The C-Terminal Domains of the PB2 Subunit of the Influenza A Virus RNA Polymerase Directly Interact with Cellular GTPase Rab11a.

Influenza A virus (IAV) contains a segmented RNA genome that is transcribed and replicated by the viral RNA polymerase in the cell nucleus. Replicated RNA segments are assembled with viral polymerase and oligomeric nucleoprotein into viral ribonucleoprotein (vRNP) complexes which are exported from the nucleus and transported across the cytoplasm to be packaged into progeny virions. Host GTPase Rab11a associated with recycling endosomes is believed to contribute to this process by mediating the cytoplasmic transport of vRNPs. However, how vRNPs interact with Rab11a remains poorly understood. In this study, we utilized a combination of biochemical, proteomic, and biophysical approaches to characterize the interaction between the viral polymerase and Rab11a. Using pulldown assays, we showed that vRNPs but not complementary RNPs (cRNPs) from infected cell lysates bind to Rab11a. We also showed that the viral polymerase directly interacts with Rab11a and that the C-terminal two-thirds of the PB2 polymerase subunit (PB2-C) comprising the cap-binding, mid-link, 627, and nuclear localization signal (NLS) domains mediate this interaction. Small-angle X-ray scattering (SAXS) experiments confirmed that PB2-C associates with Rab11a in solution forming a compact folded complex with a 1:1 stoichiometry. Furthermore, we demonstrate that the switch I region of Rab11a, which has been shown to be important for binding Rab11 family-interacting proteins (Rab11-FIPs), is also important for PB2-C binding, suggesting that IAV polymerase and Rab11-FIPs compete for the same binding site. Our findings expand our understanding of the interaction between the IAV polymerase and Rab11a in the cytoplasmic transport of vRNPs. IMPORTANCE The influenza virus RNA genome segments are replicated in the cell nucleus and are assembled into viral ribonucleoprotein (vRNP) complexes with viral RNA polymerase and nucleoprotein (NP). Replicated vRNPs need to be exported from the nucleus and trafficked across the cytoplasm to the cell membrane, where virion assembly takes place. The host GTPase Rab11a plays a role in vRNP trafficking. In this study, we showed that the viral polymerase directly interacts with Rab11a mediating the interaction between vRNPs and Rab11a. We mapped this interaction to the C-terminal domains of the PB2 polymerase subunit and the switch I region of Rab11a. Identifying the exact site of Rab11a binding on the viral polymerase could uncover a novel target site for the development of an influenza antiviral drug.

Wake-sleep cycles are severely disrupted by diseases affecting cytoplasmic homeostasis.

The circadian clock is based on a transcriptional feedback loop with an essential time delay before feedback inhibition. Previous work has shown that PERIOD (PER) proteins generate circadian time cues through rhythmic nuclear accumulation of the inhibitor complex and subsequent interaction with the activator complex in the feedback loop. Although this temporal manifestation of the feedback inhibition is the direct consequence of PER's cytoplasmic trafficking before nuclear entry, how this spatial regulation of the pacemaker affects circadian timing has been largely unexplored. Here we show that circadian rhythms, including wake-sleep cycles, are lengthened and severely unstable if the cytoplasmic trafficking of PER is disrupted by any disease condition that leads to increased congestion in the cytoplasm. Furthermore, we found that the time delay and robustness in the circadian clock are seamlessly generated by delayed and collective phosphorylation of PER molecules, followed by synchronous nuclear entry. These results provide clear mechanistic insight into why circadian and sleep disorders arise in such clinical conditions as metabolic and neurodegenerative diseases and aging, in which the cytoplasm is congested.

ZmTE1 promotes plant height by regulating intercalary meristem formation and internode cell elongation in maize.

Maize height is determined by the number of nodes and the length of internodes. Node number is driven by intercalary meristem formation and internode length by intercalary cell elongation, respectively. However, mechanisms regulating establishment of nodes and internode growth are unclear. We screened EMS-induced maize mutants and identified a dwarf mutant zm66, linked to a single base change in TERMINAL EAR 1 (ZmTE1). Detailed phenotypic analysis revealed that zm66 (zmte1-2) has shorter internodes and increased node numbers, caused by decreased cell elongation and disordered intercalary meristem formation, respectively. Transcriptome analysis showed that auxin signalling genes are also dysregulated in zmte1-2, as are cell elongation and cell cycle-related genes. This argues that ZmTE1 regulates auxin signalling, cell division, and cell elongation. We found that the ZmWEE1 kinase phosphorylates ZmTE1, thus confining it to the nucleus and probably reducing cell division. In contrast, the ZmPP2Ac-2 phosphatase promotes dephosphorylation and cytoplasmic localization of ZmTE1, as well as cell division. Taken together, ZmTE1, a key regulator of plant height, is responsible for maintaining organized formation of internode meristems and rapid cell elongation. ZmWEE1 and ZmPP2Ac-2 might balance ZmTE1 activity, controlling cell division and elongation to maintain normal maize growth.

