Dual Localization Research Articles

APRF1 Interactome Reveals HSP90 as a New Player in the Complex That Epigenetically Regulates Flowering Time in Arabidopsis thaliana.

WD40 repeat proteins (WDRs) are present in all eukaryotes and include members that are implicated in numerous cellular activities. They act as scaffold proteins and thus as molecular "hubs" for protein-protein interactions, which mediate the assembly of multifunctional complexes that regulate key developmental processes in Arabidopsis thaliana, such as flowering time, hormonal signaling, and stress responses. Despite their importance, many aspects of their putative functions have not been elucidated yet. Here, we show that the late-flowering phenotype of the anthesis promoting factor 1 (aprf1) mutants is temperature-dependent and can be suppressed when plants are grown under mild heat stress conditions. To gain further insight into the mechanism of APRF1 function, we employed a co-immunoprecipitation (Co-IP) approach to identify its interaction partners. We provide the first interactome of APRF1, which includes proteins that are localized in several subcellular compartments and are implicated in diverse cellular functions. The dual nucleocytoplasmic localization of ARRF1, which was validated through the interaction of APRF1 with HEAT SHOCK PROTEIN 1 (HSP90.1) in the nucleus and with HSP90.2 in the cytoplasm, indicates a dynamic and versatile involvement of APRF1 in multiple biological processes. The specific interaction of APRF1 with the chaperon HSP90.1 in the nucleus expands our knowledge regarding the epigenetic regulation of flowering time in A. thaliana and further suggests the existence of a delicate thermoregulated mechanism during anthesis.

Ultrasound Activated Nanobowls with Deep Penetration for Enhancing Sonodynamic Therapy of Orthotopic Liver Cancer.

Owing to the high penetration ability and the safety of ultrasound (US) of sonodynamic therapy (SDT), it has gained significant attention in tumor treatment. However, its therapeutic efficiency depends on the performance of the sonosensitizers. The hypoxic microenvironment and abnormal stromal matrix restrict the full potential of sonosensitizers. In this study, a US-activated bowl-shaped nanobomb (APBN) is designed as a novel sonosensitizer to enhance the SDT effect through various means. This enhancement strategy combines three major characteristics: relieving tumor hypoxia, amplifying bubble cavitation damage, and US-movement-enhanced permeation. The unique bowl-shaped structure of APBN provides more favorable attachment sites for the generated oxygen gas bubbles. Thus, when catalase-like APBN catalyzes endogenous hydrogen peroxide to produce oxygen, bubbles accumulate at the groove, preventing the dissipation of oxygen and increasing the number of cavitation nuclei to improve the acoustic cavitation effect. This approach differs from traditional SDT strategies because it couples the sonodynamic effect with reactive oxygen species generation and bubble cavitation damage rather than a single action. Additionally, the asymmetric bowl-shaped structure generates a driving force under the US field, improving the distribution of sonosensitizers in the tumors. Using US and photoacoustic imaging for dual localization, these sonosensitizers can improve the accuracy of orthotopic liver tumor treatment, which presents a promising avenue for the treatment of deep tumors.

‘Solving’ the Paradox of the Odyssean Ethiopians’ Twin Dual Localization

Abstract This paper considers the narrative significance of localization in Homer’s Odyssey, in particular singular places that are associated with multiple spaces (identified here as dual localization). Our reading posits that spatial features hold narrative significance and, once uncovered, this resolves ‘problematic’ issues that may arise from spatial paradoxes. The chosen case study is the Odyssean land of the Ethiopians with its twin dual localization: (1) it is located simultaneously at the eastern and western peripheries of the world while also located singularly ‘with Poseidon’; and (2) this peripheral localization contrasts with a later ‘real world’ setting in Egypt/North Africa. While this people’s localization has been the subject of prior study, this paper presents a novel analysis: these localizations serve a characterizing function which, in turn, relates to the thematic function of Menelaus’ embedded narrative as foreshadowing the primary narrative of Odysseus’ nostos.

