The presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart.

PURPOSE: Is the presence of cytoplasmic strings (CS) in human blastocysts associated with the probability of clinical pregnancy with fetal heart (CPFH) after transfer. METHODS: This case-control study involved 300 single blastocyst transfers. 150 of these resulted in a CPFH (cases) while 150 did not (controls). All embryos were cultured in Embryoscope+ and AI software (IVY) was used to select the blastocyst with the highest score from the cohort for transfer. An embryologist, blind to the transfer outcome, recorded the CS number, location, and duration of their activity. RESULTS: There was a significant difference in the number of blastocysts that contained CS, with 97.3% of women's blastocysts resulting in +CPFH containing the CS compared to 88.7% of blastocysts in women who did not have a pregnancy (p = 0.007, OR; 4.67, CI 95% 1.5-14.2). CS appeared 2.4 h earlier in embryo development in the +CPFH group compared to their negative counterparts (p = 0.007). There was a significant difference in the average number of CS/blastocyst with a higher number being present in those that achieved a clinical pregnancy (mean: 6.2, SD 2.9) compared to those that did not (mean: 4.6, SD 3.0) (p ≤ 0.0001). There was a significant increase in the number of vesicles seen traveling along the CS with more seen in the blastocysts resulting in a +CPFH (mean: 4.3 SD 2.1) compared to those in the -CPFH group (mean: 3.1, SD 2.1). CONCLUSION: This study has shown that the presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart.

Multi-scale 3D Cryo-Correlative Microscopy for Vitrified Cells.

Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labeled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-hydrated specimens in a variety of healthy and diseased conditions with and without treatments.

EXOSC9 depletion attenuates P-body formation, stress resistance, and tumorigenicity of cancer cells.

Cancer cells adapt to various stress conditions by optimizing gene expression profiles via transcriptional and translational regulation. However, whether and how EXOSC9, a component of the RNA exosome complex, regulates adaptation to stress conditions and tumorigenicity in cancer cells remain unclear. Here, we examined the effects of EXOSC9 depletion on cancer cell growth under various stress conditions. EXOSC9 depletion attenuated growth and survival under various stress conditions in cancer cells. Interestingly, this also decreased the number of P-bodies, which are messenger ribonucleoprotein particles (mRNPs) required for stress adaptation. Meanwhile, EXOSC2/EXOSC4 depletion also attenuated P-body formation and stress resistance with decreased EXOSC9 protein. EXOSC9-mediated stress resistance and P-body formation were found to depend on the intact RNA-binding motif of this protein. Further, RNA-seq analyses identified 343 EXOSC9-target genes, among which, APOBEC3G contributed to defects in stress resistance and P-body formation in MDA-MB-231 cells. Finally, EXOSC9 also promoted xenografted tumor growth of MDA-MB-231 cells in an intact RNA-binding motif-dependent manner. Database analyses further showed that higher EXOSC9 activity, estimated based on the expression of 343 target genes, was correlated with poorer prognosis in some cancer patients. Thus, drugs targeting activity of the RNA exosome complex or EXOSC9 might be useful for cancer treatment.

RPS28B mRNA acts as a scaffold promoting cis-translational interaction of proteins driving P-body assembly.

P-bodies (PBs) are cytoplasmic mRNA-protein (mRNP) granules conserved throughout eukaryotes which are implicated in the repression, storage and degradation of mRNAs. PB assembly is driven by proteins with self-interacting and low-complexity domains. Non-translating mRNA also stimulates PB assembly, however no studies to date have explored whether particular mRNA transcripts are more critical than others in facilitating PB assembly. Previous work revealed that rps28bΔ (small ribosomal subunit-28B) mutants do not form PBs under normal growth conditions. Here, we demonstrate that the RPS28B 3'UTR is important for PB assembly, consistent with it harboring a binding site for the PB assembly protein Edc3. However, expression of the RPS28B 3'UTR alone is insufficient to drive PB assembly. Intriguingly, chimeric mRNA studies revealed that Rps28 protein, translated in cis from an mRNA bearing the RPS28B 3'UTR, physically interacts more strongly with Edc3 than Rps28 protein synthesized in trans. This Edc3-Rps28 interaction in turn facilitates PB assembly. Our work indicates that PB assembly may be nucleated by specific RNA 'scaffolds'. Furthermore, this is the first description in yeast to our knowledge of a cis-translated protein interacting with another protein in the 3'UTR of the mRNA which encoded it, which in turn stimulates assembly of cellular structures.

