Macromolecular condensation organizes nucleolar sub-phases to set up a pH gradient.

Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.

Emergent microenvironments of nucleoli.

In higher eukaryotes, the nucleolus harbors at least three sub-phases that facilitate multiple functionalities including ribosome biogenesis. The three prominent coexisting sub-phases are the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC). Here, we review recent efforts in profiling sub-phase compositions that shed light on the types of physicochemical properties that emerge from compositional biases and territorial organization of specific types of macromolecules. We highlight roles played by molecular grammars which refers to protein sequence features including the substrate binding domains, the sequence features of intrinsically disordered regions, and the multivalence of these distinct types of domains / regions. We introduce the concept of a barcode of emergent physicochemical properties of nucleoli. Although our knowledge of the full barcode remains incomplete, we hope that the concept prompts investigations into undiscovered emergent properties and engenders an appreciation for how and why unique microenvironments control biochemical reactions.

Dynamical control enables the formation of demixed biomolecular condensates.

Cellular matter can be organized into compositionally distinct biomolecular condensates. For example, in Ashbya gossypii, the RNA-binding protein Whi3 forms distinct condensates with different RNA molecules. Using criteria derived from a physical framework for explaining how compositionally distinct condensates can form spontaneously via thermodynamic considerations, we find that condensates in vitro form mainly via heterotypic interactions in binary mixtures of Whi3 and RNA. However, within these condensates, RNA molecules become dynamically arrested. As a result, in ternary systems, simultaneous additions of Whi3 and pairs of distinct RNA molecules lead to well-mixed condensates, whereas delayed addition of an RNA component results in compositional distinctness. Therefore, compositional identities of condensates can be achieved via dynamical control, being driven, at least partially, by the dynamical arrest of RNA molecules. Finally, we show that synchronizing the production of different RNAs leads to more well-mixed, as opposed to compositionally distinct condensates in vivo.

Mechanosensitive ion channels MSL8, MSL9, and MSL10 have environmentally sensitive intrinsically disordered regions with distinct biophysical characteristics in vitro.

Intrinsically disordered protein regions (IDRs) are highly dynamic sequences that rapidly sample a collection of conformations over time. In the past several decades, IDRs have emerged as a major component of many proteomes, comprising ~30% of all eukaryotic protein sequences. Proteins with IDRs function in a wide range of biological pathways and are notably enriched in signaling cascades that respond to environmental stresses. Here, we identify and characterize intrinsic disorder in the soluble cytoplasmic N-terminal domains of MSL8, MSL9, and MSL10, three members of the MscS-like (MSL) family of mechanosensitive ion channels. In plants, MSL channels are proposed to mediate cell and organelle osmotic homeostasis. Bioinformatic tools unanimously predicted that the cytosolic N-termini of MSL channels are intrinsically disordered. We examined the N-terminus of MSL10 (MSL10N) as an exemplar of these IDRs and circular dichroism spectroscopy confirms its disorder. MSL10N adopted a predominately helical structure when exposed to the helix-inducing compound trifluoroethanol (TFE). Furthermore, in the presence of molecular crowding agents, MSL10N underwent structural changes and exhibited alterations to its homotypic interaction favorability. Lastly, interrogations of collective behavior via in vitro imaging of condensates indicated that MSL8N, MSL9N, and MSL10N have sharply differing propensities for self-assembly into condensates, both inherently and in response to salt, temperature, and molecular crowding. Taken together, these data establish the N-termini of MSL channels as intrinsically disordered regions with distinct biophysical properties and the potential to respond uniquely to changes in their physiochemical environment.

Acentrosomal spindles assemble from branching microtubule nucleation near chromosomes in Xenopus laevis egg extract.

Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstitute microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and find that chromosomes alone can form spindles. We visualize microtubule nucleation near chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. By inhibiting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient that spatially biases branching microtbule nucleation. In the presence of motors, these branched networks are ultimately organized into functional spindles, where the number of emergent spindle poles scales with the number of chromosomes and total chromatin area.

Augmin is a Ran-regulated spindle assembly factor.

