G-quartet self-assembly under osmotic pressure: remote control by vesicle shrinking rather than stress

Does osmotic pressure stimulate assembly or disassembly of supramolecules in vesicles? Self‐assembly was conceivable as intravesicular response to osmotic shrinking upon application of extravesicular overpressure, whereas disassembly was conceivable as a response to bilayer stress in hyperosmotic vesicles. Self‐assembly of guanosine 5′‐monophosphates (GMPs) into G‐quartets was selected to investigate the nature of remote control of supramolecular chemistry within vesicles by osmotic pressure. Using circular dichroism spectroscopy to selectively detect G‐quartets, we found that extravesicular overpressure stimulates intravesicular self‐assembly, whereas underpressure stimulates disassembly. G‐quartet self‐assembly by osmotic pressure exhibited ion‐selective metal‐cation templation, as expected. The key conclusions are that supramolecular chemistry within vesicles is governed by vesicle shape rather than vesicle stress and that detection of osmotic pressure by CD spectroscopy is an interesting alternative to the commonly used methods based on fluorescence self‐quenching. Chirality 15:766–771, 2003. © 2003 Wiley‐Liss, Inc.     

Self-organization and complexity in historical landscape patterns

Self‐organization describes the evolution process of complex structures where systems emerge spontaneously, driven internally by variations of the system itself. Self‐organization to the critical state is manifested by scale‐free behavior across many orders of magnitude (Bak et al. 1987, Bak 1996, Solé et al. 1999). Spatial scale‐free behavior implies fractal properties and is quantified by the fractal dimension. Temporal scale‐free behavior is evident in power spectra of fluctuations that obey power laws. Self‐organized criticality is a universal phenomenon that likely produces some of the fractals and power laws observed in nature. We investigated the historical landscape of southern Wisconsin (USA) (60,000 km²) for self‐organization and complexity. The landscape is patterned into prairies, savannas, and open and closed forests, using data from the United States General Land Office Surveys that were conducted during the 19th century, at a time prior to Euro‐American settlement. We applied a two‐dimensional cellular automaton model with one adjustable parameter. Model evolution replaces a cell that dies at random times by a cell chosen randomly from within a circular radius r, where r typically takes values between 1 (local) and 10 units (regional). Cluster probability is used to measure the degree of organization. The model landscape self‐organizes to a realistic critical state if neighborhoods of intermediate size (r=3) are chosen, indicating that (a) no particular time or space scale for the clusters is singled out, i.e. the spatial dependence is fractal, and temporal fluctuations in the cluster probability exhibit power laws; (b) a simple model suffices to replicate the landscape pattern resulting from complex spatial and temporal interactions. Measures of comparison between the observed and the simulated landscape show good agreement: fractal dimensions for simulated (1.6) and observed landscapes (1.64), cluster probabilities for simulated (32.3%) and observed (32.6%) landscapes, and algorithmic complexity for simulated (6792 bytes) and observed (6205 bytes) landscapes. The results are robust towards variation of initial and boundary conditions as well as perturbations.     

Dynamin GTPase regulation is altered by PH domain mutations found in centronuclear myopathy patients

The large GTPase dynamin has an important membrane scission function in receptor‐mediated endocytosis and other cellular processes. Self‐assembly on phosphoinositide‐containing membranes stimulates dynamin GTPase activity, which is crucial for its function. Although the pleckstrin‐homology (PH) domain is known to mediate phosphoinositide binding by dynamin, it remains unclear how this promotes activation. Here, we describe studies of dynamin PH domain mutations found in centronuclear myopathy (CNM) that increase dynamin's GTPase activity without altering phosphoinositide binding. CNM mutations in the PH domain C‐terminal α‐helix appear to cause conformational changes in dynamin that alter control of the GTP hydrolysis cycle. These mutations either ‘sensitize’ dynamin to lipid stimulation or elevate basal GTPase rates by promoting self‐assembly and thus rendering dynamin no longer lipid responsive. We also describe a low‐resolution structure of dimeric dynamin from small‐angle X‐ray scattering that reveals conformational changes induced by CNM mutations, and defines requirements for domain rearrangement upon dynamin self‐assembly at membrane surfaces. Our data suggest that changes in the PH domain may couple lipid binding to dynamin GTPase activation at sites of vesicle invagination.     