A Dual Scalar Magnetic Target Localization Method Using Anderson Basis Functions and Cuckoo-Search-Based Spiral Update

A Dual Scalar Magnetic Target Localization Method Using Anderson Basis Functions and Cuckoo-Search-Based Spiral Update

DSIL4IMR: A dual step-based initial localization method for industrial mobile robot

This paper introduces a dual-step based initial localization method for industrial mobile robots (DSIL4IMR) that operates without user intervention at start-up. Utilizing a differential chassis equipped with a radar sensor, the method combines feature extraction and spatial information matching steps to locate the robot’s initial position. A unique metric, degree of stability, is developed to identify a feature map whether it is a dynamic or erroneous feature. DSIL4IMR does not require artificial landmarks and outperforms existing WiFi fingerprinting and map filter methods in both accuracy and efficiency. DSIL4IMR triples the localization efficiency compared to traditional approaches while achieving centimetre-level precision.

A modified procedure for separating yeast peroxisomes from mitochondria.

Mitochondria and peroxisomes are mutually dependent organelles that share several membrane proteins that carry out the same function in both organelles. To study the unique features of these dually localized proteins in each of the two organelles, it is essential to separate mitochondria from peroxisomes. Isolating organelles from cells of Baker's yeast, Saccharomyces cerevisiae, is crucial for our understanding of the biogenesis and functions of proteins. Traditionally, subcellular fractionation and isolation of individual organelles by differential centrifugation benefit from the specific and unique density of each organelle. However, when yeast cells are grown under normal conditions, certain organelles like mitochondria and peroxisomes share strikingly similar densities. This similarity challenges the separation of these organelles from one another. In this chapter, we describe an optimized procedure to address this task. We depict growth conditions that would favor stimulation of peroxisomes to increase their number and density, and portray organellar isolation followed by gradient centrifugation, enabling an improved separation of both organelles. Additionally, we illustrate the advantage of the procedure to study the dual localization of the membrane protein Fis1.

Clickable APEX2 Probes for Enhanced RNA Proximity Labeling in Live Cells.

Although APEX2-mediated proximity labeling has been extensively implemented for studying RNA subcellular localization in live cells, the biotin-phenoxyl radical used for labeling RNAs has a relatively low efficiency, which can limit its compatibility with other profiling methods. Herein, a set of phenol derivatives were designed as APEX2 probes through balancing reactivity, hydrophilicity, and lipophilicity. Among these derivatives, Ph_N3 exhibited reliable labeling ability and enabled two biotinylation routes for downstream analysis. As a proof of concept, we used APEX2/Ph_N3 labeling with high-throughput sequencing analysis to examine the transcriptomes in the mitochondrial matrix, demonstrating high sensitivity and specificity. To further expand the utility of Ph_N3, we employed mechanistically orthogonal APEX2 and singlet oxygen (1O2)-mediated strategies for dual location labeling in live cells. Specifically, DRAQ5, a DNA-intercalating photosensitizer, was applied for nucleus-restricted 1O2 labeling. We validated the orthogonality of APEX2/Ph_N3 and DRAQ5-1O2 at the imaging level, providing an attractive and feasible approach for future studies of RNA translocation in live cells.

Developing Connections Between LINC00298 RNA and Alzheimer's Disease Through Mapping Its Interactome and Through Biochemical Characterization.

Long non-coding RNAs are ubiquitous throughout the human system, yet many of their biological functions remain unknown. LINC00298 RNA, a long intergenic non-coding RNA, has been shown to have preferential expression in the central nervous system where it contributes to neuronal differentiation and development. Furthermore, previous research has indicated that LINC00298 RNA is known to be a genetic risk factor for the development of Alzheimer's disease. To biochemically characterize LINC00298 RNA and to elucidate its biological function within hippocampal neuronal cells, thereby providing a greater understanding of its role in Alzheimer's disease pathogenesis. LINC00298 RNA was in vitro transcribed and then subjected to structural analysis using circular dichroism, and UV-Vis spectroscopy. Additionally, affinity column chromatography was used to capture LINC00298 RNA's protein binding partners from hippocampal neuronal cells, which were then identified using liquid chromatography and mass spectrometry (LC/MS). LINC00298 RNA is comprised of stem-loop secondary structural elements, with a cylindrical tertiary structure that has highly dynamic regions, which result in high positional entropy. LC/MS identified 24 proteins within the interactome of LINC00298 RNA. Through analysis of LINC00298 RNA's 24 protein binding partners, it was determined that LINC00298 RNA may play significant roles in neuronal development, proliferation, and cellular organization. Furthermore, analysis of LINC00298 RNA's interactome indicated that LINC00298 RNA is capable of intracellular motility with dual localization in the nucleus and the cytosol. This biochemical characterization of LINC00298 RNA has shed light on its role in Alzheimer's disease pathogenesis.