Mitotic spindles are composed of microtubules (MTs) that must nucleate at the right place and time. Ran regulates this process by directly controlling the release of spindle assembly factors (SAFs) from nucleocytoplasmic shuttle proteins importin-αß and subsequently forms a biochemical gradient of SAFs localized around chromosomes. The majority of spindle MTs are generated by branching MT nucleation, which has been shown to require an eight-subunit protein complex known as augmin. In Xenopus laevis, Ran can control branching through a canonical SAF, TPX2, which is nonessential in Drosophila melanogaster embryos and HeLa cells. Thus, how Ran regulates branching MT nucleation when TPX2 is not required remains unknown. Here, we use in vitro pulldowns and total internal reflection fluorescence microscopy to show that augmin is a Ran-regulated SAF. We demonstrate that augmin directly interacts with both importin-α and importin-ß through two nuclear localization sequences on the Haus8 subunit, which overlap with the MT-binding site. Moreover, we show that Ran controls localization of augmin to MTs in both Xenopus egg extract and in vitro. Our results demonstrate that RanGTP directly regulates augmin, which establishes a new way by which Ran controls branching MT nucleation and spindle assembly both in the absence and presence of TPX2.

Attempted Cardiopulmonary Bypass Venous Cannula Extraction Catheter Extraction of a Rare Intracardiac Myoepithelioma.

Myoepithelioma of the soft tissue is a rare entity that can mimic myxoma when presenting within the heart. We present a case where cardiopulmonary bypass venous cannula extraction catheter removal of an intracardiac myoepithelioma was attempted with minimal debulking and subsequently required minimally invasive open-heart surgery with cardiopulmonary bypass. (Level of Difficulty: Advanced.).

Dynamical control enables the formation of demixed biomolecular condensates.

Macromolecular phase separation underlies the regulated formation and dissolution of biomolecular condensates. What is unclear is how condensates of distinct and shared macromolecular compositions form and coexist within cellular milieus. Here, we use theory and computation to establish thermodynamic criteria that must be satisfied to achieve compositionally distinct condensates. We applied these criteria to an archetypal ribonucleoprotein condensate and discovered that demixing into distinct protein-RNA condensates cannot be the result of purely thermodynamic considerations. Instead, demixed, compositionally distinct condensates arise due to asynchronies in timescales that emerge from differences in long-lived protein-RNA and RNA-RNA crosslinks. This type of dynamical control is also found to be active in live cells whereby asynchronous production of molecules is required for realizing demixed protein-RNA condensates. We find that interactions that exert dynamical control provide a versatile and generalizable way to influence the compositions of coexisting condensates in live cells.

Single fluorogen imaging reveals distinct environmental and structural features of biomolecular condensates.

Recent computations suggest that biomolecular condensates that form via macromolecular phase separation are network fluids featuring spatially inhomogeneous organization of the underlying molecules. Computations also point to unique conformations of molecules at condensate interfaces. Here, we test these predictions using high-resolution structural characterizations of condensates formed by intrinsically disordered prion-like low complexity domains (PLCDs). We leveraged the localization and orientational preferences of freely diffusing fluorogens and the solvatochromic effect whereby specific fluorogens are turned on in response to the physic-chemical properties of condensate microenvironments to facilitate single-molecule tracking and super-resolution imaging. We deployed three different fluorogens to probe internal microenvironments and molecular organization of PLCD condensates. The spatiotemporal resolution and environmental sensitivity afforded by single-fluorogen imaging shows that the internal environments of condensates are more hydrophobic than coexisting dilute phases. Molecules within condensates are organized in a spatially inhomogeneous manner featuring slow-moving nanoscale molecular clusters or hubs that coexist with fast-moving molecules. Finally, molecules at interfaces of condensates are found to have distinct orientational preferences when compared to the interiors. Our findings, which affirm computational predictions, help provide a structural basis for condensate viscoelasticity and dispel the notion of protein condensates being isotropic liquids defined by uniform internal densities.

Dynamical control enables the formation of demixed biomolecular condensates.

Macromolecular phase separation underlies the regulated formation and dissolution of biomolecular condensates. What is unclear is how condensates of distinct and shared macromolecular compositions form and coexist within cellular milieus. Here, we use theory and computation to establish thermodynamic criteria that must be satisfied to achieve compositionally distinct condensates. We applied these criteria to an archetypal ribonucleoprotein condensate and discovered that demixing into distinct protein-RNA condensates cannot be the result of purely thermodynamic considerations. Instead, demixed, compositionally distinct condensates arise due to asynchronies in timescales that emerge from differences in long-lived protein-RNA and RNA-RNA crosslinks. This type of dynamical control is also found to be active in live cells whereby asynchronous production of molecules is required for realizing demixed protein-RNA condensates. We find that interactions that exert dynamical control provide a versatile and generalizable way to influence the compositions of coexisting condensates in live cells.

Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus-ends.

Growing microtubule ends organize end-tracking proteins into comets of mixed composition. Here using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid-phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions may facilitate non-stoichiometric accumulation of cargo Tip1. We found that two disordered protein regions in Mal3 are required for the formation of droplets and motor-dependent accumulation of Tip1, while autonomous Mal3 comet formation requires only one of them. Using theoretical modelling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends. We conclude that microtubule ends may act as platforms where multivalent interactions condense microtubule-associated proteins into large multi-protein complexes.

Interaction of spindle assembly factor TPX2 with importins-α/ß 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/ß until the onset of mitosis, yet the molecular nature of this regulation remains unclear. Here we demonstrate that TPX2 interacts with importins-α/ß 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-ß via dispersed, weak interactions. We show that interactions of both importin-α and -ß 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.

Insulin deficiency, but not resistance, exaggerates cognitive deficits in transgenic mice expressing human amyloid and tau proteins. Reversal by Exendin-4 treatment.

Epidemiological studies have pointed at diabetes as a risk factor for Alzheimer's disease (AD) and this has been supported by several studies in animal models of both type 1 and type 2 diabetes. However, side-by-side comparison of the two types of diabetes is limited. We investigated the role of insulin deficiency and insulin resistance in the development of memory impairments and the effect of Exendin-4 (Ex4) treatment in a mouse model of AD. Three-4-month-old female wild type (WT) mice and mice overexpressing human tau and amyloid precursor protein (TAPP) were injected with streptozotocin (STZ) or fed a high-fat diet (HFD). A second study was performed in TAPP-STZ mice treated with Ex4, a long-lasting analog of GLP-1. Plasma and brain were collected at study termination for ELISA, Western blot, and immunohistochemistry analysis. Learning and memory deficits were impaired in TAPP transgenic mice compared with WT mice at the end of the study. Deficits were exaggerated by insulin deficiency in TAPP mice but 12 weeks of insulin resistance did not affect memory performances in either WT or TAPP mice. Levels of phosphorylated tau were increased in the brain of WT-STZ and TAPP-STZ mice but not in the brain of WT or TAPP mice on HFD. In the TAPP-STZ mice, treatment with Ex4 initiated after established cognitive deficits ameliorated learning, but not memory, impairments. This was accompanied by the reduction of amyloid ß and phosphorylated tau expression. Theses studies support the role of Ex4 in AD, independently from its actions on diabetes.

Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation.

Phase separation of substrates and effectors is proposed to enhance biological reaction rates and efficiency. Targeting protein for Xklp2 (TPX2) is an effector of branching microtubule nucleation in spindles and functions with the substrate tubulin by an unknown mechanism. Here we show that TPX2 phase separates into a co-condensate with tubulin, which mediates microtubule nucleation in vitro and in isolated cytosol. TPX2-tubulin co-condensation preferentially occurs on pre-existing microtubules, the site of branching microtubule nucleation, at the endogenous and physiologically relevant concentration of TPX2. Truncation and chimera versions of TPX2 suggest that TPX2-tubulin co-condensation enhances the efficiency of TPX2-mediated branching microtubule nucleation. Finally, the known inhibitor of TPX2, the importin-α/ß heterodimer, regulates TPX2 condensation in vitro and, consequently, branching microtubule nucleation activity in isolated cytosol. Our study demonstrates how regulated phase separation can simultaneously enhance reaction efficiency and spatially coordinate microtubule nucleation, which may facilitate rapid and accurate spindle formation.

Mechanism of how augmin directly targets the γ-tubulin ring complex to microtubules.

Microtubules (MTs) must be generated from precise locations to form the structural frameworks required for cell shape and function. MTs are nucleated by the γ-tubulin ring complex (γ-TuRC), but it remains unclear how γ-TuRC gets to the right location. Augmin has been suggested to be a γ-TuRC targeting factor and is required for MT nucleation from preexisting MTs. To determine augmin's architecture and function, we purified Xenopus laevis augmin from insect cells. We demonstrate that augmin is sufficient to target γ-TuRC to MTs by in vitro reconstitution. Augmin is composed of two functional parts. One module (tetramer-II) is necessary for MT binding, whereas the other (tetramer-III) interacts with γ-TuRC. Negative-stain electron microscopy reveals that both tetramers fit into the Y-shape of augmin, and MT branching assays reveal that both are necessary for MT nucleation. The finding that augmin can directly bridge MTs with γ-TuRC via these two tetramers adds to our mechanistic understanding of how MTs can be nucleated from preexisting MTs.