Limited proteolysis analysis of the ribosome is affected by subunit association

Our understanding of the structural organization of ribosome assembly intermediates, in particular those intermediates that result from misfolding leading to their eventual degradation within the cell, is limited because of the lack of methods available to characterize assembly intermediate structures. Because conventional structural approaches, such as NMR, X‐ray crystallography, and cryo‐EM, are not ideally suited to characterize the structural organization of these flexible and sometimes heterogeneous assembly intermediates, we have set out to develop an approach combining limited proteolysis with matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) that might be applicable to ribonucleoprotein complexes as large as the ribosome. This study focuses on the limited proteolysis behavior of appropriately assembled ribosome subunits. Isolated subunits were analyzed using limited proteolysis and MALDI‐MS and the results were compared with previous data obtained from 70S ribosomes. Generally, ribosomal proteins were found to be more stable in 70S ribosomes than in their isolated subunits, consistent with a reduction in conformational flexibility on subunit assembly. This approach demonstrates that limited proteolysis combined with MALDI‐MS can reveal structural changes to ribosomes on subunit assembly or disassembly, and provides the appropriate benchmark data from 30S, 50S, and 70S proteins to enable studies of ribosome assembly intermediates. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 410–422, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Nanoscale elongating control of the self‐assembled protein filament with the cysteine‐introduced building blocks

Self‐assembly of artificially designed proteins is extremely desirable for nanomaterials. Here we show a novel strategy for the creation of self‐assembling proteins, named “Nanolego.” Nanolego consists of “structural elements” of a structurally stable symmetrical homo‐oligomeric protein and “binding elements,” which are multiple heterointeraction proteins with relatively weak affinity. We have established two key technologies for Nanolego, a stabilization method and a method for terminating the self‐assembly process. The stabilization method is mediated by disulfide bonds between Cysteine‐residues incorporated into the binding elements, and the termination method uses “capping Nanolegos,” in which some of the binding elements in the Nanolego are absent for the self‐assembled ends. With these technologies, we successfully constructed timing‐controlled and size‐regulated filament‐shape complexes via Nanolego self‐assembly. The Nanolego concept and these technologies should pave the way for regulated nanoarchitecture using designed proteins.     

Multifractal growth in periphyton communities

Periphyton is an aquatic community composed by algae, bacteria, fungi, and other microorganisms that can develop a complex architecture comparable to tropical forests. We analyzed the spatial pattern of a periphyton community along a succession developed in experimental tanks. Our aim was to identify regularities that may help us to explain the patchiness of this community. Therefore, we estimated the spatial pattern of periphyton biomass using a non‐destructive image analysis technique to obtain a temporal series of the spatial distribution. These were analyzed using multifractal techniques. Multifractals are analogous to fractals but they look at the geometry of quantities instead of the geometry of pattern. To use these techniques the object of study must show scale invariance and then can be characterized by a spectra of fractal dimensions. Self‐organization describes the evolution of complex structures that emerge spontaneously driven internally by variations of the system itself. The spatial distribution of biomass showed scale invariance at all stages of succession and as the periphyton developed in a homogeneous landscape, in a demonstration of self‐organized behavior. Self‐organization to a critical state (SOC) is presented in the complex systems literature as a general explanation for scale invariance in nature. SOC requires a mechanism where the history of past events in a place influence the actual dynamics, this was termed ecological memory. The scale invariance was found from the very beginning of the succession thus self‐organized criticality is a very improbable explanation for the pattern because there would be not enough time for the build‐up of ecological memory. Positive interactions between algae and bacteria, and the existence of different spatial scales of colonization and growth are the likely causes of this pattern. Our work is a demonstration of how large scale patterns emerge from local biotic interactions.     

Modeling the competition between aggregation and self‐assembly during virus‐like particle processing

Understanding and controlling aggregation is an essential aspect in the development of pharmaceutical proteins to improve product yield, potency and quality consistency. Even a minute quantity of aggregates may be reactogenic and can render the final product unusable. Self‐assembly processing of virus‐like particles (VLPs) is an efficient method to quicken the delivery of safe and efficacious vaccines to the market at low cost. VLP production, as with the manufacture of many biotherapeutics, is susceptible to aggregation, which may be minimized through the use of accurate and practical mathematical models. However, existing models for virus assembly are idealized, and do not predict the non‐native aggregation behavior of self‐assembling viral subunits in a tractable nor useful way. Here we present a mechanistic mathematical model describing VLP self‐assembly that accounts for partitioning of reactive subunits between the correct and aggregation pathways. Our results show that unproductive aggregation causes up to 38% product loss by competing favorably with the productive nucleation of self‐assembling subunits, therefore limiting the availability of nuclei for subsequent capsid growth. The protein subunit aggregation reaction exhibits an apparent second‐order concentration dependence, suggesting a dimerization‐controlled agglomeration pathway. Despite the plethora of possible assembly intermediates and aggregation pathways, protein aggregation behavior may be predicted by a relatively simple yet realistic model. More importantly, we have shown that our bioengineering model is amenable to different reactor formats, thus opening the way to rational scale‐up strategies for products that comprise biomolecular assemblies. Biotechnol. Bioeng. 2010;107: 550–560. © 2010 Wiley Periodicals, Inc.     

