The seed storage proteins of soybean (Glycine max) are composed mainly of glycinin (11S globulin) and β-conglycinin (7S globulin). The subunits of glycinin (A1aB1b, A1bB2, A2B1a, A3B4, and A5A4B3) are synthesized as a single polypeptide precursor. These precursors are assembled into trimers with a random combination of subunits in the endoplasmic reticulum, and are sorted to the protein storage vacuoles. Proteins destined for transport to protein storage vacuoles possess a vacuolar sorting determinant, and in this regard, the A1aB1b subunit contains a C-terminal peptide that is sufficient for its sorting to protein storage vacuoles. The A3B4 subunit, however, lacks a corresponding C-terminal sorting determinant. In this study, we found that, unlike the A1aB1b subunit, the A3B4 subunit does not bind to previously reported vacuolar sorting receptors. Despite this difference, we observed that the A3B4 subunit is sorted to protein storage vacuoles in a transgenic soybean line expressing the A3B4 subunit of glycinin. These results indicate that a protein storage vacuolar sorting mechanism that functions independently of the known vacuolar sorting receptors in seeds might be present in soybean seeds.

Saccharomyces cerevisiae is a single-celled fungal microorganism. S. cerevisiae-derived vacuoles are closely related to mammalian lysosomes, which play a role in the degradation of macromolecules by various hydrolytic enzymes. This study evaluated the anti-inflammatory efficacy of S. cerevisiae-vacuoles by inhibiting inflammatory mediators induced by lipopolysaccharide (LPS). The results showed that treatment with 5, 10, and 20 µg/mL of S. cerevisiae-derived vacuoles almost completely inhibited the LPS-induced expression of iNOS protein and mRNA. Moreover, vacuoles significantly reduced the mRNA expression of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6) in LPS-stimulated macrophages compared to the control cells. The immunofluorescence analysis confirmed that S. cerevisiae-derived vacuoles inhibited the translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in LPS-stimulated cells. Taken together, the treatment with S. cerevisiae-derived vacuoles alone activated macrophages, but LPS-activated macrophages modulated pro-inflammatory mediators by downregulating the NF-κB pathway. These results suggest that S. cerevisiae-derived vacuoles may have therapeutic potential in the treatment of inflammatory diseases. In conclusion, our study provides new insights into the immunomodulatory effects of S. cerevisiae-derived vacuoles and their potential as a novel anti-inflammatory agent. IMPORTANCE This study investigates the potential of using vacuoles derived from the yeast Saccharomyces cerevisiae as a new anti-inflammatory therapy. Inflammation is a natural response of the immune system to invading pathogens, but when it is dysregulated, it can lead to chronic diseases. The researchers found that treating macrophages with vacuoles significantly reduced the production of pro-inflammatory cytokines and iNOS, markers of inflammation when they were stimulated with lipopolysaccharide. The study also showed that vacuoles inhibited the NF-κB signaling pathway, which is involved in the induction of pro-inflammatory cytokines in macrophages. These findings suggest that S. cerevisiae-derived vacuoles may have potential as a new therapeutic agent for regulating the inflammatory response in various diseases. Further studies are needed to evaluate the efficacy and safety of vacuoles in vivo and to elucidate the underlying mechanisms of their anti-inflammatory effects.

In this retrospective study of 873 men enrolled for assisted reproduction techniques, relationships between sperm quality parameters, motile sperm organelle morphology examination (MSOME), DNA damage and live birth rate were evaluated. The presence of vacuoles in the sperm heads was detected by MSOME. Either chromatin decondensation or DNA fragmentation was used to study DNA damage. Results show that age significantly affected some of the examined parameters. In particular, sperm concentration was positively correlated (R = 0.088; P = 0.01) and chromatin decondensation was negatively correlated (R = -0.102; P = 0.003) with age. Furthermore, live birth rate was significantly lower in men aged 40 years or older (P < 0.02) compared with the younger age groups. The presence of sperm head vacuoles was not associated with head morphology, main sperm quality parameters, DNA fragmentation and live birth rate. Considering sperm heads in relation to the shape (normal/abnormal) and vacuoles (presence/absence), no significant variations in the occurrence of vacuoles in either normal or abnormal heads were found. These data suggest that vacuoles are physiological features that do not alter sperm functionality, and it seems that MSOME is not necessary for increasing the success of assisted reproduction techniques.

