Nucleocytoplasmic Partitioning Research Articles

Spatial memory in Alzheimer's disease 5XFAD mice is enhanced by XPO1 inhibitor KPT-330.

The proteostatic decline in Alzheimer's disease is well established and improvement in proteostasis could potentially delay cognitive impairment. One emerging entry point to modulate proteostasis is the regulation of nucleo-cytoplasmic partitioning of proteins across the nuclear pore via karyopherins. The nuclear exportin XPO1 is a key regulator of proteostasis by driving the assembly of ribosomes and by modulating the process of autophagy. We recently found that XPO1 inhibitor KPT-330 (Selinexor), an FDA approved drug against multiple myelomas, enhances proteostasis, leading to benefits in models of neurodegenerative diseases in C. elegans and Drosophila. Here, we find that KPT-330 increases autophagy in murine neuronal cells and improves spatial memory performance in a murine model of Alzheimer's disease (5XFAD). Unexpectedly, general amyloid deposition in several brain regions was significantly increased by KPT-330, but specific regions, especially the thalamus, displayed significantly lower deposition, suggesting that XPO1 inhibition has regional-specific effects on proteostasis and amyloid plaque formation. Altogether, we conclude that XPO1 inhibition can improve cognition via spatially-specific reductions in amyloid deposition.

MCTP controls nucleocytoplasmic partitioning of AUXIN RESPONSE FACTORs during lateral root development.

The plant hormone auxin orchestrates almost all aspects of plant growth and development. AUXIN RESPONSE FACTORs (ARFs) control the transcription of auxin-responsive genes, forming cytoplasmic condensates to modulate auxin sensitivity and diversify auxin response regulation. However, the dynamic control of ARF distribution across different subcellular compartments remains largely obscure. Here, we show that three MULTIPLE C2 DOMAIN AND TRANSMEMBRANE REGION PROTEINs (MCTPs), MCTP3, MCTP4, and MCTP6, control ARF nucleocytoplasmic partitioning and determine lateral root development. MCTP3/4/6 are highly expressed in lateral roots and specifically interact with ARF7 and ARF19 to dissolve their cytoplasmic condensates. This promotes ARF nuclear localization in lateral root primordia and enhances auxin signaling during lateral root formation. Our findings confer MCTP as a key switch to modulate auxin responses and outline an MCTP-ARF signaling cascade that is crucial for the establishment of the plant root system.

Formation of HopQ1:14-3-3 complex in the host cytoplasm modulates nuclear import rate of Pseudomonas syringae effector in Nicotiana benthamiana cells.

HopQ1, a type three effector from Pseudomonas syringae upon phosphorylation coopts plant 14-3-3 proteins to control its stability and subcellular localization. Mass spectrometry of the cytoplasm-restricted effector revealed that HopQ1 already in this subcellular compartment undergoes phosphorylation at serine 51 within the canonical 14-3-3 binding motif and within the second putative 14-3-3 binding site, 24RTPSES29. Our analyses revealed that the stoichiometry of the HopQ1:14-3-3a complex is 1:2 indicating that both binding sites of HopQ1 are involved in the interaction. Notably, 24RTPSES29 comprises a putative nuclear translocation signal (NTS). Although a peptide containing NTS mediates nuclear import of a Cargo protein suggesting its role in the nuclear trafficking of HopQ1, a deletion of 25TPS27 does not change HopQ1 distribution. In contrast, elimination of 14-3-3 binding site, accelerates nuclear trafficking the effector. Collectively, we show that formation of the HopQ1:14-3-3 complex occurs in the host cytoplasm and slows down the effector translocation into the nucleus. These results provide a mechanism that maintains the proper nucleocytoplasmic partitioning of HopQ1, and at the same time is responsible for the relocation of 14-3-3s from the nucleus to cytoplasm in the presence of the effector.

Nucleocytoplasmic transport at the crossroads of proteostasis, neurodegeneration and neuroprotection.

Nucleocytoplasmic transport comprises the multistep assembly, transport and disassembly of protein and RNA cargoes entering and exiting nuclear pores. Accruing evidence supports that impairments to nucleocytoplasmic transport are a hallmark of neurodegenerative diseases. These impairments cause dysregulations in nucleocytoplasmic partitioning and proteostasis of nuclear transport receptors and client substrates that promote intracellular deposits - another hallmark of neurodegeneration. Disturbances in liquid-liquid phase separation (LLPS) between dense and dilute phases of biomolecules implicated in nucleocytoplasmic transport promote micrometer-scale coacervates, leading to proteinaceous aggregates. This Review provides historical and emerging principles of LLPS at the interface of nucleocytoplasmic transport, proteostasis, aging and noxious insults, whose dysregulations promote intracellular aggregates. E3 SUMO-protein ligase Ranbp2 constitutes the cytoplasmic filaments of nuclear pores, where it acts as a molecular hub for rate-limiting steps of nucleocytoplasmic transport. A vignette is provided on the roles of Ranbp2 in nucleocytoplasmic transport and at the intersection of proteostasis in the survival of photoreceptor and motor neurons under homeostatic and pathophysiological environments. Current unmet clinical needs are highlighted, including therapeutics aiming to manipulate aggregation-dissolution models of purported neurotoxicity in neurodegeneration.

