The Small Heat Shock Protein Hsp27 Affects Assembly Dynamics and?Structure of Keratin Intermediate Filament Networks

The mechanical properties of living cells are essential for many processes. They are defined by the cytoskeleton, a composite network of protein fibers. Thus, the precise control of its architecture is of paramount importance. Our knowledge about the molecular and physical mechanisms defining the network structure remains scarce, especially for the intermediate filament cytoskeleton. Here, we investigate the effect of small heat shock proteins on the keratin 8/18 intermediate filament cytoskeleton using a well-controlled model system of reconstituted keratin networks. We demonstrate that Hsp27 severely alters the structure of such networks by changing their assembly dynamics. Furthermore, the C-terminal tail domain of keratin 8 is shown to be essential for this effect. Combining results from fluorescence and electron microscopy with data from analytical ultracentrifugation reveals the crucial role of kinetic trapping in keratin network formation.     

The Small Heat Shock Protein Hsp27 Affects Assembly Dynamics and Structure of Keratin Intermediate Filament Networks

The mechanical properties of living cells are essential for many processes. They are defined by the cytoskeleton, a composite network of protein fibers. Thus, the precise control of its architecture is of paramount importance. Our knowledge about the molecular and physical mechanisms defining the network structure remains scarce, especially for the intermediate filament cytoskeleton. Here, we investigate the effect of small heat shock proteins on the keratin 8/18 intermediate filament cytoskeleton using a well-controlled model system of reconstituted keratin networks. We demonstrate that Hsp27 severely alters the structure of such networks by changing their assembly dynamics. Furthermore, the C-terminal tail domain of keratin 8 is shown to be essential for this effect. Combining results from fluorescence and electron microscopy with data from analytical ultracentrifugation reveals the crucial role of kinetic trapping in keratin network formation.     

Retrovirus-mediated transgenic keratin expression in cultured fibroblasts: specific domain functions in keratin stabilization and filament formation.

With retrovirus-mediated gene transfer, we used intact and deleted keratin proteins to investigate the molecular basis of intermediate filament function. Three levels of assembly show a different stringency for the involvement of individual keratin domains: protein accumulation requires the alpha helix domains; stable filament formation additionally requires both N- and C-terminal domains of either one of the two interacting keratins, suggesting that head to tail homotypic interaction is important for effective elongation; and higher order organization of the cytoplasmic network depends on correct type I-type II pairing of keratins. The presence of two distinct interaction sites along potentially different axes may explain the characteristic morphology of keratin intermediate filament networks.     

Properties of intermediate filament networks assembled from keratin 8 and 18 in the presence of mg(2+)

The mechanical properties of epithelial cells are modulated by structural changes in keratin intermediate filament networks. To investigate the relationship between network architecture and viscoelasticity, we assembled keratin filaments from recombinant keratin proteins 8 (K8) and 18 (K18) in the presence of divalent ions (Mg²⁺). We probed the viscoelastic modulus of the network by tracking the movement of microspheres embedded in the network during assembly, and studied the network architecture using scanning electron microscopy. Addition of Mg²⁺ at physiological concentrations (<1 mM) resulted in networks whose structure was similar to that of keratin networks in epithelial cells. Moreover, the elastic moduli of networks assembled in vitro were found to be within the same magnitude as those measured in keratin networks of detergent-extracted epithelial cells. These findings suggest that Mg²⁺-induced filament cross-linking represents a valid model for studying the cytoskeletal mechanics of keratin networks.     

Modeling the self-organization property of keratin intermediate filaments

Keratin intermediate filaments (IFs) fulfill an important function of structural support in epithelial cells. The necessary mechanical attributes require that IFs be organized into a crosslinked network and accordingly, keratin IFs are typically organized into large bundles in surface epithelia. For IFs comprised of keratins 5 and 14 (K5, K14), found in basal keratinocytes of epidermis, bundling can be self-driven through interactions between K14's carboxy-terminal tail domain and two regions in the central α-helical rod domain of K5. Here, we exploit theoretical principles and computational modeling to investigate how such cis-acting determinants best promote IF crosslinking. We develop a simple model where keratin IFs are treated as rigid rods to apply Brownian dynamics simulation. Our findings suggest that long-range interactions between IFs are required to initiate the formation of bundlelike configurations, while tail domain-mediated binding events act to stabilize them. Our model explains the differences observed in the mechanical properties of wild-type versus disease-causing, defective IF networks. This effort extends the notion that the structural support function of keratin IFs necessitates a combination of intrinsic and extrinsic determinants, and makes specific predictions about the mechanisms involved in the formation of crosslinked keratin networks in vivo.     

