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Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a quality control system called ER-associated protein degradation (ERAD). However, it is unknown how misfolded proteins in the inner nuclear membrane (INM), a specialized ER subdomain, are degraded. We used a quantitative proteomics approach to reveal an ERAD branch required for INM protein quality control in yeast. This branch involved the integral membrane proteins Asi1, Asi2, and Asi3, which assembled into an Asi complex. Besides INM misfolded proteins, the Asi complex promoted the degradation of functional regulators of sterol biosynthesis. Asi-mediated ERAD was required for ER homeostasis, which suggests that spatial segregation of protein quality control systems contributes to ER function.

Piwi protein Miwi is shown to be a small RNA-guided RNase in mice; disrupting the catalytic activity of Miwi results in increased accumulation of LINE1 retrotransposon transcripts and male infertility. Transposon silencing by Piwi proteins The combination of Piwi proteins and their associated Piwi-interacting RNAs (piRNAs) mediates epigenetic transposon silencing in animal germlines. Piwi proteins are predicted to be endonucleases, but the significance of this activity had not been demonstrated in vivo . The laboratories of Dónal O'Carroll and Ramesh Pillai have now made mouse models in which residues expected to be critical for nuclease activity in the three mouse Piwi homologues, Mili, Miwi and Miwi2, are mutated. The mutant mice show phenotypic differences. The Mili and Miwi mutants are defective in piRNA production, transposon silencing and fertility, whereas the Miwi2 mutant has normal piRNA levels, seems to undergo piRNA amplification and silences transposons. These studies highlight distinctions between the murine enzymes responsible for piRNA biogenesis. Repetitive-element-derived Piwi-interacting RNAs (piRNAs) 1 , 2 act together with Piwi proteins Mili (also known as Piwil2) and Miwi2 (also known as Piwil4) in a genome defence mechanism that initiates transposon silencing via DNA methylation in the mouse male embryonic germ line. This silencing depends on the participation of the Piwi proteins in a slicer-dependent piRNA amplification pathway and is essential for male fertility 3 , 4 . A third Piwi family member, Miwi (also known as Piwil1), is expressed in specific postnatal germ cells and associates with a unique set of piRNAs of unknown function 5 , 6 , 7 . Here we show that Miwi is a small RNA-guided RNase (slicer) that requires extensive complementarity for target cleavage in vitro . Disruption of its catalytic activity in mice by a single point mutation causes male infertility, and mutant germ cells show increased accumulation of LINE1 retrotransposon transcripts. We provide evidence for Miwi slicer activity directly cleaving transposon messenger RNAs, offering an explanation for the continued maintenance of repeat-derived piRNAs long after transposon silencing is established in germline stem cells. Furthermore, our study supports a slicer-dependent silencing mechanism that functions without piRNA amplification. Thus, Piwi proteins seem to act in a two-pronged mammalian transposon silencing strategy: one promotes transcriptional repression in the embryo, the other reinforces silencing at the post-transcriptional level after birth.

Centrosomes are essential organelles with functions in microtubule organization that duplicate once per cell cycle. The first step of centrosome duplication is the daughter centriole formation followed by the pericentriolar material recruitment to this centriole. This maturation step was termed centriole-to-centrosome conversion. It was proposed that CEP295-dependent recruitment of pericentriolar proteins drives centriole conversion. Here we show, based on the analysis of proteins that promote centriole biogenesis, that the developing centriole structure helps drive centriole conversion. Depletion of the luminal centriole protein CEP44 that binds to the A-microtubules and interacts with POC1B affecting centriole structure and centriole conversion, despite CEP295 binding to centrioles. Impairment of POC1B, TUBE1 or TUBD1, which disturbs integrity of centriole microtubules, also prevents centriole-to-centrosome conversion. We propose that the CEP295, CEP44, POC1B, TUBE1 and TUBD1 centriole biogenesis pathway that functions in the centriole lumen and on the cytoplasmic side is essential for the centriole-to-centrosome conversion. During cell division, centrosomes duplicate and newly formed centrioles must undergo centriole-to-centrosome conversion, but the molecular details are unclear. Here, the authors report that the centriole microtubule-triplet 9-fold structure scaffolds pericentriolar proteins and permits the conversion of centrioles to fully functional centrosomes.