The nuclear transporter SAD2 plays a role in calcium- and H2 O2 -mediated cell death in Arabidopsis.

In response to pathogens, plant cells exhibit a rapid increase in the intracellular calcium concentration and a burst of reactive oxygen species (ROS). The cytosolic increase in Ca2+ and the accumulation of ROS are critical for inducing programmed cell death (PCD), but the molecular mechanism is not fully understood. We screened an Arabidopsis mutant, sad2-5, which harbours a T-DNA insertion in the 18th exon of the importin beta-like gene, SAD2. The H2 O2 -induced increase in the [Ca2+ ]cyt of the sad2-5 mutant was greater than that of the wild type, and the sad2-5 mutant showed clear cell death phenotypes and abnormal H2 O2 accumulation under fumonisin-B1 (FB1) treatment. CaCl2 could enhance the FB1-induced cell death of the sad2-5 mutant, whereas lanthanum chloride (LaCl3 ), a broad-spectrum calcium channel blocker, could restore the FB1-induced PCD phenotype of sad2-5. The sad2-5 fbr11-1 double mutant exhibited the same FB1-insensitive phenotype as fbr11-1, which plays a critical role in novo sphingolipid synthesis, indicating that SAD2 works downstream of FBR11. These results suggest the important role of nuclear transporters in calcium- and ROS-mediated PCD response as well as provide an important theoretical basis for further analysis of the molecular mechanism of SAD2 function in PCD and for improvement of the resistance of crops to adverse environments.

Developmentally Regulated RNA-binding Protein 1 (Drb1)/RNA-binding Motif Protein 45 (RBM45), a Nuclear-Cytoplasmic Trafficking Protein, Forms TAR DNA-binding Protein 43 (TDP-43)-mediated Cytoplasmic Aggregates.

Cytoplasmic protein aggregates are one of the pathological hallmarks of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Several RNA-binding proteins have been identified as components of inclusion bodies. Developmentally regulated RNA-binding protein 1 (Drb1)/RNA-binding motif protein 45 is an RNA-binding protein that was recently described as a component in ALS- and FTLD-related inclusion bodies. However, the molecular mechanism underlying cytoplasmic Drb1 aggregation remains unclear. Here, using an in vitro cellular model, we demonstrated that Drb1 co-localizes with cytoplasmic aggregates mediated by TAR DNA-binding protein 43, a major component of ALS and FTLD-related inclusion bodies. We also defined the domains involved in the subcellular localization of Drb1 to clarify the role of Drb1 in the formation of cytoplasmic aggregates in ALS and FTLD. Drb1 predominantly localized in the nucleus via a classical nuclear localization signal in its carboxyl terminus and is a shuttling protein between the nucleus and cytoplasm. Furthermore, we identify a double leucine motif serving as a nuclear export signal. The Drb1 mutant, presenting mutations in both nuclear localization signal and nuclear export signal, is prone to aggregate in the cytoplasm. The mutant Drb1-induced cytoplasmic aggregates not only recruit TAR DNA-binding protein 43 but also decrease the mitochondrial membrane potential. Taken together, these results indicate that perturbation of Drb1 nuclear-cytoplasmic trafficking induces toxic cytoplasmic aggregates, suggesting that mislocalization of Drb1 is involved in the cause of cytotoxicity in neuronal cells.

Comment on Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights by Róna et al. (2013).

The authors comment on the article by Róna et al. [(2013), Acta Cryst. D69, 2495-2505].

Factors influencing nucleo-cytoplasmic trafficking: which matter? Response to Alvisi & Jans' comment on Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights.

The authors respond to a comment by Alvisi & Jans [(2014), Acta Cryst. D70, 2775-2776] on the article Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights [Róna et al. (2013), Acta Cryst. D69, 2495-2505].

Cell-Penetrating Peptides as Keys to Endosomal Escape and Intracellular Trafficking in Nanomedicine Delivery.