G3BP1 Is a Tunable Switch that Triggers Phase Separation to Assemble Stress Granules.

The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.

Yb body assembly on the flamenco piRNA precursor transcripts reduces genic piRNA production.

In Drosophila ovarian somatic cells, PIWI-interacting small RNAs (piRNAs) against transposable elements are mainly produced from the ∼180-kb flamenco (flam) locus. flam transcripts are gathered into foci, located close to the nuclear envelope, and processed into piRNAs in the cytoplasmic Yb bodies. The mechanism of Yb body formation remains unknown. Using RNA fluorescence in situ hybridization, we found that in the follicle cells of ovaries the 5'-ends of flam transcripts are usually located in close proximity to the nuclear envelope and outside of Yb bodies, whereas their extended downstream regions mostly overlap with Yb bodies. In flamKG mutant ovaries, flam transcripts containing the first and, partially, second exons but lacking downstream regions are gathered into foci at the nuclear envelope, but Yb bodies are not assembled. Strikingly, piRNAs from the protein-coding gene transcripts accumulate at higher levels in flamKG ovaries indicating that piRNA biogenesis may occur without Yb bodies. We propose that normally in follicle cells, flam downstream transcript regions function not only as a substrate for generation of piRNAs but also as a scaffold for Yb body assembly, which competitively decreases piRNA production from the protein-coding gene transcripts. By contrast, in ovarian somatic cap and escort cells Yb body assembly does not require flam transcription.

Proteasome-Rich PaCS as an Oncofetal UPS Structure Handling Cytosolic Polyubiquitinated Proteins. In Vivo Occurrence, in Vitro Induction, and Biological Role.

In this article, we outline and discuss available information on the cellular site and mechanism of proteasome interaction with cytosolic polyubiquitinated proteins and heat-shock molecules. The particulate cytoplasmic structure (PaCS) formed by barrel-like particles, closely reproducing in vivo the high-resolution structure of 26S proteasome as isolated in vitro, has been detected in a variety of fetal and neoplastic cells, from living tissue or cultured cell lines. Specific trophic factors and interleukins were found to induce PaCS during in vitro differentiation of dendritic, natural killer (NK), or megakaryoblastic cells, apparently through activation of the MAPK-ERK pathway. Direct interaction of CagA bacterial oncoprotein with proteasome was shown inside the PaCSs of a Helicobacter pylori-infected gastric epithelium, a finding suggesting a role for PaCS in CagA-mediated gastric carcinogenesis. PaCS dissolution and autophagy were seen after withdrawal of inducing factors. PaCS-filled cell blebs and ectosomes were found in some cells and may represent a potential intercellular discharge and transport system of polyubiquitinated antigenic proteins. PaCS differs substantially from the inclusion bodies, sequestosomes, and aggresomes reported in proteinopathies like Huntington or Parkinson diseases, which usually lack PaCS. The latter seems more linked to conditions of increased cell proliferation/differentiation, implying an increased functional demand to the ubiquitin⁻proteasome system.

Aggregation-prone Regions in HYPK Help It to Form Sequestration Complex for Toxic Protein Aggregates.

Protein aggregates result from altered structural conformations and they can perturb cellular homeostasis. Prevention mechanisms, which function against protein aggregation by modulatory processes, are diverse and redundant. In this study, we have characterized Huntingtin interacting protein K (HYPK) as a global aggregation-regulatory protein. We report the mechanistic details of how HYPK's aggregation-prone regions allow it to sense and prevent other toxic protein's aggregation by forming unique annular-shaped sequestration complexes. Screenings for interacting partners of different aggregation-prone proteins identify HYPK as a global interacting partner/regulator of Huntingtin97Qexon1, α-Synuclein-A53T and Superoxide dismutase1-G93A. C-terminal hydrophobic region in HYPK makes direct contacts with aggregates to initiate the formation of sequestration complexes. HYPK acts as aggregate sensor by existing in a seeded amyloid-like state which also favors its own concentration-dependent self-oligomerization. Oligomerization of HYPK leads to annular and non-fibrillar/amorphous aggregates. Two hydrophobic segments in the C-terminus of HYPK are responsible for its own aggregations. Self-association of HYPK follows seed nucleation, in which oligomeric HYPK seeds nucleate to annular structures. Annular oligomers of HYPK fuse with each other to form amorphous aggregates. HYPK shows differential interactions with aggregation-prone and non-aggregating proteins, as it preferentially binds to aggregation-prone proteins with higher affinity than native/non-aggregating proteins. This favors the formation of HYPK's sequestration complexes both in cytosol and in ribosome. Besides having aggregation-preventive property, HYPK also reduces the cellular level of toxic proteins. In vivo, HYPK sequestration complexes prevent the formation of toxic protein aggregates to physiologically show positive impact on cell survival and restoration of normal cell physiology.

