RanGTP and importin ß regulate meiosis I spindle assembly and function in mouse oocytes.

Homologous chromosome segregation during meiosis I (MI) in mammalian oocytes is carried out by the acentrosomal MI spindles. Whereas studies in human oocytes identified Ran GTPase as a crucial regulator of the MI spindle function, experiments in mouse oocytes questioned the generality of this notion. Here, we use live-cell imaging with fluorescent probes and Förster resonance energy transfer (FRET) biosensors to monitor the changes in Ran and importin ß signaling induced by perturbations of Ran in mouse oocytes while examining the MI spindle dynamics. We show that unlike RanT24N employed in previous studies, a RanT24N, T42A double mutant inhibits RanGEF without perturbing cargo binding to importin ß and disrupts MI spindle function in chromosome segregation. Roles of Ran and importin ß in the coalescence of microtubule organizing centers (MTOCs) and MI spindle assembly are further supported by the use of the chemical inhibitor importazole, whose effects are partially rescued by the GTP hydrolysis-resistant RanQ69L mutant. These results indicate that RanGTP is essential for MI spindle assembly and function both in humans and mice.

Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking.

The nuclear pore complex (NPC) mediates all exchange between the cytoplasm and the nucleus. Small molecules can passively diffuse through the NPC, whereas larger cargos require transport receptors to translocate. How the NPC facilitates the translocation of transport receptor/cargo complexes remains unclear. To investigate this process, we tracked single protein-functionalized quantum dot cargos as they moved through human NPCs. Here we show that import proceeds by successive substeps comprising cargo capture, filtering and translocation, and release into the nucleus. Most quantum dots are rejected at one of these steps and return to the cytoplasm, including very large cargos that abort at a size-selective barrier. Cargo movement in the central channel is subdiffusive and cargos that can bind more transport receptors diffuse more freely. Without Ran GTPase, a critical regulator of transport directionality, cargos still explore the entire NPC, but have a markedly reduced probability of exit into the nucleus, suggesting that NPC entry and exit steps are not equivalent and that the pore is functionally asymmetric to importing cargos. The overall selectivity of the NPC seems to arise from the cumulative action of multiple reversible substeps and a final irreversible exit step.

Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts.

During cell division the genetic material on chromosomes is distributed to daughter cells by a dynamic microtubule structure called the mitotic spindle. Here we establish a reconstitution system to assess the contribution of individual chromosome proteins to mitotic spindle formation around single 10 µm diameter porous glass beads in Xenopus egg extracts. We find that Regulator of Chromosome Condensation 1 (RCC1), the Guanine Nucleotide Exchange Factor (GEF) for the small GTPase Ran, can induce bipolar spindle formation. Remarkably, RCC1 beads oscillate within spindles from pole to pole, a behavior that could be converted to a more typical, stable association by the addition of a kinesin together with RCC1. These results identify two activities sufficient to mimic chromatin-mediated spindle assembly, and establish a foundation for future experiments to reconstitute spindle assembly entirely from purified components.

Coordinated in confined migration: crosstalk between the nucleus and ion channel-mediated mechanosensation.

Cell surface and intracellular mechanosensors enable cells to perceive different geometric, topographical, and physical cues. Mechanosensitive ion channels (MICs) localized at the cell surface and on the nuclear envelope (NE) are among the first to sense and transduce these signals. Beyond compartmentalizing the genome of the cell and its transcription, the nucleus also serves as a mechanical gauge of different physical and topographical features of the tissue microenvironment. In this review, we delve into the intricate mechanisms by which the nucleus and different ion channels regulate cell migration in confinement. We review evidence suggesting an interplay between macromolecular nuclear-cytoplasmic transport (NCT) and ionic transport across the cell membrane during confined migration. We also discuss the roles of the nucleus and ion channel-mediated mechanosensation, whether acting independently or in tandem, in orchestrating migratory mechanoresponses. Understanding nuclear and ion channel sensing, and their crosstalk, is critical to advancing our knowledge of cell migration in health and disease.

Multivalent GU-rich oligonucleotides sequester TDP-43 in the nucleus by inducing high molecular weight RNP complexes.

