Subcellular Compartments Research Articles

Nucleoporin downregulation modulates progenitor differentiation independent of nuclear pore numbers

Nucleoporins (NUPs) comprise nuclear pore complexes, gateways for nucleocytoplasmic transport. As primary human keratinocytes switch from the progenitor state towards differentiation, most NUPs are strongly downregulated, with NUP93 being the most downregulated NUP in this process. To determine if this NUP downregulation is accompanied by a reduction in nuclear pore numbers, we leveraged Stochastic Optical Reconstruction Microscopy. No significant changes in nuclear pore numbers were detected using three independent NUP antibodies; however, NUP reduction in other subcellular compartments such as the cytoplasm was identified. To investigate how NUP reduction influences keratinocyte differentiation, we knocked down NUP93 in keratinocytes in the progenitor-state culture condition. NUP93 knockdown diminished keratinocytes’ clonogenicity and epidermal regenerative capacity, without drastically affecting nuclear pore numbers or permeability. Using transcriptome profiling, we identified that NUP93 knockdown induces differentiation genes related to both mechanical and immune barrier functions, including the activation of known NF-κB target genes. Consistently, keratinocytes with NUP93 knockdown exhibited increased nuclear localization of the NF-κB p65/p50 transcription factors, and increased NF-κB reporter activity. Taken together, these findings highlight the gene regulatory roles contributed by differential NUP expression levels in keratinocyte differentiation, independent of nuclear pore numbers.

Insights from the protein interaction Universe of the multifunctional "Goldilocks" kinase DYRK1A.

Human Dual specificity tyrosine (Y)-Regulated Kinase 1A (DYRK1A) is encoded by a dosage-dependent gene located in the Down syndrome critical region of human chromosome 21. The known substrates of DYRK1A include proteins involved in transcription, cell cycle control, DNA repair and other processes. However, the function and regulation of this kinase is not fully understood, and the current knowledge does not fully explain the dosage-dependent function of this kinase. Several recent proteomic studies identified DYRK1A interacting proteins in several human cell lines. Interestingly, several of known protein substrates of DYRK1A were undetectable in these studies, likely due to a transient nature of the kinase-substrate interaction. It is possible that the stronger-binding DYRK1A interacting proteins, many of which are poorly characterized, are involved in regulatory functions by recruiting DYRK1A to the specific subcellular compartments or distinct signaling pathways. Better understanding of these DYRK1A-interacting proteins could help to decode the cellular processes regulated by this important protein kinase during embryonic development and in the adult organism. Here, we review the current knowledge of the biochemical and functional characterization of the DYRK1A protein-protein interaction network and discuss its involvement in human disease.

Recent Advances and Future Challenges of Nano-based Drug Delivery System

Nano drug delivery systems play a crucial role in addressing key challenges encountered in conventional drug delivery methodologies, making them of significant importance in the field of medicine. These systems offer enhanced drug bioavailability by improving drug solubility and stability, thereby maximizing therapeutic efficacy. Their ability to achieve targeted drug delivery to specific tissues, cells, or subcellular compartments minimizes off-target effects and reduces systemic toxicity. Additionally, nano drug delivery systems present opportunities for combination therapy, where multiple drugs or therapeutic agents can be encapsulated within a single nanoparticle, promoting synergistic effects and personalized treatment strategies. Overall, the utilization of nano drug delivery systems holds great promise in enhancing therapeutic outcomes, mitigating adverse effects, and advancing the field of precision medicine. Nano drug delivery systems have demonstrated remarkable potential through various examples, mesoporous nanoparticles, nanomaterials, polymeric nanoparticles, carbon nanotubes, dendrimers, liposomes, metallic nanoparticles, and nanomedicine etc.

