Phase-separated bienzyme compartmentalization as artificial intracellular membraneless organelles for cell repair

Implanting artificial organelles in living cells is capable of correcting cellular dysfunctionalities for cell repair and biomedical applications. In this work, phase-separated bienzyme-loaded coacervate microdroplets are established as a model of artificial membraneless organelles in endothelial dysfunctional cells for the cascade enzymatic production of nitric oxide(NO) with a purpose of correcting cellular NO deficiency. We prepared the coacervate microdroplets via liquid-liquid phase separation of oppositely charged polyelectrolytes, in which glucose oxidase/horseradish peroxidase-mediated cascade reaction was compartmented. After the coacervate microdroplets were implanted in NO-deficient dysfunctional cells, the compartments maintained a phase-separated liquid droplet structure, which facilitated a significant enhancement of NO production in the dysfunctional cells. The recovery of NO production was further exploited to inhibit clot formation in blood plasma located in the cell suspension. This demonstrated a proof-of-concept design of artificial organelles in dysfunctional cells for cell repair and anticoagulation-related medical applications. Our results demonstrate an approach for the construction of coacervate droplets through phase separation for the generation of artificial membraneless organelles, which can be designed to provide an array of functionalities in living organisms that have the potential to be used in the field of cell engineering and medical therapy.

Plant peroxisomes:Small organelles with versatility and complexity

Peroxisomes are small,highly dynamic,and multifunctional organelles in eukaryotes.Essential to plant survival,peroxisomes house various crucial metabolic activities,such as degradation of hydrogen peroxide(H2O2),lipid metabolism,photorespiration,and hormone biosynthesis and catabolism,and remodel their proteome in response to developmental and environmental changes(Hu et al.2012;Pan and Hu 2018).The four reviews and three research articles in this special issue on plant peroxisomes provide new insights into the diverse roles and dynamics of these structurally simple but functionally complicated organelles,raising exciting new questions for future investigations.

Compartmentalized regulation of organelle integrity in neurodegenerative diseases:lessons from the Drosophila motor neuron

Neurons are highly polarized,morphologically asymmetric,and functionally compartmentalized cells that contain long axons extending from the cell body.For this reason,their maintenance relies on spatiotemporal regulation of organelle distribution between the somatodendritic and axonal domains.Although some organelles,such as mitochondria and smooth endoplasmic reticulum,are widely distributed throughout the neuron,others are segregated to either the somatodendritic or axonal compartment.For example,Golgi outposts and acidified lysosomes are predominantly present in the somatodendritic domain and rarely distributed along the axon,whereas newly formed autophagosomes and synaptic vesicles are mainly distributed in the distal axon(Britt et al.,2016).

Recent Advances of AIEgens for Targeted Imaging of Subcellular Organelles

Fluorescence imaging based on luminogens with aggregation-induced emission(AIE)effect has drawn great attention in recent two decades,due to their superior advantages to overcome the technical difficulties.Thus,the AIE-active bioprobes with targeted ability at the subcellular level have been widely investigated to visualize the subcellular structures and monitor the biological processes.Considering the very rapid developments and the significance of selective imaging of subcellular structures,we summarize the recent two-year achievements about the AIEgens for targeted imaging of subcellular organelles including nuclei,membranes,lipid droplets(LDs),endoplasmic reticulum(ER),lysosomes,mitochondria and cytoplasm.The designed protocols and advantages of AIEgens,their mechanisms for targeted staining at organelles and the imaging performance are discussed.These AIE bioprobes exhibit great potentials for early diagnosis and therapeutics of diseases that related to subcellular organelles.Finally,the perspectives about AIEgens for these applications are also discussed.

Role of plant myosins in motile organelles: Is a direct interaction required?

Plant organelles are highly motile, with speed values of 3-71μm/s in cells of land plants and about 20-60 μm/s in characean algal cells. This movement is believed to be important for rapid distribution of materials around the cell, for the plant＇s ability to respond to environmental biotic and abiotic signals and for proper growth. The main machinery that propels motility of organelles within plant cells is based on the actin cytoskeleton and its motor proteins the myosins.Most plants express multiple members of two main classes： myosin VIII and myosin XI. While myosin VIII has been characterized as a slow motor protein, myosins from class XI were found to be the fastest motor proteins known in all kingdoms. Paradoxically, while it was found that myosins from class XI regulate most organelle movement, it is not quite clear how or even if these motor proteins attach to the organelles whose movement they regulate.

