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.     

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.     

The dynamic phosphorylation of the human intermediate filament keratin 1 chain

The major sites of phosphorylation have been determined on the human keratin intermediate filament keratin 1 (type II) chain expressed in terminally differentiating epidermis. A total of nine phosphate sites were found, involving 1 threonine and 8 serine residues, and were localized to end domain sequences. The sites identified corresponded to major sites of phosphorylation as determined by direct quantitation of O-phosphoserine. Since the tissue was cultured with [32 P] orthophosphate only briefly, labeling occurred primarily by turnover, so that information on the dynamics of phosphorylation was also obtained. The degrees and specific activities (that is, turnover rates) of phosphorylation of these sites varied widely between different isoelectric variants (phosphate isomers) of keratin 1 chains and correlated with their locations on the chain: those sites on the more exposed E1 and E2 subdomains were fully phosphorylated and turning over at high rates, while a site near the end of the rod domain in a presumably more confined location was only slightly phosphorylated and turning over at low rate. The nature of the sequences around the phosphorylated residues indicates that cAMP-dependent and probably other protein kinase activities operate simultaneously in intact normal epidermal tissue. The correlation between the degrees and rates of turnover of phosphorylation with the locations on the chain may have an important bearing on the functional role of phosphorylation of the keratin intermediate filaments in this tissue.     

Assembly characteristics of plant keratin intermediate filamentsin vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembledin vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filamentsin vitro. In higher mcation images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24–25 nm periodic structural repeat alone the axis of both the 10 nm filaments and protofilarnents. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments.     

Assembly characteristics of plant keratin intermediate filaments in vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembledin vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filamentsin vitro. In higher mcation images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24–25 nm periodic structural repeat alone the axis of both the 10 nm filaments and protofilarnents. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments. Project supported by the National Natural Science Foundation of China (Grant No. 39370352) and the Doctor Foundation of Minishy of Education of China.     

Assembly characteristics of plant keratin intermediate filaments in vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembled in vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filaments in vitro. In higher magnification images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24—25 nm periodic structural repeat alone the axis of beth the 10 nm filaments and protofilaments. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments.     

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.     

Cytoskeleton: Anatomy of an organizing center

One component of the yeast spindle pole body, Spc42p, has been found to form a crystalline array within one of the central layers of the structure; the Spc42p crystal might provide a scaffold around which the spindle pole body is assembled, and could be involved in regulating the size of the spindle pole body.     

Assemblies of psoriatic keratin and their relation to normal intermediate filament structures

Protein extracts from normal human epidermis reassemble in vitro into 8-10 nm diameter filaments characteristic of intermediate filaments, whereas extracts from psoriatic epidermal scales reassemble, under identical conditions, into a variety of paracrystalline bundles. Optical diffraction and image analysis of these paracrystalline bundles reveal an axial repeat of 16.5 nm, which subdivides into three bands of 5.5 nm, and a lateral spacing of 5.1 nm. This information, together with available sequence studies of intermediate filaments and biochemical data, suggests that the subunit of psoriatic keratin is made up essentially from the coiled-coil alpha-helical rod domain of the normal keratin subunits, whereas the random coil domains are missing or greatly reduced in size.     

Evidence for an intermediate in tau filament formation

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule-associated protein tau. In vitro, fibrillization of full-length, unphosphorylated recombinant tau can be induced under near-physiological conditions by treatment with various agents, including anionic surfactants. Here we examine the pathway through which anionic surfactants promote tau fibrillization using a combination of electron microscopy and fluorescence spectroscopy. Protein and surfactant first interacted in solution to form micelles, which then provided negatively charged surfaces that accumulated tau aggregates. Surface aggregation of tau protein was followed by the time-dependent appearance of a thioflavin S reactive intermediate that accumulated over a period of hours. The intermediate was unstable in the absence of anionic surfaces, suggesting it was not filamentous. Fibrillization proceeded after intermediate formation with classic nucleation-dependent kinetics, consisting of lag phase followed by the exponential increase in filament lengths, followed by an equilibrium phase reached in approximately 24 h. The pathway did not require protein insertion into the micelle hydrophobic core or conformational change arising from mixed micelle formation, because anionic microspheres constructed from impermeable polystyrene were capable of qualitatively reproducing all aspects of the fibrillization reaction. It is proposed that the progression from amorphous aggregation through intermediate formation and fibrillization may underlie the activity of other inducers such as hyperphosphorylation and may be operative in vivo.     

Amphioxus type I keratin cDNA and the evolution of intermediate filament genes.

We report the cloning of an intermediate filament (IF) cDNA from the cephalochordate amphioxus that encodes a protein assignable to the type I keratin group. This is the first type I keratin reported from an invertebrate. Molecular phylogenetic analyses reveal that amphioxus also possesses a type II keratin, and that the genes encoding short-rod IF proteins underwent different patterns of duplication in vertebrates and their closest relatives, the cephalochordates. Extensive IF gene duplication and divergence may have facilitated the origin of new specialised cell types in vertebrates.     