Cytoskeleton in motion: the dynamics of keratin intermediate filaments in epithelia

Epithelia are exposed to multiple forms of stress. Keratin intermediate filaments are abundant in epithelia and form cytoskeletal networks that contribute to cell type-specific functions, such as adhesion, migration, and metabolism. A perpetual keratin filament turnover cycle supports these functions. This multistep process keeps the cytoskeleton in motion, facilitating rapid and protein biosynthesis-independent network remodeling while maintaining an intact network. The current challenge is to unravel the molecular mechanisms underlying the regulation of the keratin cycle in relation to actin and microtubule networks and in the context of epithelial tissue function.     

p38 MAPK-dependent shaping of the keratin cytoskeleton in cultured cells

Plasticity of the resilient keratin intermediate filament cytoskeleton is an important prerequisite for epithelial tissue homeostasis. Here, the contribution of stress-activated p38 MAPK to keratin network organization was examined in cultured cells. It was observed that phosphorylated p38 colocalized with keratin granules that were rapidly formed in response to orthovanadate. The same p38(p) recruitment was noted during mitosis, in various stress situations and in cells producing mutant keratins. In all these situations keratin 8 became phosphorylated on S73, a well-known p38 target site. To demonstrate that p38-dependent keratin phosphorylation determines keratin organization, p38 activity was pharmacologically and genetically modulated: up-regulation induced keratin granule formation, whereas down-regulation prevented keratin filament network disassembly. Furthermore, transient p38 inhibition also inhibited keratin filament precursor formation and mutant keratin granule dissolution. Collectively, the rapid and reversible effects of p38 activity on keratin phosphorylation and organization in diverse physiological, stress, and pathological situations identify p38-dependent signalling as a major intermediate filament-regulating pathway.     

Stochastic S-system modeling of gene regulatory network

Microarray gene expression data can provide insights into biological processes at a system-wide level and is commonly used for reverse engineering gene regulatory networks (GRN). Due to the amalgamation of noise from different sources, microarray expression profiles become inherently noisy leading to significant impact on the GRN reconstruction process. Microarray replicates (both biological and technical), generated to increase the reliability of data obtained under noisy conditions, have limited influence in enhancing the accuracy of reconstruction . Therefore, instead of the conventional GRN modeling approaches which are deterministic, stochastic techniques are becoming increasingly necessary for inferring GRN from noisy microarray data. In this paper, we propose a new stochastic GRN model by investigating incorporation of various standard noise measurements in the deterministic S-system model. Experimental evaluations performed for varying sizes of synthetic network, representing different stochastic processes, demonstrate the effect of noise on the accuracy of genetic network modeling and the significance of stochastic modeling for GRN reconstruction . The proposed stochastic model is subsequently applied to infer the regulations among genes in two real life networks: (1) the well-studied IRMA network, a real-life in-vivo synthetic network constructed within the Saccharomycescerevisiae yeast, and (2) the SOS DNA repair network in Escherichiacoli.     

Plectin deficiency affects precursor formation and dynamics of vimentin networks

Plectin is a typical cytolinker protein that connects intermediate filaments to the other cytoskeletal filament systems and anchors them at membrane-associated junctional sites. One of the most important binding partners of plectin in fibroblasts is the intermediate filament subunit protein vimentin. Previous studies have demonstrated that vimentin networks are highly dynamic structures whose assembly and disassembly is accomplished stepwise via several intermediates. The precursor forms as well as polymerized (filamentous) vimentin are found in the cells in a dynamic equilibrium characterized by the turnover of the subunits within the polymer and the movement of the smaller precursors. To examine whether plectin plays a role in intermediate filament dynamics, we studied vimentin filament formation in plectin-deficient compared to wild-type fibroblasts using GFP-tagged vimentin. Monitoring vimentin and plectin in spreading and dividing cells, we demonstrate that plectin is associated with vimentin from the early stages of assembly and is required for vimentin motility as well as for the stepwise formation of stable filaments. Furthermore, plectin prevents vimentin networks from complete disassembly during mitosis, facilitating the rebuilding of the intermediate filament network in daughter cells.     

