Intracellular assembly and sorting of intermediate filament proteins: role of the 42 amino acid lamin insert

Nuclear and cytoplasmic intermediate filament (IF) proteins segregate into two independent cellular networks by mechanisms that are poorly understood. We examined the role of a 42 amino acid (aa) insert unique to vertebrate lamin rod domains in the coassembly of nuclear and cytoplasmic IF proteins by overexpressing chimeric IF proteins in human SW13+ and SW13- cells, which contain and lack endogenous cytoplasmic IF proteins, respectively. The chimeric IF proteins consisted of the rod domain of human nuclear lamin A/C protein fused to the amino and carboxyl-terminal domains of the mouse neurofilament light subunit (NF-L), which contained or lacked the 42 aa insert. Immunofluorescence microscopy was used to follow assembly and targeting of the proteins in cells. Chimeric proteins that lacked the 42 aa insert colocalized with vimentin, whereas those that contained the 42 aa insert did not. When overexpressed in SW13- cells, chimeric proteins containing the 42 aa formed very short or broken cytoplasmic filaments, whereas chimeric proteins that lacked the insert assembled efficiently into long, stable cytoplasmic filaments. To examine the roles of other structural motifs in intracellular targeting, we added two additional sequences to the chimera, a nuclear localization signal (NLS) and a CAAX motif, which are found in nuclear IF proteins. Addition of an NLS alone or an NLS in combination with the CAAX motif to the chimera with the 42 aa insert resulted in cagelike filament that assembled close to the nuclear envelope and nuclear lamina-like targeting, respectively. Our results suggest that the rod domains of eukaryotic nuclear and cytoplasmic IF proteins, which are related to each other, are still compatible upon deletion of the 42 aa insert of coassembly. In addition, NF-L end domains can substitute for the corresponding lamin domains in nuclear lamina targeting.     

Intermediate filament networks: in vitro and in vivo assembly models

We propose two systems of ordinary differential equations modeling the assembly of intermediate filament networks. The first one describes the in vitro intermediate filament assembly dynamics. The second one deals with the in vivo evolution of cytokeratin, which is the intermediate filament protein expressed by epithelial cells. The in vitro model is then briefly analyzed in a simplified case.     

Desmosome and intermediate filament assembly during differentiation and stratification of epithelial cells

In the present investigation the sequential expression and organization of keratin intermediate filament proteins were studied in the developing rat palatal epithelia starting from early gestation period to the adult. The distribution and organization of keratin proteins were correlated with the formation and elaboration of desmosornes during differentiation and stratification of the epithelia.     

Characterization of mammalian synemin,an intermediate filament protein present in all four classes of muscle cells and some neuroglial cells: co-localization and interaction with type III intermediate filament proteins and keratins

Using a monoclonal antibody, we have detected a high molecular weight muscle protein, co-localized and co-isolating with desmin. Searching a human cDNA database with partial amino acid sequences of the protein, we found a cDNA clone encoding a 1565-amino-acid polypeptide, identified as a mammalian (human) synemin, a member of the intermediate filament (IF) protein family. Immunoblotting showed the presence of a 180-kDa polypeptide in skeletal muscle and 180- and 200-kDa polypeptides in cardiac and smooth muscles. Interestingly, synemin was also found in myoepithelial cells, which have keratin filaments instead of desmin. Moreover, synemin was also found in astrocytes of optic nerves and non-myelin-forming Schwann cells, together with glial fibrillary acidic protein (GFAP) and vimentin. Blot overlays pointed to molecular interactions of synemin with desmin, vimentin, GFAP and keratin 5 and 6, but not with keratin 14. The experimental data also suggested a possible link with nebulin, a skeletal muscle protein. Purified synemin was coassembled with desmin in different molar ratios, and at 1:25, as typically found in vivo, IFs were formed which were comparable in length to desmin filaments. However, at molar ratios of 3:25 and 6:25, much shorter and irregular shaped filamentous polymers were generated. The fact that synemin is present in all four classes of muscle cells and a specific type of glial cells is indicative of important functions. Its incorporation may give structural and functional versatility to the IF cytoskeleton.This work was supported by grants from the Ministry of Education, Science, and Culture of Japan.     

Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis

Intestinal cells of C. elegans show an unexpectedly high complexity of cytoplasmic intermediate filament (IF) proteins. Of the 11 known IF genes six are coexpressed in the intestine, i.e. genes B2, C1, C2, D1, D2, and E1. Specific antibodies and GFP-promoter constructs show that genes B2, D1, D2, and E1 are exclusively expressed in intestinal cells. Using RNA interference (RNAi) by microinjection at 25 degrees C rather than at 20 degrees C we observe for the first time lethal phenotypes for C1 and D2. RNAi at 25 degrees C also shows that the known A1 phenotype occurs already in the late embryo after microinjection and is also observed by feeding which was not the case at 20 degrees C. Thus, RNAi at 25 degrees C may also be useful for the future analysis of other nematode genes. Finally, we show that triple RNAi at 20 degrees C is necessary for the combinations B2, D1, E1 and B2, D1, D2 to obtain a phenotype. Together with earlier results on genes A1, A2, A3, B1, and C2 RNAi phenotypes are now established for all 11IF genes except for the A4 gene. RNAi phenotypes except for A2 (early larval lethality) and C2 (adult phenotype) relate to the late embryo. We conclude that in C. elegans cytoplasmic IFs are required for tissue integrity including late embryonic stages. This is in strong contrast to the mouse, where ablation of IF genes apparently does not affect the embryo proper.     

The nanomechanical properties of rat fibroblasts are modulated by interfering with the vimentin intermediate filament system

The contribution of the intermediate filament (IF) network to the mechanical response of cells has so far received little attention, possibly because the assembly and regulation of IFs are not as well understood as that of the actin cytoskeleton or of microtubules. The mechanical role of IFs has been mostly inferred from measurements performed on individual filaments or gels in vitro. In this study we employ atomic force microscopy (AFM) to examine the contribution of vimentin IFs to the nanomechanical properties of living cells under native conditions. To specifically target and modulate the vimentin network, Rat-2 fibroblasts were transfected with GFP-desmin variants. Cells expressing desmin variants were identified by the fluorescence microscopy extension of the AFM instrument. This allowed us to directly compare the nanomechanical response of transfected and untransfected cells at high spatial resolution by means of AFM. Depending on the variant desmin, transfectants were either softer or stiffer than untransfected fibroblasts. Expression of the non-filament forming GFP-DesL345P mutant led to a collapse of the endogenous vimentin network in the perinuclear region that was accompanied by localized stiffening. Correlative confocal microscopy indicates that the expression of desmin variants specifically targets the endogenous vimentin IF network without major rearrangements of other cytoskeletal components. By measuring functional changes caused by IF rearrangements in intact cells, we show that IFs play a crucial role in mechanical behavior not only at large deformations but also in the nanomechanical response of individual cells.     

Immunohistochemical distribution of simple-epithelial-type keratins and other intermediate filament proteins in the developing human pituitary gland

Summary An immunohistochemical study of the production of the intermediate filaments [vimentin, cytokeratin, and glial filament acidic protein (GFAP)] during development of the pituitary gland was made by use of fetal and adult human pituitary tissue. Among these intermediate filament proteins in the anterior and intermediate lobes of the pituitary, cytokeratin is the first to appear, followed by GFAP and vimentin. However, only cytokeratin is seen during the period of morphogenesis of the pituitary gland, with the type-II subfamily cytokeratin 8 being the earliest to appear. Among the simple-epithelial-type cytokeratins, cytokeratins 8 and 19 were observed within the pituitary primordium during morphogenesis. Cells immunoreactive for cytokeratins 8 and 19 showed a heterogeneous three-dimensional distribution pattern in Rathke's pouch. Both cytokeratins 8 and 19 tended to be strongly positive at sites in the pituitary primordium where cells had become more loosely arranged (i.e., areas far from the diencephalon) but were only weakly positive in areas in which the epithelial cells were densely packed (i.e., areas closely associated with the diencephalon). It is concluded that, during the period of morphogenesis, Rathke's pouch has the intermediate filaments characteristic of simple epithelium and shows different immunoreactivity for simple-epithelial-type cytokeratins from place to place according to the extent of cellular differentiation.     

