The superfamily of intermediate filament (IF) proteins contains at least 65
distinct proteins in man, which all assemble into ∼10 nm wide filaments and
are principal structural elements both in the ...nucleus and the cytoplasm with
essential scaffolding functions in metazoan cells. At present, we have only
circumstantial evidence of how the highly divergent primary sequences of IF
proteins lead to the formation of seemingly similar polymers and how this
correlates with their function in individual cells and tissues. Point mutations
in IF proteins, particularly in lamins, have been demonstrated to lead to
severe, inheritable multi-systemic diseases, thus underlining their importance
at several functional levels. Recent structural work has now begun to shed some
light onto the complex fine tuning of structure and function in these fibrous,
coiled coil forming multidomain proteins and their contribution to cellular
physiology and gene regulation.
Intermediate filaments (IFs) are structural elements of eukaryotic cells with distinct mechanical properties. Tissue integrity is severely impaired, in particular in skin and muscle, when IFs are ...either absent or malfunctioning due to mutations. Our knowledge on the mechanical properties of IFs is mainly based on tensile testing of macroscopic fibers and on the rheology of IF networks. At the single filament level, the only piece of data available is a measure of the persistence length of vimentin IFs.
Here, we have employed an atomic force microscopy (AFM) based protocol to directly probe the mechanical properties of single cytoplasmic IFs when adsorbed to a solid support in physiological buffer environment. Three IF types were studied
in vitro: recombinant murine desmin, recombinant human keratin K5/K14 and neurofilaments isolated from rat brains, which are composed of the neurofilament triplet proteins NF-L, NF-M and NF-H. Depending on the experimental conditions, the AFM tip was used to laterally displace or to stretch single IFs on the support they had been adsorbed to. Upon applying force, IFs were stretched on average 2.6-fold. The maximum stretching that we encountered was 3.6-fold. A large reduction of the apparent filament diameter was observed concomitantly. The observed mechanical properties therefore suggest that IFs may indeed function as mechanical shock absorbers
in vivo.
Summary Objective Functional cartilage tissue engineering aims to generate grafts with a functional surface, similar to that of authentic cartilage. Bioreactors that stimulate cell-scaffold ...constructs by simulating natural joint movements hold great potential to generate cartilage with adequate surface properties. In this study two methods based on atomic force microscopy (AFM) were applied to obtain information about the quality of engineered graft surfaces. For better understanding of the molecule–function relationships, AFM was complemented with immunohistochemistry. Methods Bovine chondrocytes were seeded into polyurethane scaffolds and subjected to dynamic compression, applied by a ceramic ball, for 1 h daily loading group 1 (LG1). In loading group 2 (LG2), the ball additionally oscillated over the scaffold, generating sliding surface motion. After 3 weeks, the surfaces of the engineered constructs were analyzed by friction force and indentation-type AFM (IT-AFM). Results were complemented and compared to immunohistochemical analyses. Results The loading type significantly influenced the mechanical and histological outcomes. Constructs of LG2 exhibited lowest friction coefficient and highest micro- and nanostiffness. Collagen type II and aggrecan staining were readily observed in all constructs and appeared to reach deeper areas in loaded (LG1, LG2) compared to unloaded scaffolds. Lubricin was specifically detected at the top surface of LG2. Conclusions This study proposes a quantitative AFM-based functional analysis at the micrometer- and nanometer scale to evaluate the quality of cartilage surfaces. Mechanical testing (load-bearing) combined with friction analysis (gliding) can provide important information. Notably, sliding-type biomechanical stimuli may favor (re-)generation and maintenance of functional articular surfaces and support the development of mechanically competent engineered cartilage.
Eukaryotic cells contain three cytoskeletal filament systems that exhibit very distinct assembly properties, supramolecular architectures, dynamic behaviour and mechanical properties. Microtubules ...and microfilaments are relatively stiff polar structures whose assembly is modulated by the state of hydrolysis of the bound nucleotide. In contrast, intermediate filaments (IFs) are more flexible apolar structures assembled from a ∼45 nm long coiled-coil dimer as the elementary building block. The differences in flexibility that exist among the three filament systems have been described qualitatively by comparing electron micrographs of negatively stained dehydrated filaments and by directly measuring the persistence length of F-actin filaments (∼3–10 μm) and microtubules (∼1–8 mm) by various physical methods. However, quantitative data on the persistence length of IFs are still missing.