AUXIN RESPONSE FACTOR protein accumulation and function.

Auxin is a key regulator of plant developmental processes. Its effects on transcription are mediated by the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. ARFs tightly control specific auxin responses necessary for proper plant growth and development. Recent research has revealed that regulated ARF protein accumulation and ARF nucleo-cytoplasmic partitioning can determine auxin transcriptional outputs. In this review, we explore these recent findings and consider the potential for regulated ARF accumulation in driving auxin responses in plants.

LEAFY homeostasis is regulated via ubiquitin-dependent degradation and sequestration in cytoplasmic condensates

The transcription factor LEAFY (LFY) plays crucial roles in flower development by activating floral homeotic genes. Activation of LFY targets requires the combined action of LFY and the E3 ubiquitin ligase UFO, although the precise underlying mechanism remains unclear. Here, we show that LFY accumulates in biomolecular condensates within the cytoplasm, while recombinant LFY forms condensates with similar properties invitro. UFO interacts with LFY within these condensates and marks it for degradation. LFY levels in the nucleus are buffered against changes in total LFY levels induced by proteasome inhibition, UFO overexpression, or mutation of lysine residues in a disordered region of LFY. Perturbation of cytoplasmic LFY condensates by 1,6-hexanediol treatment induces the relocalization of LFY to the nucleus and the subsequent activation of the LFY target AP3 in flowers. Our data suggest that nucleocytoplasmic partitioning, condensation, and ubiquitin-dependent degradation regulate LFY levels in the nucleus to control its activity.

Identification of the Karyopherin Superfamily in Maize and Its Functional Cues in Plant Development.

Appropriate nucleo-cytoplasmic partitioning of proteins is a vital regulatory mechanism in phytohormone signaling and plant development. However, how this is achieved remains incompletely understood. The Karyopherin (KAP) superfamily is critical for separating the biological processes in the nucleus from those in the cytoplasm. The KAP superfamily is divided into Importin α (IMPα) and Importin β (IMPβ) families and includes the core components in mediating nucleocytoplasmic transport. Recent reports suggest the KAPs play crucial regulatory roles in Arabidopsis development and stress response by regulating the nucleo-cytoplasmic transport of members in hormone signaling. However, the KAP members and their associated molecular mechanisms are still poorly understood in maize. Therefore, we first identified seven IMPα and twenty-seven IMPβ genes in the maize genome and described their evolution traits and the recognition rules for substrates with nuclear localization signals (NLSs) or nuclear export signals (NESs) in plants. Next, we searched for the protein interaction partners of the ZmKAPs and selected the ones with Arabidopsis orthologs functioning in auxin biosynthesis, transport, and signaling to predict their potential function. Finally, we found that several ZmKAPs share similar expression patterns with their interacting proteins, implying their function in root development. Overall, this article focuses on the Karyopherin superfamily in maize and starts with this entry point by systematically comprehending the KAP-mediated nucleo-cytoplasmic transport process in plants, and then predicts the function of the ZmKAPs during maize development, with a perspective on a closely associated regulatory mechanism between the nucleo-cytoplasmic transport and the phytohormone network.

Global Transcriptome Analyses of Cellular and Viral mRNAs during HAdV-C5 Infection Highlight New Aspects of Viral mRNA Biogenesis and Cytoplasmic Viral mRNA Accumulations.