The identification and localization of two intermediate filament proteins in the tunic of Styela plicata (Tunicata, Styelidae)

The intermediate filament (IF) proteins Styela C and Styela D from the tunicate Styela (Urochordata) are co-expressed in all epidermal cells and they are thought to behave as type I and type II keratins. These two IF proteins, Styela C and Styela D, were identified in immunoblots of proteins isolated from the tunic of Styela plicata. The occurrence and distribution of these proteins within the tunic of this ascidian was examined by means of immunofluorescence and immunoperoxidase techniques, using anti-Styela C and anti-Styela D antibodies. In addition, immuno-electron microscopy of the tunic showed that the two proteins are located in the cuticle layer and in the tunic matrix. These results represent the first data about the presence of IF proteins in the tunic of adult ascidian S. plicata. The possible involvement of these IF proteins in reinforcing the integrity of the tunic, that represents the interface between the animal body and the external environment, is discussed.     

Deletion mutagenesis of the amino-terminal head domain of vimentin reveals dispensability of large internal regions for intermediate filament assembly and stability

Previous studies have shown that the non-alpha-helical head domain of vimentin is required for polymerization of intermediate filaments (IFs) and, furthermore, a nonapeptide highly conserved among type III IF subunit proteins at their extreme amino-terminus is essential for this process. Recombinant DNA technology was employed to produce specific vimentin deletion mutant proteins (for in vitro studies) or vimentin protein expression plasmids (for in vivo studies), which were used to identify other regions of the vimentin head domain important for polymerization. Various vimentin proteins lacking either residues 25-38, 44-95, or 40-95 polymerized into wild-type or largely normal IFs, both in vitro and in vivo. Vimentin proteins lacking residues 44-69 or 25-63 failed to form IFs in vitro, but assembled into IFs in vivo. Vimentin proteins lacking residues 25-68, 44-103, or 88-103 failed to form IFs in vitro or in vivo. Taken together with previous results, these data demonstrate that the middle of the vimentin non-alpha-helical head domain, which is known to be the site of nucleic acid binding, is completely dispensable for IF formation, whereas both ends of the vimentin non-alpha-helical head domain are required for IF formation. The simplest explanation for these results is that the middle of the vimentin non-alpha-helical head domain loops out, thereby permitting the juxtaposition of the ends of the head domain and their productive interaction with other protein domains (probably the C-terminus of the rod domain) during IF polymerization. The ability of some of the mutant proteins to form IFs in vivo, but not in vitro, suggests that as-yet-unknown cellular proteins may interact with and, in some cases, enable polymerization of IFs, even though they are not absolutely required for IF formation by wild-type vimentin.     

Fine structure and assembly in vitro of nuclear lamina in plant cells

The fine structure of the nuclear lamina (NL) in sperm cells ofGinkgo biloba was visualised using high resolution low-voltage scanning electron microscopy (LVSEM). It was shown that the nuclear lamina was composed of 10 nm filaments which formed a fine network. Lamins were purified from cultured carrot suspension cells and assembledin vitro. Long 8–12 nm diameter filaments were seen and sometimes subfilaments could be distinguished. Western blot of filament preparations showed that these contained the 66 and 84 ku lamins. These data demonstrate that plant lamins are capable of assembling into filamentsin vitro. Project supported by the National Natural Science Foundation of China (Grant No. 3500073).     