Group-specific functional patterns of mitochondrion-related organelles shed light on their multiple transitions from mitochondria in ciliated protists

Adaptations of ciliates to hypoxic environments have arisen independently several times.Studies on mitochondrion-related organelle(MRO)metabolisms from distinct anaerobic ciliate groups provide evidence for understanding the transitions from mitochondria to MROs within eukaryotes.To deepen our knowledge about the evolutionary patterns of ciliate anaerobiosis,mass-culture and single-cell transcriptomes of two anaerobic species,Metopus laminarius(class Armophorea)and Plagiopyla cf.narasimhamurtii(class Plagiopylea),were sequenced and their MRO metabolic maps were compared.In addition,we carried out comparisons using publicly available predicted MRO proteomes from other ciliate classes(i.e.,Armophorea,Litostomatea,Muranotrichea,Oligohymenophorea,Parablepharismea and Plagiopylea).We found that single-cell transcriptomes were similarly comparable to their mass-culture counterparts in predicting MRO metabolic pathways of ciliates.The patterns of the components of the MRO metabolic pathways might be divergent among anaerobic ciliates,even among closely related species.Notably,our findings indicate the existence of group-specific functional relics of electron transport chains(ETCs).Detailed group-specific ETC functional patterns are as follows:full oxidative phosphorylation in Oligohymenophorea and Muranotrichea;only electron-transfer machinery in Armophorea;either of these functional types in Parablepharismea;and ETC functional absence in Litostomatea and Plagiopylea.These findings suggest that adaptation of ciliates to anaerobic conditions is group-specific and has occurred multiple times.Our results also show the potential and the limitations of detecting ciliate MRO proteins using single-cell transcriptomes and improve the understanding of the multiple transitions from mitochondria to MROs within ciliates.

ATF6 aggravates apoptosis in early porcine embryonic development by regulating organelle homeostasis under high-temperature conditions

Activating transcription factor 6(ATF6),one of the three sensor proteins in the endoplasmic reticulum(ER),is an important regulator of ER stress-induced apoptosis.ATF6 resides in the ER and,upon activation,is translocated to the Golgi apparatus,where it is cleaved by site-1 protease(S1P)to generate an amino-terminal cytoplasmic fragment.Although recent studies have made progress in elucidating the regulatory mechanisms of ATF6,its function during early porcine embryonic development under high-temperature(HT)stress remains unclear.In this study,zygotes were divided into four groups:control,HT,HT+ATF6 knockdown,and HT+PF(S1P inhibitor).Results showed that HT exposure induced ER stress,which increased ATF6 protein expression and led to a decrease in the blastocyst rate.Next,ATF6 expression was knocked down in HT embryos under microinjection of ATF6 double-stranded RNA(dsRNA).Results revealed that ATF6 knockdown(ATF6-KD)attenuated the increased expression of CHOP,an ER stress marker,and Ca2+release induced by HT.In addition,ATF6-KD alleviated homeostasis dysregulation among organelles caused by HT-induced ER stress,and further reduced Golgi apparatus and mitochondrial dysfunction in HT embryos.AIFM2 is an important downstream effector of ATF6.Results showed that ATF6-KD reduced the occurrence of AIFM2-mediated embryonic apoptosis at HT.Taken together,our findings suggest that ATF6 is a crucial mediator of apoptosis during early porcine embryonic development,resulting from HT-induced ER stress and disruption of organelle homeostasis.