Гігієна планування та забудови населених місць як безпекова детермінанта громадського здоров’я в умовах воєнного стану

Introduction: Martial law continues to operate in Ukraine, in which the primary task of the state is to preserve the health and life of the population. The priority in this regard is the prevention of the harmful influence of martial law conditions on the formation of children's bodies and the creation of conditions for their upbringing and education. Proceeding from the above, the solution to the issues regarding collective civil protection should be carried out through the construction/reconstruction of civil protection facilities, taking into account the specific conditions for both the adult population and the children's contingent. Objective: there is the development of sanitary and hygienic requirements for civil defense structures of public and residential buildings during the period of martial law and their use in the post-war state - as dual-purpose structures, taking into account the experience of warring countries. Research methods: research methods are used in the work: bibliosemantic, analytical, hygienic and sanitary-epidemiological examination of construction projects. The materials of the research were the sanitary, urban planning and educational legislations of Ukraine and other countries in the field of designing civil defense structures of residential and public buildings, construction projects of public and residential buildings. Results: the article highlights the activities of the Laboratory, which is aimed at studying the hygienic urban planning problems of the comprehensive restoration of settlements (territories) that suffered as a result of hostilities, terrorist acts, and the development of hygienic criteria for their reconstruction in order to create safe living conditions of the population, in particular ensuring the conditions of collective civil protection in civil protection facilities of public and residential buildings. Conclusion: The activities of urban planning specialists in the direction of hygienic assessment of urban planning documentation of settlements affected by hostilities and the development of requirements for safe living of the population should be aimed at strengthening the safety determinant of public health by creating optimal conditions for collective civil protection of the population in civil protection structures of public and residential buildings - for the period of martial law and construction - the use of these structures in a dual location.

Dual localization of receptor-type adenylate cyclases and cAMP response protein 3 unveils the presence of two putative signaling microdomains in Trypanosoma cruzi.

Trypanosoma cruzi is the etiologic agent of Chagas disease, a leading cause of disability and premature death in the Americas. This parasite spends its life between a triatomine insect and a mammalian host, transitioning between developmental stages in response to microenvironmental changes. Among the second messengers driving differentiation in T. cruzi, cAMP has been shown to mediate metacyclogenesis and response to osmotic stress, but this signaling pathway remains largely unexplored in this parasite. Adenylate cyclases (ACs) catalyze the conversion of ATP to cAMP. They comprise a multigene family encoding putative receptor-type ACs in T. cruzi. Using protein sequence alignment, we classified them into five groups and chose a representative member from each group to study their localization (TcAC1-TcAC5). We expressed an HA-tagged version of each protein in T. cruzi and performed immunofluorescence analysis. A peculiar dual localization of TcAC1 and TcAC2 was observed in the flagellar distal domain and in the contractile vacuole complex (CVC), and their enzymatic activity was confirmed by gene complementation in yeast. Furthermore, TcAC1 overexpressing parasites showed an increased metacyclogenesis, a defect in host cell invasion, and a reduced intracellular replication, highlighting the importance of this protein throughout T. cruzi life cycle. These mutants were more tolerant to hypoosmotic stress and showed a higher adhesion capacity during in vitro metacyclogenesis, whereas the wild-type phenotype was restored after disrupting TcAC1 localization. Finally, TcAC1 was found to interact with cAMP response protein 3 (TcCARP3), co-localizing with this protein in the flagellar tip and CVC. IMPORTANCE We identified three components of the cAMP signaling pathway (TcAC1, TcAC2, and TcCARP3) with dual localization in Trypanosoma cruzi: the flagellar distal domain and the CVC, structures involved in cell adhesion and osmoregulation, respectively. We found evidence on the role of TcAC1 in both cellular processes, as well as in metacyclogenesis. Our data suggest that TcACs act as signal sensors and transducers through cAMP synthesis in membrane microdomains. We propose a model in which TcACs sense the harsh conditions in the triatomine hindgut (nutrient deprivation, acidic pH, osmotic stress, ionic composition, hydrophobic interactions) and become active. Synthesis of cAMP then triggers cell adhesion prior completion of metacyclogenesis, while mediating a response to osmotic stress in the parasite. These results shed light into the mechanisms driving cAMP-mediated cell differentiation in T. cruzi, while raising new questions on the activation of TcACs and the role of downstream components of this pathway.