不同培养体系对鼠-猪异种嵌合胚胎发育和嵌合能力的比较

近年来,随着干细胞分化与再生医学研究的不断深入,异种嵌合已成为当前干细胞和再生医学领域的热点问题,并有望为未来解决器官移植供体来源严重短缺等再生医学难题开辟新的方向。异种嵌合以及异种器官再造过程中面临众多科学问题和技术难题,而异种嵌合过程中嵌合胚胎时期的选择,后续培养液的选择以及这些环节所造成的供体细胞与受体胚胎之间的发育平衡成为建立异种器官再造的第一个科学问题。猪由于具有与人类器官大小相似、繁殖快等特点,成为异种嵌合最适合的潜在研究对象。为了提高鼠-猪异种嵌合胚胎中小鼠供体细胞——诱导多潜能干细胞(Induced pluripotent stem cells,i PSCs)的存活率和增殖率,我们尝试以i PSCs培养液(N2B27)以及N2B27→PZM-3梯度更换的培养液(N2B27(3.5 h))作为研究异种嵌合胚胎体外发育培养的对象,并与猪胚胎培养液(PZM-3,Porcine zygotic medium)体系下发育进行比较,从而评价了这3种培养液在8-细胞和囊胚期注射后,对嵌合胚胎后续发育的影响及嵌合情况。结果显示,8-细胞期注射后,PZM-3不仅对嵌合胚胎的后续发育较为有利,更有利于小鼠i PS嵌合到猪胚胎中;囊胚期注射后3种培养体系下GFP阳性嵌合率差异不显著,但其嵌合率显著低于8-细胞期嵌合率。结果表明,PZM-3培养体系更有利于鼠-猪异种嵌合胚胎的体外发育,对8-细胞期胚胎进行嵌合操作有益于提高鼠-猪异种嵌合后胚胎的嵌合率。     

In vitro modulation of filament bundling in f-actin and keratins by annexin II and calcium

Summary In our preliminary subcellular localization experiment we demonstrated that annexin II co-localized with submembranous actin in subpopulations of both cultured fibroblasts and keratinocytes. To investigate the physical interaction between annexin II and actin at the cell periphery, in vitro reconstitution experiments were carried out with keratins used as a control. Annexin II, isolated by immunoaffinity column chromatography, was found to exist as globular structures measuring 10 to 25 nm in diameter by rotary shadowing, similar to a previous report. We believe that these structures represent its polymeric forms. By negative staining, monomeric annexin II was detectable as tapered rods, measuring 6 nm in length and 1 to 2 nm in diameter. When annexin II was mixed with actin in 3 mM piperazine-N, N-bis-2-ethanesulfonic acid (PIPES) buffer with 10 mM NaCl₂, 2 mM MgCl₂ and 0.1 mM CaCl₂, thick twisting actin bundles formed, confirming previous reports. This bundling was much reduced when calcium was removed. In the presence of 5 mM ethylenediamine tetra-acetic acid (EDTA) in 5 mM tris, pH 7.2, keratins were found to form a network of filaments, which began to disassemble when the chelator was removed and became fragmented when 0.1 mM CaCl₂ was added. Keratins under the same conditions did not fragment when annexin II was present. These results suggest that annexin II, in conjunction with Ca²⁺, may be involved in a flexible system accommodating changes in the membrane cytoskeletal framework at the cell periphery in keratinocytes.     

