Assembly Kinetics of Vimentin Tetramers to Unit-Length Filaments: A Stopped-Flow Study.

Intermediate filaments (IFs) are principal components of the cytoskeleton, a dynamic integrated system of structural proteins that provides the functional architecture of metazoan cells. They are major contributors to the elasticity of cells and tissues due to their high mechanical stability and intrinsic flexibility. The basic building block for the assembly of IFs is a rod-like, 60-nm-long tetrameric complex made from two antiparallel, half-staggered coiled coils. In low ionic strength, tetramers form stable complexes that rapidly assemble into filaments upon raising the ionic strength. The first assembly products, "frozen" by instantaneous chemical fixation and viewed by electron microscopy, are 60-nm-long "unit-length" filaments (ULFs) that apparently form by lateral in-register association of tetramers. ULFs are the active elements of IF growth, undergoing longitudinal end-to-end annealing with one another and with growing filaments. Originally, we have employed quantitative time-lapse atomic force and electron microscopy to analyze the kinetics of vimentin-filament assembly starting from a few seconds to several hours. To obtain detailed quantitative insight into the productive reactions that drive ULF formation, we now introduce a "stopped-flow" approach in combination with static light-scattering measurements. Thereby, we determine the basic rate constants for lateral assembly of tetramers to ULFs. Processing of the recorded data by a global fitting procedure enables us to describe the hierarchical steps of IF formation. Specifically, we propose that tetramers are consumed within milliseconds to yield octamers that are obligatory intermediates toward ULF formation. Although the interaction of tetramers is diffusion controlled, it is strongly driven by their geometry to mediate effective subunit targeting. Importantly, our model conclusively reflects the previously described occurrence of polymorphic ULF and mature filaments in terms of their number of tetramers per cross section.

Contributions of the lower dimer to supramolecular actin patterning revealed by TIRF microscopy.

Two distinct dimers are formed during the initial steps of actin polymerization. The first one, referred to as the 'lower dimer' (LD) was discovered many years ago by means of chemical crosslinking. Owing to its transient nature, a biological relevance had long been precluded when, using LD-specific antibodies, we detected LD-like contacts in actin assemblies that are associated with the endolysosomal compartment in a number of different cell lines. Moreover, immunofluorescence showed the presence of LD-related structures at the cell periphery of migrating fibroblasts, in the nucleus, and in association with the centrosome of interphase cells. Here, we explore contributions of the LD to the assembly of supramolecular actin structures in real time by total internal reflection fluorescence (TIRF) microscopy. Our data shows that while LD on its own cannot polymerize under filament forming conditions, it is able to incorporate into growing F-actin filaments. This incorporation of LD triggers the formation of X-shaped filament assemblies with barbed ends that are pointing in the same direction in the majority of cases. Similarly, an increased frequency of junction sites was observed when filaments were assembled in the presence of oxidized actin. This data suggests that a disulfide bridge between Cys374 residues might stabilize LD-contacts. Based on our findings, we propose two possible models for the molecular mechanism underlying the supramolecular actin patterning in LD-related structures.

Atomic structure of the vimentin central α-helical domain and its implications for intermediate filament assembly.

Together with actin filaments and microtubules, intermediate filaments (IFs) are the basic cytoskeletal components of metazoan cells. Over 80 human diseases have been linked to mutations in various IF proteins to date. However, the filament structure is far from being resolved at the atomic level, which hampers rational understanding of IF pathologies. The elementary building block of all IF proteins is a dimer consisting of an α-helical coiled-coil (CC) "rod" domain flanked by the flexible head and tail domains. Here we present three crystal structures of overlapping human vimentin fragments that comprise the first half of its rod domain. Given the previously solved fragments, a nearly complete atomic structure of the vimentin rod has become available. It consists of three α-helical segments (coils 1A, 1B, and 2) interconnected by linkers (L1 and L12). Most of the CC structure has a left-handed twist with heptad repeats, but both coil 1B and coil 2 also exhibit untwisted, parallel stretches with hendecad repeats. In the crystal structure, linker L1 was found to be α-helical without being involved in the CC formation. The available data allow us to construct an atomic model of the antiparallel tetramer representing the second level of vimentin assembly. Although the presence of the nonhelical head domains is essential for proper tetramer stabilization, the precise alignment of the dimers forming the tetramer appears to depend on the complementarity of their surface charge distribution patterns, while the structural plasticity of linker L1 and coil 1A plays a role in the subsequent IF assembly process.