Toward this goal, we have carried out atomic force microscopy (AFM) in physiological buffer to characterise the morphology of individual vimentin IFs adsorbed to different solid supports. In addition, we compared these images with those obtained by transmission electron microscopy (TEM) of negatively stained dehydrated filaments. For each support, we could accurately measure the apparent persistence length of the filaments, yielding values ranging between 0.3 μm and 1 μm. Making simple assumptions concerning the adsorption mechanism, we could estimate the persistence length of an IF in a dilute solution to be ∼1 μm, indicating that the lower measured values reflect constraints induced by the adsorption process of the filaments on the corresponding support.
Based on our knowledge of the structural organisation and mechanical properties of IFs, we reason that the lower persistence length of IFs compared to that of F-actin filaments is caused by the presence of flexible linker regions within the coiled-coil dimer and by postulating the occurrence of axial slipping between dimers within IFs.
Intermediate filaments (IFs), together with actin filaments and microtubules, compose the cytoskeleton. Among other functions, IFs impart mechanical stability to cells when exposed to mechanical ...stress and act as a support when the other cytoskeletal filaments cannot keep the structural integrity of the cells. Here we present a study on the bending properties of single vimentin IFs in which we used an atomic force microscopy (AFM) tip to elastically deform single filaments hanging over a porous membrane. We obtained a value for the bending modulus of non-stabilized IFs between 300 MPa and 400 MPa. Our results together with previous ones suggest that IFs present axial sliding between their constitutive building blocks and therefore have a bending modulus that depends on the filament length. Measurements of glutaraldehyde-stabilized filaments were also performed to reduce the axial sliding between subunits and therefore provide a lower limit estimate of the Young's modulus of the filaments. The results show an increment of two to three times in the bending modulus for the stabilized IFs with respect to the non-stabilized ones, suggesting that the Young's modulus of vimentin IFs should be around 900 MPa or higher.
For many years the existence of actin in the nucleus has been doubted because of the lack of phalloidin staining as well as the failure to document nuclear actin filaments by electron microscopy. ...More recent findings reveal actin to be a component of chromatin remodeling complexes and of the machinery involved in RNA synthesis and transport. With distinct functions for nuclear actin emerging, the quest for its conformation and oligomeric/polymeric structure in the nucleus has resumed importance. We used chemically cross-linked ‘lower dimer’ (LD) to generate mouse monoclonal antibodies specific for different actin conformations. One of the resulting antibodies, termed 1C7, recognizes an epitope that is buried in the F-actin filament, but is surface-exposed in G-actin as well as in the LD. In immunofluorescence studies with different cell lines, 1C7 selectively reacts with non-filamentous actin in the cytoplasm. In addition, it detects a discrete form of actin in the nucleus, which is different from the nuclear actin revealed by the previously described 2G2 Gonsior, S.M., Platz, S., Buchmeier, S., Scheer, U., Jockusch, B.M., Hinssen, H., 1999. J. Cell Sci. 112, 797. Upon latrunculin-induced disassembly of the filamentous cytoskeleton in Rat2 fibroblasts, we observed a perinuclear accumulation of the 1C7-reactive actin conformation. In addition, latrunculin treatment led to the assembly of phalloidin-staining actin structures in chromatin-free regions of the nucleus in these cells. Our results indicate that distinct actin conformations and/or structures are present in the nucleus and the cytoplasm of different cell types and that their distribution varies in response to external signals.
To date, over 20 peptides or proteins have been identified that can form amyloid fibrils in the body and are thought to cause disease. The mechanism by which amyloid peptides cause the cytotoxicity ...observed and disease is not understood. However, one of the major hypotheses is that amyloid peptides cause membrane perturbation. Hence, we have studied the interaction between lipid bilayers and the 37 amino acid residue polypeptide amylin, which is the primary constituent of the pancreatic amyloid associated with type 2 diabetes. Using a dye release assay we confirmed that the amyloidogenic human amylin peptide causes membrane disruption; however, time-lapse atomic force microscopy revealed that this did not occur by the formation of defined pores. On the contrary, the peptide induced the formation of small defects spreading over the lipid surface. We also found that rat amylin, which has 84% identity with human amylin but cannot form amyloid fibrils, could also induce similar lesions to supported lipid bilayers. The effect, however, for rat amylin but not human amylin, was inhibited under high ionic conditions. These data provide an alternative theory to pore formation, and how amyloid peptides may cause membrane disruption and possibly cytotoxicity.