It is well established that human adenoviruses such as species C, types 2 and 5 (HAdV-C2 and HAdV-C5), induce a nearly complete shutoff of host-cell protein synthesis in the infected cell, simultaneously directing very efficient production of viral proteins. Such preferential expression of viral over cellular genes is thought to be controlled by selective nucleocytoplasmic export and translation of viral mRNA. While detailed knowledge of the regulatory mechanisms responsible for the translation of viral mRNA is available, the viral or cellular mechanisms of mRNA biogenesis are not completely understood. To identify parameters that control the differential export of viral and cellular mRNAs, we performed global transcriptome analyses (RNAseq) and monitored temporal nucleocytoplasmic partitioning of viral and cellular mRNAs during HAdV-C5 infection of A549 cells. Our analyses confirmed previously reported features of the viral mRNA expression program, as a clear shift in viral early to late mRNA accumulation was observed upon transition from the early to the late phase of viral replication. The progression into the late phase of infection, however, did not result in abrogation of cellular mRNA export; rather, viral late mRNAs outnumbered viral early and most cellular mRNAs by several orders of magnitude during the late phase, revealing that viral late mRNAs are not selectively exported but outcompete cellular mRNA biogenesis.

HOP1 and HOP2 are involved in salt tolerance by facilitating the brassinosteroid-related nucleo-cytoplasmic partitioning of the HSP90-BIN2 complex.

The co-chaperoneheat shock protein(HSP)70-HSP90 organizing protein (HOP) is involved in plant thermotolerance. However, its function in plant salinity tolerance was not yet studied. We found that Arabidopsis HOP1 and HOP2 play critical roles in salt tolerance by affecting the nucleo-cytoplasmic partitioning of HSP90and brassinosteroid-insensitive 2 (BIN2). A hop1/2 double mutant was hypersensitive to salt-stress. Interestingly, this sensitivity was remedied by exogenous brassinolide application, while the application of brassinazole impeded growth of both wild-type (WT) and hop1/2 plants under normal and salt stress conditions. This suggested that the insufficient brassinosteroid (BR) content was responsible for the salt-sensitivity of hop1/2. After WT was transferred to salt stress conditions, HOP1/2, BIN2 and HSP90 accumulated in the nucleus, brassinazole-resistant 1 (BZR1) was phosphorylated and accumulated in the cytoplasm, and BR content significantly increased. This initial response resulted in dephosphorylation of BZR1 and BR response. This dynamic regulation of BR content was impeded in salt-stressed hop1/2. Thus, we propose that HOP1 and HOP2 are involved in salt tolerance by affecting BR signalling.

Regulation of AUXIN RESPONSE FACTOR condensation and nucleo-cytoplasmic partitioning

Auxin critically regulates plant growth and development. Auxin-driven transcriptional responses are mediated through the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. ARF protein condensation attenuates ARF activity, resulting in dramatic shifts in the auxin transcriptional landscape. Here, we perform a forward genetics screen for ARF hypercondensation, identifying an F-box protein, which we named AUXIN RESPONSE FACTOR F-BOX1 (AFF1). Functional characterization of SCFAFF1 revealed that this E3 ubiquitin ligase directly interacts with ARF19 and ARF7 to regulate their accumulation, condensation, and nucleo-cytoplasmic partitioning. Mutants defective in AFF1 display attenuated auxin responsiveness, and developmental defects, suggesting that SCFAFF1 -mediated regulation of ARF protein drives aspects of auxin response and plant development.

Phytochrome A in plants comprises two structurally and functionally distinct populations-water-soluble phyA' and amphiphilic phyA″.

Photoreceptor phytochrome A (phyA) plays a key role both in the individual development and in the evolution of higher plants. It acts in three distinct modes - far-red light-induced very low fluence responses (VLFRs), high irradiance responses (HIRs), and red/far-red-reversible low fluence responses (LFRs). Signal transduction from phyA includes its transportation from the cytoplasm into the nucleus and activation of light-responsive genes there. It is also active in the cytoplasm. Two types of phyA speckles were detected upon its light-induced nucleocytoplasmic partitioning and a fraction remained in the cytoplasm. In this review, we present a concept that this complex picture of the phyA action is due, at least partially, to the existence of two phyA types in the cell differing by the structure of the N-terminus, probably, by its serine phosphorylation. These are phosphorylated water-soluble phyA' and underphosphorylated ambiquitous phyA″ represented by two fractions - water-soluble and membrane-associated. From the analysis of the phyA pools' activity in the regulation of phyA synthesis, seed germination, seedling establishment, and (proto)chlorophyll biosynthesis it is concluded that phyA″ is responsible for the regulation of seed germination, whereas in seedlings phyA' mediates the VLFRs, and the water-soluble phyA″ fraction, the HIRs. The membrane-associated phyA″ is likely to be active in cytoplasmic photoregulatory events. Functional interaction between phyA and the defense-related hormone jasmonic acid is also considered.

Understanding the role of mechanics in nucleocytoplasmic transport.

