Supercoiled DNA and non-equilibrium formation of protein complexes: A quantitative model of the nucleoprotein ParBS partition complex

ParABS, the most widespread bacterial DNA segregation system, is composed of a centromeric sequence, parS, and two proteins, the ParA ATPase and the ParB DNA binding proteins. Hundreds of ParB proteins assemble dynamically to form nucleoprotein parS-anchored complexes that serve as substrates for ParA molecules to catalyze positioning and segregation events. The exact nature of this ParBS complex has remained elusive, what we address here by revisiting the Stochastic Binding model (SBM) introduced to explain the non-specific binding profile of ParB in the vicinity of parS. In the SBM, DNA loops stochastically bring loci inside a sharp cluster of ParB. However, previous SBM versions did not include the negative supercoiling of bacterial DNA, leading to use unphysically small DNA persistences to explain the ParB binding profiles. In addition, recent super-resolution microscopy experiments have revealed a ParB cluster that is significantly smaller than previous estimations and suggest that it results from a liquid-liquid like phase separation. Here, by simulating the folding of long (≥ 30 kb) supercoiled DNA molecules calibrated with realistic DNA parameters and by considering different possibilities for the physics of the ParB cluster assembly, we show that the SBM can quantitatively explain the ChIP-seq ParB binding profiles without any fitting parameter, aside from the supercoiling density of DNA, which, remarkably, is in accord with independent measurements. We also predict that ParB assembly results from a non-equilibrium, stationary balance between an influx of produced proteins and an outflux of excess proteins, i.e., ParB clusters behave like liquid-like protein condensates with unconventional “leaky” boundaries.     

The UNI1 and UNI2 Genes Function in the Transition of Triplet to Doublet Microtubules between the Centriole and Cilium in Chlamydomonas

One fundamental role of the centriole in eukaryotic cells is to nucleate the growth of cilia. The unicellular alga Chlamydomonas reinhardtii provides a simple genetic system to study the role of the centriole in ciliogenesis. Wild-type cells are biflagellate, but “uni” mutations result in failure of some centrioles (basal bodies) to assemble cilia (flagella). Serial transverse sections through basal bodies in uni1 and uni2 single and double mutant cells revealed a previously undescribed defect in the transition of triplet microtubules to doublet microtubules, a defect correlated with failure to assemble flagella. Phosphorylation of the Uni2 protein is reduced in uni1 mutant cells. Immunogold electron microscopy showed that the Uni2 protein localizes at the distal end of the basal body where microtubule transition occurs. These results provide the first mechanistic insights into the function of UNI1 and UNI2 genes in the pathway mediating assembly of doublet microtubules in the axoneme from triplet microtubules in the basal body template.     

Microtubules form by progressively faster tubulin accretion,not by nucleation–elongation

Microtubules are dynamic polymers that play fundamental roles in all eukaryotes. Despite their importance, how new microtubules form is poorly understood. Textbooks have focused on variations of a nucleation–elongation mechanism in which monomers rapidly equilibrate with an unstable oligomer (nucleus) that limits the rate of polymer formation; once formed, the polymer then elongates efficiently from this nucleus by monomer addition. Such models faithfully describe actin assembly, but they fail to account for how more complex polymers like hollow microtubules assemble. Here, we articulate a new model for microtubule formation that has three key features: (1) microtubules initiate via rectangular, sheet-like structures that grow faster the larger they become; (2) the dominant pathway proceeds via accretion, the stepwise addition of longitudinal or lateral layers; and (3) a “straightening penalty” to account for the energetic cost of tubulin’s curved-to-straight conformational transition. This model can quantitatively fit experimental assembly data, providing new insights into biochemical determinants and assembly pathways for microtubule nucleation.     