Production of enzymes for the removal of odorous substances in plant biomass

Residual plant biomass collected from agricultural, technical or biopharmaceutical processes contains odorous substances. The latter are often unacceptable for customers if the biomass is used in sustainable products such as building materials, paints, glues or flame-resistant foils. The objective of this study was to identify enzymes that can prevent the formation or facilitate the degradation of odorous substances such as butanol, eugenol or ethyl acetate and their derivatives in residual biomass. We used plant cell packs (PCPs) as a small-scale screening platform to assess the expression of enzymes that break down odorous substances in tobacco biomass. First, we compiled a list of volatile compounds in residual plant biomass that may give rise to undesirable odors, refining the list to 10 diverse compounds representing a range of odors. We then selected five monomeric enzymes (a eugenol oxidase, laccase, oxidase, alkane mono-oxidase and ethyl acetate hydrolase) with the potential to degrade these substances. We transiently expressed the proteins in PCPs, targeting different subcellular compartments to identify optimal production conditions. The maximum yield we achieved was ∼20 mg kg−1 for Trametes hirsute laccase targeted to the chloroplast. Our results confirm that enzymes for the removal of odorous substances can be produced in plant systems, facilitating the upcycling of residual biomass as an ingredient for sustainable products.

In vitro modeling of polyclonal infection dynamics within the human airways by Haemophilus influenzae differential fluorescent labeling.

Standardized clinical procedures for antibiotic administration rely on pathogen identification and antibiotic susceptibility testing, often performed on single-colony bacterial isolates. For respiratory pathogens, this could be questionable, as chronic patients may be persistently colonized by multiple clones or lineages from the same bacterial pathogen species. Indeed, multiple strains of nontypeable Haemophilus influenzae, with different antibiotic susceptibility profiles, can be co-isolated from cystic fibrosis and chronic obstructive pulmonary disease sputum specimens. Despite this clinical evidence, we lack information about the dynamics of H. influenzae polyclonal infections, which limits the optimization of therapeutics. Here, we present the engineering and validation of a plasmid toolkit (pTBH, toolbox for Haemophilus), with standardized modules consisting of six reporter genes for fluorescent or bioluminescent labeling of H. influenzae. This plasmid set was independently introduced in a panel of genomically and phenotypically different H. influenzae strains, and two of them were used as a proof of principle to analyze mixed biofilm growth architecture and antibiotic efficacy, and to visualize the dynamics of alveolar epithelial co-infection. The mixed biofilms showed a bilayer architecture, and antibiotic efficacy correlated with the antibiotic susceptibility of the respective single-species strains. Furthermore, differential kinetics of bacterial intracellular location within subcellular acidic compartments were quantified upon co-infection of cultured airway epithelial cells. Overall, we present a panel of novel plasmid tools and quantitative image analysis methods with the potential to be used in a whole range of bacterial host species, assay types, and/or conditions and generate meaningful information for clinically relevant settings. IMPORTANCE Genomic diversity of nontypeable H. influenzae strains confers phenotypic heterogeneity. Multiple strains of H. influenzae can be simultaneously isolated from clinical specimens, but we lack detailed information about polyclonal infection dynamics by this pathogen. A long-term barrier to our understanding of this host-pathogen interplay is the lack of genetic tools for strain engineering and differential labeling. Here, we present a novel plasmid toolkit named pTBH (toolbox for Haemophilus), with standardized modules for fluorescent or bioluminescent labeling, adapted to H. influenzae requirements but designed to be versatile so it can be utilized in other bacterial species. We present detailed experimental and quantitative image analysis methods, together with proof-of-principle examples, and show the ample possibilities of 3D microscopy, combined with quantitative image analysis, to model H. influenzae polyclonal infection lifestyles and unravel the co-habitation and co-infection dynamics of this respiratory pathogen.

A viral protein targets mitochondria and chloroplasts by subverting general import pathways and specific receptors.

Many plant proteins and some proteins from plant pathogens are dually targeted to chloroplasts and mitochondria, and are supposed to be transported along the general pathways for organellar protein import, but this issue has not been explored yet. Moreover, organellar translocon receptors exist as families of several members whose functional specialization in different cargos is supposed but not thoroughly studied. This article provides novel insights into such topics showing for the first time that an exogenous protein, the melon necrotic spot virus coat protein, exploits the common Toc/Tom import systems to enter both mitochondria and chloroplasts while identifying the involved specific receptors.

Antidepressants enter cells, organelles, and membranes.

