Assembly and architecture of invertebrate cytoplasmic intermediate filaments reconcile features of vertebrate cytoplasmic and nuclear lamin-type intermediate filaments

The two major intermediate filament (IF) proteins from the esophagus epithelium of the snail Helix pomatia and the two major IF proteins from muscle tissue of the nematode Ascaris suum were investigated under a variety of assembly conditions. The lowest-order complexes from each of the four protostomic invertebrate (p-INV) IF proteins are parallel, unstaggered dimers involving two-stranded alpha-helical coiled coil formation of their approximately 350 amino acid residue central rod domain (i.e. long-rod). In the electron microscope these are readily recognized by their distinct approximately 56 nm long rod with two globular domains (i.e. representing the non-helical carboxy-terminal tail domain of the p-INV IF proteins) attached at one end, closely resembling vertebrate lamin dimers. The next-higher-order oligomers are tetramers, which are easily recognized by their two pairs of globular tail domains attached at either end of a approximately 72 nm long central rod portion. According to their size and shape, these tetramers are built from two dimers associated laterally in an antiparallel, approximately half-staggered fashion via the amino-terminal halves of their rod domains. This is similar to the NN-type tetramers found as the most abundant oligomer species in all types of vertebrate cytoplasmic IF proteins, which contain a approximately 310 amino acid residue central rod domain (i.e. short-rod). As a first step toward filament formation, the p-INV IF tetramers anneal longitudinally into protofilaments by antiparallel CC-type association of the carboxy-terminal halves of their dimer rods. The next step involves radial growth, occurring initially through lateral association of two four-chain protofilaments into octameric subfibrils, which then further associate into mature, full-width filaments. Head-to-tail polymers of dimers and paracrystalline fibers commonly observed with vertebrate lamins were only rarely seen with p-INV IF proteins. The globular domains residing at the carboxy-terminal end of p-INV IF dimers were studding the surface of the filaments at regular, approximately 24.5 nm intervals, thereby giving them a "beaded" appearance with an axial periodicity of about 24.5 nm, which is approximately 3 nm longer than the corresponding approximately 21.5 nm repeat pattern exhibited by short-rod vertebrate IFs.     

Molecular phylogeny of metazoan intermediate filament proteins

We have cloned cytoplasmic intermediate filament (IF) proteins from a large number of invertebrate phyla using cDNA probes, the monoclonal antibody IFA, peptide sequence information, and various RT-PCR procedures. Novel IF protein sequences reported here include the urochordata and nine protostomic phyla, i.e., Annelida, Brachiopoda, Chaetognatha, Echiura, Nematomorpha, Nemertea, Platyhelminthes, Phoronida, and Sipuncula. Taken together with the wealth of data on IF proteins of vertebrates and the results on IF proteins of Cephalochordata, Mollusca, Annelida, and Nematoda, two IF prototypes emerge. The L-type, which includes 35 sequences from 11 protostomic phyla, shares with the nuclear lamins the long version of the coil 1b subdomain and, in most cases, a homology segment of some 120 residues in the carboxyterminal tail domain. The S-type, which includes all four subfamilies (types I to IV) of vertebrate IF proteins, lacks 42 residues in the coil 1b subdomain and the carboxyterminal lamin homology segment. Since IF proteins from all three phyla of the chordates have the 42-residue deletion, this deletion arose in a progenitor prior to the divergence of the chordates into the urochordate, cephalochordate, and vertebrate lineages, possibly already at the origin of the deuterostomic branch. Four phyla recently placed into the protostomia on grounds of their 18S rDNA sequences (Brachiopoda, Nemertea, Phoronida, and Platyhelminthes) show IF proteins of the L-type and fit by sequence identity criteria into the lophotrochozoic branch of the protostomia.     

Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins

In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution-binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.     

The fourth armadillo repeat of plakoglobin (gamma-catenin) is required for its high affinity binding to the cytoplasmic domains of E-cadherin and desmosomal cadherin Dsg2, and the tumor suppressor APC protein