Microtubule pivoting enables mitotic spindle assembly in S. cerevisiae

To assemble a bipolar spindle, microtubules emanating from two poles must bundle into an antiparallel midzone, where plus end–directed motors generate outward pushing forces to drive pole separation. Midzone cross-linkers and motors display only modest preferences for antiparallel filaments, and duplicated poles are initially tethered together, an arrangement that instead favors parallel interactions. Pivoting of microtubules around spindle poles might help overcome this geometric bias, but the intrinsic pivoting flexibility of the microtubule–pole interface has not been directly measured, nor has its importance during early spindle assembly been tested. By measuring the pivoting of microtubules around isolated yeast spindle poles, we show that pivoting flexibility can be modified by mutating a microtubule-anchoring pole component, Spc110. By engineering mutants with different flexibilities, we establish the importance of pivoting in vivo for timely pole separation. Our results suggest that passive thermal pivoting can bring microtubules from side-by-side poles into initial contact, but active minus end–directed force generation will be needed to achieve antiparallel alignment.     

Membrane reshaping by protein condensates

Proteins can organize into dynamic, functionally important assemblies on fluid membrane surfaces. Phase separation has emerged as an important mechanism for forming such protein assemblies on the membrane during cell signaling, endocytosis, and cytoskeleton regulation. Protein-protein phase separation thus adds novel fluid mosaics to the classical Singer and Nicolson model. Protein condensates formed in this process can modulate membrane morphologies. This is evident from recent reports of protein condensate-driven membrane reshaping in processes such as endocytosis, autophagosome formation, and protein storage vacuole morphogenesis in plants. Lateral phase separation (on the membrane surface) of peripheral curvature coupling proteins can modulate such membrane morphological transitions. Additionally, three-dimensional protein phase separation can result in droplets that through adhesion can affect membrane shape changes. How do these condensate-driven curvature generation mechanisms contrast with the classically recognized scaffolding and amphipathic helix insertion activities of specific membrane remodeling proteins? A salient feature of these condensate-driven membrane activities is that they depend upon both macroscopic features (such as interfacial energies of the condensate, membrane, and cytosol) as well as microscopic, molecular-level interactions (such as protein-lipid binding). This review highlights the current understanding of the mechanisms underlying curvature generation by protein condensates in various biological pathways.     

Interaction of spindle assembly factor TPX2 with importins-α/β inhibits protein phase separation

The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed via several microtubule nucleation pathways. Targeting Protein for XKlp2 (TPX2) stimulates the branching microtubule nucleation pathway, where new microtubules are nucleated from preexisting ones within mitotic or meiotic spindles. TPX2, like other spindle assembly factors, is sequestered by binding to nuclear importins-α/β until the onset of mitosis, yet the molecular nature of this regulation remains unclear. Here we demonstrate that TPX2 interacts with importins-α/β with nanomolar affinity in a 1:1:1 monodispersed trimer. We also identify a new nuclear localization sequence in TPX2 that contributes to its high-affinity interaction with importin-α. In addition, we establish that TPX2 interacts with importin-β via dispersed, weak interactions. We show that interactions of both importin-α and -β with TPX2 inhibit its ability to undergo phase separation, which was recently shown to enhance the kinetics of branching microtubule nucleation. In summary, our study informs how importins regulate TPX2 to facilitate spindle assembly, and provides novel insight into the functional regulation of protein phase separation.     

Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes

In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which “amplifies” spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle.     

Inhibition of Cell Migration and Cell Division Correlates with Distinct Effects of Microtubule Inhibiting Drugs

Drugs that target microtubules are thought to inhibit cell division and cell migration by suppressing dynamic instability, a “search and capture” behavior that allows microtubules to probe their environment. Here, we report that subtoxic drug concentrations are sufficient to inhibit plus-end microtubule dynamic instability and cell migration without affecting cell division or microtubule assembly. The higher drug concentrations needed to inhibit cell division act through a novel mechanism that generates microtubule fragments by stimulating microtubule minus-end detachment from their organizing centers. The frequency of microtubule detachment in untreated cells increases at prophase suggesting that it is a regulated cellular process important for spindle assembly and function. We conclude that drugs produce differential dose-dependent effects at microtubule plus and minus-ends to inhibit different microtubule-mediated functions.     