Live-Cell Mitochondrial Targeted NIR Fluorescent Covalent Labeling of Specific Proteins Using a Dual Localization Effect.

Here, our designed water-soluble NIR fluorescent unsymmetrical Cy-5-Mal/TPP+ consists of a lipophilic cationic TPP+ subunit that can selectively target and accumulate in a live-cell inner mitochondrial matrix where a maleimide residue of the probe undergoes faster chemoselective and site-specific covalent attachment with the exposed Cys residue of mitochondrion-specific proteins. On the basis of this dual localization effect, Cy-5-Mal/TPP+ molecules remain for a longer time period even after membrane depolarization, enabling long-term live-cell mitochondrial imaging. Due to the adequate concentration of Cy-5-Mal/TPP+ reached in live-cell mitochondria, it facilitates site-selective NIR fluorescent covalent labeling with Cys-exposed proteins, which are identified by the in-gel fluorescence assay and LC-MS/MS-based proteomics and supported by a computational method. This dual targeting approach with admirable photostability, narrow NIR absorption/emission bands, bright emission, long fluorescence lifetime, and insignificant cytotoxicity has been shown to improve real-time live-cell mitochondrial tracking including dynamics and interorganelle crosstalk with multicolor imaging applications.

Systematic Approaches to Study Eclipsed Targeting of Proteins Uncover a New Family of Mitochondrial Proteins.

Dual localization or dual targeting refers to the phenomenon by which identical, or almost identical, proteins are localized to two (or more) separate compartments of the cell. From previous work in the field, we had estimated that a third of the mitochondrial proteome is dual-targeted to extra-mitochondrial locations and suggested that this abundant dual targeting presents an evolutionary advantage. Here, we set out to study how many additional proteins whose main activity is outside mitochondria are also localized, albeit at low levels, to mitochondria (eclipsed). To do this, we employed two complementary approaches utilizing the α-complementation assay in yeast to uncover the extent of such an eclipsed distribution: one systematic and unbiased and the other based on mitochondrial targeting signal (MTS) predictions. Using these approaches, we suggest 280 new eclipsed distributed protein candidates. Interestingly, these proteins are enriched for distinctive properties compared to their exclusively mitochondrial-targeted counterparts. We focus on one unexpected eclipsed protein family of the Triose-phosphate DeHydrogenases (TDH) and prove that, indeed, their eclipsed distribution in mitochondria is important for mitochondrial activity. Our work provides a paradigm of deliberate eclipsed mitochondrial localization, targeting and function, and should expand our understanding of mitochondrial function in health and disease.

An exonuclease V homologue is expressed predominantly during early megasporogenesis in apomictic Brachiaria brizantha.

An exonuclease V homologue from apomictic Brachiaria brizantha is expressed and localized in nucellar cells at key moments when these cells differentiate to give rise to unreduced gametophytes. Brachiaria is a genus of forage grasses with economical and agricultural importance to Brazil. Brachiaria reproduces by aposporic apomixis, in which unreduced embryo sacs, derived from nucellar cells, other than the megaspore mother cell (MMC), are formed. The unreduced embryo sacs produce an embryo without fertilization resulting in clones of the mother plant. Comparative gene expression analysis in ovaries of sexual and apomictic Brachiaria spp. revealed a sequence from B. brizantha that showed a distinct pattern of expression in ovaries of sexual and apomictic plants. In this work, we describe a gene named BbrizExoV with strong identity to exonuclease V (Exo V) genes from other grasses. Sequence analysis in signal prediction tools showed that BbrizExoV might have dual localization, depending on the translation point. A longer form to the nucleus and a shorter form which would be directed to the chloroplast. This is also the case for monocot sequences analyzed from other species. The long form of BbrizExoV protein localizes to the nucleus of onion epidermal cells. Analysis of ExoV proteins from dicot species, with exception of Arabidopsis thaliana ExoVL protein, showed only one localization. Using a template-based AlphaFold 2 modelling approach the structure of BbrizExoV in complex with metal and ssDNA was predicted based on the holo structure of the human counterpart. Features predicted to define ssDNA binding but a lack of sequence specificity are shared between the human enzyme and BbrizExoV. Expression analyses indicated the precise site and timing of transcript accumulation during ovule development, which coincides with the differentiation of nucelar cells to form the typical aposporic four-celled unreduced gametophyte. A putative function for this protein is proposed based on its homology and expression pattern.