Cell nuclei are submitted to mechanical forces, which in turn affect nuclear and cell functions. Recent evidence shows that a crucial mechanically regulated nuclear function is nucleocytoplasmic transport, mediated by nuclear pore complexes (NPCs). Mechanical regulation occurs at two levels: first, by force application to the nucleus, which increases NPC permeability likely through NPC stretch. Second, by the mechanical properties of the transported proteins themselves, as mechanically labile proteins translocate through NPCs faster than mechanically stiff ones. In this perspective, we discuss this evidence and the associated mechanisms by which mechanics can regulate the nucleo-cytoplasmic partitioning of proteins. Finally, we analyze how mechanical regulation of nucleocytoplasmic transport can provide a systematic approach to the study of mechanobiology and open new avenues both in fundamental and applied research.

Modeling the role for nuclear import dynamics in the early embryonic cell cycle

Nuclear composition determines nuclear function. The early embryos of many species begin life with large pools of maternally provided components that become rapidly imported into an increasing number of nuclei as the cells undergo repeated cleavage divisions. Because early cell cycles are too fast for nuclei to achieve steady-state nucleocytoplasmic partitioning, the composition of cleavage stage nuclei is likely dominated by nuclear import. The end of the rapid cleavage stage and onset of major zygotic transcription, known as the mid-blastula transition (MBT), is controlled by the ratio of nuclei/cytoplasm, indicating that changes in nuclear composition likely mediate MBT timing. Here, we explore how different nuclear import regimes can affect protein accumulation in the nucleus in the early Drosophila embryo. We find that nuclear import differs dramatically for a general nuclear cargo (NLS (nuclear localization signal)-mRFP) and a proposed MBT regulator (histone H3). We show that nuclear import rates of NLS-mRFP in a given nucleus remain relatively unchanged throughout the cleavage cycles, whereas those of H3 halve with each cycle. We model these two distinct modes of nuclear import as “nucleus-limited” and “import-limited” and examine how the two different modes can contribute to different protein accumulation dynamics. Finally, we incorporate these distinct modes of nuclear import into a model for cell-cycle regulation at the MBT and find that the import-limited H3 dynamics contribute to increased robustness and allow for stepwise cell-cycle slowing at the MBT.

Cover Image: Nucleo‐cytoplasmic partitioning of HDA6

Most of the epigenetic regulations occur within nucleus. Whether and how nucleo–cytoplasmic partitioning participates in chromatin regulation is largely unknown. In this issue, Zhu et al. (1243–1254) identified XPO1A as an anti–silencing factor through preventing DNA hypermethylation. XPO1A affects DNA methylation by guiding the nucleo–cytoplasm partitioning of histone deacetylase HDA6, thereby connecting the regulation of DNA methylation and transcriptional gene silencing with nucleo–cytoplasmic trafficking.

Compensatory Mutations in GI and ZTL May Modulate Temperature Compensation in the Circadian Clock.

Circadian systems share the three properties of entrainment, free-running period, and temperature compensation (TC). TC ensures nearly the same period over a broad range of physiologically relevant temperatures; however, the mechanisms behind TC remain poorly understood. Here, we identify single point mutations in two key elements of the Arabidopsis circadian clock, GIGANTEA (GI) and ZEITLUPE (ZTL), which likely act as compensatory substitutions to establish a remarkably constant free-running period over a wide range of temperatures. Using near-isogenic lines generated from the introgression of the Cape Verde Islands (Cvi) alleles of GI and ZTL into the Landsberg erecta (Ler) background, we show how longer periods in the Cvi background at higher temperatures correlate with a difference in strength of the GI/ZTL interaction. Pairwise interaction testing of all GI/ZTL allelic combinations shows similar affinities for isogenic alleles at 22°C, but very poor interaction between GI (Cvi) and ZTL (Cvi) at higher temperature. In vivo, this would result in lower ZTL levels at high temperatures leading to longer periods in the Cvi background. Mismatched allelic combinations result in extremely strong or weak GI/ZTL interactions, indicating how the corresponding natural variants likely became fixed through epistatic selection. Additionally, molecular characterization of GI (Cvi) reveals a novel functional motif that can modulate the GI/ZTL interaction as well as nucleocytoplasmic partitioning. Taken together, these results identify a plausible temperature-dependent molecular mechanism, which contributes to the robustness of TC through natural variation in GI and ZTL alleles found on the Cape Verde Islands.

LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants.