Sorting Tubules Regulate Blood-Brain Barrier Transcytosis.

Transcytosis across the blood-brain barrier (BBB) regulates key processes of the brain, but the intracellular sorting mechanisms that determine successful receptor-mediated transcytosis in brain endothelial cells (BECs) remain unidentified. Here, we used Transferrin receptor-based Brain Shuttle constructs to investigate intracellular transport in BECs, and we uncovered a pathway for the regulation of receptor-mediated transcytosis. By combining live-cell imaging and mathematical modeling in vitro with super-resolution microscopy of the BBB, we show that intracellular tubules promote transcytosis across the BBB. A monovalent construct (sFab) sorted for transcytosis was localized to intracellular tubules, whereas a bivalent construct (dFab) sorted for degradation formed clusters with impaired transport along tubules. Manipulating tubule biogenesis by overexpressing the small GTPase Rab17 increased dFab transport into tubules and induced its transcytosis in BECs. We propose that sorting tubules regulate transcytosis in BECs and may be a general mechanism for receptor-mediated transport across the BBB.

Loukoumasomes Are Distinct Subcellular Structures from Rods and Rings and Are Structurally Associated with MAP2 and the Nuclear Envelope in Retinal Cells.

"Rods and rings" (RR) and loukoumasomes are similarly shaped, subcellular macromolecular structures with as yet unknown function. RR, so named because of their shape, are formed in response to inhibition in the GTP or CTP synthetic pathways and are highly enriched in the two key enzymes of the nucleotide synthetic pathway. Loukoumasomes also occur as linear and toroidal bodies and were initially inferred to be the same as RR, largely due to their shared shape and size and the fact that it was unclear if they shared the same subcomponents. In human retinoblastoma tissue and cells we have observed toroidal, perinuclear, macromolecular structures of similar size and antigenicity to those previously reported in neurons (neuronal-loukoumasomes). To further characterize the subcomponents of the retinal-loukoumasomes, confocal analysis following immunocytochemical staining for alpha-tubulin, beta-III tubulin and detyrosinated tubulin was performed. These studies indicate that retinal-loukoumasomes are enriched for beta-III tubulin and other tubulins associated with microtubules. Immunofluorescence together with the in situ proximity ligation assay (PLA), confirmed that beta-III tubulin colocalized with detyrosinated tubulin within loukoumasomes. Our results indicate that these tissues contain only loukoumasomes because these macromolecular structures are immunoreactive with an anti-tubulin antibody but are not recognized by the prototype anti-RR/inosine monophosphate dehydrogenase (IMPDH) antibody (It2006). To further compare the RR and retinal-loukoumasomes, retinoblastoma cells were exposed to the IMPDH-inhibitor ribavirin, a drug known to induce the formation of RR. In contrast to RR, the production of retinal-loukoumasomes was unaffected. Coimmunostaining of Y79 cells for beta-III tubulin and IMPDH indicate that these cells, when treated with ribavirin, can contain both retinal-loukoumasomes and RR and that these structures are antigenically distinct. Subcellular fractionation studies indicate that ribavirin increased the RR subcomponent, IMPDH, in the nuclear fraction of Y79 cells from 21.3 ± 5.8% (0 mM ribavirin) to 122.8 ± 7.9% (1 mM ribavirin) while the subcellular localization of the retinal-loukoumasome subcomponent tubulin went unaltered. Further characterization of retinal-loukoumasomes in retinoblastoma cells reveals that they are intimately associated with lamin folds within the nuclear envelope. Using immunofluorescence and the in situ PLA in this cell type, we have observed colocalization of beta-III tubulin with MAP2. As MAP2 is a microtubule-associated protein implicated in microtubule crosslinking, this supports a role for microtubule crosslinkers in the formation of retinal-loukoumasomes. Together, these results suggest that loukoumasomes and RR are distinct subcellular macromolecular structures, formed by different cellular processes and that there are other loukoumasome-like structures within retinal tissues and cells.