Comparison and phylogeny on mitochondrial genome of marine and freshwater taxa of genus Hildenbrandia(Florideophyceae,Rhodophyta)

Hildenbrandia is an early diverged lineage in Florideophyceae,Rhodophyta.The species diversity of this genus is still unresolved due to the simple morphology and limited molecular information.The mitochondrial genome of freshwater H.jigongshanensis was determined in this study.The freshwater H.jigongshanensis had a larger mitochondrial genome than the marine H.rubra and GC content was higher.Collinear alignment structure was observed between the mitochondrial genomes of H.jigongshanensis and H.rubra,except for one block that was encoded on the complement strand.More introns were found in mitochondrial genome of H.jigongshanensis than in H.rubra,and H.jigongshanensis shares the common feature with Bangiophyceae that two introns were distributed in cox1.Comparison of mitochondrial genome organization suggests that H.jigongshanensis preserves characters that could be hypothetically more similar to the ancestor of Bangiophyceae and Florideophyceae,which differ with previous studies based on chloroplast,and nuclear markers.More mitochondrial genomes and phylogenetic analyses combing nuclear,chloroplast and mitochondrial genomes are needed to clarify this discrepancy.Mitochondrion-based phylogeny in this study resulted in better solution at both the deep and recent derived nodes than single-gene phylogenies.Most protein-coding genes between H.jigongshanensis and H.rubra were identical except atp8,which was present in H.jigongshanensis while absent from H.rubra.This finding follows the trend that high Ka/Ks ratio genes are more frequently lost than low Ka/Ks ratio ones in red algae.

Phosphatidylglycerol-containing ER-transport vesicles built and restore outer mitochondrial membrane and deliver nuclear DNA translation products to generate cardiolipin in the inner mitochondrial membrane

Phosphatidylglycerol (PG) an important membrane phospholipid required for the synthesis of diphos-phatidylglycerol (DPG) commonly known as cardiolipin (CL) was identified in the fraction of endo-plasmic reticulum (ER)-derived transport vesicles which had no affinity for Golgi. The vesicles were produced in the presence of Brefeldin A (BFA), the agent known to inhibit ER-Golgi transport, and found to display affinity to mitochondria. The analysis revealed that their cargo was not containing proteins that are transported to Golgi, and that their membrane was free of phosphatidylinositol (PI) and ceramides (Cer). The incubation of PG-containing transport vesicles with mitochondria afforded incorporation of their membrane into the Outer Mito-chondrial Membrane (OMM) and formation of lyso-phosphatidylglycerol (LPG). In turn, upon further incubation with fresh transport active cytosol, the mitochondrial LPG was converted to PG. The results of analysis of the OMM, Inner Mitochondrial Mem-brane (IMM) and Inner Mitochondrial Space Components (IMSC) strongly suggest that PG-containing transport vesicles deliver nuclear DNA translation products to the IMSC and thus facilitate CL synthesis in the IMM. In summary, our studies provide evidence that ER-generated PG-enriched transport vesicles represent the general pathway for restitution of mitochondrial membranes and the delivery of nuclear DNA translation products that generate CL, and thus sustain the mitochondrial matrix CL-dependent metabolic reactions.

Glyphosate exposure deteriorates oocyte meiotic maturation via induction of organelle dysfunctions in pigs

Background:Recently,defects in mammalian oocytes maturation induced by environmental pollution results in the decreasing animal reproduction.Animal exposed to glyphosate is largely unavoidable because glyphosate is one of the most widely used herbicide worldwide due to its high-efficiency and broad-spectrum effects,which causes glyphosate an environmental contaminant found in soil,water and food.During the last few years,the growing and wider use of glyphosate has raised great concerns about its effects of reproductive toxicity.In this study,using porcine models,we investigated effects of glyphosate on organelle functions during oocyte meiosis.Results:The results showed glyphosate exposure disrupted porcine oocyte maturation.Expression levels of cumulus expansion-related genes were interfered,further indicating the meiotic defects.The damaging effects were mediated by destruction of mitochondrial distribution and functions,which induced ROS accumulation and oxidative stress,also indicated by the decreased mRNA expression of related antioxidant enzyme genes.We also found an interference of endoplasmic reticulum(ER)distribution,disturbance of Ca^(2+)homeostasis,as well as fluctuation of ER stress,showing with the reduced ER stress-related mRNA or protein expression,which could indicate the dysfunction of ER for protein processing and signal transduction in glyphosate-exposed oocytes.Moreover,glyphosate exposure induced the disruption of lysosome function for autophagy,showing with the decrease of LAMP2 expression and autophagy-related genes mRNA expression.Additionally,our data showed the distribution of Golgi apparatus and the functions of ribosome were disturbed after glyphosate exposure,which might affect protein synthesis and transport.Conclusions:Collectively,our study showed that exposed to glyphosate could affect animal reproduction by compromising the quality of oocytes through its wide toxic effects on organelle functions.