The biodistribution of self-assembling protein nanoparticles shows they are promising vaccine platforms.

BACKGROUND: Because of the need to limit side-effects, nanoparticles are increasingly being studied as drug-carrying and targeting tools. We have previously reported on a scheme to produce protein-based self-assembling nanoparticles that can act as antigen display platforms. Here we attempted to use the same system for cancer-targeting, making use of a C-terminal bombesin peptide that has high affinity for a receptor known to be overexpressed in certain tumors, as well as an N-terminal polyhistidine tag that can be used for radiolabeling with technetium tricarbonyl. RESULTS: In order to increase circulation time, we experimented with PEGylated and unPEGylated varities typo particle. We also tested the effect of incorporating different numbers of bombesins per nanoparticle. Biophysical characterization determined that all configurations assemble into regular particles with relatively monodisperse size distributions, having peaks of about 33-36 nm. The carbonyl method used for labeling produced approximately 80% labeled nanoparticles. In vitro, the nanoparticles showed high binding, both specific and non-specific, to PC-3 prostate cancer cells. In vivo, high uptake was observed for all nanoparticle types in the spleens of CD-1 nu/nu mice, decreasing significantly over the course of 24 hours. High uptake was also observed in the liver, while only low uptake was seen in both the pancreas and a tumor xenograft. CONCLUSIONS: The data suggest that the nanoparticles are non-specifically taken up by the reticuloendothelial system. Low uptake in the pancreas and tumor indicate that there is little or no specific targeting. PEGylation or increasing the amount of bombesins per nanoparticle did not significantly improve targeting. In particular, the uptake in the spleen, which is a primary organ of the immune system, highlights the potential of the nanoparticles as vaccine carriers. Also, the decrease in liver and spleen radioactivity with time implies that the nanoparticles are broken down and cleared. This is an important finding, as it shows that the nanoparticles can be safely used as a vaccine platform without the risk of prolonged side effects. Furthermore, it demonstrates that technetium carbonyl radiolabeling of our protein-based nanoparticles can be used to evaluate their pharmacokinetic properties in vivo.

Complex formation and kinetics of filament assembly exhibited by the simple epithelial keratins K8 and K18.

We have generated human recombinant keratins K8 and K18 and describe conditions to quantitatively follow their assembly into filaments. When renatured individually from 8M urea into a low ionic strength/high pH-buffer, K8 was present in a dimeric to tetrameric form as revealed by analytical ultracentrifugation. In contrast, K18 sedimented as a monomer. When mixed in 8 M urea and renatured together, K8 and K18 exhibited s-value profiles compatible with homogeneous tetrameric complexes. This finding was confirmed by sedimentation equilibrium centrifugation. Subsequently, these tetrameric starter units were subjected to assembly experiments at various protein concentrations. At low values such as 0.0025 g/l, unit-length filaments were abundantly present after 2s of assembly. During the following 5 min, filaments grew rapidly and by measuring the length of individual filaments we were able to generate time-dependent length profiles. These data revealed that keratins K8/K18 assemble several times faster than vimentin and desmin. In addition, we determined the persistence length l(p) of K8/K18 filaments to be in the range of 300 nm. Addition of 1 mM MgCl(2) increases l(p) to 480 nm indicating that magnesium ions affect the interaction of keratin subunits within the filament during assembly to some extent.

An antiparallel actin dimer is associated with the endocytic pathway in mammalian cells.

The dynamic rearrangement of the actin cytoskeleton plays a key role in several cellular processes such as cell motility, endocytosis, RNA processing and chromatin organization. However, the supramolecular actin structures involved in the different processes remain largely unknown. One of the less studied forms of actin is the lower dimer (LD). This unconventional arrangement of two actin molecules in an antiparallel orientation can be detected by chemical crosslinking at the onset of polymerization in vitro. Moreover, evidence for a transient incorporation of LD into growing filaments and its ability to inhibit nucleation of F-actin filament assembly implicate that the LD pathway contributes to supramolecular actin patterning. However, a clear link from this actin species to a specific cellular function has not yet been established. We have developed an antibody that selectively binds to LD configurations in supramolecular actin structures assembled in vitro. This antibody allowed us to unveil the LD in different mammalian cells. In particular, we show an association of the antiparallel actin arrangement with the endocytic compartment at the cellular and ultrastructural level. Taken together, our results strongly support a functional role of LD in the patterning of supramolecular actin assemblies in mammalian cells.

Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit.

The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments. Along this pathway, multiple pre-ribosomal particles are generated, which transiently associate with numerous non-ribosomal factors before mature 60S and 40S subunits are formed. However, most mechanistic details of ribosome biogenesis are still unknown. Here we identify a maturation step of the yeast pre-40S subunit that is regulated by the protein kinase Hrr25 and involves ribosomal protein Rps3. A high salt concentration releases Rps3 from isolated pre-40S particles but not from mature 40S subunits. Electron microscopy indicates that pre-40S particles lack a structural landmark present in mature 40S subunits, the 'beak'. The beak is formed by the protrusion of 18S ribosomal RNA helix 33, which is in close vicinity to Rps3. Two protein kinases Hrr25 and Rio2 are associated with pre-40S particles. Hrr25 phosphorylates Rps3 and the 40S synthesis factor Enp1. Phosphorylated Rsp3 and Enp1 readily dissociate from the pre-ribosome, whereas subsequent dephosphorylation induces formation of the beak structure and salt-resistant integration of Rps3 into the 40S subunit. In vivo depletion of Hrr25 inhibits growth and leads to the accumulation of immature 40S subunits that contain unstably bound Rps3. We conclude that the kinase activity of Hrr25 regulates the maturation of 40S ribosomal subunits.

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.

Amyloid structure and assembly: insights from scanning transmission electron microscopy.

Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR).

Tracking down the different forms of nuclear actin.

Actin is a rather uncommitted protein with a high degree of structural plasticity: it can adopt a variety of structural states, depending on the specific ionic conditions or the interaction with ligand proteins. These interactions lock actin into a distinct conformation, which specifies the oligomeric or polymeric form it can assume. The interplay between monomeric, oligomeric and polymeric forms is used by the cell to execute an enormous variety of motility processes, such as lamellipodium formation during locomotion or intracellular transport of vesicles. In these cytoplasmic events, monomeric G-actin and filamentous F-actin are the prevalent forms. However, there might be other structural states of actin in cells that have so far not received the attention they deserve. Here, we propose that specific, "unconventional" actin conformations might contribute especially to the multitude of functions executed by actin in the nucleus. We present evidence for the existence of different forms of nuclear actin, taken from studies with selected antibodies.

A nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria.

We have designed and produced a prototypic malaria vaccine based on a highly versatile self-assembling polypeptide nanoparticle (SAPN) platform that can repetitively display antigenic epitopes. We used this platform to display a tandem repeat of the B cell immunodominant repeat epitope (DPPPPNPN)(2)D of the malaria parasite Plasmodium berghei circumsporozoite protein. Administered in saline, without the need for a heterologous adjuvant, the SAPN construct P4c-Mal conferred a long-lived, protective immune response to mice with a broad range of genetically distinct immune backgrounds including the H-2(b), H-2(d), and H-2(k) alleles. Immunized mice produced a CD4(+) T cell-dependent, high-titer, long-lasting, high-avidity Ab response against the B cell epitope. Mice were protected against an initial challenge of parasites up to 6 mo after the last immunization or for up to 15 mo against a second challenge after an initial challenge of parasites had successfully been cleared. Furthermore, we demonstrate that the SAPN platform not only functions to deliver an ordered repetitive array of B cell peptide epitopes but operates as a classical immunological carrier to provide cognate help to the P4c-Mal-specific B cells.

Laminopathic mutations interfere with the assembly, localization, and dynamics of nuclear lamins.

Lamins are nuclear intermediate filament proteins and the major building blocks of the nuclear lamina. Besides providing nuclear shape and mechanical stability, lamins are required for chromatin organization, transcription regulation, DNA replication, nuclear assembly, nuclear positioning, and apoptosis. Mutations in human lamins cause many different heritable diseases, affecting various tissues and causing early aging. Although many of these mutations result in nuclear deformation, their effects on lamin filament assembly are unknown. Caenorhabditis elegans has a single evolutionarily conserved lamin protein, which can form stable 10-nm-thick filaments in vitro. To gain insight into the molecular basis of lamin filament assembly and the effects of laminopathic mutations on this process, we investigated mutations in conserved residues of the rod and tail domains that are known to cause various laminopathies in human. We show that 8 of 14 mutant lamins present WT-like assembly into filaments or paracrystals, whereas 6 mutants show assembly defects. Correspondingly, expressing these mutants in transgenic animals shows abnormal distribution of Ce-lamin, abnormal nuclear shape or change in lamin mobility. These findings help in understanding the role of individual residues and domains in laminopathy pathology and, eventually, promote the development of therapeutic interventions.