We begin by summarizing several examples of antidepressants whose therapeutic actions begin when they encounter their targets in the cytoplasm or in the lumen of an organelle. These actions contrast with the prevailing view that most neuropharmacological actions begin when drugs engage their therapeutic targets at extracellular binding sites of plasma membrane targets-ion channels, receptors, and transporters. We review the chemical, pharmacokinetic, and pharmacodynamic principles underlying the movements of drugs into subcellular compartments. We note the relationship between protonation-deprotonation events and membrane permeation of antidepressant drugs. The key properties relate to charge and hydrophobicity/lipid solubility, summarized by the parameters LogP, pKa, and LogDpH7.4. The classical metric, volume of distribution (Vd), is unusually large for some antidepressants and has both supracellular and subcellular components. A table gathers structures, LogP, PKa, LogDpH7.4, and Vd data and/or calculations for most antidepressants and antidepressant candidates. The subcellular components, which can now be measured in some cases, are dominated by membrane binding and by trapping in the lumen of acidic organelles. For common antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) and serotonin/norepinephrine reuptake inhibitors (SNRIs), the target is assumed to be the eponymous reuptake transporter(s), although in fact the compartment of target engagement is unknown. We review special aspects of the pharmacokinetics of ketamine, ketamine metabolites, and other rapidly acting antidepressants (RAADs) including methoxetamine and scopolamine, psychedelics, and neurosteroids. Therefore, the reader can assess properties that markedly affect a drug's ability to enter or cross membranes-and therefore, to interact with target sites that face the cytoplasm, the lumen of organelles, or a membrane. In the current literature, mechanisms involving intracellular targets are termed "location-biased actions" or "inside-out pharmacology". Hopefully, these general terms will eventually acquire additional mechanistic details.

Enhanced Anti-Herpetic Activity of Valacyclovir Loaded in Sulfobutyl-ether-β-cyclodextrin-decorated Chitosan Nanodroplets.

Valacyclovir (VACV) was developed as a prodrug of the most common anti-herpetic drug Acyclovir (ACV), aiming to enhance its bioavailability. Nevertheless, prolonged VACV oral treatment may lead to the development of important side effects. Nanotechnology-based formulations for vaginal administration represent a promising approach to increase the concentration of the drug at the site of infection, limiting systemic drug exposure and reducing systemic toxicity. In this study, VACV-loaded nanodroplet (ND) formulations, optimized for vaginal delivery, were designed. Cell-based assays were then carried out to evaluate the antiviral activity of VACV loaded in the ND system. The chitosan-shelled ND exhibited an average diameter of about 400 nm and a VACV encapsulation efficiency of approximately 91% and was characterized by a prolonged and sustained release of VACV. Moreover, a modification of chitosan shell with an anionic cyclodextrin, sulfobutyl ether β-cyclodextrin (SBEβCD), as a physical cross-linker, increased the stability and mucoadhesion capability of the nanosystem. Biological experiments showed that SBEβCD-chitosan NDs enhanced VACV antiviral activity against the herpes simplex viruses type 1 and 2, most likely due to the long-term controlled release of VACV loaded in the ND and an improved delivery of the drug in sub-cellular compartments.

Actomyosin-driven motility and coalescence of phase-separated viral inclusion bodies are required for efficient replication of a plant rhabdovirus.

Phase separation has emerged as a fundamental principle for organizing viral and cellular membraneless organelles. Although these subcellular compartments have been recognized for decades, their biogenesis and mechanisms of regulation are poorly understood. Here, we investigate the formation of membraneless inclusion bodies (IBs) induced during the infection of a plant rhabdovirus, tomato yellow mottle-associated virus (TYMaV). We generated recombinant TYMaV encoding a fluorescently labeled IB constituent protein and employed live-cell imaging to characterize the intracellular dynamics and maturation of viral IBs in infected Nicotiana benthamiana cells. We show that TYMaV IBs are phase-separated biomolecular condensates and that viral nucleoprotein and phosphoprotein are minimally required for IB formation invivo and invitro. TYMaV IBs move along the microfilaments, likely through the anchoring of viral phosphoprotein to myosin XIs. Furthermore, pharmacological disruption of microfilaments or inhibition of myosin XI functions suppresses IB motility, resulting in arrested IB growth and inefficient virus replication. Our study establishes phase separation as a process driving the formation of liquid viral factories and emphasizes the role of the cytoskeletal system in regulating the dynamics of condensate maturation.

Cellular reductive stress: Is plasma membrane electron transport an evolutionarily-conserved safety valve?