Extraction and long‐term storage of S‐layer proteins and flagella from Lysinibacillus sphaericus NCTC 9602: Building blocks for nanotechnology

Self‐assembling surface layer (SL) proteins of bacteria have been widely studied, in particular their use as molecularly defined, 2D coatings of technical surfaces. An important prerequisite is the availability of a sufficient amount of protein. However, a detailed and optimized protocol for the complete SL extraction is so far not available. Here, we describe the complete purification and reassembly procedure of an SL protein of Lysinibacillus sphaericus NCTC 9602, starting from the cultivation of cells, the preparation and purification of SL proteins up to the long‐term storage and in vitro self‐assembly of the proteins. All crucial steps of the procedure are assessed by different microscopic techniques, such as light microscopy, atomic force microscopy, and scanning electron microscopy as well as by SDS‐PAGE as a biochemical method. We demonstrate that storage of the protein in the presence of sodium azide or upon lyophilization allows the preservation of the self‐assembly properties for at least 9 years. Additionally, we describe a method allowing the extraction of intact flagella with lengths in the range up to 4 μm. Flagella may have applications in bio‐nanotechnology, for example as templates for metallic nanowires.     

Species divergence and trait convergence in experimental plant community assembly

Despite decades of research, it remains controversial whether ecological communities converge towards a common structure determined by environmental conditions irrespective of assembly history. Here, we show experimentally that the answer depends on the level of community organization considered. In a 9‐year grassland experiment, we manipulated initial plant composition on abandoned arable land and subsequently allowed natural colonization. Initial compositional variation caused plant communities to remain divergent in species identities, even though these same communities converged strongly in species traits. This contrast between species divergence and trait convergence could not be explained by dispersal limitation or community neutrality alone. Our results show that the simultaneous operation of trait‐based assembly rules and species‐level priority effects drives community assembly, making it both deterministic and historically contingent, but at different levels of community organization.     

Relationships between plasma membrane microdomains and HIV‐1 assembly

Advances in cell biology and biophysics revealed that cellular membranes consist of multiple microdomains with specific sets of components such as lipid rafts and TEMs (tetraspanin‐enriched microdomains). An increasing number of enveloped viruses have been shown to utilize these microdomains during their assembly. Among them, association of HIV‐1 (HIV type 1) and other retroviruses with lipid rafts and TEMs within the PM (plasma membrane) is well documented. In this review, I describe our current knowledge on interrelationships between PM microdomain organization and the HIV‐1 particle assembly process. Microdomain association during virus particle assembly may also modulate subsequent virus spread. Potential roles played by microdomains will be discussed with regard to two post‐assembly events, i.e., inhibition of virus release by a raft‐associated protein BST‐2/tetherin and cell‐to‐cell HIV‐1 transmission at virological synapses.     

From null community to non-randomly structured actual plant assemblages: parsimony analysis of species co-occurrences

Null community is a spatio‐temporal abstraction of an initial regional species pool from which local species pools and actual community assemblages are organized. Any process that causes joint responses of species with similar susceptibilities affects community assembly. Through time, sequential assembly processes change the composition of a species pool in a way analogous to the one in which evolutionary processes promote character changes from an ancestor to current species. The segregation of species occurrences in an actual community suggests that assembly processes non‐randomly structured the observed community assemblages. However, going backwards to imply the causes of a particular arrangement of species is a non‐trivial challenge. I merge these premises with the philosophical and methodological foundations of cladistics. I propound parsimony analysis of species co‐occurrences as an outstanding means of devising operational hypotheses about the assembly of any non‐randomly structured set of actual community assemblages related to a common species pool. To explore this approach, I used field data gathered in a suite of 10 wetland assemblages. First, I tested independence of 101 plant species occurrences by a null model. As significant non‐random species co‐occurrence was detected, I applied a parsimony analysis taking the species occurrences as attributes, the assemblages as terminal units, and a putative null community constituted by all the present local species as the root of the assembly suite. The analysis produced four most parsimonious trees of assembly relationships. These trees maximize the number of similarities among community assemblages that can be explained by the sole fact of sharing a common regional species pool. One most parsimonious spatio‐temporal arrangement of species occurrence changes was reconstructed on one of the trees. I interpret this reconstruction in terms of assembly events, species exclusions and recruitments, showing the potentialities of this analysis to formulate operational hypotheses about community organization.     

