Differentiating between niche and neutral assembly in metacommunities using null models of β‐diversity

The β‐null deviation measure, developed as a null model for β‐diversity, is increasingly used in empirical studies to detect the underlying structuring mechanisms in communities (e.g. niche versus neutral and stochastic versus deterministic). Despite growing use, the ecological interpretation of the presence/absence and abundance‐based versions of the β‐null diversity measure have not been tested against communities with known assembly mechanisms, and thus have not been validated as an appropriate tool for inferring assembly mechanisms. Using a mechanistic model with known assembly mechanisms, we simulated replicate metacommunities and examined β‐null deviation values 1) across a gradient of niche (species‐sorting) to neutrally structured metacommunities, 2) through time, and 3) we compared the effect of changes in assembly mechanism on the performance of the β‐null deviation measures. The impact of stochasticity on assembly outcomes was also considered. β‐null deviation measures proved to be interpretable as a measure of niche or neutral assembly. However, the presence/absence version of the β‐null deviation measure could not differentiate between niche and neutral metacommunities if demographic stochasticity were present. The abundance‐based β‐null deviation measure was successful in distinguishing between niche and neutral metacommunities and was robust to the presence of stochasticity, changes through time, and changing assembly mechanisms. However, we suggest that it is not robust to changing abundance evenness distributions or sampling of communities, and so its interpretation still requires some care. We encourage the testing of the assumptions behind null models for ecology and care in their application.     

Dissecting the Function and Assembly of Acentriolar Microtubule Organizing Centers in Drosophila Cells In Vivo

Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2—the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs.     

A quantitative kinetic model for the in vitro assembly of intermediate filaments from tetrameric vimentin

In vitro assembly of intermediate filament proteins is a very rapid process. It starts without significant delay by lateral association of tetramer complexes into unit-length filaments (ULFs) after raising the ionic strength from low salt to physiological conditions (100 mM KCl). We employed electron and scanning force microscopy complemented by mathematical modeling to investigate the kinetics of in vitro assembly of human recombinant vimentin. From the average length distributions of the resulting filaments measured at increasing assembly times we simulated filament assembly and estimated specific reaction rate parameters. We modeled eight different potential pathways for vimentin filament elongation. Comparing the numerical with the experimental data we conclude that a two-step mechanism involving rapid formation of ULFs followed by ULF and filament annealing is the most robust scenario for vimentin assembly. These findings agree with the first two steps of the previously proposed three-step assembly model (Herrmann, H., and Aebi, U. (1998) Curr. Opin. Struct. Biol. 8, 177-185). In particular, our modeling clearly demonstrates that end-to-end annealing of ULFs and filaments is obligatory for forming long filaments, whereas tetramer addition to filament ends does not contribute significantly to filament elongation.     

Mechanics of stabilized intercellular bridges

Numerous engineered and natural systems form through reinforcement and stabilization of a deformed configuration that was generated by a transient force. An important class of such structures arises during gametogenesis, when a dividing cell undergoes incomplete cytokinesis, giving rise to daughter cells that remain connected through a stabilized intercellular bridge (ICB). ICBs can form through arrest of the contractile cytokinetic furrow and its subsequent stabilization. Despite knowledge of the molecular components, the mechanics underlying robust ICB assembly and the interplay between ring contractility and stiffening are poorly understood. Here, we report joint experimental and theoretical work that explores the physics underlying robust ICB assembly. We develop a continuum mechanics model that reveals the minimal requirements for the formation of stable ICBs, and validate the model’s equilibrium predictions through a tabletop experimental analog. With insight into the equilibrium states, we turn to the dynamics: we demonstrate that contractility and stiffening are in dynamic competition and that the time intervals of their action must overlap to ensure assembly of ICBs of biologically observed proportions. Our results highlight a mechanism in which deformation and remodeling are tightly coordinated—one that is applicable to several mechanics-based applications and is a common theme in biological systems spanning several length scales.     

Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.     

Cooperativity-based modeling of heterotypic DNA nanostructure assembly

DNA origami is a robust method for the fabrication of nanoscale 2D and 3D objects with complex features and geometries. The process of DNA origami folding has been recently studied, however quantitative understanding of it is still elusive. Here, we describe a systematic quantification of the assembly process of DNA nanostructures, focusing on the heterotypic DNA junction—in which arms are unequal—as their basic building block. Using bulk fluorescence studies we tracked this process and identified multiple levels of cooperativity from the arms in a single junction to neighboring junctions in a large DNA origami object, demonstrating that cooperativity is a central underlying mechanism in the process of DNA nanostructure assembly. We show that the assembly of junctions in which the arms are consecutively ordered is more efficient than junctions with randomly-ordered components, with the latter showing assembly through several alternative trajectories as a potential mechanism explaining the lower efficiency. This highlights consecutiveness as a new design consideration that could be implemented in DNA nanotechnology CAD tools to produce more efficient and high-yield designs. Altogether, our experimental findings allowed us to devise a quantitative, cooperativity-based heuristic model for the assembly of DNA nanostructures, which is highly consistent with experimental observations.     