Lipid-Protein Microinclusions in the Morphological Structures of Organelle Membranes Studied by Fluorescent Confocal Microscopy

Peculiar properties of morphological structures of organelle membranes were studied by fluorescent confocal microscopy. The list of objects in our experiments was represented by mitochondria, chloroplasts and vacuoles. During this study, identification of lipid microinclusions having the form of such lipid-protein structural microformations as lipid-protein microdomains, vesicles and membrane tubular structures (cytoplasmic transvacuolar strands and nanotubes) located in organelle membranes or bound up with them was conducted. Such membrane probes as laurdan, DPH, ANS and bis-ANS were used. Comparison of fluorescence intensity of these membrane probes was conducted. This investigation of the morphological properties of lipid-protein structural microformations was accompanied with analysis of 1) the phase state and 2) dynamics of microviscosity variations in the membrane elements of isolated plant cell organelles. Distributions of laurdan fluorescence generalized polarization (GP) values for the membrane on the whole and for the intensively fluorescing membrane segments were obtained. It was discovered that the microviscosity of intensively fluorescing membrane segments essentially differed from the microviscosity of the rest part of the membrane. In conclusion, some results of the study of peculiar properties of lipid-protein structural microformations related to the structure of organelle membranes and the discoveries made in this investigation are discussed.

The endocytosis and intracellular fate of nanomedicines: Implication for rational design

Nanomedicines employ multiple endocytic pathways to enter cells.Their following fate is interesting,but it is not sufficient understood currently.This review introduces the endocytic pathways,presents new technologies to confirm the specific endocytic pathways and discusses factors for pathway selection.In addition,some intriguing implication about nanomedicine design based on endocytosis will also be discussed at the end.This review may provide new thoughts for the design of novel multifunctional nanomedicines.

Genomic data provides simple evidence for a single origin of life

One hundred and fifty years ago, Charles Darwin’s on the Origin of Species explained the evolution of species through evolution by natural selection. To date, there is no simple piece of evidence demonstrating this concept across species. Chargaff’s first parity rule states that comple-mentary base pairs are in equal proportion across DNA strands. Chargaff’s second parity rule, in-consistently followed across species, states that the base pairs are in equal proportion within DNA strands [G ≈ C, T ≈ A and (G + A) ≈ (C + T)]. Using genomic libraries, we analyzed the extent to which DNA samples followed Chargaff’s second parity rule. In organelle DNA, nucleotide rela-tionships were heteroskedastic. After classifying organelles into chloroplasts and mitochondria, and then into plant, vertebrate, and invertebrate I and II mitochondria, nucleotide relationships were expressed by linear regression lines. All regression lines based on nuclear and organelle DNA crossed at the same point. This is a simple demonstration of a common ancestor across species.

Ferroptosis molecular inducers:A future direction for malignant tumor chemotherapy

Iron-dependent ferroptosis is a form of cell death dependent on iron levels.Cells that undergo ferroptosis have glutathione(GSH)deficiency,reduced Glutathione peroxidase-4(GPX4)activity and intracellular lipid peroxidation,Mitochondria,lysosomes and many signal pathways are involved in the regulation of ferroptosis.More importantly,many tumor cells resistant to other cell death methods exhibit sensitivity to ferroptosis.Moreover,over recent years,a number of ferroptosis-induced drugs have been recommended for the treatment of malignant tumors.Therefore,the study of ferroptosis is of great significance for future cancer treatments.In this review,we discussed the metabolic process of ferroptosis,the role of different organelles,the typical signaling pathways involved in ferroptosis,as well as natural and synthetic compounds that can induce ferroptosis,aiming to point out new conceptual avenues for utilizing ferroptosis in future cancer treatments.