During establishment of arbuscular mycorrhizal symbioses, fungal hyphae invade root cells producing transient tree-like structures, the arbuscules, where exchange of photosynthates for soil minerals occurs. Arbuscule formation and collapse lead to rapid production and degradation of plant and fungal membranes, their spatiotemporal dynamics directly influencing nutrient exchange. We determined the ultra-structural details of both membrane surfaces and the interstitial apoplastic matrix by transmission electron microscopy tomography during growth and senescence of Rhizophagus irregularis arbuscules in rice. Invasive growth of arbuscular hyphae was associated with abundant fungal membrane tubules (memtubs) and plant peri-arbuscular membrane evaginations. Similarly, the phylogenetically distant arbuscular mycorrhizal fungus, Gigaspora rosea , and the fungal maize pathogen, Ustilago maydis , developed memtubs while invading host cells, revealing structural commonalities independent of the mutualistic or parasitic outcome of the interaction. Additionally, extracellular vesicles formed continuously in the peri-arbuscular interface from arbuscule biogenesis to senescence, suggesting an involvement in inter-organismic signal and nutrient exchange throughout the arbuscule lifespan. The symbiosis of mycorrhizal fungi and roots involves invasion of plant cells, followed by collapse of the intracellular fungal arbuscules. Ultra-structural details of this interaction show continuous formation of extracellular vesicles at the peri-arbuscular space.

One of the first steps in mitotic spindle assembly is the dissolution of the centrosome linker followed by centrosome separation driven by EG5, a tetrameric plus-end-directed member of the kinesin-5 family. However, even in the absence of the centrosome linker, the two centrosomes are kept together by an ill-defined microtubule-dependent mechanism. Here we show that KIFC3, a minus-end-directed kinesin-14, provides microtubule-based centrosome cohesion. KIFC3 forms a homotetramer that pulls the two centrosomes together via a specific microtubule network. At mitotic onset, KIFC3 activity becomes the main driving force of centrosome cohesion to prevent premature spindle formation after linker dissolution as it counteracts the increasing EG5-driven pushing forces. KIFC3 is eventually inactivated by NEver in mitosis-related Kinase 2 (NEK2) to enable EG5-driven bipolar spindle assembly. We further show that persistent centrosome cohesion in mitosis leads to chromosome mis-segregation. Our findings reveal a mechanism of spindle assembly that is evolutionary conserved from yeast to humans. Hata et al. report that minus-end-directed kinesin KIFC3 promotes centrosome cohesion at the onset of mitosis, counteracting centrosome separation driven by the plus-end-directed kinesin EG5.

Liver-generated plasma Apolipoprotein E (ApoE)-containing lipoproteins (LPs) (ApoE-LPs) play central roles in lipid transport and metabolism. Perturbations of ApoE can result in several metabolic disorders and ApoE genotypes have been associated with multiple diseases. ApoE is synthesized at the endoplasmic reticulum and transported to the Golgi apparatus for LP assembly; however, the ApoE-LPs transport pathway from there to the plasma membrane is largely unknown. Here, we established an integrative imaging approach based on a fully functional fluorescently tagged ApoE. We found that newly synthesized ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and CD63-positive intraluminal vesicles (ILVs), which are precursors of extracellular vesicles (EVs), along the late endosomal trafficking route in a microtubule-dependent manner. A fraction of ApoE-LPs associated with CD63-positive EVs appears to be co-transmitted from cell to cell. Given the important role of ApoE in viral infections, we employed as well-studied model the hepatitis C virus (HCV) and found that the viral replicase component nonstructural protein 5A (NS5A) is enriched in ApoE-containing ILVs. Interaction between NS5A and ApoE is required for the efficient release of ILVs containing HCV RNA. These vesicles are transported along the endosomal ApoE egress pathway. Taken together, our data argue for endosomal egress and transmission of hepatic ApoE-LPs, a pathway that is hijacked by HCV. Given the more general role of EV-mediated cell-to-cell communication, these insights provide new starting points for research into the pathophysiology of ApoE-related metabolic and infection-related disorders.