Non-dividing Cell Virtosomes Affect In Vitro and In Vivo Tumour Cell Replication.

In vitro studies of partially purified virtosomes from rat liver showed inhibition of cell multiplication in four normal and two tumour cell lines. In vivo, the liver virtosomes slowed tumour growth and limited metastases in rats bearing DHD/K12-PROb cell initiated tumours.

A Historical and Evolutionary Perspective on Circulating Nucleic Acids and Extracellular Vesicles: Circulating Nucleic Acids as Homeostatic Genetic Entities.

The quantitative and qualitative differences of circulating nucleic acids (cirNAs) between healthy and diseased individuals have motivated researchers to utilize these differences in the diagnosis and prognosis of various pathologies. The position maintained here is that reviewing the rather neglected early work associated with cirNAs and extracellular vesicles (EVs) is required to fully describe the nature of cirNAs. This review consists of an empirically up-to-date schematic summary of the major events that developed and integrated the concepts of heredity, genetic information and cirNAs. This reveals a clear pattern implicating cirNA as a homeostatic entity or messenger of genetic information. The schematic summary paints a picture of how cirNAs may serve as homeostatic genetic entities that promote synchrony of both adaptation and damage in tissues and organs depending on the source of the message.

Chaperone molecules concentrate together with the ubiquitin-proteasome system inside particulate cytoplasmic structures: possible role in metabolism of misfolded proteins.

Ubiquitin-proteasome system (UPS) proteins and proteolytic activity are localized in a recently identified cytoplasmic structure characterized by accumulation of barrel-like particles, which is known as the particulate cytoplasmic structure (PaCS). PaCSs have been detected in neoplastic, preneoplastic, chronically infected, and fetal cells, which produce high amounts of misfolded proteins to be degraded by the UPS. Chaperone molecules are crucial in the early stages of handling misfolded proteins; therefore, we searched for these molecules in PaCSs. Heat shock proteins (Hsp), Hsp90, Hsp70, Hsp40, and Bcl-2-associated athanogene (Bag)3 chaperones, although not Bag6, were selectively concentrated into PaCSs of several cell lines and ex vivo fetal or neoplastic cells. Present findings point to PaCSs as an integrated, active UPS center well equipped for metabolism of misfolded proteins, especially in cells under physiological (fetal development) or pathological (neoplasia or inflammation) stress.

Plasma membrane-associated platforms: dynamic scaffolds that organize membrane-associated events.

Specialized regions of the plasma membrane dedicated to diverse cellular processes, such as vesicle exocytosis, extracellular matrix remodeling, and cell migration, share a few cytosolic scaffold proteins that associate to form large plasma membrane-associated platforms (PMAPs). PMAPs organize signaling events and trafficking of membranes and molecules at specific membrane domains. On the basis of the intrinsic disorder of the proteins constituting the core of these PMAPs and of the dynamics of these structures at the periphery of motile cells, we propose a working model for the assembly and turnover of these platforms.

Somatic insulin signaling regulates a germline starvation response in Drosophila egg chambers.

Egg chambers from starved Drosophila females contain large aggregates of processing (P) bodies and cortically enriched microtubules. As this response to starvation is rapidly reversed upon re-feeding females or culturing egg chambers with exogenous bovine insulin, we examined the role of endogenous insulin signaling in mediating the starvation response. We found that systemic Drosophila insulin-like peptides (dILPs) activate the insulin pathway in follicle cells, which then regulate both microtubule and P body organization in the underlying germline cells. This organization is modulated by the motor proteins Dynein and Kinesin. Dynein activity is required for microtubule and P body organization during starvation, while Kinesin activity is required during nutrient-rich conditions. Blocking the ability of egg chambers to form P body aggregates in response to starvation correlated with reduced progeny survival. These data suggest a potential mechanism to maximize fecundity even during periods of poor nutrient availability, by mounting a protective response in immature egg chambers.

Assemblages: functional units formed by cellular phase separation.

The partitioning of intracellular space beyond membrane-bound organelles can be achieved with collections of proteins that are multivalent or contain low-complexity, intrinsically disordered regions. These proteins can undergo a physical phase change to form functional granules or other entities within the cytoplasm or nucleoplasm that collectively we term "assemblage." Intrinsically disordered proteins (IDPs) play an important role in forming a subset of cellular assemblages by promoting phase separation. Recent work points to an involvement of assemblages in disease states, indicating that intrinsic disorder and phase transitions should be considered in the development of therapeutics.