Cellular respiration is highly regulated, changes dynamically in response to the microenvironment of individual cells and during differentiation and differs between cell and tissue types. Too little cell respiration can cause an accumulation of reductants, leading to reductive stress, while inefficient respiration, that causes a build-up of reactive oxygen species (ROS), can result in oxidative stress. Most of the discussion of this central redox dichotomy has centred around oxidative stress because the damaging effects of cellular oxidants on DNA, lipids and proteins are well-established, and have been shown to contribute to health issues including, mitochondrial and cardiovascular diseases, tumorigenesis, and to the effects of ageing. Much less attention has been paid to cellular reductive stress. Nevertheless, excessive levels of key cellular reductants including NADH, NADPH and glutathione, as well as an imbalance in protein thiols, and insufficient levels of ROS to maintain cell signalling pathways, can be harmful to cells and result in poor health outcomes. Recently, cellular mechanisms that sense and regulate cellular reductive stress associated with low ROS levels have been identified. In addition, plasma membrane electron transport has been shown to be a key player in cellular redox homeostasis involving NAD(P)H/NAD(P)+ ratios. It is now well-established that the plasma membrane contains coenzyme Q-mediated electron transport pathways capable of oxidizing intracellular NAD(P)H and reducing extracellular electron acceptors such as molecular oxygen. A better understanding of the origins, cellular and subcellular compartmentalization and regulation of cellular reductants could lead to the development of new anticancer strategies.

Alteration in the expression of tomato sucrose transporter gene SlSUT4 modulates sucrose subcellular compartmentation and affects responses of plants to drought stress

Sucrose transporters (SUTs) are involved in the transmembrane transport of sucrose. The roles of SUTs in regulating stress tolerance of plants have been reported, but differential results were observed in different plants, implying the need for more studies in different plants. Moreover, the mechanism of SUTs in regulating stress tolerance is still poorly understood. Here, we investigated the role of SlSUT4 from tomato in regulating drought tolerance by using SlSUT4-overexpressed and -RNAi plants. The results showed that SlSUT4 overexpression decreased the drought tolerance of plants, as demonstrated by reduced seed germination, decreased plant growth, photosynthesis and relative water content, and higher oxidative damage under osmotic stress or drought stress; whereas silencing of SlSUT4 increased the stress tolerance. In the control conditions, the leaf sucrose export rate was reduced in the overexpression plants, but promoted in the RNAi plants, and all transgenic lines had higher leaf sucrose concentrations than the wild type. Accumulation of soluble sugars was more obvious in SlSUT4-overexpressed plants and the wild type than the RNAi plants under drought. Overexpressing SlSUT4 decreased the proportion of sucrose in the vacuole in the control conditions, whereas the RNAi plants showed an opposite change. These results suggest that alteration in SlSUT4 expression modulates sucrose vacuole-cytosol compartmentation, thus affecting the responses of tomato plants to drought stress.

Development of a plant-based oral vaccine candidate against the bovine respiratory pathogen Mannheimia haemolytica.

Bovine respiratory disease (BRD) affects feedlot cattle across North America, resulting in economic losses due to animal treatment and reduced performance. In an effort to develop a vaccine candidate targeting a primary bacterial agent contributing to BRD, we produced a tripartite antigen consisting of segments of the virulence factor Leukotoxin A (LktA) and lipoprotein PlpE from Mannheimia haemolytica, fused to a cholera toxin mucosal adjuvant (CTB). This recombinant subunit vaccine candidate was expressed in the leaves of Nicotiana benthamiana plants, with accumulation tested in five subcellular compartments. The recombinant protein was found to accumulate highest in the endoplasmic reticulum, but targeting to the chloroplast was employed for scaling up production due the absence of post-translational modification while still producing feasible levels. Leaves were freeze dried, then orally administered to mice to determine its immunogenicity. Sera from mice immunized with leaf tissue expressing the recombinant antigen contained IgG antibodies, specifically recognizing both LktA and PlpE. These mice also had a mucosal immune response to the CTB+LktA+PlpE protein as measured by the presence of LktA- and PlpE-specific IgA antibodies in lung and fecal material. Moreover, the antigen remained stable at room temperature with limited deterioration for up to one year when stored as lyophilized plant material. This study demonstrated that a recombinant antigen expressed in plant tissue elicited both humoral and mucosal immune responses when fed to mice, and warrants evaluation in cattle.