Neurofilament light chain (NfL), released during central nervous injury, has evolved as a powerful serum marker of disease severity in many neurological disorders, including infectious diseases. So far NfL has not been assessed in cerebral malaria in human or its rodent model experimental cerebral malaria (ECM), a disease that can lead to fatal brain edema or reversible brain edema. In this study we assessed if NfL serum levels can also grade disease severity in an ECM mouse model with reversible (n = 11) and irreversible edema (n = 10). Blood–brain-barrier disruption and brain volume were determined by magnetic resonance imaging. Neurofilament density volume as well as structural integrity were examined by electron microscopy in regions of most severe brain damage (olfactory bulb (OB), cortex and brainstem). NfL plasma levels in mice with irreversible edema (317.0 ± 45.01 pg/ml) or reversible edema (528.3 ± 125.4 pg/ml) were significantly increased compared to controls (103.4 ± 25.78 pg/ml) by three to five fold, but did not differ significantly in mice with reversible or irreversible edema. In both reversible and irreversible edema, the brain region most affected was the OB with highest level of blood–brain-barrier disruption and most pronounced decrease in neurofilament density volume, which correlated with NfL plasma levels (r = − 0.68, p = 0.045). In cortical and brainstem regions neurofilament density was only decreased in mice with irreversible edema and strongest in the brainstem. In reversible edema NfL plasma levels, MRI findings and neurofilament volume density normalized at 3 months’ follow-up. In conclusion, NfL plasma levels are elevated during ECM confirming brain damage. However, NfL plasma levels fail short on reliably indicating on the final outcomes in the acute disease stage that could be either fatal or reversible. Increased levels of plasma NfL during the acute disease stage are thus likely driven by the anatomical location of brain damage, the olfactory bulb, a region that serves as cerebral draining pathway into the nasal lymphatics.

Unicellular organisms or cells of metazoans often change their morphology during development or life cycle progression to adapt to environmental changes. Malaria parasites undergo a striking range of morphological transformations as they navigate through the different environments of mammalian hosts and mosquito vectors. These developmental transitions are accompanied by changes in the subcellular organelles. Here, this work introduces an unbiased approach using volume electron microscopy (vEM) to facilitate cluster analyses of morphometric parameters during developmental transformation. Investigating the transformation of fertilized Plasmodium zygotes into the motile ookinetes with three complementary vEM techniques revealed intimate mitochondrion‐nucleus interactions, different microtubule arrangements, elongated shapes of micronemes and their close interaction with the apicoplast. The presented data and approach provide an open‐access subcellular atlas for ookinete development to aid mechanistic molecular insights from reverse genetic studies and a framework for the ultrastructural study of other parasite stages and developmental transitions in general. This article presents a detailed ultrastructural cell atlas of Plasmodium ookinete development using advanced volume electron microscopy (v) techniques. It defines seven distinct developmental stages, revealing complex organelle interactions and offering new insights into parasite differentiation. The work highlights the usage of morphometric parameters obtained from vEM for cell stage determination.