This review article discusses the challenges of delivering cargoes to the cytoplasm, for example, proteins, peptides, and nucleic acids, and the mechanisms involved in endosomal escape. Endocytosis, endosomal maturation, and exocytosis pose significant barriers to effective cytoplasmic delivery. The article explores various endosomal escape mechanisms, such as the proton sponge effect, osmotic lysis, membrane fusion, pore formation, membrane destabilization/ disruption, and vesicle budding and collapse. Additionally, it discusses the role of lysosomes, glycocalyx, and molecular crowding in the cytoplasmic delivery process. Despite the recent advances in nonviral delivery systems, there is still a need to improve cytoplasmic delivery. Strategies such as fusogenic peptides, endosomolytic polymers, and cell-penetrating peptides have shown promise in improving endosomal escape and cytoplasmic delivery. More research is needed to refine these strategies and make them safer and more effective. In conclusion, the article highlights the challenges associated with cytoplasmic delivery and the importance of understanding the mechanisms involved in endosomal escape. A better understanding of these processes could result in the creation of greater effectiveness and safe delivery systems for various cargoes, including proteins, peptides, and nucleic acids.

The rice RING finger E3 ligase, OsHCI1, drives nuclear export of multiple substrate proteins and its heterogeneous overexpression enhances acquired thermotolerance.

Thermotolerance is very important for plant survival when plants are subjected to lethally high temperature. However, thus far little is known about the functions of RING E3 ligase in response to heat shock in plants. This study found that one rice gene encoding the RING finger protein was specifically induced by heat and cold stress treatments but not by salinity or dehydration and named it OsHCI1 (Oryza sativa heat and cold induced 1). Subcellular localization results showed that OsHCI1 was mainly associated with the Golgi apparatus and moved rapidly and extensively along the cytoskeleton. In contrast, OsHCI1 may have accumulated in the nucleus under high temperatures. OsHCI1 physically interacted with nuclear substrate proteins including a basic helix-loop-helix transcription factor. Transient co-overexpression of OsHCI1 and each of three nuclear proteins showed that their fluorescent signals moved into the cytoplasm as punctuate formations. Heterogeneous overexpression of OsHCI1 in Arabidopsis highly increased survival rate through acquired thermotolerance. It is proposed that OsHCI1 mediates nuclear-cytoplasmic trafficking of nuclear substrate proteins via monoubiquitination and drives an inactivation device for the nuclear proteins under heat shock.

RNA Binding Protein OsTZF7 Traffics Between the Nucleus and Processing Bodies/Stress Granules and Positively Regulates Drought Stress in Rice.

Tandem CCCH zinc finger (TZF) proteins are the essential components of processing bodies (PBs) and stress granules (SGs), which play critical roles in growth development and stress response in both animals and plants through posttranscriptional regulation of target mRNA. In this study, we characterized the biological and molecular functions of a novel tandem zinc finger protein, OsTZF7. The expression of OsTZF7 was upregulated by abiotic stresses, including polyethylene glycol (PEG) 4000, NaCl, and abscisic acid (ABA) in rice. Accordingly, the overexpression of OsTZF7 increased drought tolerance and enhanced sensitivity to exogenous ABA in rice, whereas the knockdown of OsTZF7 resulted in the opposite phenotype. RNA-seq analysis revealed that genes related to "response to stress," "abscisic acid signaling," "methylated histone binding," and "cytoplasmic mRNA processing body" are regulated by OsTZF7. We demonstrated that OsTZF7 can traffic between the nucleus and PBs/SGs, and the leucine-rich nuclear export signal (NES) mediates the nuclear export of OsTZF7. Additionally, we revealed that OsTZF7 can bind adenine- and uridine-rich (AU-rich) element (ARE) or ARE-like motifs within the 3' untranslated region of downregulated mRNAs, and interact with PWWP family proteins in vitro. Together, these results indicate that OsTZF7 positively regulates drought response in rice via ABA signaling and may be involved in mRNA turnover.

Histone H2A Nuclear/Cytoplasmic Trafficking Is Essential for Negative Regulation of Antiviral Immune Response and Lysosomal Degradation of TBK1 and IRF3.