Opportunistic attachment assembles plant–pollinator networks

Species and interactions are being lost at alarming rates and it is imperative to understand how communities assemble if we have to prevent their collapse and restore lost interactions. Using an 8‐year dataset comprising nearly 20 000 pollinator visitation records, we explore the assembly of plant–pollinator communities at native plant restoration sites in an agricultural landscape. We find that species occupy highly dynamic network positions through time, causing the assembly process to be punctuated by major network reorganisations. The most persistent pollinator species are also the most variable in their network positions, contrary to what preferential attachment – the most widely studied theory of ecological network assembly – predicts. Instead, we suggest assembly occurs via an opportunistic attachment process. Our results contribute to our understanding of how communities assembly and how species interactions change through time while helping to inform efforts to reassemble robust communities.     

The interplay of the N- and C-terminal domains of MCAK control microtubule depolymerization activity and spindle assembly

Spindle assembly and accurate chromosome segregation require the proper regulation of microtubule dynamics. MCAK, a Kinesin-13, catalytically depolymerizes microtubules, regulates physiological microtubule dynamics, and is the major catastrophe factor in egg extracts. Purified GFP-tagged MCAK domain mutants were assayed to address how the different MCAK domains contribute to in vitro microtubule depolymerization activity and physiological spindle assembly activity in egg extracts. Our biochemical results demonstrate that both the neck and the C-terminal domain are necessary for robust in vitro microtubule depolymerization activity. In particular, the neck is essential for microtubule end binding, and the C-terminal domain is essential for tight microtubule binding in the presence of excess tubulin heterodimer. Our physiological results illustrate that the N-terminal domain is essential for regulating microtubule dynamics, stimulating spindle bipolarity, and kinetochore targeting; whereas the C-terminal domain is necessary for robust microtubule depolymerization activity, limiting spindle bipolarity, and enhancing kinetochore targeting. Unexpectedly, robust MCAK microtubule (MT) depolymerization activity is not needed for sperm-induced spindle assembly. However, high activity is necessary for proper physiological MT dynamics as assayed by Ran-induced aster assembly. We propose that MCAK activity is spatially controlled by an interplay between the N- and C-terminal domains during spindle assembly.     

A label-free light-scattering method to resolve assembly and disassembly of DNA nanostructures

DNA self-assembly, and in particular DNA origami, has evolved into a reliable workhorse for organizing organic and inorganic materials with nanometer precision and with exactly controlled stoichiometry. To ensure the intended performance of a given DNA structure, it is beneficial to determine its folding temperature, which in turn yields the best possible assembly of all DNA strands. Here, we show that temperature-controlled sample holders and standard fluorescence spectrometers or dynamic light-scattering setups in a static light-scattering configuration allow for monitoring the assembly progress in real time. With this robust label-free technique, we determine the folding and melting temperatures of a set of different DNA origami structures without the need for more tedious protocols. In addition, we use the method to follow digestion of DNA structures in the presence of DNase I and find strikingly different resistances toward enzymatic degradation depending on the structural design of the DNA object.     

Examining how the spatial organization of chromatin signals influences metaphase spindle assembly

During cell division, the proper assembly of a microtubule-based bipolar spindle depends on signals from chromatin. However, it is unknown how the spatial organization of chromatin signals affects spindle morphology. Here, we use paramagnetic chromatin beads, and magnetic fields for their alignment in cell-free extracts, to examine the spatial components of signals that regulate spindle assembly. We find that for linear chromatin-bead arrays that vary by eightfold in length, metaphase spindle size and shape are constant. Our findings indicate that, although chromatin provides cues for microtubule formation, metaphase spindle organization, which is controlled by microtubule-based motors, is robust to changes in the shape of chromatin signals.     

Engineering of interlocked DNA G-quadruplexes as a robust scaffold

Interlock is a structural element in DNA G-quadruplexes that can be compared with the commonly used complementary binding of ‘sticky ends’ in DNA duplexes. G-quadruplex interlocking can be a basis for the assembly of higher-order structures. In this study, we formulated a rule to engineer (3 + 1) interlocked dimeric G-quadruplexes and established the folding topology of the designed DNA sequences by nuclear magnetic resonance spectroscopy. These interlocked G-quadruplexes are very stable and can serve as compact robust scaffolds for various applications. Different structural elements can be engineered in these robust scaffolds. We demonstrated the anti-HIV inhibition activity of the newly designed DNA sequences.     