Small-molecule modulators of INAVA cytosolic condensate and cell–cell junction assemblies

Epithelial cells lining mucosal surfaces distinctively express the inflammatory bowel disease risk gene INAVA. We previously found that INAVA has dual and competing functions: one at lateral membranes where it affects mucosal barrier function and the other in the cytosol where INAVA enhances IL-1β signal transduction and protein ubiquitination and forms puncta. We now find that IL-1β–induced INAVA puncta are biomolecular condensates that rapidly assemble and physiologically resolve. The condensates contain ubiquitin and the E3 ligase βTrCP2, and their formation correlates with amplified ubiquitination, suggesting function in regulation of cellular proteostasis. Accordingly, a small-molecule screen identified ROS inducers, proteasome inhibitors, and inhibitors of the protein folding chaperone HSP90 as potent agonists for INAVA condensate formation. Notably, inhibitors of the p38α and mTOR pathways enhanced resolution of the condensates, and inhibitors of the Rho–ROCK pathway induced INAVA’s competing function by recruiting INAVA to newly assembled intercellular junctions in cells where none existed before.     

Adsorption kinetics of laterally and polarly flagellated Vibrio.

The adsorption of laterally and polarly flagellated bacteria to chitin was measured, and from the data obtained, a modified Langmuir adsorption isotherm was derived. Results indicated that the adsorption of laterally flagellated Vibrio parahaemolyticus follows the Langmuir adsorption isotherm, a type of adsorption referred to as surface saturation kinetics, when conditions are favorable for the production of lateral flagella. When conditions were not favorable for the production of lateral flagella, bacterial adsorption did not follow the Langmuir adsorption isotherm; instead, proportional adsorption kinetics were observed. The adsorption of some polarly flagellated bacteria exhibited surface saturation kinetics. However, the binding index (the product of the number of binding sites and bacterial affinity to the surface) of polarly flagellated bacteria differed significantly from that of laterally flagellated bacteria, suggesting that polarly flagellated bacteria adsorb to chitin by a different mechanism from that used by the laterally flagellated bacteria. From the results of dual-label adsorption competition experiments, in which polarly flagellated V. cholerae competed with increasing concentrations of laterally flagellated V. parahaemolyticus, it was observed that laterally flagellated bacteria inhibited the adsorption of polarly flagellated bacteria. In contrast, polarly flagellated bacteria enhanced the adsorption of V. cholerae. In competition experiments, where V. parahaemolyticus competed against increasing concentrations of other bacteria, polarly flagellated bacteria enhanced V. parahaemolyticus adsorption significantly, whereas laterally flagellated bacteria only slightly enhanced the process. The direct correlation observed between surface saturation kinetics, the production of lateral flagella, and the ability of laterally flagellated bacteria to inhibit the adsorption of polarly flagellated bacteria suggests that lateral flagella represent a component of bacterial structure that is important in the adsorption of laterally flagellated bacteria to surfaces. A model for adsorption events of laterally flagellated bacteria is proposed, based on the evidence presented.     

Molecular crowding and RNA synergize to promote phase separation,microtubule interaction,and seeding of Tau condensates

Biomolecular condensation of the neuronal microtubule‐associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate‐mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate‐like density for nuclear‐envelope Tau. These findings suggest that a complex interplay between interaction partners, post‐translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding‐competent Tau and lead to distinct cellular Tau accumulations.     

Competitive adsorption of vanillin and syringaldehyde on a macro-mesopore polymeric resin: modeling

Vanillin and syringaldehyde are widely used as flavoring and fragrance agents in the food products. The potential of a macro-mesoporous adsorption resin was assessed for separation of these binary mixtures. This work focuses on modeling of the competitive adsorption behaviors and exploration of the adsorption mechanism. The characterization results showed the resin had a large BET surface area and specific pore structure with hydrophobic properties. By analysis of the physicochemical properties of the solutes and the resin, the separation mechanism was mainly contributed by hydrophobic effect. Subsequently, the competitive Langmuir isotherm model was used to fit the competitive adsorption isotherms. The pore diffusion coefficient was obtained by macropore diffusion model. Afterwards, a mathematical model was established to predict the breakthrough curves of the binary mixture at various operating conditions. The data and model presented are valuable for design and simulation of the continuous chromatographic separation process.