Complete organelle genomes of Sinapis arvensis and their evolutionary implications

Sinapis arvensis,belonging to the genus Sinapis of the family Brassicaceae,has good agronomic characters that make it a valuable genetic resource for crop improvement and is a cytoplasmic source of heterologous cytoplasmic male sterility(CMS).In addition,S.arvensis has played an important role in the evolution of the six major cultivated Brassica species involved in the triangle of U.Using next-generation sequencing,we assembled and revealed the gene composition of S.arvensis cytoplasmic genome.The chloroplast genome comprises 153,590 bp,with 112 individual genes,including 4 r RNA,29 t RNA,and 79 proteincoding genes.The mitochondrial genome comprises 240,024 bp,with 54 genes,including 18 t RNA,three r RNA and 33 protein-coding genes.Genome structure and evolutionary analysis indicated that the sequences of the S.arvensis organellar genomes were more similar to those of Brassica nigra and B.carinata than to those of other Brassicaceae species.Four mitochondrial open reading frames displaying chimeric structural features and encoding hypothetical proteins with transmembrane domains may account for the infertility of Nsa CMS previously derived from somatic cell hybridization between B.napus and S.arvensis.These results will not only contribute to utilize the germplasm resource of S.arvensis,and comprehend the evolution of organelle genomes within the Brassicaceae family,but also help to identify genes conditioning the alloplasmic male sterility of Nsa CMS in B.napus.

Codon evolution in double-stranded organelle DNA: strong regulation of homonucleotides and their analog alternations

In our previous study, complete single DNA strands which were obtained from nuclei, chloroplasts and plant mitochondria obeyed Chargaff’s second parity rule, although those which were obtained from animal mitochondria deviated from the rule. On the other hand, plant mitochondria obeyed another different rule after their classification. Complete single DNA strand sequences obtained from chloroplasts, plant mitochondria, and animal mitochondria, were divided into the coding and non-coding regions. The non-coding region, which was the complementary coding region on the reverse strand, was incorporated as a coding region in the forward strand. When the nucleotide contents of the coding region or non-coding regions were plotted against the composition of the four nucleotides in the complete single DNA strand, it was determined that chloroplast and plant mitochondrial DNA obeyed Chargaff’s second parity rule in both the coding and non-coding regions. However, animal mitochondrial DNA deviated from this rule. In chloroplast and plant mitochondrial DNA, which obey Chargaff’s second parity rule, the lines of regression for G (purine) and C (pyrimidine) intersected with regression lines for A (purine) and T (pyrimidines), respectively, at around 0.250 in all cases. On the other hand, in animal mitochondrial DNA, which deviates from Chargaff’s second parity rule, only regression lines due to the content of homonucleotides or their analogs in the coding or non-coding region against those in the complete single DNA strand intersected at around 0.250 at the horizontal axis. Conversely, the intersection of the two lines of regression (G and A or C and T) against the contents of heteronucleotides or their analogs shifted from 0.25 in both coding and non-coding regions. Nucleotide alternations in chloroplasts and plant mitochondria are strictly regulated, not only by the proportion of homonucleotides and their analogs, but also by the heteronucleotides and their analogs. They are strictly regulated in animal mitochondria only by the content of homonucleotides and their analogs.

Evolution based on genome structure: the “diagonal genome universe”

The ratios of amino acid to the total amino acids and those of nucleotides to the total nucleotides in genes or genomes are suitable indexes to compare whole gene or genome characteristics based on the large number of nucleotides rather than their sequences. As these ratios are strictly calculated from nucleotide sequences, the values are independent of experimental errors. In the present mini-review, the following themes are approached according to the ratios of amino acids and nucleotides to their total numbers in the genome: prebiotic evolution, the chronological precedence of protein and codon formations, genome evolution, Chargaff’s second pa- rity rule, and the origins of life. Amino acid formation might have initially occurred during pre- biotic evolution, the “amino acid world”, and amino acid polymerization might chronologically precede codon formation at the end of prebiotic evolution. All nucleotide alterations occurred synchronously over the genome during biolo- gical evolution. After establishing primitive lives, all nucleotide alterations have been governed by linear formulae in nuclear and organelle genomes consisting of the double-stranded DNA. When the four nucleotide contents against each individual nucleotide content in organelles are expressed by four linear regression lines representing the diagonal lines of a 0.5 square – the “Diagonal Genome Universe”, evolution obeys Chargaff’s second parity rule. The fact that linear regression lines intersect at a single point su- ggests that all species originated from a single life source.