High-throughput,cost-effective verification of structural DNA assembly

DNA ‘assembly’ from ‘building blocks’ remains a cornerstone in synthetic biology, whether it be for gene synthesis (∼1 kb), pathway engineering (∼10 kb) or synthetic genomes (>100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at <$1 per sample.     

Protein fabrication automation

Facile "writing" of DNA fragments that encode entire gene sequences potentially has widespread applications in biological analysis and engineering. Rapid writing of open reading frames (ORFs) for expressed proteins could transform protein engineering and production for protein design, synthetic biology, and structural analysis. Here we present a process, protein fabrication automation (PFA), which facilitates the rapid de novo construction of any desired ORF from oligonucleotides with low effort, high speed, and little human interaction. PFA comprises software for sequence design, data management, and the generation of instruction sets for liquid-handling robotics, a liquid-handling robot, a robust PCR scheme for gene assembly from synthetic oligonucleotides, and a genetic selection system to enrich correctly assembled full-length synthetic ORFs. The process is robust and scalable.     

Aurora A kinase and its substrate TACC3 are required for central spindle assembly

Cell division entails a marked reorganization of the microtubule network to form the spindle, a molecular machine that ensures accurate chromosome segregation to the daughter cells. Spindle organization is highly dynamic throughout mitosis and requires the activity of several kinases and complex regulatory mechanisms. Aurora A (AurA) kinase is essential for the assembly of the metaphase bipolar spindle and, thus, it has been difficult to address its function during the last phases of mitosis. Here, we examine the consequences of inhibiting AurA in cells undergoing anaphase, and show that AurA kinase activity is necessary for the assembly of a robust central spindle during anaphase. We also identify TACC3 as an AurA substrate essential in central spindle formation.     

Interrelationships between yeast ribosomal protein assembly events and transient ribosome biogenesis factors interactions in early pre-ribosomes

Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.     

Assembly of the 30S ribosomal subunit

The assembly of ribosomes requires a significant fraction of the energy expenditure for rapidly growing bacteria. The ribosome is composed of three large RNA molecules and over 50 small proteins that must be rapidly and efficiently assembled into the molecular machine responsible for protein synthesis. For over 30 years, the 30S ribosome has been a key model system for understanding the process of ribosome biogenesis through in vitro assembly experiments. We have recently developed an isotope pulse-chase experiment using quantitative mass spectrometry that permits assembly kinetics to be measured in real time. Kinetic studies have revealed an assembly energy landscape that ensures efficient assembly by a flexible and robust pathway.     

Three wise centromere functions: see no error,hear no break,speak no delay

The main function of the centromere is to promote kinetochore assembly for spindle microtubule attachment. Two additional functions of the centromere, however, are becoming increasingly clear: facilitation of robust sister‐chromatid cohesion at pericentromeres and advancement of replication of centromeric regions. The combination of these three centromere functions ensures correct chromosome segregation during mitosis. Here, we review the mechanisms of the kinetochore–microtubule interaction, focusing on sister‐kinetochore bi‐orientation (or chromosome bi‐orientation). We also discuss the biological importance of robust pericentromeric cohesion and early centromere replication, as well as the mechanisms orchestrating these two functions at the microtubule attachment site.     

The synaptonemal complex is assembled by a polySUMOylation-driven feedback mechanism in yeast

During meiotic prophase I, proteinaceous structures called synaptonemal complexes (SCs) connect homologous chromosomes along their lengths via polymeric arrays of transverse filaments (TFs). Thus, control of TF polymerization is central to SC formation. Using budding yeast, we show that efficiency of TF polymerization closely correlates with the extent of SUMO conjugation to Ecm11, a component of SCs. HyperSUMOylation of Ecm11 leads to highly aggregative TFs, causing frequent assembly of extrachromosomal structures. In contrast, hypoSUMOylation leads to discontinuous, fragmented SCs, indicative of defective TF polymerization. We further show that the N terminus of the yeast TF, Zip1, serves as an activator for Ecm11 SUMOylation. Coexpression of the Zip1 N terminus and Gmc2, a binding partner of Ecm11, is sufficient to induce robust polySUMOylation of Ecm11 in nonmeiotic cells. Because TF assembly is mediated through N-terminal head-to-head associations, our results suggest that mutual activation between TF assembly and Ecm11 polySUMOylation acts as a positive feedback loop that underpins SC assembly.