     

Coordination of adjacent domains mediates TACC3–ch-TOG–clathrin assembly and mitotic spindle binding

Acomplex of transforming acidic coiled-coil protein 3 (TACC3), colonic and hepatic tumor overexpressed gene (ch-TOG), and clathrin has been implicated in mitotic spindle assembly and in the stabilization of kinetochore fibers by cross-linking microtubules. It is unclear how this complex binds microtubules and how the proteins in the complex interact with one another. TACC3 and clathrin have each been proposed to be the spindle recruitment factor. We have mapped the interactions within the complex and show that TACC3 and clathrin were interdependent for spindle recruitment, having to interact in order for either to be recruited to the spindle. The N-terminal domain of clathrin and the TACC domain of TACC3 in tandem made a microtubule interaction surface, coordinated by TACC3–clathrin binding. A dileucine motif and Aurora A–phosphorylated serine 558 on TACC3 bound to the “ankle” of clathrin. The other interaction within the complex involved a stutter in the TACC3 coiled-coil and a proposed novel sixth TOG domain in ch-TOG, which was required for microtubule localization of ch-TOG but not TACC3–clathrin.     

Ecological rules for the assembly of microbiome communities

Humans and many other hosts establish a diverse community of beneficial microbes anew each generation. The order and identity of incoming symbionts is critical for health, but what determines the success of the assembly process remains poorly understood. Here we develop ecological theory to identify factors important for microbial community assembly. Our method maps out all feasible pathways for the assembly of a given microbiome—with analogies to the mutational maps underlying fitness landscapes in evolutionary biology. Building these “assembly maps” reveals a tradeoff at the heart of the assembly process. Ecological dependencies between members of the microbiota make assembly predictable—and can provide metabolic benefits to the host—but these dependencies may also create barriers to assembly. This effect occurs because interdependent species can fail to establish when each relies on the other to colonize first. We support our predictions with published data from the assembly of the preterm infant microbiota, where we find that ecological dependence is associated with a predictable order of arrival. Our models also suggest that hosts can overcome barriers to assembly via mechanisms that either promote the uptake of multiple symbiont species in one step or feed early colonizers. This predicted importance of host feeding is supported by published data on the impacts of breast milk in the assembly of the human microbiome. We conclude that both microbe to microbe and host to microbe interactions are important for the trajectory of microbiome assembly.

Humans and many other hosts establish a diverse community of beneficial microbes anew each generation, but what determines the success of the assembly process remains poorly understood. This study develops ecological theory that reveals the rules underlying the assembly of such host-associated microbiota.     

Multivalent protein–protein interactions are pivotal regulators of eukaryotic Hsp70 complexes

Heat shock protein 70 (Hsp70) is a molecular chaperone and central regulator of protein homeostasis (proteostasis). Paramount to this role is Hsp70’s binding to client proteins and co-chaperones to produce distinct complexes, such that understanding the protein–protein interactions (PPIs) of Hsp70 is foundational to describing its function and dysfunction in disease. Mounting evidence suggests that these PPIs include both “canonical” interactions, which are universally conserved, and “non-canonical” (or “secondary”) contacts that seem to have emerged in eukaryotes. These two categories of interactions involve discrete binding surfaces, such that some clients and co-chaperones engage Hsp70 with at least two points of contact. While the contributions of canonical interactions to chaperone function are becoming increasingly clear, it can be challenging to deconvolute the roles of secondary interactions. Here, we review what is known about non-canonical contacts and highlight examples where their contributions have been parsed, giving rise to a model in which Hsp70’s secondary contacts are not simply sites of additional avidity but are necessary and sufficient to impart unique functions. From this perspective, we propose that further exploration of non-canonical contacts will generate important insights into the evolution of Hsp70 systems and inspire new approaches for developing small molecules that tune Hsp70-mediated proteostasis.