De Novo Organelle Biogenesis in the Cyanobacterium TDX16 Released from the Green Alga <i>Haematococcus pluvialis</i>

It is believed that eukaryotes arise from prokaryotes, which means that organelles can form de novo in prokaryotes. Such events, however, had not been observed previously. Here, we report the biogenesis of organelles in the endosymbiotic cyanobacterium TDX16 (prokaryote) that was released from its senescent/necrotic host cell of green alga Haematococcus pluvialis (eukaryote). Microscopic observations showed that organelle biogenesis in TDX16 initiated with cytoplasm compartmentalization, followed by de-compartmentalization, DNA allocation, and re-compartmentalization, as such two composite organelles-the primitive chloroplast and primitive nucleus sequestering minor and major fractions of cellular DNA respectively were formed. Thereafter, the eukaryotic cytoplasmic matrix was built up from the matrix extruded from the primitive nucleus;mitochondria were assembled in and segregated from the primitive chloroplast, whereby the primitive nucleus and primitive chloroplast matured into the nucleus and chloroplast respectively. While mitochondria subsequently turned into double-membraned vacuoles after matrix degradation. Results of pigment analyses, 16S rRNA and genome sequencing revealed that TDX16 is a phycocyanin-containing cyanobacterium resembling Chroococcidiopsis thermalis, which had acquired 9,017,401 bp DNAs with 10,301 genes from its host. Accordingly, we conclude that organelle biogenesis in TDX16 is achieved by hybridizing the acquired eukaryotic DNAs with its own one and expressing the hybrid genome. The formation of organelles in cyanobacterium TDX16 is the first case of organelle biogenesis in prokaryotes observed so far, which sheds an unprecedented light on eukaryotes and their connections with prokaryotes, and thus has broad implications on biology.

Development of organelle single nucleotide polymorphism (SNP) markers and their application for the identification of cytoplasmic inheritance patterns in Pyropia yezoensis (Bangiales,Rhodophyta)

The genus Pyropia contains several important cultivated species.Genetic research in nori species has mainly focused on the cell nucleus,with few studies on organelles(chloroplast and mitochondria).Due to the high copy numbers of organelles in cells,which influence the development and traits of algae,it is necessary to study their genetic mechanism.In this study,the marine red alga Pyropia yezoensis,an important economic macroalga,was selected as the study object.To investigate organelle(chloroplast and mitochondria)inheritance in P.yezoensis,the wild type RZ(maternal strain)was crossed with the red mutant HT(paternal strain)and 30 color-sectors from 11 F1 gametophytic blades were examined.The complete chloroplast and mitochondrial genomes of the red mutant(HT)were assembled for the first time.One reliable and stable single nucleotide polymorphism(SNP)loci filtrated by bioinformatics analysis was used as a molecular marker for chloroplast and mitochondrial DNA,respectively,in subsequent experiments.PCR amplification and sequence analysis showed that the haplotypes of color-sectors detected were consistent with those of the maternal parent,confirming that both chloroplast and mitochondrial genomes were inherited maternally in P.yezoensis.The inheritance pattern of organelles in P.yezoensis can be used to guide the hybridization and breeding of nori.Additionally,the organelle SNP markers developed in this study can be applied in subsequent genetic research.

A Simple Method for Isolating Chloroplast DNA and Mitochondria DNA from the Same Rapeseed Green Leaf Tissue

In the study, we present a fast, simple and inexpensive protocol for isolating chloroplast and mitochondrial DNA from one rapeseed leaf tissue sample. The chloroplast and mitochondria were separated from the same green leaf tissue by differential centrifugations. The protocol is the first report that isolates plant chloroplast DNA （cpDNA） and mitochondrial DNA （mtDNA） from the same sample homogenate. The organelle DNA yield is 2-10 micrograms per gram of tissue; the DNA was fully restrictable and was successfully used for sequencing.