Micro- and nanomechanical analysis of articular cartilage by indentation-type atomic force microscopy: validation with a gel-microfiber composite.

As documented previously, articular cartilage exhibits a scale-dependent dynamic stiffness when probed by indentation-type atomic force microscopy (IT-AFM). In this study, a micrometer-size spherical tip revealed an unimodal stiffness distribution (which we refer to as microstiffness), whereas probing articular cartilage with a nanometer-size pyramidal tip resulted in a bimodal nanostiffness distribution. We concluded that indentation of the cartilage's soft proteoglycan (PG) gel gave rise to the lower nanostiffness peak, whereas deformation of its collagen fibrils yielded the higher nanostiffness peak. To test our hypothesis, we produced a gel-microfiber composite consisting of a chondroitin sulfate-containing agarose gel and a fibrillar poly(ethylene glycol)-terephthalate/poly(butylene)-terephthalate block copolymer. In striking analogy to articular cartilage, the microstiffness distribution of the synthetic composite was unimodal, whereas its nanostiffness exhibited a bimodal distribution. Also, similar to the case with cartilage, addition of the negatively charged chondroitin sulfate rendered the gel-microfiber composite's water content responsive to salt. When the ionic strength of the surrounding buffer solution increased from 0.15 to 2 M NaCl, the cartilage's microstiffness increased by 21%, whereas that of the synthetic biomaterial went up by 31%. When the nanostiffness was measured after the ionic strength was raised by the same amount, the cartilage's lower peak increased by 28%, whereas that of the synthetic biomaterial went up by 34%. Of interest, the higher peak values remained unchanged for both materials. Taken together, these results demonstrate that the nanoscale lower peak is a measure of the soft PG gel, and the nanoscale higher peak measures collagen fibril stiffness. In contrast, the micrometer-scale measurements fail to resolve separate stiffness values for the PG and collagen fibril moieties. Therefore, we propose to use nanostiffness as a new biomarker to analyze structure-function relationships in normal, diseased, and engineered cartilage.

Atomic structure of vimentin coil 2.

Intermediate filaments (IFs) are essential cytoskeletal components in metazoan cells. They assemble from elementary dimers that are built around the central alpha-helical coiled-coil rod domain representing the IF 'signature'. The rod consists of two similarly-sized parts, coil 1 and coil 2, connected by a non-alpha-helical linker L12. Coil 2 is absolutely conserved in length across all IF types and was initially predicted to consist of a short coiled-coil segment 2A based on a heptad pattern of hydrophobic residues, another linker L2 and a coiled-coil segment 2B. Here we present the crystal structure of human vimentin fragment including residues 261-335 i.e. approximately the first half of coil 2. The N-terminal part of this fragment reveals a parallel alpha-helical bundle characterized by 3.5 consecutive hendecad repeats. It is immediately followed by a regular left-handed coiled coil. The distinct non-helical linker L2 is therefore not observed. Together with the previously determined crystal structure of the major part of segment 2B (Strelkov et al., 2002), we can now build a complete atomic model of the 21nm long vimentin coil 2 dimer being a relatively rigid rod.

Towards monitoring transport of single cargos across individual nuclear pore complexes by time-lapse atomic force microscopy.

A new preparation procedure was developed for the stable adsorption of either the cytoplasmic or the nuclear face of native (i.e. in physiological buffer without detergent extraction and in the absence of chemical fixatives) Xenopus oocyte nuclear envelopes (NEs) onto silicon (Si) surfaces. This yields optimal structural preservation of the nuclear pore complexes (NPCs) without compromising their functional properties. The functional viability of thus prepared NPCs was documented by time-lapse atomic force microscopy (AFM) of the reversible calcium-mediated opening (i.e. +Ca(2+)) and closing (i.e. -Ca(2+)) of the iris diaphragm-like distal ring topping the NPCs' nuclear baskets. Moreover, site-specific single colloidal gold particle detection was documented by AFM imaging one and the same NPC before and after immuno-gold labeling the sample with a nucleoporin-specific antibody. With this new preparation protocol at hand, we should eventually be able to follow by time-lapse AFM transport of single gold-conjugated cargos across individual NPCs.