Multiscale Asymptotic Analysis Reveals How Cell Growth and Subcellular Compartments Affect Tissue-Scale Hormone Transport

Determining how cell-scale processes lead to tissue-scale patterns is key to understanding how hormones and morphogens are distributed within biological tissues and control developmental processes. In this article, we use multiscale asymptotic analysis to derive a continuum approximation for hormone transport in a long file of cells to determine how subcellular compartments and cell growth and division affect tissue-scale hormone transport. Focusing our study on plant tissues, we begin by presenting a discrete multicellular ODE model tracking the hormone concentration in each cell’s cytoplasm, subcellular vacuole, and surrounding apoplast, represented by separate compartments in the cell-file geometry. We allow the cells to grow at a rate that can depend both on space and time, accounting for both cytoplasmic and vacuolar expansion. Multiscale asymptotic analysis enables us to systematically derive the corresponding continuum model, obtaining an effective reaction–advection–diffusion equation and revealing how the effective diffusivity, effective advective velocity, and the effective sink term depend on the parameters in the cell-scale model. The continuum approximation reveals how subcellular compartments, such as vacuoles, can act as storage vessels, that significantly alter the effective properties of hormone transport, such as the effective diffusivity and the induced effective velocity. Furthermore, we show how cell growth and spatial variance across cell lengths affect the effective diffusivity and the induced effective velocity, and how these affect the tissue-scale hormone distribution. In particular, we find that cell growth naturally induces an effective velocity in the direction of growth, whereas spatial variance across cell lengths induces effective velocity due to the presence of an extra compartment, such as the apoplast and the vacuole, and variations in the relative sizes between the compartments across the file of cells. It is revealed that hormone transport is faster across cells of decreasing lengths than cells with increasing lengths. We also investigate the effect of cell division on transport dynamics, assuming that each cell divides as soon as it doubles in size, and find that increasing the time between successive cell divisions decreases the growth rate, which enhances the effect of cell division in slowing hormone transport. Motivated by recent experimental discoveries, we discuss particular applications for transport of gibberellic acid (GA), an important growth hormone, within the Arabidopsis root. The model reveals precisely how membrane proteins that mediate facilitated GA transport affect the effective tissue-scale transport. However, the results are general enough to be relevant to other plant hormones, or other substances that are transported in a similar way in any type of cells.

The human cellular protein NoL12 is a specific partner of the HIV-1 nucleocapsid protein NCp7.

The human immunodeficiency virus-1 (HIV-1) nucleocapsid protein (NCp7) is a nucleic acid chaperone protein with two highly conserved zinc fingers. To exert its key roles in the viral cycle, NCp7 interacts with several host proteins. Among them, the human NoL12 protein (hNoL12) was previously identified in genome wide screens as a potential partner of NCp7. hNoL12 is a highly conserved 25 kDa nucleolar RNA-binding protein implicated in the 5'end processing of ribosomal RNA in the nucleolus and thus in the assembly and maturation of ribosomes. In this work, we confirmed the NCp7/hNoL12 interaction in cells by Förster resonance energy transfer visualized by Fluorescence Lifetime Imaging Microscopy and co-immunoprecipitation. The interaction between NCp7 and hNoL12 was found to strongly depend on their both binding to RNA, as shown by the loss of interaction when the cell lysates were pretreated with RNase. Deletion mutants of hNoL12 were tested for their co-immunoprecipitation with NCp7, leading to the identification of the exonuclease domain of hNoL12 as the binding domain for NCp7. Finally, the interaction with hNoL12 was found to be specific of the mature NCp7 and to require NCp7 basic residues. IMPORTANCE HIV-1 mature nucleocapsid (NCp7) results from the maturation of the Gag precursor in the viral particle and is thus mostly abundant in the first phase of the infection which ends with the genomic viral DNA integration in the cell genome. Most if not all the nucleocapsid partners identified so far are not specific of the mature form. We described here the specific interaction in the nucleolus between NCp7 and the human nucleolar protein 12, a protein implicated in ribosomal RNA maturation and DNA damage response. This interaction takes place in the cell nucleolus, a subcellular compartment where NCp7 accumulates. The absence of binding between hNoL12 and Gag makes hNoL12 one of the few known specific cellular partners of NCp7.