Nanostructures from the self‐assembly of α‐helical peptide amphiphiles

Self‐assembly of PAs composed of palmitic acid and several repeated heptad peptide sequences, C₁₅H₃₁CO‐(IEEYTKK)_n‐NH₂ (n = 1–4, represented by PA1–PA4), was investigated systematically. The secondary structures of the PAs were characterized by CD. PA3 and PA4 (n = 3 and 4, respectively) showed an α‐helical structure, whereas PA1 and PA2 (n = 1 and 2, respectively) did not display an α‐helical conformations under the tested conditions. The morphology of the self‐assembled peptides in aqueous medium was studied by transmission electron microscopy. As the number of heptad repeats in the PAs increased, the nanostructure of the self‐assembled peptides changed from nanofibers to nanovesicles. Changes of the secondary structures and the self‐assembly morphologies of PA3 and PA4 in aqueous medium with various cations were also studied. The critical micelle concentrations were determined using a pyrene fluorescence probe. In conclusion, this method may be used to design new peptide nanomaterials. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

The Rise of the Cartwheel: Seeding the Centriole Organelle

The cartwheel is a striking structure critical for building the centriole, a microtubule‐based organelle fundamental for organizing centrosomes, cilia, and flagella. Over the last 50 years, the cartwheel has been described in many systems using electron microscopy, but the molecular nature of its constituent building blocks and their assembly mechanisms have long remained mysterious. Here, we review discoveries that led to the current understanding of cartwheel structure, assembly, and function. We focus on the key role of SAS‐6 protein self‐organization, both for building the signature ring‐like structure with hub and spokes, as well as for their vertical stacking. The resemblance of assembly intermediates in vitro and in vivo leads us to propose a novel registration step in cartwheel biogenesis, whereby stacked SAS‐6‐containing rings are put in register through interactions with peripheral elements anchored to microtubules. We conclude by evoking some avenues for further uncovering cartwheel and centriole assembly mechanisms.     

Finding Nemo’s Genes: A chromosome‐scale reference assembly of the genome of the orange clownfish Amphiprion percula

The iconic orange clownfish, Amphiprion percula, is a model organism for studying the ecology and evolution of reef fishes, including patterns of population connectivity, sex change, social organization, habitat selection and adaptation to climate change. Notably, the orange clownfish is the only reef fish for which a complete larval dispersal kernel has been established and was the first fish species for which it was demonstrated that antipredator responses of reef fishes could be impaired by ocean acidification. Despite its importance, molecular resources for this species remain scarce and until now it lacked a reference genome assembly. Here, we present a de novo chromosome‐scale assembly of the genome of the orange clownfish Amphiprion percula. We utilized single‐molecule real‐time sequencing technology from Pacific Biosciences to produce an initial polished assembly comprised of 1,414 contigs, with a contig N50 length of 1.86 Mb. Using Hi‐C‐based chromatin contact maps, 98% of the genome assembly were placed into 24 chromosomes, resulting in a final assembly of 908.8 Mb in length with contig and scaffold N50s of 3.12 and 38.4 Mb, respectively. This makes it one of the most contiguous and complete fish genome assemblies currently available. The genome was annotated with 26,597 protein‐coding genes and contains 96% of the core set of conserved actinopterygian orthologs. The availability of this reference genome assembly as a community resource will further strengthen the role of the orange clownfish as a model species for research on the ecology and evolution of reef fishes.     

Enhanced stiffness of silk‐like fibers by loop formation in the corona leads to stronger gels

We study the self‐assembly of protein polymers consisting of a silk‐like block flanked by two hydrophilic blocks, with a cysteine residue attached to the C‐terminal end. The silk blocks self‐assemble to form fibers while the hydrophilic blocks form a stabilizing corona. Entanglement of the fibers leads to the formation of hydrogels. Under oxidizing conditions the cysteine residues form disulfide bridges, effectively connecting two corona chains at their ends to form a loop. We find that this leads to a significant increase in the elastic modulus of the gels. Using atomic force microscopy, we show that this stiffening is due to an increase of the persistence length of the fibers. Self‐consistent‐field calculations indicate a slight decrease of the lateral pressure in the corona upon loop formation. We argue that this small decrease in the repulsive interactions affects the stacking of the silk‐like blocks in the core, resulting in a more rigid fiber.     