Characterization of intermediate filaments and their structural organization during epithelium formation in pigmented epithelial cells of the retina in vitro

Summary Retinal pigmented epithelial cells of chicken have circumferential microfilament bundles (CMBs) at the zonula adherens region. Isolated CMBs are polygons filled with a meshwork composed primarily of intermediate filaments; they show three major components of 200000, 55000, and 42000 daltons in SDS-gel electrophoresis. Here we have characterized the 55000-dalton protein immunochemically and ultrastructurally. Immunoblotting and immunofluorescence microscopy have shown that the 55000-dalton protein is an intermediate filament protein, vimentin.Vimentin filaments changed their distribution during differentiation of pigmented epithelial cells in culture. The protein in the elongated cells showed a fibroblast-type pattern of intermediate filaments. During epithelium formation, the filaments were uniformly distributed and formed a finer meshwork at the apical level. In pigmented epithelial cells that differentiated and matured in culture, vimentin and actin exhibited their characteristic behavior after treatment with colcemid. In the central to basal region of the cell, intermediate filaments formed thick perinuclear bundles. In the apical region, however, intermediate filaments changed in organization from a nonpolarized meshwork to a polarized bundle-like structure. Simultaneously, new actin bundles were formed, running parallel to the intermediate filaments. This suggests that there is some interaction between microfilaments and intermediate filaments in the apical region of these cells.     

Characterization of the in vitro co-assembly process of the intermediate filament proteins vimentin and desmin: mixed polymers at all stages of assembly

We have investigated the co-assembly properties of the intermediate filament (IF) proteins vimentin and desmin. First, the soluble complexes formed by both proteins separately in 5 mM Tris-HCl, pH 8.4, were characterized by analytical ultracentrifugation. In both cases, s-values of around 5 S were obtained corresponding to the formation of tetramers. However, at pH 7.5 and in the presence of 1 mM EDTA, both proteins behaved quite differently; whereas vimentin sedimented at 7.2 S, desmin assembled into much larger complexes of about 13 S. A mixture of equimolar amounts of vimentin and desmin in 8 M urea yielded, after reconstitution into 5 mM Tris-HCl, pH 7.5, and 1 mM EDTA, complexes exhibiting a sharp peak at 10.9 S. This intermediate s-value indicated that co-assembly into a distinct new set of complexes had occurred. As judged by electron microscopy and viscometry, these mixtures assembled into IFs with characteristics similar to those of pure vimentin and desmin. Furthermore, when vimentin and desmin tetramers were mixed in 5 mM Tris-HCl, pH 8.4, and subsequently subjected to IF assembly conditions, again "hybrid" filaments were obtained. Most interestingly, after 10 min of assembly, mass-per-length (MPL) measurements by scanning transmission electron microscopy yielded IFs with an MPL-peak value of 36 +/- 5 kDa/nm, hence closer to that of vimentin IFs (33 +/- 4 kDa/nm) than to that of desmin IFs (48 +/- 8 kDa/nm). Finally, when unit length-filaments (ULF) of vimentin and desmin were mixed and assembled further, the diameters of individual mature IFs formed exhibited a significantly higher degree of width inhomogeneity along their length than vimentin and desmin IFs as might be expected for a modular mode of assembly. Last but not least, atomic force microscopy provided further direct evidence that desmin IFs are able to fuse end-to-end with vimentin IFs. In summary, we have shown that vimentin and desmin are able to co-assemble at the dimer, tetramer, ULF and even the mature IF level.     

The axial alpha-helices and radial spokes in the core of the cryo-negatively stained complex flagellar filament of Pseudomonas rhodos: recovering high-resolution details from a flexible helical assembly

Of the two known "complex" flagellar filaments, those of Pseudomonas are far more flexible than those of Rhizobium. Their diameter is larger and their outer three-start ridges and grooves are more prominent. Although the symmetry of both complex filaments is similar, the polymer's linear mass density and the flagellin molecular mass of the latter are lower. A recent comparison of a three-dimensional reconstruction of the filament of Pseudomonas rhodos to that of Rhizobium lupini indicates that the outer flagellin domain (D3) is missing in R.lupini. Here, we concentrate on the structure of the inner core of the filament of P.rhodos using field emission cryo-negative staining electron microscopy and a hybrid helical/single particle reconstruction technique. Averaging 158 filaments caused the density band corresponding to the radial spokes to nearly average out due to their variability and inferred flexibility. Treating the Z=0 cross-sections through the aligned individual three-dimensional density maps as images, classifying them by correspondence analysis (using a mask containing the radial spokes domain) and re-averaging the subclasses (using helical reconstruction techniques) allowed a recovery of the radial spokes and resolved the alpha-helices in domain D0 and the triple alpha-helical bundles in domain D1 at a resolution of 1/7A(-1). Although the perturbed components of the helical lattice are present along the entire filament's radius, the interior of the complex filament is similar to that of the plain one, whereas it's exterior is altered. Reconstructions of vitrified and cryo-negatively stained plain, right-handed filaments of Salmonella typhimurium SJW1655 prepared and imaged under conditions identical with those used for P.rhodos confirm the similarity of their inner cores and that the secondary structures in the interior of the flagellar filament can, under critical conditions of image recording and correction, be resolved in negative stain.     