AKIR-1 regulates proteasome subcellular function in Caenorhabditis elegans

Polyubiquitinated proteins are primarily degraded by the ubiquitin-proteasome system (UPS). Proteasomes are present both in the cytoplasm and nucleus. Here, we investigated mechanisms coordinating proteasome subcellular localization and activity in a multicellular organism. We identified the nuclear protein-encoding gene akir-1 as a proteasome regulator in a genome-wide Caenorhabditis elegans RNAi screen. We demonstrate that depletion of akir-1 causes nuclear accumulation of endogenous polyubiquitinated proteins in intestinal cells, concomitant with slower invivo proteasomal degradation in this subcellular compartment. Remarkably, akir-1 is essential for nuclear localization of proteasomes both in oocytes and intestinal cells but affects differentially the subcellular distribution of polyubiquitinated proteins. We further reveal that importin ima-3 genetically interacts with akir-1 and influences nuclear localization of a polyubiquitin-binding reporter. Our study shows that the conserved AKIR-1 is an important regulator of the subcellular function of proteasomes in a multicellular organism, suggesting a role for AKIR-1 in proteostasis maintenance.

Genome-wide identification, phylogenomics, and expression analysis of benzoxazinoids gene family in rice (Oryza sativa)

Benzoxazinoids (BXs) are potent secondary metabolites that affect plants' biotic and abiotic interactions. Extensive studies on the functions of benzoxazinoids in mediating biotic and abiotic stressors have been reported in maize, wheat, and rye. However, little is known about BXs biosynthesis in rice. This study presents a genome-wide analysis of forty-three Oryzae sativa BXs genes that form diphyletic clusters in a neighbor-joining tree. The first cluster comprised genes that encode the first four steps in BXs biosynthesis (BX2–BX4), leading to the production of 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA). The second cluster mainly comprised BX6 and BX7 genes responsible for BXs glycosylation. Furthermore, BX proteins harboring similar conserved motifs were found to group according to their phylogenetic clustering. Whereas the P450 superfamily protein is conserved in BX1-BX4 proteins, the UDP-glucosyltransferase is conserved in BX7 members. These proteins were found to be strategically localized in subcellular compartments where their catalytic activities are executed. BX1 proteins are localized in the chloroplasts, where they encode the production of indole. BX6 were identified as cytoplasmic proteins, where they are involved in the hydroxylation of DIBOA-Glycoside to 2-(2,4,7-trihydroxy-8-methoxy-1,4-benzoxazine-3-one)-β-d-glucopyranose (TRIBOA-Glycoside). Further investigation of the molecular addresses showed that BX proteins tend to localize together on chromosomes based on their functions. Moreover, the expression profiles of these proteins vary at various developmental stages in rice tissues and organs, highlighting the potential for biotic and abiotic interactions. The prospects of BXs genes in growth induction were also investigated by analyzing their responsiveness to plant hormones and nutrient treatment. BX gene expression is affected by exogenous treatment with auxin and gibberellin as well as nitrogen, potassium, and phosphorus contents, suggesting a possible role in growth mediation. This study lays the foundation for further studies to elucidate the functions of BX genes in rice.

RNA damage: the forgotten target of clinical compounds

Unlike DNA, RNA can be found in every sub-cellular compartment, where it is used to impart the genetic code or perform essential catalytic activities. As a result, damage to RNA is more spatially pervasive than damage to DNA and can have profound effects on gene expression and RNA-dependent activities. The past decade has seen the pathways involved in detecting and responding to damage of specific RNAs defined. These studies largely used high concentrations of tool compounds or deletion of essential factors for the response to RNA damage to study its effects. RNA is damaged by both endogenous and exogenous agents, with the effect of exogenous agents administered as therapeutics the focus of this review. In an effort to formalise studies into clinical RNA damage biology we propose 4 types of RNA damaging drug that we divide into 2 broad classes. Class 1 drugs result from synthesis using non-canonical nucleotides, which are incorporated into RNA in place of the canonical nucleotides. This class is subdivided depending on the outcome of this misincorporation on the nascent transcript. Class 2 drugs result in covalent ligation of moieties that alter RNA structure. This class is subdivided according to the functionality of the covalent ligation—class 2a are monovalent while class 2b are divalent. We discuss the evidence for and mechanisms of RNA damage as well as highlighting the unknown factors that require further investigation to determine the molecular mechanisms of these drugs.

Synapse-Enriched m6A-Modified Malat1 Interacts with the Novel m6A Reader, DPYSL2, and Is Required for Fear-Extinction Memory.