TDP-43 nuclear clearance and cytoplasmic aggregation are hallmarks of TDP-43 proteinopathies. We recently demonstrated that binding to endogenous nuclear GU-rich RNAs sequesters TDP-43 in the nucleus by restricting its passive nuclear export. Here, we tested the feasibility of synthetic RNA oligonucleotide-mediated augmentation of TDP-43 nuclear localization. Using biochemical assays, we compared the ability of GU-rich oligonucleotides to engage in multivalent, RRM-dependent binding with TDP-43. When transfected into cells, (GU)16 attenuated TDP-43 mislocalization induced by transcriptional blockade or RanGAP1 ablation. Clip34nt and (GU)16 accelerated TDP-43 nuclear re-import after cytoplasmic mislocalization. RNA pulldowns confirmed that multivalent GU-oligonucleotides induced high molecular weight RNP complexes, incorporating TDP-43 and possibly other GU-binding proteins. Transfected GU-repeat oligos disrupted TDP-43 cryptic exon repression, likely by diverting TDP-43 from endogenous RNAs, except for Clip34nt which contains interspersed A and C. Thus, exogenous multivalent GU-RNAs can promote TDP-43 nuclear localization, though pure GU-repeat motifs impair TDP-43 function.

Multivalent GU-rich oligonucleotides sequester TDP-43 in the nucleus by inducing high molecular weight RNP complexes.

TDP-43 nuclear clearance and cytoplasmic aggregation are hallmarks of TDP-43 proteinopathies. We recently demonstrated that binding to endogenous nuclear GU-rich RNAs sequesters TDP-43 in the nucleus by restricting its passive nuclear export. Here, we tested the feasibility of synthetic RNA oligonucleotide-mediated augmentation of TDP-43 nuclear localization. Using biochemical assays, we compared the ability of GU-rich oligonucleotides to engage in multivalent, RRM-dependent binding with TDP-43. When transfected into cells, (GU)16 attenuated TDP-43 mislocalization induced by transcriptional blockade or RanGAP1 ablation. Clip34nt and (GU)16 accelerated TDP-43 nuclear re-import after cytoplasmic mislocalization. RNA pulldowns confirmed that multivalent GU-oligonucleotides induced high molecular weight RNP complexes, incorporating TDP-43 and possibly other GU-binding proteins. Transfected GU-repeat oligos disrupted TDP-43 cryptic exon repression, likely by diverting TDP-43 from endogenous RNAs, except for Clip34nt that contains interspersed A and C. Thus, exogenous multivalent GU-RNAs can promote TDP-43 nuclear localization, though pure GU-repeat motifs impair TDP-43 function.

Cytoplasmic accumulation and plasma membrane association of anillin and Ect2 promote confined migration and invasion.

Cells migrating in confinement experience mechanical challenges whose consequences on cell migration machinery remain only partially understood. Here, we demonstrate that a pool of the cytokinesis regulatory protein anillin is retained during interphase in the cytoplasm of different cell types. Confinement induces recruitment of cytoplasmic anillin to plasma membrane at the poles of migrating cells, which is further enhanced upon nuclear envelope (NE) rupture(s). Rupture events also enable the cytoplasmic egress of predominantly nuclear RhoGEF Ect2. Anillin and Ect2 redistributions scale with microenvironmental stiffness and confinement, and are observed in confined cells in vitro and in invading tumor cells in vivo. Anillin, which binds actomyosin at the cell poles, and Ect2, which activates RhoA, cooperate additively to promote myosin II contractility, and promote efficient invasion and extravasation. Overall, our work provides a mechanistic understanding of how cytokinesis regulators mediate RhoA/ROCK/myosin II-dependent mechanoadaptation during confined migration and invasive cancer progression.

Cytokinesis machinery promotes cell dissociation from collectively migrating strands in confinement.

Cells tune adherens junction dynamics to regulate epithelial integrity in diverse (patho)physiological processes, including cancer metastasis. We hypothesized that the spatially confining architecture of peritumor stroma promotes metastatic cell dissemination by remodeling cell-cell adhesive interactions. By combining microfluidics with live-cell imaging, FLIM/FRET biosensors, and optogenetic tools, we show that confinement induces leader cell dissociation from cohesive ensembles. Cell dissociation is triggered by myosin IIA (MIIA) dismantling of E-cadherin cell-cell junctions, as recapitulated by a mathematical model. Elevated MIIA contractility is controlled by RhoA/ROCK activation, which requires distinct guanine nucleotide exchange factors (GEFs). Confinement activates RhoA via nucleocytoplasmic shuttling of the cytokinesis-regulatory proteins RacGAP1 and Ect2 and increased microtubule dynamics, which results in the release of active GEF-H1. Thus, confining microenvironments are sufficient to induce cell dissemination from primary tumors by remodeling E-cadherin cell junctions via the interplay of microtubules, nuclear trafficking, and RhoA/ROCK/MIIA pathway and not by down-regulating E-cadherin expression.