Plakoglobin is a member of a protein family with a repeated amino acid motif, the armadillo repeat, and is a cytoplasmic protein found in both adherens junctions and desmosomes. Plakoglobin has been shown to form distinct complexes with cadherins or desmosomal cadherins. Also, plakoglobin has been shown to complex with APC, the tumor suppressor gene product. Recently we isolated a cDNA clone encoding plakoglobin lacking the fourth armadillo repeat of the original 13-repeat protein [Ozawa et al. (1995) J. Biochem. 118, 836-840]. In this study, we established an in vitro assay system to study the molecular interaction of plakoglobin with cadherins, the APC gene product, and alpha-catenin. Establishment of the system and cloning of an alternate form of plakoglobin cDNA allowed us to examine the biological activity of plakoglobin lacking the fourth armadillo repeat. Experiments with the bacterially expressed 12-repeat plakoglobin revealed that the protein binds to E-cadherin, desmoglein (Dsg2), and APC with lower affinity than the 13-repeat form does. Consistent with the observation that the affinity of alpha-catenin for these two alternate forms was similar, we found amino acid residues 104 to 145 of plakoglobin, the residues present in both isoforms, are sufficient for its binding to alpha-catenin.     

Integrin cytoplasmic domains as connectors to the cell's signal transduction apparatus

Integrins mediate the bidirectional transfer of signals across the plasma membrane. Integrin cytoplasmic domains provide one pathway linking integrin engagement with the cell's signal transduction apparatus. Recent structure-function studies have defined regions of beta cytoplasmic domains required for integrin function and have identified distinct roles for individual alpha cytoplasmic domains in regulating cell behavior. Newly identified proteins that bind to integrin alpha and beta cytoplasmic domains have provided new insights and new questions into the mechanisms involved in integrin signaling.     

Motile properties of vimentin intermediate filament networks in living cells

PURPOSE: To evaluate the clinical utility and morphologic accuracy of gadolinium-enhanced excretory magnetic resonance (MR) urography after low-dose diuretic injection and to correlate the results with those of conventional urography. MATERIALS AND METHODS: In 71 patients with urologic symptoms, excretory MR urography was performed after intravenous injection of 5-10 mg furosemide and, 30-60 seconds later, 0.1 mmol of gadopentetate dimeglumine per kilogram of body weight. The MR urograms were interpreted by three radiologists, who were blinded to the clinical outcome, and subsequently compared with conventional urograms. RESULTS: Injection of furosemide before contrast material led to rapid, uniform gadolinium distribution inside a sufficiently distended collecting system such that there was no excessive concentration of gadolinium in the urine. In patients with normal or moderately reduced excretory function, this effect allowed complete visualization of the urinary tract within 5-20 minutes of contrast material injection while minimizing gadolinium-related endoluminal T2* effects. The clinical course helped verify almost all MR urographic results. The MR urographic technique was significantly superior to conventional urography in the assessment of the ureters and bladder (P < .0001). Delineation of small caliceal abnormalities is still problematic. The best depiction of the pelvicaliceal system was obtained with fat-suppressed MR imaging, although it was still slightly inferior to conventional urography (P < .05). CONCLUSION: Gadolinium-enhanced excretory MR urography performed after low-dose diuretic injection is a promising and accurate alternative to conventional excretory urography for imaging the morphology of the urinary tract.     

Spatial control of actin filament assembly: lessons from Listeria

STUDY OBJECTIVE: To determine the effect of the CHAMPUS Reform Initiative (CRI) on emergency department use and charges, and to ascertain whether any reductions were concentrated among repeat users of the ED, those with less serious ED diagnoses, or those with selected chronic medical conditions. METHODS: Participants were approximately 1.2 million beneficiaries of the Civilian Health and Medical Program of Uniformed Services (CHAMPUS) residing within either 11 military hospital catchment areas in California and Hawaii ("demonstration areas") or 11 matched control areas in other parts of the United States. Under CRI, participants were offered a choice of the standard CHAMPUS indemnity plan, a Preferred Provider Organization-type plan, or a network-model Health Maintenance Organization plan. Beneficiaries were encouraged to use alternatives to the ED for nonemergency conditions. Visits to civilian EDs during two 12-month periods, before and after institution of CRI, were compared. RESULTS: Under CRI, the number of CHAMPUS ED visits decreased by approximately 40% relative to the control, and allowed charges fell by almost 50%. Relative reductions in ED use under CRI were seen among both frequent and infrequent users of the ED. ED case-mix severity increased modestly relative to control (+3.5% versus +.9%). ED use among patients with diabetes, hypertension, and asthma fell sharply in the demonstration areas (by 14% to 41%) but rose in control areas (by 4% to 9%). CONCLUSION: In one of the largest managed care demonstrations ever conducted, a nonintrusive use management program and improved access to outpatient care appeared to reduce ED use, allowed charges, and costs to the government. Reductions in ED use were concentrated to some extent among repeat users and patients with less severe illnesses and were effected without capitation of provider groups or strict gatekeeping requirements.     

MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains

The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins.     

Formation and localization of cytoplasmic domains in leech and ascidian zygotes

Leech and ascidian embryos are well suited for the study of certain developmental processes. Although leeches and ascidians belong to different bilateralia groups (protostomes and deuterostomes, respectively) they share important developmental features and, in particular, the determinate character of their embryogenesis. In both types of embryos this property is related to the presence of specific cytoplasmic domains that are selectively allocated to different blastomeres during cleavage. In this review leech and ascidian eggs and zygotes are compared in terms of the structure of these cytoplasmic domains and of the cellular mechanisms involved in their formation and localization. During meiosis the zygote of leeches and ascidians undergo stereotypic actin-dependent contraction movements related to both the emission of the polar bodies and the formation and relocalization of cytoplasmic domains. After completion of meiosis, during first interphase, monaster microtubules nucleated from the sperm-derived centrosome play a key role in pronuclear migration. In addition, these astral microtubules direct the relocalization of cytoplasmic domains and the translocation and accumulation of organelles in the interior of the zygote. Microtubules and microfilaments, on the other hand, are involved in cortical reorganizations and organelle translocations in both zygote species during interphase and cleavage divisions. In the case of leech zygotes, this process leads to formation of characteristic polar cytoplasmic domains called teloplasms. These domains are selectively inherited by teloblasts, precursor stem cells of ectodermal and mesodermal tissues in the leech embryo. In the ascidian zygote, the cytoplasmic movements observed during interphase and mitosis lead to relocalization of the bulk of a mitochondria-rich domain, called the myoplasm, along with an endoplasmic reticulum-rich domain towards the future posterior pole of the embryo. The myoplasm is inherited by a subset of posterior blastomeres committed to become the primary muscle cells of the ascidian tadpole.     

Complete sequence of a hair-like intermediate filament type II keratin gene

The Intermediate Filament (IF) superfamily comprises several multigene families, of which the two keratin families are the largest. The keratin IF genes are expressed in epithelial tissues in differentiation-specific patterns and recently we reported the sequence and expression of a hair IF type II keratin gene (KRT2.9). Two related genes were present in the cosmid containing KRT2.9 and we have now sequenced one of them and found that it encodes a hair-like IF type II protein (KRT2.13). However, KRT2.13 is not expressed in the hair follicle. Interestingly there is significant sequence homology between introns 1, 5 and 6 of KRT2.13 and KRT2.9 to suggest gene conversion of these regions or possibly conservation of functional sequences.     

Formation of CLC-0 chloride channels from separated transmembrane and cytoplasmic domains

CLC-0, a member of the CLC family of Cl(-)-conducting ion channels, consists of an N-terminal hydrophobic core and a C-terminal region that is thought to be cytoplasmic. This study provides evidence that the C-terminal region is a mechanistically relevant cytoplasmic domain of the CLC-0 ion channel. Both a point mutation and a 37-residue deletion in this region cause drastic alterations in voltage-dependent gating of CLC-0 current expressed in Xenopus oocytes. CLC-0 current is not observed when the entire C-terminal region is deleted, but functional channels are efficiently reconstituted by co-injection of separate cRNA constructs encoding the N-terminal transmembrane and the C-terminal cytoplasmic domains. Moreover, reconstitution of CLC-0 can be achieved by co-injection of cRNA encoding the transmembrane domain along with Escherichia coli-expressed C-terminal domain polypeptide.     

Primary sequence, secondary structure, gene structure, and assembly properties suggests that the lens-specific cytoskeletal protein filensin represents a novel class of intermediate filament protein

The ocular lens fiber cell assembles a novel cytoskeletal element, the Beaded Filament, from CP49 and filensin, two proteins expressed only in the differentiated lens fiber cell. We report the primary sequence, secondary structural analysis, gene structure and Yeast Two Hybrid interaction data for human filensin, and develop a consensus model of filensin from the human and previously reported bovine and chicken filensin sequences. This consensus model, combined with gene structure and Yeast Two Hybrid studies establish that filensin is a member of the Intermediate Filament family of proteins. Specifically, filensin exhibits (1) divergence at amino acid sequence motifs otherwise highly conserved among intermediate filament proteins, (2) a loss of 29 amino acids from the central rod domain which is unique among cytoplasmic intermediate filament proteins, (3) an absence of sequence identity with any existing class of intermediate filament protein, (4) a gene structure unique among intermediate filament family, (5) an inability to dimerize with representatives of Type I, II, and III intermediate filament proteins. Thus, at each level of analysis, we find that filensin is similar to the consensus model of intermediate filament proteins, supporting our conclusion that filensin's relatedness to the IF family is not the consequence of convergent evolution. However, filensin also shows unique or extreme distinctions from the consensus intermediate filament protein at each level of analysis, indicating that filensin constitutes a novel class of IF protein. Some of filensin's unique features are incompatible with current models of IF assembly. Analysis of filensin gene structure suggests that the 29 amino acid reduction in the central rod domain was not the result of a single splice site mutation, the mechanism suggested for the transition between nuclear lamins and cytoplasmic intermediate filament proteins.     