A 'hot-spot' mutation alters the mechanical properties of keratin filament networks

Keratins 5 and 14 polymerize to form the intermediate filament network in the progenitor basal cells of many stratified epithelia including epidermis, where it provides crucial mechanical support. Inherited mutations in K5 or K14 result in epidermolysis bullosa simplex (EBS), a skin-fragility disorder. The impact that such mutations exert on the intrinsic mechanical properties of K5/K14 filaments is unknown. Here we show, by using differential interference contrast microscopy, that a 'hot-spot' mutation in K14 greatly reduces the ability of reconstituted mutant filaments to bundle under crosslinking conditions. Rheological assays measure similar small-deformation mechanical responses for crosslinked solutions of wild-type and mutant keratins. The mutation, however, markedly reduces the resilience of crosslinked networks against large deformations. Single-particle tracking, which probes the local organization of filament networks, shows that the mutant polymer exhibits highly heterogeneous structures compared to those of wild-type filaments. Our results indicate that the fragility of epithelial cells expressing mutant keratin may result from an impaired ability of keratin polymers to be crosslinked into a functional network.     

上皮细胞分裂过程中中等纤维的变化

Immunofluorescence microscopy was used to follow the rearrangement of keratin filaments and vimentin filaments during mitosis in Vero and HeLa cell lines. The experiment results showed that the three dimensional organization and structure of intermediate filaments changed drastically during mitosis. The behavior of intermediate filaments was different in these two epithelial cell lines. In mitotic Vero cells the keratin filaments and vimentin filaments maintained their filamentous structure and formed a cage around the mitotic apparatus. In mitotic HeLa cells the keratin filaments and vimentin filaments reorganized extensively and formed granular cytoplasmic bodies. The ratio of granular cytoplasmic body formation changed in different mitotic phase. The interphase intermediate filament network was reconstructed after mitosis. It is proposed that the state of intermediate filament network in these cells is cell cycle-dependent and intermediate filaments may have some skeletal role in mitosis.     

A nontetrameric species is the major soluble form of keratin in Xenopus oocytes and rabbit reticulocyte lysates

Inside the interphase cell, approximately 5% of the total intermediate filament protein exists in a soluble form. Past studies using velocity gradient sedimentation (VGS) indicate that soluble intermediate filament protein exists as an approximately 7 S tetrameric species. While studying intermediate filament assembly dynamics in the Xenopus oocyte, we used both VGS and size-exclusion chromatography (SEC) to analyze the soluble form of keratin. Previous studies (Coulombe, P. A., and E. Fuchs. 1990. J. Cell Biol. 111:153) report that tetrameric keratins migrate on SEC with an apparent molecular weight of approximately 150,000; the major soluble form of keratin in the oocyte, in contrast, migrates with an apparent molecular weight of approximately 750,000. During oocyte maturation, the keratin system disassembles into a soluble form (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787) and the amount of the 750-kD keratin complex increases dramatically. Immunoprecipitation analysis of soluble keratin from matured oocytes revealed the presence of type I and type II keratins, but no other stoichiometrically associated polypeptides, suggesting that the 750-kD keratin complex is composed solely of keratin. To further study the formation of the 750-kD keratin complex, we used rabbit reticulocyte lysates (RRL). The 750-kD keratin complex was formed in RRLs contranslating type I and type II Xenopus keratins, but not when lysates translated type I or type II keratin RNAs alone. The 750-kD keratin complex could be formed posttranslationally in an ATP-independent manner when type I and type II keratin translation reactions were mixed. Under conditions of prolonged incubation, such as occur during VGS analysis, the 750-kD keratin complex disassembled into a 7 S (by VGS), 150-kD (by SEC) form. In urea denaturation studies, the 7 S/150-kD form could be further disassembled into an 80-kD species that consists of cofractionating dimeric and monomeric keratin. Based on these results, the 750-kD species appears to be a supratetrameric complex of keratins and is the major, soluble form of keratin in both prophase and M-phase oocytes, and RRL reactions.     

上皮细胞分裂过程中中等纤维的变化

应用制备的血清抗体,采用免疫细胞化学方法观察了两株培养上皮细胞的分裂过程中IF的动态变化过程。实验结果显示,在上皮细胞分裂过程中,IF形态结构及空间分布发生了显著变化,不同细胞之间存在差异,分裂的Vero细胞中角蛋白纤维和波形纤维都维持纤维形态,围绕分裂器形成纤维网罩或纤维束环,随着细胞分裂的进行,IF网的空间组织结构和外观发生动态变化;分裂的HeLa细胞中,角蛋白纤维和波形纤维广泛重组形成颗粒状胞质小体,分裂结束后重建IF网。实验结果表明,IF变化具有细胞周期依赖性和一定的细胞特异性。本文对IF在细胞分裂过程中的功能意义作了讨论。     

Effects of Plectin Depletion on Keratin Network Dynamics and Organization

The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin β4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and β1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells.     