Microtubule assembly by the Apc protein is regulated by importin-beta--RanGTP.

Mutations in the tumour suppressor Adenomatous polyposis coli (Apc) initiate most sporadic colorectal cancers. Apc is implicated in regulating microtubule (MT) dynamics in interphase and mitosis. However, little is known about the underlying mechanism or regulation of this Apc function. We identified importin-beta as a binding partner of Apc that regulates its effect on MTs. Apc binds importin-beta in vitro and in Xenopus egg extracts, and RanGTP inhibits this interaction. The armadillo-like repeat domain of importin-beta binds to the middle of Apc, where it can compete with beta-catenin. In addition, two independent sites in the C terminus of Apc bind the N-terminal region of importin-beta. Binding to importin-beta reduces the ability of Apc to assemble and bundle MTs in vitro and to promote assembly of microtubule asters in Xenopus egg extracts, but does not affect the binding of Apc to MTs or to EB1. Depletion of Apc decreases the formation of cold-stable spindles in Xenopus egg extracts. Importantly, the ability of purified Apc to rescue this phenotype was reduced when it was constitutively bound to importin-beta. Thus, importin-beta binds to Apc and negatively regulates the MT-assembly and spindle-promoting activity of Apc in a Ran-regulatable manner.

A centriole- and RanGTP-independent spindle assembly pathway in meiosis I of vertebrate oocytes.

Spindle formation is essential for stable inheritance of genetic material. Experiments in various systems indicate that Ran GTPase is crucial for meiotic and mitotic spindle assembly. Such an important role for Ran in chromatin-induced spindle assembly was initially demonstrated in Xenopus laevis egg extracts. However, the requirement of RanGTP in living meiotic cells has not been shown. In this study, we used a fluorescence resonance energy transfer probe to measure RanGTP-regulated release of importin beta. A RanGTP-regulated gradient was established during meiosis I and was centered on chromosomes throughout mouse meiotic maturation. Manipulating levels of RanGTP in mice and X. laevis oocytes did not inhibit assembly of functional meiosis I spindles. However, meiosis II spindle assembly did not tolerate changes in the level of RanGTP in both species. These findings suggest that a mechanism common to vertebrates promotes meiosis I spindle formation in the absence of chromatin-induced microtubule production and centriole-based microtubule organizing centers.

Analysis of a RanGTP-regulated gradient in mitotic somatic cells.

The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-beta family. The Ran-importin-beta system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran-importin-beta system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-beta by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vitro experiments and modelling show that this localized increase of free cargoes corresponds to changes in RanGTP concentration sufficient to stabilize microtubules in extracts. In cells, the Ran-importin-beta-cargo gradient kinetically promotes spindle formation but is largely dispensable once the spindle has been established. Consistent with previous reports, we observe that the Ran system also affects spindle pole formation and chromosome congression in vivo. Our results demonstrate that conserved Ran-regulated pathways are involved in multiple, parallel processes required for spindle function, but that their relative contribution differs in chromatin- versus centrosome/kinetochore-driven spindle assembly systems.

Emerging Therapies and Novel Targets for TDP-43 Proteinopathy in ALS/FTD.

Nuclear clearance and cytoplasmic mislocalization of the essential RNA binding protein, TDP-43, is a pathologic hallmark of amyotrophic lateral sclerosis, frontotemporal dementia, and related neurodegenerative disorders collectively termed "TDP-43 proteinopathies." TDP-43 mislocalization causes neurodegeneration through both loss and gain of function mechanisms. Loss of TDP-43 nuclear RNA processing function destabilizes the transcriptome by multiple mechanisms including disruption of pre-mRNA splicing, the failure of repression of cryptic exons, and retrotransposon activation. The accumulation of cytoplasmic TDP-43, which is prone to aberrant liquid-liquid phase separation and aggregation, traps TDP-43 in the cytoplasm and disrupts a host of downstream processes including the trafficking of RNA granules, local translation within axons, and mitochondrial function. In this review, we will discuss the TDP-43 therapy development pipeline, beginning with therapies in current and upcoming clinical trials, which are primarily focused on accelerating the clearance of TDP-43 aggregates. Then, we will look ahead to emerging strategies from preclinical studies, first from high-throughput genetic and pharmacologic screens, and finally from mechanistic studies focused on the upstream cause(s) of TDP-43 disruption in ALS/FTD. These include modulation of stress granule dynamics, TDP-43 nucleocytoplasmic shuttling, RNA metabolism, and correction of aberrant splicing events.

Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export.

Nuclear clearance of the RNA-binding protein TDP-43 is a hallmark of neurodegeneration and an important therapeutic target. Our current understanding of TDP-43 nucleocytoplasmic transport does not fully explain its predominantly nuclear localization or mislocalization in disease. Here, we show that TDP-43 exits nuclei by passive diffusion, independent of facilitated mRNA export. RNA polymerase II blockade and RNase treatment induce TDP-43 nuclear efflux, suggesting that nuclear RNAs sequester TDP-43 in nuclei and limit its availability for passive export. Induction of TDP-43 nuclear efflux by short, GU-rich oligomers (presumably by outcompeting TDP-43 binding to endogenous nuclear RNAs), and nuclear retention conferred by splicing inhibition, demonstrate that nuclear TDP-43 localization depends on binding to GU-rich nuclear RNAs. Indeed, RNA-binding domain mutations markedly reduce TDP-43 nuclear localization and abolish transcription blockade-induced nuclear efflux. Thus, the nuclear abundance of GU-RNAs, dictated by the balance of transcription, pre-mRNA processing, and RNA export, regulates TDP-43 nuclear localization.

Dual control of formin-nucleated actin assembly by the chromatin and ER in mouse oocytes.

The first asymmetric meiotic cell divisions in mouse oocytes are driven by formin 2 (FMN2)-nucleated actin polymerization around the spindle. In this study, we investigated how FMN2 is recruited to the spindle peripheral ER and how its activity is regulated in mouse meiosis I (MI) oocytes. We show that this process is regulated by the Ran GTPase, a conserved mediator of chromatin signal, and the ER-associated protein VAPA. FMN2 contains a nuclear localization sequence (NLS) within a domain (SLD) previously shown to be required for FMN2 localization to the spindle periphery. FMN2 NLS is bound to the importin α1/ß complex, and the disruption of this interaction by RanGTP is required for FMN2 accumulation in the area proximal to the chromatin and the MI spindle. The importin-free FMN2 is then recruited to the surface of ER around the spindle through the binding of the SLD with the ER-membrane protein VAPA. We further show that FMN2 is autoinhibited through an intramolecular interaction between the SLD with the C-terminal formin homology 2 (FH2) domain that nucleates actin filaments. VAPA binding to SLD relieves the autoinhibition of FMN2, leading to localized actin polymerization. This dual control of formin-mediated actin assembly allows actin polymerization to initiate the movement of the meiotic spindle toward the cortex, an essential step in the maturation of the mammalian female gamete.

The design of Förster (fluorescence) resonance energy transfer (FRET)-based molecular sensors for Ran GTPase.

The application of FRET-based molecular biosensors provided confirmation of the central model of Ran GTPase function and led to important new insights into its physiological role. In many fields of cell biology, methods employing FRET are a standard approach that is becoming increasingly accessible due to advances in instrumentation and available fluorophores. However, the optimal design of a FRET sensor remains to be the cornerstone of any successful FRET application. Utilizing the recent literature on FRET applications and our studies on Ran, we outline the basic considerations involved in designing molecular FRET sensors. We point to several broadly applicable principles that were used in many different FRET sensors that can detect a wide range of molecular events. Using the FRET sensors for Ran that we created as examples, we then focus on the practical aspects of FRET assays. We describe the preparation of a bipartite FRET sensor consisting of ECFP-Ran and EYFP-importin beta and its validation as a reporter for FRET-based high throughput screening in small molecule libraries. Finally, we review the design and optimization of monomolecular FRET sensors that monitor the RanGTP-RanBP1 interaction, and of sensors detecting the RanGTP-regulated importin beta cargo release.

Nuclear Transport Assays in Permeabilized Mouse Cortical Neurons.