Structure of the Drosophila projectin protein: isoforms and implication for projectin filament assembly

The rare alleles model of mutation-selection balance (MSB) hypothesis for the maintenance of genetic variation was evaluated for two quantitative traits, ovariole number and body size. Mutational variances (VM) for these traits, estimated from mutation accumulation lines, were 4.75 and 1.97 x 10(-4) times the environmental variance (VE), respectively. The mutation accumulation lines were studied in three environments to test for genotype x environment interaction (GEI) of new mutations; significant mutational GEI was found for both traits. Mutations for ovariole number have a quadratic relationship with competitive fitness, suggesting stabilizing selection for the trait; there is no significant correlation between mutations for body size and competitive fitness. Under MSB, the ratio of segregating genetic variance, VG, to mutational variance, VM, estimates the inverse of the selection coefficient against a heterozygote for a new mutation. Estimates of VG/VM for ovariole number and body size were both approximately 1.1 x 10(4). Thus, MSB can explain the level of variation, if mutations affecting these traits are under very weak selection, which is inconsistent with the empirical observation of stabilizing selection, or if the estimate of VM is biased downward by two orders of magnitude. GEI is a possible alternative explanation.     

Immunohistochemical distribution pattern of intermediate filament proteins and muscle actin in feline and human mammary carcinomas

Thirty-seven feline and 38 human spontaneous mammary gland carcinomas were studied immunohistochemically. Commercially available antibodies directed against high and low molecular weight keratins (RCK-102 and NCL-5D3), vimentin, desmin, glial fibrillary acidic protein (GFAP), neurofilament (NF) proteins and muscle actin (HHF35) were used in the avidin biotin peroxidase complex (ABC) technique on formalin-fixed paraffin wax-embedded tumour tissue samples. Healthy feline and human mammary gland tissue adjacent to the neoplasms was also examined. The distribution pattern of intermediate filament proteins and muscle actin was comparable in healthy mammary gland tissue of the two species: both RCK-102 and NCL-5D3 antibodies reacted with luminal epithelial cells of ducts and acini, but basal/myoepithelial cells were stained by RCK-102 exclusively. In addition, basal/myoepithelial cells expressed vimentin and muscle actin in both species, and GFAP was found in some feline basal/myoepithelial cells. No immunoreactivity to desmin and NF proteins was observed. Feline mammary gland carcinoma cells reacted with RCK-102 (89%), NCL-5D3 (62%), vimentin (76%) and GFAP (30%) antibodies, while human mammary gland carcinoma cells reacted with RCK-102 (95%), NCL-5D3 (100%) and vimentin (13%) antibodies. HHF35 immunoreactivity was observed in stromal cells only. These results indicate that mammary gland carcinomas of both species share a heterogeneous immunophenotype with respect to intermediate filament proteins, which adds to the list of known similarities between mammary gland carcinomas of both species.     

Effect of cellular level of FliK on flagellar hook and filament assembly in Salmonella typhimurium