Sorting out IF networks: consequences of domain swapping on IF recognition and assembly

Vimentin and keratin are coexpressed in many cells, but they segregate into two distinct intermediate filament (IF) networks. To understand the molecular basis for the sorting out of these IF subunits, we genetically engineered cDNAs encoding hybrid IF proteins composed of part vimentin and part type I keratin. When these cDNAs were transiently expressed in cells containing vimentin, keratin, or both IFs, the hybrid IF proteins all recognized one or the other or both networks. The ability to distinguish networks was dependent upon which segments of IF proteins were present in each construct. Constructs containing sequences encoding either helix 1B or helix 2B seemed to be the most critical in conferring IF recognition. At least for type I keratins, recognition was exerted at the level of dimer formation with wild-type type II keratin, as demonstrated by anion exchange chromatography. Interestingly, despite the fact that swapping of helical domains was not as deleterious to IF structure/function as deletion of helical domains, keratin/vimentin hybrids still caused structural aberrations in one or more of the cytoplasmic IF network. Thus, sequence diversity among IF proteins seems to influence not only coiled-coil but also higher ordered associations leading to 10-nm filament formation and/or IF interactions with other cellular organelles/proteins.     

The growth and form of tunnelling networks in ants

Many biological networks grow under strong spatial constraints, where the large-scale structure emerges from the extension, the branching and intersection of growing parts of the network. One example is provided by ant tunnelling networks, which represent the most common nest architecture in ants. Our goal was to understand how these network structures emerge from the tunnel growth dynamics. We used a standardized two-dimensional set-up shaped as a disk and studied the characteristics of tunnel growth in terms of initiation, propagation and termination of new digging sites and found that they can be described with simple probabilistic laws. We show that a model based on these simple laws and for which parameters were measured from the sand disks experiments can account for the emergence of several topological properties that were observed in experimental networks. In particular, the model accurately reproduced an allometric relation between the number of edges and the number of nodes, as well as an invariance of the node degree distribution. The model was then used to make predictions about the resulting networks' topology when the geometry of the sand substrate was shaped as a square. Experiments aimed at testing the model's predictions showed that the predictions were indeed validated. Both in the model and in the experiments, there was a similar trend for the node degree distribution tail to be steeper in the square sand patch than in the disk sand patch, while other characteristics such as the meshedness (i.e. how densely the network is internally connected) remained constant. Because network growth based on branching/fusion events is widespread in biological systems, this general model might provide useful insights for the study of other systems and, more generally, the evolution of spatial networks in biological systems.     

Predators temper the relative importance of stochastic processes in the assembly of prey metacommunities

Communities assemble through a combination of stochastic processes, which can make environmentally similar communities divergent (high β-diversity), and deterministic processes, which can make environmentally similar communities convergent (low β-diversity). Top predators can influence both stochasticity (e.g. colonization and extinction events) and determinism (e.g. size of the realized species pool), in community assembly, and thus their net effect is unknown. We investigated how predatory fish influenced the scaling of prey diversity in ponds at local and regional spatial scales. While fish reduced both local and regional richness, their effects were markedly more intense at the regional scale. Underlying this result was that the presence of fish made localities within metacommunities more similar in their community composition (lower β-diversity), suggesting that fish enhance the deterministic, relative to the stochastic, components of community assembly. Thus, the presence of predators can alter fundamental mechanisms of community assembly and the scaling of diversity within metacommunities.     

Evidence that subcellular flagellin pools in Caulobacter crescentus are precursors in flagellum assembly

To study the assembly of the Caulobacter crescentus flagellar filament, we have devised a fractionation protocol that separates the cellular flagellin into three compartments: soluble, membrane, and assembled. Radioactive labeling in pulse-chase and pulse-labeling experiments has demonstrated for the first time that both soluble and membrane-associated flagellin pools are precursors in the assembly of the flagellar filament. The results of these experiments also indicate that flagellar filament assembly occurs via the translocation of newly synthesized flagellins from the soluble pool to the membrane pool to the assembled flagellar filaments. It is not possible to conclude whether the soluble flagellin fraction is synthesized cytoplasmically or as a loosely associated membrane intermediate which is released during lysis. It is clear, however, that the soluble and membrane flagellins are in physically and functionally distinct pools. The implications of these findings for the study of protein secretion from cells and the invariant targeting of flagellar proteins to the stalk-distal pole of the dividing cell during flagellum morphogenesis are discussed.     

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.