Eukaryotic organisms accomplish the removal of introns to produce mature mRNAs through splicing. Nuclear and organelle splicing mechanisms are distinctively executed by spliceosome and group II intron complex, respectively. Here, we show that LEFKOTHEA, a nuclear encoded RNA-binding protein, participates in chloroplast group II intron and nuclear pre-mRNA splicing. Transiently optimized LEFKOTHEA nuclear activity is fundamental for plant growth, whereas the loss of function abruptly arrests embryogenesis. Nucleocytoplasmic partitioning and chloroplast allocation are efficiently balanced via functional motifs in LEFKOTHEA polypeptide. In the context of nuclear-chloroplast coevolution, our results provide a strong paradigm of the convergence of RNA maturation mechanisms in the nucleus and chloroplasts to coordinately regulate gene expression and effectively control plant growth.

Nucleo-cytoplasmic Partitioning of ARF Proteins Controls Auxin Responses in Arabidopsis thaliana.

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The auxin response factor (ARF) transcription factor family regulates auxin-responsive gene expression and exhibits nuclear localization in regions of high auxin responsiveness. Here we show that the ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness. We found that the intrinsically disordered middle region and the folded PB1 interaction domain of ARFs drive protein assembly formation. Mutation of a single lysine within the PB1 domain abrogates cytoplasmic assemblies, promotes ARF nuclear localization, and results in an altered transcriptome and morphological defects. Our data suggest a model in which ARF nucleo-cytoplasmic partitioning regulates auxin responsiveness, providing a mechanism for cellular competence for auxin signaling.

Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity.

The study of Hutchinson–Gilford progeria syndrome (HGPS) has provided important clues to decipher mechanisms underlying aging. Progerin, a mutant lamin A, disrupts nuclear envelope structure/function, with further impairment of multiple processes that culminate in senescence. Here, we demonstrate that the nuclear protein export pathway is exacerbated in HGPS, due to progerin‐driven overexpression of CRM1, thereby disturbing nucleocytoplasmic partitioning of CRM1‐target proteins. Enhanced nuclear export is central in HGPS, since pharmacological inhibition of CRM1 alleviates all aging hallmarks analyzed, including senescent cellular morphology, lamin B1 downregulation, loss of heterochromatin, nuclear morphology defects, and expanded nucleoli. Exogenous overexpression of CRM1 on the other hand recapitulates the HGPS cellular phenotype in normal fibroblasts. CRM1 levels/activity increases with age in fibroblasts from healthy donors, indicating that altered nuclear export is a common hallmark of pathological and physiological aging. Collectively, our findings provide novel insights into HGPS pathophysiology, identifying CRM1 as potential therapeutic target in HGPS.

The hydrogen sulfide signal enhances seed germination tolerance to high temperatures by retaining nuclear COP1 for HY5 degradation

Seed germination is a critical stage during the initiation of the plant lifecycle and is strongly affected by endogenous phytohormones and environmental stress. High temperature (HT) upregulates endogenous abscisic acid (ABA) to suppress seed germination, and ABA-INSENSITIVE 5 (ABI5) is the key positive regulator in the ABA signal-mediated modulation of seed germination. In plants, hydrogen sulfide (H2S) is a small gas messenger that participates in multiple physiological processes, but its role in seed germination thermotolerance has not been thoroughly elucidated to date. In this study, we found that H2S enhanced the seed germination rate under HT. Moreover, HT accelerates the efflux of the E3 ligase CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) from the nucleus to the cytoplasm, which results in increased nuclear accumulation of ELONG HYPCOTYL 5 (HY5) to activate the expression of ABI5 and thereby suppress seed germination. However, the H2S signal reversed the HT effect, as characterized by increased COP1 in the nucleus, which resulted in increased degradation of HY5 and reduced expression of ABI5 and thereby enhanced the seed germination thermotolerance. Thus, our findings reveal a novel role for the H2S signal in the modulation of seed germination thermotolerance through the nucleocytoplasmic partitioning of COP1 and the downstream HY5 and ABI5 pathways.

A Common Regulator Couples Nuclear and Chloroplast mRNA Splicing Mechanisms in Plants

Eukaryotic organisms accomplish the removal of introns to produce mature mRNAs through splicing. Nuclear and organelle splicing mechanisms are distinctively executed by spliceosome and group II intron complex, respectively. Here, we show that LEFKOTHEA a nuclear encoded RNA-binding protein participates in chloroplast group II intron and nuclear pre-mRNA splicing. Transiently optimized LEFKOTHEA nuclear activity is fundamental for plant growth, whereas the loss-of-function abruptly arrests embryogenesis. Nucleocytoplasmic partitioning and chloroplast allocation are efficiently balanced via functional motifs in LEFKOTHEA polypeptide. In the context of nuclear-chloroplast coevolution, our results provide a strong paradigm of the convergence of RNA maturation mechanisms in the nucleus and chloroplasts to coordinately regulate gene expression and effectively control plant growth.