Histone H2A is a nuclear molecule tightly associated in the form of the nucleosome. Our previous studies have demonstrated the antibacterial property of piscine H2A variants against gram-negative bacteria Edwardsiella piscicida and Gram-positive bacteria Streptococcus agalactiae. In this study, we show the function and mechanism of piscine H2A in the negative regulation of RLR signaling pathway and host innate immune response against spring viremia of carp virus (SVCV) infection. SVCV infection significantly inhibits the expression of histone H2A during an early stage of infection, but induces the expression of histone H2A during the late stage of infection such as at 48 and 72 hpi. Under normal physiological conditions, histone H2A is nuclear-localized. However, SVCV infection promotes the migration of histone H2A from the nucleus to the cytoplasm. The in vivo studies revealed that histone H2A overexpression led to the increased expression of SVCV gene and decreased survival rate. The overexpression of histone H2A also significantly impaired the expression levels of those genes involved in RLR antiviral signaling pathway. Furthermore, histone H2A targeted TBK1 and IRF3 to promote their protein degradation via the lysosomal pathway and impair the formation of TBK1-IRF3 functional complex. Importantly, histone H2A completely abolished TBK1-mediated antiviral activity and enormously impaired the protein expression of IRF3, especially nuclear IRF3. Further analysis demonstrated that the inhibition of histone H2A nuclear/cytoplasmic trafficking could relieve the protein degradation of TBK1 and IRF3, and blocked the negative regulation of histone H2A on the SVCV infection. Collectively, our results suggest that histone H2A nuclear/cytoplasmic trafficking is essential for negative regulation of RLR signaling pathway and antiviral immune response in response to SVCV infection.

Cytoplasmic Trafficking of Nanoparticles Delivers Plasmid DNA for Macrophage Gene-editing.

BACKGROUND: Successful delivery of gene-editing tools using nano-carriers is dependent on the ability of nanoparticles to pass through the cellular membrane, move through the cytoplasm, and cross the nuclear envelope to enter the nucleus. It is critical that intracellular nanoparticles interact with the cytoskeletal network to move toward the nucleus, and must escape degradation pathways including lysosomal digestion. Without efficient intracellular transportation and nuclear entry, nanoparticles-based gene-editing cannot be effectively used for targeted genomic modification. OBJECTIVE: We have developed nanoparticles with a low molecular weight branched polyethylenimine lipid shell and a PLGA core that can effectively deliver plasmid DNA to macrophages for gene editing while limiting toxicity. METHODS: Core-shell nanoparticles were synthesized by a modified solvent evaporation method and were loaded with plasmid DNA. Confocal microscopy was used to visualize the internalization, intracellular distribution and cytoplasmic transportation of plasmid DNA loaded nanoparticles (pDNA-NPs) in bone marrow-derived macrophages. RESULTS: Core-shell nanoparticles had a high surface charge of +56 mV and narrow size distribution. When loaded with plasmid DNA for transfection, the nanoparticles increased in size from 150 nm to 200 nm, and the zeta potential decreased to +36 mV, indicating successful encapsulation. Further, fluorescence microscopy revealed that pDNA-NPs crossed the cell membrane and interacted with actin filaments. Intracellular tracking of pDNA-NPs showed successful separation of pDNA- NPs from lysosomes, allowing entry into the nucleus at 2 hours, with further nuclear ingress up to 5 hours. Bone marrow-derived macrophages treated with pDNA/GFP-NPs exhibited high GFP expression with low cytotoxicity. CONCLUSION: Together, this data suggests pDNA-NPs are an effective delivery system for macrophage gene-editing.

Conformation of the nuclear pore in living cells is modulated by transport state.

While the static structure of the nuclear pore complex (NPC) continues to be refined with cryo-EM and x-ray crystallography, in vivo conformational changes of the NPC remain under-explored. We developed sensors that report on the orientation of NPC components by rigidly conjugating mEGFP to different NPC proteins. Our studies show conformational changes to select domains of nucleoporins (Nups) within the inner ring (Nup54, Nup58, Nup62) when transport through the NPC is perturbed and no conformational changes to Nups elsewhere in the NPC. Our results suggest that select components of the NPC are flexible and undergo conformational changes upon engaging with cargo.

The nuclear-cytoplasmic trafficking of a chromatin-modifying and remodelling protein (KMT2C), in osteosarcoma.

