaction with signaling molecules. A number of keratin in vitro and in vivo phosphorylation sites have been identified. One example is K8 Ser-73, which has been implicated as an important phosphorylation site during mitosis, cell stress, and apoptosis. We show that K8 is strongly phosphorylated on Ser-73 upon stimulation of the pro-apoptotic cytokine receptor Fas/CD95/Apo-1 in HT-29 cells. Kinase assays showed that c-Jun N-terminal kinase (JNK) was also activated with activation kinetics corresponding to that of K8 phosphorylation. Furthermore, K8 was also phosphorylated on Ser-73 by JNK in vitro, yielding similar phosphopeptide maps as the in vivo phosphorylated material. In addition, co-immunoprecipitation studies revealed that part of JNK is associated with K8 in vivo, correlating with decreased ability of JNK to phosphorylate the endogenous c-Jun. Taken together, K8 is a new cytoplasmic target for JNK in Fas receptor-mediated signaling. The functional significance of this phosphorylation could relate to regulation of JNK signaling and/or regulation of keratin dynamics.     

Subunit structure of the mouse epidermal keratin filament.

The two proteins which are the subunits of mouse epidermal keratin filaments have been isolated from fully differentiated epidermis (stratum corneum), viable differentiating cells and cells grown in culture. The proteins have molecular weights of 68 000 and 60 000, consist of families of very similar species, have common N-terminal (N-acetylserine) and C-terminal (glycine) residues, contain 35--40% alpha-helix and are immunologically cross-reacting. In mixtures, the two proteins polymerize in vitro into native-type keratin filaments that are 70--80 angstrom in diameter, up to 30 micrograms long, possess a characteristic alpha-type X-ray diffraction pattern and contain the subunits in the precise molar ratio of 1 : 2 or 2 : 1.     

Robust organizational principles of protrusive biopolymer networks in migrating living cells

Cell migration is associated with the dynamic protrusion of a thin actin-based cytoskeletal extension at the cell front, which has been shown to consist of two different substructures, the leading lamellipodium and the subsequent lamellum. While the formation of the lamellipodium is increasingly well understood, organizational principles underlying the emergence of the lamellum are just beginning to be unraveled. We report here on a 1D mathematical model which describes the reaction-diffusion processes of a polarized actin network in steady state, and reproduces essential characteristics of the lamellipodium-lamellum system. We observe a steep gradient in filament lengths at the protruding edge, a local depolymerization maximum a few microns behind the edge, as well as a differential dominance of the network destabilizer ADF/cofilin and the stabilizer tropomyosin. We identify simple and robust organizational principles giving rise to the derived network characteristics, uncoupled from the specifics of any molecular implementation, and thus plausibly valid across cell types. An analysis of network length dependence on physico-chemical system parameters implies that to limit array treadmilling to cellular dimensions, network growth has to be truncated by mechanisms other than aging-induced depolymerization, e.g., by myosin-associated network dissociation at the transition to the cell body. Our work contributes to the analytical understanding of the cytoskeletal extension's bisection into lamellipodium and lamellum and sheds light on how cells organize their molecular machinery to achieve motility.     

Identification and characterization of a novel antioxidant peptide from feather keratin hydrolysate

Objectives

To improve the potential value of feather, which is a valuable protein resource, we have separated and identified antioxidant peptide(s) from feather hydrolysate.

Results

Feather hydrolysate was prepared by fermentation with Bacillus subtilis S1–4. Antioxidative peptides were separated by sequential acid precipitation, cation exchange, and reversed-phase fast performance liquid chromatography. Finally, a peptide with antioxidative activity was identified as Ser-Asn-Leu-Cys-Arg-Pro-Cys-Gly by MALDI time-of-flight (TOF)/TOF analysis, and determined to represent a portion of feather keratin near its N-terminal. A synthesized peptide with the same sequence was used to characterize its antioxidative properties, including scavenging free radicals, reducing power, and Fe²⁺ chelation. In terms of the peptide’s amino acid composition, the antioxidative activity might be mainly attributed to Cys and other amino acid residues.

Conclusion

Feather keratin is a good source for the quantitative preparation of antioxidative peptides.