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.     

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.     

Structure of the three-chain unit of the bovine epidermal keratin filament

The characteristic α-type X-ray diffraction pattern displayed by bovine epidermal keratin filaments can be ascribed to the presence of segments of triple-chain coiled coil α-helix in the repeating three-chain unit of the filaments.Limited proteolysis of filaments polymerized in vitro or a citrate-soluble protein derived from them with crystalline trypsin releases two types of α-helix-enriched particles which provide information on the structure of the three-chain unit. The smaller, particle 2, of molecular weight 42,500, α-helix content of 92% and dimensions of 180 Å × 20 Å, consists of three chains aligned side-by-side that presumably form a coiled coil. The high yields of particle 2 allow the conclusion that all of the α-helix of the epidermal keratin filament is present in the form of these discrete three-chain α-helical segments. The larger, particle 1, recovered during the earlier stages of digestion has a molecular weight of 100,000 to 110,000, α-helix content of 75%, average dimensions of 400 Å × 20 Å and also consists of three chains aligned side-by-side. It contains two α-helical segments corresponding to particle 2 which are located at the amino -terminal and carboxyl-terminal ends and are separated by a region of non-helix. Particle 1 contains all of the α-helix and therefore is the major portion of the three-chain unit of the keratin filament. The products resulting from reaction of intact filament subunits with N-bromosuccinimide suggest that particle 1 is formed during digestion by removal of regions of non-helix from each end of this unit.The structure of the three-chain unit of the bovine epidermal keratin filament may thus be viewed as three polypeptide subunits aligned side-by-side with two discrete coiled coil α-helical segments interspersed with regions of non-helix.     

Sedimentation studies of epidermal keratins: keratin A and keratin B

Electrophoretically homogeneous keratin A and keratin B were studied in the ultracentrifuge. Both preparations revealed two fractions: one which sedimented rapidly and another which sedimented slowly. This indicated that both preparations are heterogeneous with respect to particle size.     

Mammalian epidermal keratin: isolation and characterization of the alpha-helical proteins from newborn rat.

Neutral buffer-insoluble proteins extracted from newborn rat epidermis with alkaline urea have been purified by chromatography on Sephadex G-150 columns run in the presence of sodium dodecyl sulfate. Two proteins with apparent molecular weights of 60 000 and 68 000, respectively have been isolated and characterized. Spectropolarimetric studies show both of them to be alpha-helical in contrast to the non-helical heavier and lighter species also solubilized with alkaline urea. The amino acid composition of the two proteins, their electrophoretic behavior and their immunological characteristics are essentially identical. Both proteins appear to be major constituents of rat epidermal tonofilaments.     

Mutations in the helix termination motif of mouse type I IRS keratin genes impair the assembly of keratin intermediate filament

Two classical mouse hair coat mutations, Rex (Re) and Rex wavy coat (Re(wc)), are linked to the type I inner root sheath (IRS) keratin genes of chromosome 11. An N-ethyl-N-nitrosourea-induced mutation, M100573, also maps close to the type I IRS keratin genes. In this study, we demonstrate that Re and M100573 mice bear mutations in the type I IRS gene Krt25; Re(wc) mice bear an additional mutation in the type I IRS gene Krt27. These three mutations are located in the helix termination motif of the 2B alpha-helical rod domain of a type I IRS keratin protein. Immunohistological analysis revealed abnormal foam-like immunoreactivity with an antibody raised to type II IRS keratin K71 in the IRS of Re/+ mice. These results suggest that the helix termination motif is essential for the proper assembly of types I and II IRS keratin protein complexes and the formation of keratin intermediate filaments.     

De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin

Poly(A)+ RNA isolated from bovine muzzle epidermis was microinjected into nonepithelial cells containing only intermediate-sized filaments of the vimentin type. In recipient cells keratin polypeptides are synthesized and assemble into intermediate-sized filaments at multiple dispersed sites. We describe the time course and the pattern of de novo assembly of keratin filaments within living cells. These filaments were indistinguishable, by immunofluorescence and immunoelectron microscopic criteria, from keratin filament arrays present in true epithelial cells. The presence of extended keratin fibril meshworks in these injected cells is compatible with cell growth and mitosis. Double immunolabeling revealed that newly assembled keratin was not codistributed with microfilament bundles, microtubules or vimentin filaments. We suggest that assembly mechanisms exist which in vivo sort out newly synthesized cytokeratin polypeptides from vimentin.     

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.     

Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro

To investigate the sequences important for assembly of keratins into 10- nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.     

Sak 57, an intermediate filament keratin present in intercellular bridges of rat primary spermatocytes

We have previously reported the purification of Sak 57 (for spermatogenic cell/sperm-associated keratin of molecular mass 57 kDa) from outer dense fibers of rat sperm tails. Internal protein sequence analysis of Sak 57 revealed 70–100% homology to the 1A and 2A regions of the α-helical rod domain of human, mouse, and rat keratins. A multiple antigen peptide was synthesized using the KQYEDIAQK sequence corresponding to the 2A region and a polyclonal antibody was produced in rabbit to detect Sak 57. During spermiogenesis, Sak 57 associates with the microtubular manchette before becoming a component of para-axonemal keratin structures of the developing tail. We now report that during late meiotic prophase, intercellular bridges linking late pachytene-diplotene spermatocytes display a distinct ribbon containing a Sak 57/β-tubulin complex, separated by a nonimmunoreactive midzone. Indirect immunofluorescence demonstrates that the ribbon is the final stage of a three-step developmental sequence: (1) a spindlelike arrangement radiating from equidistant spherical centers in early pachytene spermatocytes, (2) an ectoplasmic shell like framework in mid-to-late pachytene spermatocytes, and (3) a Sak 57/β-tubulin-containing ribbon found in intercellular bridges linking adjacent late pachytene-diplotene spermatocytes. Shear forces causing a breakdown of one of the conjoined spermatocytes do not disrupt the cytoskeletal ribbon. Results of this work, together with previous observations during spermiogenesis, show that Sak 57 associates with cytoplasmic microtubules in a timely fashion. Upon completion of late meiotic prophase, the Sak 57/microtubule complex behaves as an intercellular ligament and contributes to both the strength of intercellular bridges and the cohesiveness of members of a spermatocyte lineage. © 1996 Wiley-Liss, Inc.     