Evolution of sentinel lymph node biopsy for breast cancer patients in a rural setting: 10 years' experience.

Sentinel lymph node (SLN) biopsy is the standard axillary staging procedure of early breast cancer. Superparamagnetic iron oxide (SPIO) nanoparticles have been found to be comparable to, while overcoming many of the limitations associated with, the current standard of care for SLN biopsies (dual localisation with radioisotope and patent blue dye). Here, SPIO dual localisation (Sienna+® and blue dye) is compared to blue dye alone for SLN biopsies in a rural centre where radioisotope techniques are not readily available. Sienna+® dual localisation is shown to be more likely to detect nodes (detection rate of 99% compared to 90% when using blue dye alone), and detect more nodes, than blue dye alone. The use of Magseed, a magnetic tracer, was not found to influence node detection. The results from this work show that Sienna+® dual localisation is superior to blue dye alone for detecting SLN, suggesting that it is an excellent alternative to dual localisation of radioisotope and blue dye for small centres lacking easy access to a nuclear medicine department.

Is the use of blue dye really necessary in axillary sentinel lymph node biopsy in staging of breast cancer?

Sentinel lymph node biopsy (SLNB) is the standard of care for staging the clinically node-negative axilla in early breast cancer. Evidence guiding current practice describes dual localization technique using Patent blue dye and radioisotope (99mTc). Adverse effects of blue dye include 1:1000 risk of anaphylaxis, skin staining and loss of plane visibility, which may increase operative time and reduce resectional accuracy. The risk to a patient posed by anaphylaxis may be greater when operating in a unit without on-site ITU support - a situation more common with recent restructuring during the COVID-19 pandemic. Aim is to quantify the benefit of blue dye above radioisotope alone in identifying nodal disease. This is a retrospective analysis of prospectively collected sentinel node data including all consecutive sentinel node biopsies in a single center during the period 2016-2019.In terms of results, 760 sentinel nodes were taken in 435 patients. 59 nodes (7.8%) were detected by blue dye alone; 120 (15.8%) 'hot' only, 581 (76.5%) hot and blue. 4 of the blue only nodes contained macrometastases but 3 of these patients had further hot nodes excised that also contained macrometastases. 1 out of 435 patients (0.2%) had macro metastatic disease identified as a result of blue dye alone which would have been missed had it not been used. In conclusion, the use of blue dye carries risk and offers little benefit in terms of staging in SLNB and its use may be unnecessary in the hands of the skilled surgeon. This study supports the omission of blue dye, which may be advisable if operating in units without ITU support. If larger studies support these figures, it may become as outdated.

METTL16 promotes translation and lung tumorigenesis by sequestering cytoplasmic eIF4E2

N6-methyladenosine (m6A) plays crucial roles in regulating RNA metabolisms. METTL16 identified as a single-component methyltransferase catalyzes m6A formation in the nucleus; whether it regulates cytoplasmic RNA fate remains unknown. Here, we detected the dual localization of METTL16 in the nucleus and cytoplasm. METTL16 depletion attenuates protein synthesis, but the methyltransferase activity is not required for its translation-promoting function. Mechanistically, we identified an interactor of METTL16, eIF4E2, which represses translation by acting as a competitor of eIF4E. The METTL16-eIF4E2 interaction impedes the recruitment of eIF4E2 to 5' cap structure, promoting the cap recognition by eIF4E and selective protein synthesis. Depletion of METTL16 suppresses lung tumorigenesis by downregulating the translation of key oncogenes. Collectively, our study reports a role of METTL16 in modulating translation and provides a therapeutic target for lung cancer treatment.