In vivo and in vitro evidence that the four essential intermediate filament (IF) proteins A1, A2, A3 and B1 of the nematode Caenorhabditis elegans form an obligate heteropolymeric IF system

The in vitro polymerization and tissue-specific expression patterns of the four essential intermediate filament (IF) proteins (A1, A2, A3, B1) and the non-essential IF protein A4 were analyzed. Recombinant B1, used as a probe in blot overlay assays of the 11 Caenorhabditis elegans IF proteins, reacted strongly with proteins A1 to A4, indicating a heterotypic interaction. Obligate heteropolymeric filament assembly in vitro was confirmed by electron microscopy. Protein B1 formed long IF when mixed with an equimolar amount of A1, A2 or A3. Developmentally regulated coexpression of B1 and one or more members of the A family was found with GFP-promoter reporters. This coexpression pattern argues for a heteropolymer system in vivo. One or both splice variants of the B1 gene are always coexpressed in a tissue-specific manner with at least one member of the A family in hypodermis, pharynx, pharyngeal-intestinal valve, excretory cells, uterus, vulva and rectum. Interestingly, while the intestine normally lacks a B1/A pair, the dauer larva shows intestinal B1 and A4. These results are in line with similar postembryonic phenotypes of the hypodermis induced by RNA interference (RNAi) of genes B1, A2 and A3. Similarly, defects of the pharynx and its A1-GFP containing tonofilaments observed in the postembryonic B1 RNAi phenotype are consistent with the coexpression of B1 and A1 in the marginal cells. Thus RNAi analyses provide independent evidence for the existence of the B1/A obligate heteropolymer system in vivo. Proteins A1 and B1 have a similar and rather slow turnover rate in photobleaching experiments of the pharynx tonofilaments.     

Structural characterization of human vimentin rod 1 and the sequencing of assembly steps in intermediate filament formation in vitro using site-directed spin labeling and electron paramagnetic resonance

We have previously established the utility of site-directed spin labeling and electron paramagnetic resonance to determine structural relationships among proteins in intact intermediate filaments. Using this same approach we have introduced spin labels at 21 residues between amino acids 169 and 193 in rod domain 1 of human vimentin. The electron paramagnetic resonance spectra provide direct evidence for the coiled coil nature of the vimentin dimer in this region. This finding is consistent with predictions but has never been demonstrated previously. In a previous study we identified residue 348 in the rod domain 2 as one point of overlap between adjacent dimers in intact filaments. In the present study we defined residue 191 in the rod domain 1 as a second point of overlap and established that the dimers are arranged in an anti-parallel and staggered orientation at this site. Finally, by isolating spin-labeled samples at successive stages during the dialysis that lead to filament assembly in vitro, we have been able to establish a sequence of interactions that occurs during in vitro assembly, starting with the alpha helix and loose coiled coil dimer formation, then the formation of tetrameric species centered on residue 191, followed by interactions centered on residue 348 suggestive of octamer or higher order multimer formation. A continuation of this strategy revealed that both 191-191 and 348-348 interactions are present in low ionic strength Tris buffers when vimentin is maintained at the "protofilament" stage of assembly.