Accumulation of heme oxygenase-1 (HSP32) in Xenopus laevis A6 kidney epithelial cells treated with sodium arsenite, cadmium chloride or proteasomal inhibitors.

The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats.

CCHCR1 interacts with EDC4, suggesting its localization in P-bodies.

Coiled-coil alpha-helical rod protein 1 (CCHCR1) is suggested as a candidate biomarker for psoriasis for more than a decade but its function remains poorly understood because of the inconsistent findings in the literature. CCHCR1 protein is suggested to be localized in the cytoplasm, nucleus, mitochondria, or centrosome and to regulate various cellular functions, including steroidogenesis, proliferation, differentiation, and cytoskeleton organization. In this study, we attempted to find a consensus between these findings by identifying the interaction partners of CCHCR1 using co-immunoprecipiation with a stable cell line expressing EGFP-tagged CCHCR1. Out of more than 100 co-immunoprecipitants identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), the enhancer of mRNA-decapping protein 4 (EDC4), which is a processing body (P-body) component, was particularly found to be the major interacting partner of CCHCR1. Confocal imaging confirmed the localization of CCHCR1 in P-bodies and its N-terminus is required for this subcellular localization, suggesting that CCHCR1 is a novel P-body component. As P-bodies are the site for mRNA metabolism, our findings provide a molecular basis for the function of CCHCR1, any disruption of which may affect the transcriptome of the cell, and causing abnormal cell functions.

LMKB/MARF1 localizes to mRNA processing bodies, interacts with Ge-1, and regulates IFI44L gene expression.

The mRNA processing body (P-body) is a cellular structure that regulates the stability of cytoplasmic mRNA. MARF1 is a murine oocyte RNA-binding protein that is associated with maintenance of mRNA homeostasis and genomic stability. In this study, autoantibodies were used to identify Limkain B (LMKB), the human orthologue of MARF1, as a P-body component. Indirect immunofluorescence demonstrated that Ge-1 (a central component of the mammalian core-decapping complex) co-localized with LMKB in P-bodies. Two-hybrid and co-immunoprecipitation assays were used to demonstrate interaction between Ge-1 and LMKB. The C-terminal 120 amino acids of LMKB mediated interaction with Ge-1 and the N-terminal 1094 amino acids of Ge-1 were required for interaction with LMKB. LMKB is the first protein identified to date that interacts with this portion of Ge-1. LMKB was expressed in human B and T lymphocyte cell lines; depletion of LMKB increased expression of IFI44L, a gene that has been implicated in the cellular response to Type I interferons. The interaction between LMKB/MARF1, a protein that contains RNA-binding domains, and Ge-1, which interacts with core-decapping proteins, suggests that LMKB has a role in the regulation of mRNA stability. LMKB appears to have different functions in different cell types: maintenance of genomic stability in developing oocytes and possible dampening of the inflammatory response in B and T cells.

Cytoplasmic capes are nuclear envelope intrusions that are enriched in endosomal proteins and depend upon ßH-spectrin and Annexin B9.

It is increasingly recognized that non-erythroid spectrins have roles remote from the plasma membrane, notably in endomembrane trafficking. The large spectrin isoform, ßH, partners with Annexin B9 to modulate endosomal processing of internalized proteins. This modulation is focused on the early endosome through multivesicular body steps of endocytic processing and loss of either protein appears to cause a traffic jam before removal of ubiquitin at the multivesicular body. We previously reported that ßH/Annexin B9 influenced EGF receptor signaling. While investigating this effect we noticed that mSptiz, the membrane bound precursor of the secreted EGF receptor ligand sSpitz, is located in striking intrusions of the nuclear membrane. Here we characterize these structures and identify them as 'cytoplasmic capes', which were previously identified in old ultrastructural studies and probably coincide with recently recognized sites of non-nuclear-pore RNA export. We show that cytoplasmic capes contain multiple endosomal markers and that their existence is dependent upon ßH and Annexin B9. Diminution of these structures does not lead to a change in mSpitz processing. These results extend the endosomal influence of ßH and its partner Annexin B9 to this unusual compartment at the nuclear envelope.