Conservation of land plant-specific receptor-like cytoplasmic kinase subfamily XI possessing a unique kinase insert domain.

The number of genes encoding receptor-like kinases (RLKs) has expanded in the plant lineage. Their expansion has resulted in the emergence of diverse domain architectures that function in signaling cascades related to growth, development, and stress response. In this study, we focused on receptor-like cytoplasmic kinase subfamily XI (RLCK XI) in plants. We discovered an exceptionally long kinase insert domain (KID), averaging 280 amino acids, between subdomains VII and VIII of the conserved protein kinase domain. Using sequence homology search, we identified members of RLCK XI with the unique KID architecture in terrestrial plants, up to a single copy in several hornwort and liverwort species. The KID shows a high propensity for being disordered, resembling the activation segment in the model kinase domain. Several conserved sequence motifs were annotated along the length of the KID. Of note, the KID harbors repetitive nuclear localization signals capable of mediating RLCK XI translocation from the plasma membrane to the nucleus. The possible physiological implication of dual localization of RLCK XI members is discussed. The presence of a KID in RLCK XI represents a unique domain architecture among RLKs specific to land plants.

Dual Location Area Based Distributed Location Management for Hybrid LEO/MEO Mega Satellite Networks

Location management is the basis for ensuring service continuity in low earth orbit (LEO) satellite mobile communication systems. The dual mobility of LEO satellites and mobile users triggers frequent location update procedures, which will increase the network location management overhead and bring great challenges to network location management, especially in the mega satellite networks. In this paper, we propose a location management technology solution for mega satellite networks, including location management architecture, location area design, location update, and paging procedures. A medium earth orbit (MEO) satellite-assisted location management architecture is proposed to improve the efficiency of location management. It extends the location management control plane from the ground to space and adopts a distributed location management mechanism. A dual location area, including the satellite location area and user location area, is designed to balance the network location update and paging overhead. Based on the dual location area, we systematically design low-overhead location update and paging procedures. Simulation results have verified that the proposed location management technology solution can reduce the total location management overhead and delay by at least 45.8% and 20.8%, respectively.

An Interplay between Mitochondrial and ER Targeting of a Bacterial Signal Peptide in Plants.

Protein targeting is essential in eukaryotic cells to maintain cell function and organelle identity. Signal peptides are a major type of targeting sequences containing a tripartite structure, which is conserved across all domains in life. They are frequently included in recombinant protein design in plants to increase yields by directing them to the endoplasmic reticulum (ER) or apoplast. The processing of bacterial signal peptides by plant cells is not well understood but could aid in the design of efficient heterologous expression systems. Here we analysed the signal peptide of the enzyme PmoB from methanotrophic bacteria. In plant cells, the PmoB signal peptide targeted proteins to both mitochondria and the ER. This dual localisation was still observed in a mutated version of the signal peptide sequence with enhanced mitochondrial targeting efficiency. Mitochondrial targeting was shown to be dependent on a hydrophobic region involved in transport to the ER. We, therefore, suggest that the dual localisation could be due to an ER-SURF pathway recently characterised in yeast. This work thus sheds light on the processing of bacterial signal peptides by plant cells and proposes a novel pathway for mitochondrial targeting in plants.

Dual localization of the carboxy-terminal tail of GLR3.3 in sieve element–companion cell complex

ABSTRACT Glutamate receptor-like (GLR) 3.3 and 3.6 proteins are required for mediating wound-induced leaf-to-leaf electrical signaling. In the previous study, we found that the carboxy-terminal tail of GLR3.3 contains key residues that are indispensable for its action in electrical signaling. In the present work, we generated plants that expressed the truncated C-tail fraction of GLR3.3. To our expectation, the truncated C-tail itself was not functional in propagating leaf-to-leaf signals. However, we identified that the C-tail-mVENUS fusion proteins had dual localization patterns in sieve elements and companion cells. In companion cells, the fusion proteins overlapped largely with the nucleus. We speculated that a possible nuclear localization signal is present in the C-tail of GLR3.3, paralleling the C-tails of the ionotropic glutamate receptors in animal cells. Our further findings on the C-tail of GLR3.3 open up new possibilities for the regulatory roles of the C-tails to GLR proteins.