Frameshift mutations in the fliK gene of Salmonella result in abnormal elongation of the hook and the failure to assemble filament (polyhook phenotype). Second-site suppressor mutations restore filament assembly, but the cells often remain defective in hook-length control (polyhook-filament phenotype). Where the suppressor mutations are intragenic, the second mutation restores the original frame, generating a region of frameshifted sequence, but restoring the natural C terminus. Some of these frameshifted sequences contain a UGA (opal) termination codon. These cells have few flagella and swarm poorly. We suspected that readthrough of UGA by tRNATrp might be the reason for the partial function. When the UGA codon was changed to the Trp codon UGG, flagellar assembly and function were restored to wild-type levels. Conversely, underexpression of the wild-type fliK gene, achieved by changing the sole Trp codon in the sequence (Trp271) to UGA, decreased both the number of flagella and the ability to swarm. These results validate the readthrough hypothesis and indicate that low levels of FliK sustain some degree of flagellation and motility. At low levels of FliK, most flagella had polyhooks. With increasing amounts, the morphology progressively changed to polyhook-filament, and eventually to wild-type hook-filament. When FliK was overproduced, the hook length was slightly shorter (46(+/-7) nm) than that of the wild-type strain (55(+/-9) nm). FliK levels were measured by immunoblotting. Wild-type levels were about 40 to 80 molecules/cell. FliK synthesized by UGA readthrough could be detected when overproduced from plasmid fliK-W271opal, and the levels indicated a probability of readthrough of 0.002 to 0.01. This value was used to estimate the cellular level of underexpressed FliK, which could partly restore function to a fliK mutant, at about 0.07 to 0.8 molecule/cell. These results suggest that FliK does not form a large structure in the cytoplasm and may function as a regulatory protein for protein export. A model for hook-length control is presented that involves feedback from the assembly point to the export apparatus.     

In vitro reconstitution of an intermediate assembly stage of vaccinia virus

The effects of chronic exogenous testosterone treatment on the synthesis and/or secretion of two sturgeon gonadotropins (stGTH I and stGTH II) were assessed in 2-year-old juvenile white sturgeon (Acipenser transmontanus) surgically implanted with silastic capsules filled with 75 mg of testosterone and in previtellogenic female white sturgeon females implanted with 150 mg of testosterone. In groups of juvenile white sturgeon sacrificed 30, 60, 90, or 442 days postimplantation, pituitary concentrations of stGTH I were significantly greater in testosterone-treated fish (P < 0.01) when compared to those of controls. Pituitary concentrations of stGTH II were significantly higher (P < 0.01) in juvenile fish treated 60, 90, or 442 days with testosterone when compared to those of controls. Exogenous testosterone had no effect on plasma concentrations of either stGTH. Additional testosterone-treated juvenile sturgeon which were injected intraperitoneally 90 or 442 days postimplantation with 10 microg/kg of the gonadotropin releasing hormone analog d-Ala6-des-Gly10-GnRH ethylamide (GnRHa) also showed no change in plasma concentrations of stGTHs. Similar results were obtained for previtellogenic white sturgeon, as pituitary concentrations of stGTH I and stGTH II were significantly greater (P < 0.01) after 60 days of testosterone treatment compared to those of controls. A second group of 60-day testosterone-treated previtellogenic females also failed to exhibit increases of plasma stGTHs when administered 10 microg/kg of GnRHa. These results indicate that long-term testosterone treatment stimulates the accumulation of pituitary stGTHs in both juvenile and previtellogenic white sturgeon but does not affect basal or GnRHa-induced stGTH secretion.     

Role of adhesion molecule cytoplasmic domains in mediating interactions with the cytoskeleton

The past ten years have seen significant progress in cell biology research aimed at understanding how cytoskeletal filaments interact with the plasma membrane. Considerable evidence suggests that both actin microfilaments and intermediate filaments attach to the membrane via the cytoplasmic domains of various membrane proteins including adhesion molecules. Interactions between the cytoskeleton and adhesion molecules appear to be essential for a variety of cellular functions, including cell-cell and cell-extracellular matrix (ECM) interactions, cell motility, receptor-ligand interactions, and receptor internalization. Recently, many of the detailed molecular mechanisms which mediate the associations between actin filaments and adhesion molecules have been identified. Among adhesion molecules that support the attachment of cytoskeletal filaments to their cytoplasmic domains are members of the integrin and cadherin families, the intracellular adhesion molecule-1 (ICAM-1, an immunoglobulin family member), and the glycoprotein Ib/IX complex in platelets. A general conclusion emerging from these studies is that physical associations between cytoskeletal filaments and transmembrane glycoproteins do not occur directly between the filaments and the cytoplasmic tails of adhesion molecules. Instead, these interactions appear to be indirect and involve a complex ensemble of intermediary linker proteins. The severe effects of cytoplasmic domain deletion and mutagenesis on adhesion-dependent functions support the view that receptor cytoplasmic domains play a vital role in regulating receptor function and in mediating communication across the membrane. Transfection studies with mutant and chimeric adhesion molecules, along with protein-binding studies, are clarifying the mechanisms which physically link the cytoskeleton to transmembrane proteins, regulate cytoskeletal organization, mediate signaling across the cell membrane, and regulate the ligand specificity and binding affinity of surface receptors.