The RNA modification N6-methyladenosine (m6A) regulates the interaction between RNA and various RNA binding proteins within the nucleus and other subcellular compartments and has recently been shown to be involved in experience-dependent plasticity, learning, and memory. Using m6A RNA-sequencing, we have discovered a distinct population of learning-related m6A- modified RNAs at the synapse, which includes the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1). RNA immunoprecipitation and mass spectrometry revealed 12 new synapse-specific learning-induced m6A readers in the mPFC of male C57/BL6 mice, with m6A-modified Malat1 binding to a subset of these, including CYFIP2 and DPYSL2. In addition, a cell type- and synapse-specific, and state-dependent, reduction of m6A on Malat1 impairs fear-extinction memory; an effect that likely occurs through a disruption in the interaction between Malat1 and DPYSL2 and an associated decrease in dendritic spine formation. These findings highlight the critical role of m6A in regulating the functional state of RNA during the consolidation of fear-extinction memory, and expand the repertoire of experience-dependent m6A readers in the synaptic compartment.SIGNIFICANCE STATEMENT We have discovered that learning-induced m6A-modified RNA (including the long noncoding RNA, Malat1) accumulates in the synaptic compartment. We have identified several new m6A readers that are associated with fear extinction learning and demonstrate a causal relationship between m6A-modified Malat1 and the formation of fear-extinction memory. These findings highlight the role of m6A in regulating the functional state of an RNA during memory formation and expand the repertoire of experience-dependent m6A readers in the synaptic compartment.

Label-Free Intracellular Multi-Specificity in Yeast Cells by Phase-Contrast Tomographic Flow Cytometry.

In-flow phase-contrast tomography provides a 3D refractive index of label-free cells in cytometry systems. Its major limitation, as with any quantitative phase imaging approach, is the lack of specificity compared to fluorescence microscopy, thus restraining its huge potentialities in single-cell analysis and diagnostics. Remarkable results in introducing specificity are obtained through artificial intelligence (AI), but only for adherent cells. However, accessing the 3D fluorescence ground truth and obtaining accurate voxel-level co-registration of image pairs for AI training is not viable for high-throughput cytometry. The recent statistical inference approach is a significant step forward for label-free specificity but remains limited to cells' nuclei. Here, a generalized computational strategy based on a self-consistent statistical inference to achieve intracellular multi-specificity is shown. Various subcellular compartments (i.e., nuclei, cytoplasmic vacuoles, the peri-vacuolar membrane area, cytoplasm, vacuole-nucleus contact site) can be identified and characterized quantitatively at different phases of the cells life cycle by using yeast cells as a biological model. Moreover, for the first time, virtual reality is introduced for handling the information content of multi-specificity in single cells. Full fruition is proofed for exploring and interacting with 3D quantitative biophysical parameters of the identified compartments on demand, thus opening the route to a metaverse for 3D microscopy.

Downregulation of Zebrafish Cytosolic Sialidase Neu3.2 Affects Skeletal Muscle Development.

Sialidases remove terminal sialic acids residues from the non-reducing ends of glycoconjugates. They have been recognized as catabolic enzymes that work within different subcellular compartments and can ensure the proper turn-over of glycoconjugates. Four mammalian sialidases (NEU1-4) exist, with different subcellular localization, pH optimum and substrate specificity. In zebrafish, seven different sialidases, with high homology to mammalian counterparts, have been identified. Zebrafish Neu3.2 is similar to the human cytosolic sialidase NEU2, which is involved in skeletal muscle differentiation and exhibits a broad substrate specificity toward gangliosides and glycoproteins. In zebrafish neu3.2, mRNA is expressed during somite development, and its enzymatic activity has been detected in the skeletal muscle and heart of adult animals. In this paper, 1-4-cell-stage embryos injected with neu3.2 splice-blocking morpholino showed severe embryonic defects, mainly in somites, heart and anterior-posterior axis formation. Myog and myod1 expressions were altered in morphants, and impaired musculature formation was associated with a defective locomotor behavior. Finally, the co-injection of Neu2 mouse mRNA in morphants rescued the phenotype. These data are consistent with the involvement of cytosolic sialidase in pathologies related to muscle formation and support the validity of the model to investigate the pathogenesis of the diseases.