A role for disulfide bonding in keratin intermediate filament organization and dynamics in skin keratinocytes

We recently reported that a trans-dimer, homotypic disulfide bond involving Cys367 in keratin 14 (K14) occurs in an atomic-resolution structure of the interacting K5/K14 2B domains and in keratinocyte cell lines. Here we show that a sizable fraction of the K14 and K5 protein pools participates in interkeratin disulfide bonding in primary cultures of mouse skin keratinocytes. By comparing the properties of wild-type K14 with a completely cysteine-free variant thereof, we found that K14-dependent disulfide bonding limited filament elongation during polymerization in vitro but was necessary for the genesis of a perinuclear-concentrated network of keratin filaments, normal keratin cycling, and the sessile behavior of the nucleus and whole cell in keratinocytes studied by live imaging. Many of these phenotypes were rescued when analyzing a K14 variant harboring a single Cys residue at position 367. These findings establish disulfide bonding as a novel and important mechanism regulating the assembly, intracellular organization, and dynamics of K14-containing intermediate filaments in skin keratinocytes.     

Assembly and exchange of intermediate filament proteins of neurons: neurofilaments are dynamic structures

We have explored the dynamics of intermediate filament assembly and subunit exchange using fluorescently labeled neurofilament proteins and a fluorescence resonance energy transfer assay. Neurofilaments (NFs) are assembled from three highly phosphorylated proteins with molecular masses of 180 (NF-H), 130 (NF-M), and 66 kD (NF-L) of which NF-L forms the structural core. The core component, NF-L, was stoichiometrically labeled at cysteine 321 with fluorescein, coumarin, or biotin-maleimide to produce assembly-competent fluorescent or biotinylated derivatives, respectively. Using coumarin-labeled NF-L as fluorescence donor and fluorescein-labeled NF-L as the fluorescence acceptor, assembly of NF filaments was induced by rapidly raising the NaCl concentration to 170 mM, and the kinetics was followed by the decrease in the donor fluorescence. Assembly of NF-L subunits into filaments does not require nucleotide binding or hydrolysis but is strongly dependent on ionic strength, pH, and temperature. The critical concentration of NF-L, that concentration that remains unassembled at equilibrium with fully formed filaments, is 38 micrograms/ml or 0.6 microM. Under physiological salt conditions NF-L filaments also undergo extensive subunit exchange. Kinetic analysis and evaluation of several possible mechanisms indicate that subunit exchange is preceded by dissociation of subunits from the filament and generation of a kinetically active pool of soluble subunits. Given the concentration of NF-L found in nerve cells and the possibility of regulating this pool, these results provide the first information that intermediate filaments are dynamic structures and that NF-L within the NF complex is in dynamic equilibrium with a small but kinetically active pool of unassembled NF-L units.     

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.     

Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks

We previously demonstrated that truncated desmoplakin I (DP I) molecules containing the carboxyl terminus specifically coalign with and disrupt both keratin and vimentin intermediate filament (IF) networks when overexpressed in tissue culture cells (Stappenbeck, T. S., and K. J. Green. J. Cell Biol. 116:1197-1209). These experiments suggested that the DP carboxyl-terminal domain is involved either directly or indirectly in linking IF with the desmosome. Using a similar approach, we have now investigated the behavior of ectopically expressed full-length DP I in cultured cells. In addition, we have further dissected the functional sequences in the carboxyl terminus of DP I that facilitate the interaction with IF networks. Transient transfection of a clone encoding full-length DP I into COS-7 cells produced protein that appeared in some cells to associate with desmosomes and in others to coalign with and disrupt IF. Deletion of the carboxyl terminus from this clone resulted in protein that still appeared capable of associating with desmosomes but not interacting with IF networks. As the amino terminus appeared to be dispensable for IF interaction, we made finer deletions in the carboxyl terminus of DP based on blocks of sequence similarity with the related molecules bullous pemphigoid antigen and plectin. We found a sequence at the very carboxyl terminus of DP that was necessary for coalignment with and disruption of keratin IF but not vimentin IF. Furthermore, the coalignment of specific DP proteins along keratin IF but not vimentin IF was correlated with resistance to extraction by Triton. The striking uncoupling resulting from the deletion of specific DP sequences suggests that the carboxyl terminus of DP interacts differentially with keratin and vimentin IF networks.     

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.     

Organization of coiled-coil molecules in native mouse keratin 1/keratin 10 intermediate filaments: evidence for alternating rows of antiparallel in-register and antiparallel staggered molecules.

There is considerable diversity of opinion in the literature concerning the organization of two-chain coiled-coil molecules in intermediate filaments. I have reexplored this issue using the limited proteolysis paradigm with native mouse epidermal keratin intermediate filaments (KIF), consisting of keratins 1 and 10. KIF were harvested as cytoskeletal pellets, dissociated into subfilamentous forms at pH 9.8, 9.0, or 2.6, and were subjected to limited proteolytic digestion to recover alpha-helix-enriched particles that derived from the rod domains of the constituent chains, using conditions that do not promote reorganization of the constituent protein chains or coiled-coil molecules. The multichain particles were subjected to physicochemical analyses, amino acid sequencing, and electron microscopy in order to determine their composition, structure, and organization within the intact KIF. The results predict two principal modes of alignment: neighboring molecules may be aligned in register and antiparallel or staggered and antiparallel. From known structural constraints, this permits construction of a two-dimensional surface lattice for KIF which consists of alternating antiparallel rows of in-register and staggered molecules. These data establish the level of hierarchy at which the well-known antiparallelity and staggered features of KIF are introduced. This model supports the proposals of KIF structure based on theoretical considerations of ionic interactions scores (Crewther et al., 1983). When the KIF are dissociated at extremes of pH, this structural model allows for disruption along alternate axes; the in-register antiparallel alignment is seen only when KIF are dissociated at high pH values; below pH 9, only the staggered antiparallel alignment is seen. The process of molecule realignment especially in concentrated urea solutions indicates that the staggered antiparallel alignment is the more thermodynamically stable form in solution.