Fast quantitative single-molecule detection at ultralow concentrations.

The applicability of single-molecule fluorescence assays in liquids is limited by diffusion to concentrations in the low picomolar range. Here, we demonstrate quantitative single-molecule detection at attomolar concentrations within 1 min by excitation and detection of fluorescence through a single-mode optical fiber in presence of turbulent flow. The combination of high detectability and short measurement times promises applications in ultrasensitive assays, sensors, and point-of-care medical diagnostics.

An RGD-restricted substrate interface is sufficient for the adhesion, growth and cartilage forming capacity of human chondrocytes.

This study aimed at testing whether an RGD-restricted substrate interface is sufficient for adhesion and growth of human articular chondrocytes (HAC), and whether it enhances their post expansion chondrogenic capacity. HAC/substrate interaction was restricted to RGD by modifying tissue culture polystyrene (TCPS) with a poly(ethylene glycol) (PEG) based copolymer system that renders the surface resistant to protein adsorption while at the same time presenting the bioactive RGD-containing peptide GCRGYGRGDSPG (RGD). As compared to TCPS, HAC cultured on RGD spread faster (1.9-fold), maintained higher type II collagen mRNA expression (4.9-fold) and displayed a 19% lower spreading area. On RGD, HAC attachment efficiency (66±10%) and proliferation rate (0.56±0.04 doublings/day), as well as type II collagen mRNA expression in the subsequent chondrogenic differentiation phase, were similar to those of cells cultured on TCPS. In contrast, cartilaginous matrix deposition by HAC expanded on RGD was slightly but consistently higher (15% higher glycosaminoglycan-to-DNA ratio). RDG (bioinactive peptide) and PEG (no peptide ligand) controls yielded drastically reduced attachment efficiency (lower than 11%) and proliferation (lower than 0.20 doublings/day). Collectively, these data indicate that restriction of HAC interaction with a substrate through RGD peptides is sufficient to support their adhesion, growth and maintenance of cartilage forming capacity. The concept could thus be implemented in materials for cartilage repair, whereby in situ recruited/infiltrated chondroprogenitor cells would proliferate while maintaining their ability to differentiate and generate cartilage tissue.

Ca2+ -dependent interaction of S100A1 with F1-ATPase leads to an increased ATP content in cardiomyocytes.

S100A1, a Ca(2+)-sensing protein of the EF-hand family that is expressed predominantly in cardiac muscle, plays a pivotal role in cardiac contractility in vitro and in vivo. It has recently been demonstrated that by restoring Ca(2+) homeostasis, S100A1 was able to rescue contractile dysfunction in failing rat hearts. Myocardial contractility is regulated not only by Ca(2+) homeostasis but also by energy metabolism, in particular the production of ATP. Here, we report a novel interaction of S100A1 with mitochondrial F(1)-ATPase, which affects F(1)-ATPase activity and cellular ATP production. In particular, cardiomyocytes that overexpress S100A1 exhibited a higher ATP content than control cells, whereas knockdown of S100A1 expression decreased ATP levels. In pull-down experiments, we identified the alpha- and beta-chain of F(1)-ATPase to interact with S100A1 in a Ca(2+)-dependent manner. The interaction was confirmed by colocalization studies of S100A1 and F(1)-ATPase and the analysis of the S100A1-F(1)-ATPase complex by gel filtration chromatography. The functional impact of this association is highlighted by an S100A1-mediated increase of F(1)-ATPase activity. Consistently, ATP synthase activity is reduced in cardiomyocytes from S100A1 knockout mice. Our data indicate that S100A1 might play a key role in cardiac energy metabolism.

Towards an integrated understanding of the structure and mechanics of the cell nucleus.

Changes in the shape and structural organization of the cell nucleus occur during many fundamental processes including development, differentiation and aging. In many of these processes, the cell responds to physical forces by altering gene expression within the nucleus. How the nucleus itself senses and responds to such mechanical cues is not well understood. In addition to these external forces, epigenetic modifications of chromatin structure inside the nucleus could also alter its physical properties. To achieve a better understanding, we need to elucidate the relationship between nuclear structure and material properties. Recently, new approaches have been developed to systematically investigate nuclear mechanical properties. These experiments provide important new insights into the disease mechanism of a growing class of tissue-specific disorders termed 'nuclear envelopathies'. Here we review our current understanding of what determines the shape and mechanical properties of the cell nucleus.