The RNA-binding protein Rumpelstiltskin antagonizes gypsy chromatin insulator function in a tissue-specific manner.

Chromatin insulators are DNA-protein complexes that are situated throughout the genome that are proposed to contribute to higher-order organization and demarcation into distinct transcriptional domains. Mounting evidence in different species implicates RNA and RNA-binding proteins as regulators of chromatin insulator activities. Here, we identify the Drosophila hnRNP M homolog Rumpelstiltskin (Rump) as an antagonist of gypsy chromatin insulator enhancer-blocking and barrier activities. Despite ubiquitous expression of Rump, decreasing Rump levels leads to improvement of barrier activity only in tissues outside of the central nervous system (CNS). Furthermore, rump mutants restore insulator body localization in an insulator mutant background only in non-CNS tissues. Rump associates physically with core gypsy insulator proteins, and chromatin immunoprecipitation and sequencing analysis of Rump demonstrates extensive colocalization with a subset of insulator sites across the genome. The genome-wide binding profile and tissue specificity of Rump contrast with that of Shep, a recently identified RNA-binding protein that antagonizes gypsy insulator activity primarily in the CNS. Our findings indicate parallel roles for RNA-binding proteins in mediating tissue-specific regulation of chromatin insulator activity.

Role of insulin signaling impairment, adiponectin and dyslipidemia in peripheral and central neuropathy in mice.

One of the tissues or organs affected by diabetes is the nervous system, predominantly the peripheral system (peripheral polyneuropathy and/or painful peripheral neuropathy) but also the central system with impaired learning, memory and mental flexibility. The aim of this study was to test the hypothesis that the pre-diabetic or diabetic condition caused by a high-fat diet (HFD) can damage both the peripheral and central nervous systems. Groups of C57BL6 and Swiss Webster mice were fed a diet containing 60% fat for 8 months and compared to control and streptozotocin (STZ)-induced diabetic groups that were fed a standard diet containing 10% fat. Aspects of peripheral nerve function (conduction velocity, thermal sensitivity) and central nervous system function (learning ability, memory) were measured at assorted times during the study. Both strains of mice on HFD developed impaired glucose tolerance, indicative of insulin resistance, but only the C57BL6 mice showed statistically significant hyperglycemia. STZ-diabetic C57BL6 mice developed learning deficits in the Barnes maze after 8 weeks of diabetes, whereas neither C57BL6 nor Swiss Webster mice fed a HFD showed signs of defects at that time point. By 6 months on HFD, Swiss Webster mice developed learning and memory deficits in the Barnes maze test, whereas their peripheral nervous system remained normal. In contrast, C57BL6 mice fed the HFD developed peripheral nerve dysfunction, as indicated by nerve conduction slowing and thermal hyperalgesia, but showed normal learning and memory functions. Our data indicate that STZ-induced diabetes or a HFD can damage both peripheral and central nervous systems, but learning deficits develop more rapidly in insulin-deficient than in insulin-resistant conditions and only in Swiss Webster mice. In addition to insulin impairment, dyslipidemia or adiponectinemia might determine the neuropathy phenotype.

Glycogen synthase kinase-3 inhibition prevents learning deficits in diabetic mice.

There is an increasing awareness that diabetes has an impact on the central nervous system, with reports of impaired learning, memory, and mental flexibility being more common in diabetic subjects than in the general population. Insulin-deficient diabetic mice also display learning deficits associated with defective insulin-signaling in the brain and increased activity of GSK3. In the present study, AR-A014418, a GSK3ß inhibitor, and TX14(A), a neurotrophic factor with GSK3 inhibitory properties, were tested against the development of learning deficits in mice with insulin-deficient diabetes. Treatments were started at onset of diabetes and continued for 10 weeks. Treatment with AR-A014418 or TX14(A) prevented the development of learning deficits, assessed by the Barnes maze, but only AR-A014418 prevented memory deficits, as assessed by the object recognition test. Diabetes-induced increased levels of amyloid ß protein and phosphorylated tau were not significantly affected by the treatments. However, the diabetes-induced decrease in synaptophysin, a presynaptic protein marker of hippocampal plasticity, was partially prevented by both treatments. These results suggest a role for GSK3 and/or reduced neurotrophic support in the development of cognitive deficits in diabetic mice that are associated with synaptic damage.