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures. In our initial attempts, we observed the rapid loss of nuclear membrane integrity in primary mouse cortical neurons exposed to even low concentrations of digitonin. We hypothesized that neuronal nuclear membranes may be uniquely vulnerable to the loss of cytoplasmic support. After testing multiple approaches to improve nuclear stability, we observed optimal nuclear integrity following hypotonic lysis in the presence of a concentrated bovine serum albumin cushion. Neuronal nuclei prepared by this approach reliably import recombinant fluorescent cargo in an energy-dependent manner, facilitating analysis of nuclear import by high content microscopy with automated analysis. We anticipate that this method will be broadly applicable to studies of passive and active nuclear transport in primary neurons.

C9orf72 arginine-rich dipeptide repeat proteins disrupt karyopherin-mediated nuclear import.

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases, including ALS caused by a C9orf72 hexanucleotide repeat expansion. However, the mechanism(s) remain unclear. Karyopherins, including importin ß and its cargo adaptors, have been shown to co-precipitate with the C9orf72 arginine-containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs interact with importin ß, disrupt its cargo loading, and inhibit nuclear import of importin ß, importin α/ß, and transportin cargoes in permeabilized mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs induce widespread protein aggregation in this in vitro system, transport disruption is not due to nucleocytoplasmic transport protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore complex. Our results support a model in which R-DPRs interfere with cargo loading on karyopherins.

Symmetry breaking in hydrodynamic forces drives meiotic spindle rotation in mammalian oocytes.

Patterned cell divisions require a precisely oriented spindle that segregates chromosomes and determines the cytokinetic plane. In this study, we investigated how the meiotic spindle orients through an obligatory rotation during meiotic division in mouse oocytes. We show that spindle rotation occurs at the completion of chromosome segregation, whereby the separated chromosome clusters each define a cortical actomyosin domain that produces cytoplasmic streaming, resulting in hydrodynamic forces on the spindle. These forces are initially balanced but become unbalanced to drive spindle rotation. This force imbalance is associated with spontaneous symmetry breaking in the distribution of the Arp2/3 complex and myosin-II on the cortex, brought about by feedback loops comprising Ran guanosine triphosphatase signaling, Arp2/3 complex activity, and myosin-II contractility. The torque produced by the unbalanced hydrodynamic forces, coupled with a pivot point at the spindle midzone cortical contract, constitutes a unique mechanical system for meiotic spindle rotation.

Dynamic organelle distribution initiates actin-based spindle migration in mouse oocytes.

Migration of meiosis-I (MI) spindle from the cell center to a sub-cortical location is a critical step for mouse oocytes to undergo asymmetric meiotic cell division. In this study, we investigate the mechanism by which formin-2 (FMN2) orchestrates the initial movement of MI spindle. By defining protein domains responsible for targeting FMN2, we show that spindle-periphery localized FMN2 is required for spindle migration. The spindle-peripheral FMN2 nucleates short actin bundles from vesicles derived likely from the endoplasmic reticulum (ER) and concentrated in a layer outside the spindle. This layer is in turn surrounded by mitochondria. A model based on polymerizing actin filaments pushing against mitochondria, thus generating a counter force on the spindle, demonstrated an inherent ability of this system to break symmetry and evolve directional spindle motion. The model is further supported through experiments involving spatially biasing actin nucleation via optogenetics and disruption of mitochondrial distribution and dynamics.

Dorsoventral polarity directs cell responses to migration track geometries.

How migrating cells differentially adapt and respond to extracellular track geometries remains unknown. Using intravital imaging, we demonstrate that invading cells exhibit dorsoventral (top-to-bottom) polarity in vivo. To investigate the impact of dorsoventral polarity on cell locomotion through different confining geometries, we fabricated microchannels of fixed cross-sectional area, albeit with distinct aspect ratios. Vertical confinement, exerted along the dorsoventral polarity axis, induces myosin II-dependent nuclear stiffening, which results in RhoA hyperactivation at the cell poles and slow bleb-based migration. In lateral confinement, directed perpendicularly to the dorsoventral polarity axis, the absence of perinuclear myosin II fails to increase nuclear stiffness. Hence, cells maintain basal RhoA activity and display faster mesenchymal migration. In summary, by integrating microfabrication, imaging techniques, and intravital microscopy, we demonstrate that dorsoventral polarity, observed in vivo and in vitro, directs cell responses in confinement by spatially tuning RhoA activity, which controls bleb-based versus mesenchymal migration.