Chromosome‐level assembly of wild Bactrian camel genome reveals organization of immune gene loci

Camelids are characterized by their unique adaptive immune system that exhibits the generation of homodimeric heavy‐chain immunoglobulins, somatic hypermutation of T‐cell receptors, and low genetic diversity of major histocompatibility complex (MHC) genes. However, short‐read assemblies are typically highly fragmented in these gene loci owing to their repetitive and polymorphic nature. Here, we constructed a chromosome‐level assembly of wild Bactrian camel genome based on high‐coverage long‐read sequencing and chromatin interaction mapping. The assembly with a contig N50 of 5.37 Mb and a scaffold N50 of 76.03 Mb, represents the most contiguous camelid genome to date. The genomic organization of immunoglobulin heavy‐chain locus was similar between the wild Bactrian camel and alpaca, and genes encoding for conventional and heavy‐chain antibodies were intermixed. The organizations of two immunoglobulin light‐chain loci and four T cell receptor loci were also fully deciphered using the new assembly. Additionally, the complete classical MHC region was resolved into a single contig. The high‐quality assembly presented here provides an essential reference for future investigations examining the camelid immune system.     

Self‐organization and emergent individuality of favositid corals adapted to live on soft substrates

Seilacher, A. & Thomas, R.D.K. 2011: Self‐organization and emergent individuality of favositid corals adapted to live on soft substrates. Lethaia, Vol. 45, pp. 2–13. Secondary soft‐bottom dwellers share the problem that their ancestors, attached to hard substrates, had lost their mobility. On soft substrates, only a limited number of alternative tricks are available to maintain sessile organisms in life position or to right them following disturbance. Consequently, convergent adaptations have emerged in unrelated members of this ecological guild. Those of favositid corals are of particular interest because, on account of their small polyps, they were always colonial. A comparative analysis of forms adopted by favositids on soft substrates shows that key elements of their adaptive paradigms could have been achieved by self‐organization. This arose without centralized control by means of inherited reaction norms of individual polyps to local environmental conditions. The unique spiral growth habit of Favosites turbinatus Billings and its emergent individuality at the colony‐level of organization are explained in these terms. ‘Suicidal’ lids entombed altruistic marginal polyps, forming a secondary epitheca as the growing colony settled into the sediment. These lids also record the size and spacing of soft tentacles in this species. □Coloniality, Devonian, Favositida, Favosites turbinatus, morphogenesis, self‐organization, Tabulata.     

Unraveling Charge Separation and Transport Mechanisms in Aqueous‐Processed Polymer/CdTe Nanocrystal Hybrid Solar Cells

Recently great progress has been achieved in highly effective hybrid solar cells fabricated using aqueous materials. The state‐of‐the‐art energy conversion efficiency has been close to 5% with high photocurrent. However, charge separation and transport mechanism in the aqueous‐processed hybrid solar cells are rarely reported and are usually assumed to be similar to oil‐phase processed systems; that is, self‐assembly polymers are mainly responsible for charge separation and carrier transport. To date, this assumption has prohibited further improvement of the conversion efficiency in aqueous‐processed hybrid systems by adopting any appropriate technique routes. Here, ultrafast carrier dynamics in these hybrid solar cells consisting of poly(p‐phenylenevinylene) (PPV)‐based aqueous polymers and water‐solution CdTe nanocrystals (NCs) are investigated in detail. Self‐charge separation in grown CdTe NC partly capped CdS shell layers after anneal treatment is unambiguously identified. Different from their oil‐soluble counterparts, these core/shell nanocrystals do not have the restrictions of quantum confinement and surface ligands, form effective charge transport networks, and play a dominant role in the charge separation and carrier transport processes. These findings provide a greater understanding on the fundamental photophysics in aqueous‐processed hybrid systems.     

The global biogeography of semi‐arid periodic vegetation patterns

Aim Vegetation exhibiting landscape‐scale regular spatial patterns has been reported for arid and semi‐arid areas world‐wide. Recent theories state that such structures are bound to low‐productivity environments and result from a self‐organization process. Our objective was to test this relationship between periodic pattern occurrence and environmental factors at a global scale and to parametrize a predictive distribution model. Location Arid and semi‐arid areas world‐wide. Methods We trained an empirical predictive model (Maxent) for the occurrence of periodic vegetation patterns, based on environmental predictors and known occurrences verified on Landsat satellite images. Results This model allowed us to discover previously unreported pattern locations, and to report the first ever examples of spotted patterns in natural systems. Relationships to the main environmental drivers are discussed. Main conclusions These results confirm that periodic patterned vegetations are ubiquitous at the interface between arid and semi‐arid regions. Self‐organized patterning appears therefore to be a biome‐scale response to environmental conditions, including soil and topography. The set of correlations between vegetation patterns and their environmental conditions presented in this study will need to be reproduced in future modelling attempts.