The importin‐alpha/beta heterodimer and the GTPase Ran play key roles in nuclear protein import. Importin binds the nuclear localization signal (NLS). Translocation of the resulting import ligand ...complex through the nuclear pore complex (NPC) requires Ran and is terminated at the nucleoplasmic side by its disassembly. The principal GTP exchange factor for Ran is the nuclear protein RCC1, whereas the major RanGAP is cytoplasmic, predicting that nuclear Ran is mainly in the GTP form and cytoplasmic Ran is in the GDP‐bound form. Here, we show that nuclear import depends on cytoplasmic RanGDP and free GTP, and that RanGDP binds to the NPC. Therefore, import might involve nucleotide exchange and GTP hydrolysis on NPC‐bound Ran. RanGDP binding to the NPC is not mediated by the Ran binding sites of importin‐beta, suggesting that translocation is not driven from these sites. Consistently, a mutant importin‐beta deficient in Ran binding can deliver its cargo up to the nucleoplasmic side of the NPC. However, the mutant is unable to release the import substrate into the nucleoplasm. Thus, binding of nucleoplasmic RanGTP to importin‐beta probably triggers termination, i.e. the dissociation of importin‐alpha from importin‐beta and the subsequent release of the import substrate into the nucleoplasm.
Amyloiddeposits of fibrillar human amylin (hA) in the pancreas may be a causative factor in type-2 diabetes. A detailed comparison of in vitro fibril formation by full-length hA(1–37) versus ...fragments of this peptide—hA(8–37) and hA(20–29)—is presented. Circular dichroism spectroscopy revealed that fibril formation was accompanied by a conformational change: random coil to β-sheet/α-helical structure. Fibril morphologies were visualized by electron microscopy and displayed a remarkable diversity. hA(20–29) formed flat ribbons consisting of numerous 3.6-nm-wide protofibrils. In contrast, hA(1–37) and hA(8–37) formed polymorphic higher order fibrils by lateral association and/or coiling together of 5.0-nm-wide protofibril subunits. For full-length hA(1–37), the predominant fibril type contained three protofibrils and for hA(8–37), the predominant type contained two protofibrils. Polymerization was also monitored with the thioflavin-T binding assay, which revealed different kinetics of assembly for hA(1–37) and hA(8–37) fibrils. hA(20–29) fibrils did not bind thioflavin-T. Together the results demonstrate that the N-terminal region of the hA peptide influences the relative frequencies of the various higher order fibril types and thereby the overall kinetics of fibril formation. Furthermore, while residues 20–29 contribute to the fibrils' β-sheet core, the flanking C- and N-terminal regions of the hA peptide determine the interactions involved in the formation of higher order coiled polymorphic superstructures.
The asymmetric unit membrane (AUM) forms the apical plaques of mammalian urothelium and is believed to play a role in strengthening the urothelial apical surface thus preventing the cells from ...rupturing during bladder distention. We have shown previously that purified bovine AUMs contain four major integral membrane proteins: the uroplakins Ia (27 kDa), Ib (28 kDa), II (15 kDa), and III (47 kDa). This contradicts some previous reports indicating that some of these proteins are absent in AUMs of several species. Using an improved procedure, we isolated AUMs from, in addition to cattle, eight mammalian species (human, monkey, sheep, pig, dog, rabbit, rat, and mouse). The AUMs of these species appear morphologically similar bearing crystalline patches of 12-nm protein particles with a center-to-center spacing of 16.5 nm. Using antibodies raised against synthetic oligopeptides or individual bovine uroplakins, we established by immunoblotting that the four uroplakins are present in AUMs of all these species. The DNA-deduced amino acid sequences of bovine and mouse uroplakin II revealed 83% identity. These results indicate that uroplakins Ia, Ib, II, and III are the major protein components of probably all mammalian urothelial plaques, and that the sequence and three-dimensional structure of uroplakin molecules are highly conserved during mammalian evolution.
PDF