Liver sinusoidal endothelial cells (LSECs) critically regulate homeostatic liver function and liver pathogenesis. However, the isolation of LSECs remains a major technological bottleneck in studying molecular mechanisms governing LSEC functions. Current techniques to isolate LSECs, relying on perfusion-dependent liver digestion, are cumbersome with limited throughput. We here describe a perfusion-independent high-throughput procedure to isolate LSECs with high purity. Indifferently from previous perfusion-independent approaches, chopped liver tissue was incubated in the digestion mix for 30 minutes with intermittent mixing with a serological pipette. This led to the safeguarding of LSEC integrity and yielded 10 ± 1.0 million LSECs per adult mouse liver, which is far higher than previous perfusion-independent protocols and comparable yield to established perfusion-dependent protocols for isolating LSECs. Combining magnetic and fluorescence-activated cell sorting (FACS), LSECs from different zones of the hepatic sinusoid can now be isolated in high numbers in less than two hours for downstream applications including proteomics. Our protocol enables the isolation of LSECs from fibrotic liver tissues from mice and healthy liver tissues from higher vertebrate species (pigs), where traditional perfusion-based digestion protocols have very limited application. In conclusion, these technical advancements reduce post-mortem changes in the LSEC state and aid in reliable investigation of LSEC functions. A perfusion-independent method to isolate ultrapure liver sinusoidal endothelial cells for downstream applications, including primary cultures and hepatic zone-specific endothelial cell isolations.

Distal renal tubular acidosis (dRTA) is a common kidney dysfunction characterized by impaired acid secretion via urine. Previous studies revealed that α-intercalated cells of dRTA patients express mutated forms of human kidney anion exchanger 1 (kAE1) which result in inefficient plasma membrane targeting or diminished expression levels of kAE1. However, the precise dRTA-causing processes are inadequately understood, and alternative model systems are helpful tools to address kAE1-related questions in a fast and inexpensive way. In contrast to a previous study, we successfully expressed full-length kAE1 in Saccharomyces cerevisiae . Using advanced microscopy techniques as well as different biochemical and functionality assays, plasma membrane localization and biological activity were confirmed for the heterologously expressed anion transporter. These findings represent first important steps to use the potential of yeast as a model organism for studying trafficking, activity, and degradation of kAE1 and its mutant variants in the future. Saccharomyces cerevisiae has been frequently used to study biogenesis, functionality, and intracellular transport of various renal proteins, including ion channels, solute transporters, and aquaporins. Specific mutations in genes encoding most of these renal proteins affect kidney function in such a way that various disease phenotypes ultimately occur. In this context, human kidney anion exchanger 1 (kAE1) represents an important bicarbonate/chloride exchanger which maintains the acid-base homeostasis in the human body. Malfunctions in kAE1 lead to a pathological phenotype known as distal renal tubular acidosis (dRTA). Here, we evaluated the potential of baker's yeast as a model system to investigate different cellular aspects of kAE1 physiology. For the first time, we successfully expressed yeast codon-optimized full-length versions of tagged and untagged wild-type kAE1 and demonstrated their partial localization at the yeast plasma membrane (PM). Finally, pH and chloride measurements further suggest biological activity of full-length kAE1, emphasizing the potential of S. cerevisiae as a model system for studying trafficking, activity, and/or degradation of mammalian ion channels and transporters such as kAE1 in the future. IMPORTANCE Distal renal tubular acidosis (dRTA) is a common kidney dysfunction characterized by impaired acid secretion via urine. Previous studies revealed that α-intercalated cells of dRTA patients express mutated forms of human kidney anion exchanger 1 (kAE1) which result in inefficient plasma membrane targeting or diminished expression levels of kAE1. However, the precise dRTA-causing processes are inadequately understood, and alternative model systems are helpful tools to address kAE1-related questions in a fast and inexpensive way. In contrast to a previous study, we successfully expressed full-length kAE1 in Saccharomyces cerevisiae . Using advanced microscopy techniques as well as different biochemical and functionality assays, plasma membrane localization and biological activity were confirmed for the heterologously expressed anion transporter. These findings represent first important steps to use the potential of yeast as a model organism for studying trafficking, activity, and degradation of kAE1 and its mutant variants in the future.