The cDNA sequence of a human epidermal keratin: divergence of sequence but conservation of structure among intermediate filament proteins

We have determined the DNA sequence of a cloned cDNA that is complementary to the mRNA for the 50 kilodalton (kd) human epidermal keratin. This provides the first amino acid sequence for a cytoskeletal keratin. Comparison of this sequence with those of other keratins reveals an evolutionary relationship between the cytoskeletal and the microfibrillar keratins, but shows no homology to matrix or feather keratins. The 50 kd keratin shares 28%-30% homology with partial sequences of other intermediate filament proteins, which suggests that keratins may be the most distantly related members of this class of fibrous proteins. Our computer analyses predict that the 50 kd keratin contains two long alpha-helical domains separated by a cluster of helix-inhibitory residues in the middle of the protein. These findings indicate that despite major sequence divergence among intermediate filament proteins, they retain sequences compatible with secondary structural features that appear to be common to all of them.     

Molecular modeling of vimentin filament assembly

Intermediate-filament forming proteins are known to form rod-shaped dimers that are calculated to be 45 nm in length. Molecular modeling indicates that the dimerization is promoted by interchain hydrophobic interactions between sections of α helix β and helix. Further aggregation involves the formation of tetramers in which two dimers are anti-parallel and staggered to two characteristic degrees of overlap. Modeling indicated that the degrees of stagger are dictated by the association of sections of α helix in 4-chain bundles, in which hydrophobic side chains are sequestered from contact with water. The staggered arrangement of two dimers produces a tetramer having sections of 2-chain rod in which hydrophobic side chains are exposed to water. Extension of the tetramer to form protofilaments may be driven by associations with the 2-chain regions that reduce aqueous exposure of the hydrophobic side chains. Exposure of hydrophobic groups may be reduced by the 2-chain regions folding back upon themselves so that the entire tetramer becomes a 4-chain conformation. This prediction is in line with electron microscope data showing that mixtures of the lower oligomers contain rods of uniform thickness ranging upwards from 45 nm in a series having incremental increases in length. Data from previous chemical crosslinking studies support this model and also the idea that the completed intermediate filaments each consist of seven 4-chain protofilaments. Proteins 26:472–478 © 1996 Wiley-Liss, Inc.     

Focal adhesions are hotspots for keratin filament precursor formation

Recent studies showed that keratin filament (KF) formation originates primarily from sites close to the actin-rich cell cortex. To further characterize these sites, we performed multicolor fluorescence imaging of living cells and found drastically increased KF assembly in regions of elevated actin turnover, i.e., in lamellipodia. Abundant KF precursors (KFPs) appeared within these areas at the distal tips of actin stress fibers, moving alongside the stress fibers until their integration into the peripheral KF network. The earliest KFPs were detected next to actin-anchoring focal adhesions (FAs) and were only seen after the establishment of FAs in emerging lamellipodia. Tight spatiotemporal coupling of FAs and KFP formation were not restricted to epithelial cells, but also occurred in nonepithelial cells and cells producing mutant keratins. Finally, interference with FA formation by talin short hairpin RNA led to KFP depletion. Collectively, our results support a major regulatory function of FAs for KF assembly, thereby providing the basis for coordinated shaping of the entire cytoskeleton during cell relocation and rearrangement.     

Filament-guided filament assembly provides structural memory of filament alignment during cytokinesis

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Dynamics of keratin assembly: exogenous type I keratin rapidly associates with type II keratin in vivo

Keratin intermediate filaments (IF) are obligate heteropolymers containing equal amounts of type I and type II keratin. We have previously shown that microinjected biotinylated type I keratin is rapidly incorporated into endogenous bundles of keratin IF (tonofilaments) of PtK2 cells. In this study we show that the earliest steps in the assembly of keratin subunits into tonofilaments involve the extremely rapid formation of discrete aggregates of microinjected keratin. These are seen as fluorescent spots containing both type I and type II keratins within 1 min post-injection as determined by double label immunofluorescence. These observations suggest that endogenous type II keratin subunits can be rapidly mobilized from their endogenous state to form complexes with the injected type I protein. Furthermore, confocal microscopy and immunogold electron microscopy suggest that the type I-type II keratin spots from in close association with the endogenous keratin IF network. When the biotinylated protein is injected at concentrations of 0.3-0.5 mg/ml, the organization of the endogenous network of tonofilaments remains undisturbed during incorporation into tonofilaments. However, microinjection of 1.5-2.0 mg/ml of biotinylated type I results in significant alterations in the organization and assembly state of the endogenous keratin IF network soon after microinjection. The results of this study are consistent with the existence of a state of equilibrium between keratin subunits and polymerized keratin IF in epithelial cells, and provide further proof that IF are dynamic elements of the cytoskeleton of mammalian cells.