Osteosarcoma is the most common paediatric primary non-hematopoietic bone tumor; the survival is related to the response to chemotherapy and development of metastases. KMT2C is a chromatin-modifying and remodelling protein and its expression has never been studied in osteosarcoma. The aim of this study was to understand the role of KMT2C in the osteosarcoma carcinogenesis and metastatic progression to identify a new molecular target and to provide new therapeutic approach. We performed the immunohistochemical and gene expression analysis of KMT2C in 32 samples of patients with diagnosis of osteosarcoma with known clinic-pathological data and we analysed the expression of genes involved in the metastatic pathway in four osteosarcoma cell lines by blocking the KMT2C expression using siRNA. We found a nuclear-cytoplamic trafficking of KMT2C and the cytoplasmic localization was higher than the nuclear localization (p < 0.0001). Moreover, the percentage of cells with cytoplasmic positivity increased from low grade primary tissue to metastatic tissues. The cytoplasmic localization of KMT2C could lead to a change in its function supporting osteosarcoma carcinogenesis and progression. Our hypothesis is that KMT2C could affect the enhancer activity of genes influencing the invasive properties and metastatic potential of osteosarcoma.

Spatiotemporal dynamics of the calcineurin target CrzA.

The response of Aspergilli to elevated concentrations of extracellular calcium and manganese, or environmental alkalinization is mediated by CrzA, a calcineurin-responsive transcription factor (TF). CrzA is the effector of a signaling pathway which includes the apical protein's calmodulin and calcineurin, and the protein kinases GskA and CkiA. Preferentially located in the cytoplasm, CrzA is the only element of the pathway modifying its localization under those stress conditions, being imported into nuclei. Remarkably, there is a direct relationship between the nature/intensity of the stimulus and the pace of nuclear import and time of nuclear permanence of CrzA. Alkalinity caused a transient nuclear accumulation of CrzA while high Ca2+ and Mn2+ concentrations generated a long-lasting accumulation. Furthermore, Ca2+ concentrations (below 5mM) that are non-toxic for a crzAΔ mutant promoted full signaling of CrzA. However, micromolar concentrations or a mutation disrupting the interaction of CrzA with the phosphatase complex calcineurin, permitted the visualization of a transient and polarized nuclear accumulation of the TF in a tip-to-base gradient. Overall, these results support a model in which nucleo-cytoplasmic dynamics and transcriptional activity of CrzA are driven by apical signals transmitted by calmodulin and calcineurin. This communication is essential to understand Ca+2-induced stress response in fungi.

How successful is nuclear targeting by nanocarriers?

The nucleus is ultimately the final target for many therapeutics treating various disorders including cancers, heart dysfunction and brain disorders. Owing to their specialized cell uptake and trafficking mechanisms, nanoparticles (NPs) allow drug targeting where degradation sensitive therapeutics could be delivered to their target tissues and cell in active form and sufficient concentration. However, it has recently become increasingly obvious that cytosolic internalization of a drug molecule does not entail its interaction with its subcellular target and hence careful nanoparticle design and optimization is required to enable nuclear targeting. This review, discusses the barriers to NP nuclear delivery; crossing the cell membrane, endo/lysosomal escape, cytoplasmic trafficking and finally nuclear entry focusing on how NP synthesis and modification could allow for bypassing each of the aforementioned barriers and successfully reaching the nucleus. Examples of nuclear targeted NPs are also discussed, stressing on the critical aspects of nuclear targeting and pointing out how the disease state might change the normal NP path and how such change could be exploited to increase efficiency of nuclear targeting. Finally, the criteria set for the evaluation of nanocarriers for nuclear delivery are discussed highlighting that quantitative rather than qualitative evaluation is required to evaluate how successful nanocarriers for nuclear delivery are, particularly with regards to the amount of drug delivered and released in the nucleus.

Traversing the Cell: Agrobacterium T-DNA's Journey to the Host Genome.

The genus Agrobacterium is unique in its ability to conduct interkingdom genetic exchange. Virulent Agrobacterium strains transfer single-strand forms of T-DNA (T-strands) and several Virulence effector proteins through a bacterial type IV secretion system into plant host cells. T-strands must traverse the plant wall and plasma membrane, traffic through the cytoplasm, enter the nucleus, and ultimately target host chromatin for stable integration. Because any DNA sequence placed between T-DNA "borders" can be transferred to plants and integrated into the plant genome, the transfer and intracellular trafficking processes must be mediated by bacterial and host proteins that form complexes with T-strands. This review summarizes current knowledge of proteins that interact with T-strands in the plant cell, and discusses several models of T-complex (T-strand and associated proteins) trafficking. A detailed understanding of how these macromolecular complexes enter the host cell and traverse the plant cytoplasm will require development of novel technologies to follow molecules from their bacterial site of synthesis into the plant cell, and how these transferred molecules interact with host proteins and sub-cellular structures within the host cytoplasm and nucleus.