Bacterial pathogens like Salmonella and Legionella establish intracellular niches in host cells known as bacteria-containing vacuoles. In these vacuoles, bacteria can survive and replicate. Ubiquitin-dependent selective autophagy is a host defense mechanism to counteract infection by invading pathogens. The Legionella effector protein RavZ interferes with autophagy by irreversibly deconjugating LC3, an autophagy-related ubiquitin-like protein, from a phosphoglycolipid phosphatidylethanolamine. Using a co-infection system with Salmonella, we show here that Legionella RavZ interferes with ubiquitin recruitment to the Salmonella-containing vacuoles. The inhibitory activity is dependent on the same catalytic residue of RavZ that is involved in LC3 deconjugation. In semi-permeabilized cells infected with Salmonella, external addition of purified RavZ protein, but not of its catalytic mutant, induced removal of ubiquitin associated with Salmonella-containing vacuoles. The RavZ-mediated restriction of ubiquitin recruitment to Salmonella-containing vacuoles took place in the absence of the host system required for LC3 conjugation. These observations suggest the possibility that the targets of RavZ deconjugation activity include not only LC3, but also ubiquitin.

Plant cells may contain two functionally distinct vacuolar compartments. Membranes of protein storage vacuoles (PSV) are marked by the presence of alpha-tonoplast intrinsic protein (TIP), whereas lytic vacuoles (LV) are marked by the presence of gamma-TIP. Mechanisms for sorting integral membrane proteins to the different vacuoles have not been elucidated. Here we study a chimeric integral membrane reporter protein expressed in tobacco suspension culture protoplasts whose traffic was assessed biochemically by following acquisition of complex Asn-linked glycan modifications and proteolytic processing, and whose intracellular localization was determined with confocal immunofluorescence. We show that the transmembrane domain of the plant vacuolar sorting receptor BP-80 directs the reporter protein via the Golgi to the LV prevacuolar compartment, and attaching the cytoplasmic tail (CT) of gamma-TIP did not alter this traffic. In contrast, the alpha-TIP CT prevented traffic of the reporter protein through the Golgi and caused it to be localized in organelles separate from ER and from Golgi and LV prevacuolar compartment markers. These organelles had a buoyant density consistent with vacuoles, and alpha-TIP protein colocalized in them with the alpha-TIP CT reporter protein when the two were expressed together in protoplasts. These results are consistent with two separate pathways to vacuoles for membrane proteins: a direct ER to PSV pathway, and a separate pathway via the Golgi to the LV.

Many of the mechanisms by which organelles are inherited by spores during meiosis are not well understood. Dramatic chromosome motion and bouquet formation are evolutionarily conserved characteristics of meiotic chromosomes. The budding yeast bouquet genes (NDJ1, MPS3, CSM4) mediate these movements via telomere attachment to the nuclear envelope (NE). Here, we report that during meiosis the NE is in direct contact with vacuoles via nucleus-vacuole junctions (NVJs). We show that in meiosis NVJs are assembled through the interaction of the outer NE-protein Nvj1 and the vacuolar membrane protein Vac8. Notably, NVJs function as diffusion barriers that exclude the nuclear pore complexes, the bouquet protein Mps3 and NE-tethered telomeres from the outer nuclear membrane and nuclear ER, resulting in distorted NEs during early meiosis. An increase in NVJ area resulting from Nvj1-GFP overexpression produced a moderate bouquet mutant-like phenotype in wild-type cells. NVJs, as the vacuolar contact sites of the nucleus, were found to undergo scission alongside the NE during meiotic nuclear division. The zygotic NE and NVJs were partly segregated into 4 spores. Lastly, new NVJs were also revealed to be synthesized de novo to rejoin the zygotic NE with the newly synthesized vacuoles in the mature spores. In conclusion, our results revealed that budding yeast nuclei and vacuoles exhibit dynamic interorganelle interactions and different inheritance patterns in meiosis, and also suggested that nvj1Δ mutant cells may be useful to resolve the technical challenges pertaining to the isolation of intact nuclei for the biochemical study of meiotic nuclear proteins.

When exposed to sublethal high temperatures, budding yeast cells can survive for a period of time; however, a sufficient amount of ubiquitin is necessary for this survival. To understand the nature of the stress, we examined the morphological changes in yeast cells, focusing on the vacuoles. Changes in vacuolar morphology were notable, and ruffled vacuolar membranes, accelerated invaginations of vacuolar membranes, and vesicle-like formations were observed. These changes occurred in the absence of Atg1, Atg9 or Ivy1 but appeared to require endosomal sorting proteins, such as Vps23, Vps24 or Pep12. Furthermore, the serial sections of the vacuoles analysed using an electron microscopic analysis revealed that spherical invaginated structures were linked together in a vacuole. Because degradation of cell surface proteins is induced from heat stress, fusion of endosomal and vacuolar membranes might occur frequently in heat-stressed cells, and yeast cells might be able to cope with a rapid increase in vacuolar surface area by such invaginations.

Selective autophagy of damaged organelles is important to maintain cellular homeostasis. The mechanisms how autophagy selects specific targets is often poorly understood. Rabaptin5 was previously known as a major regulator of early endosome identity and maturation. Here, we identify two novel Rabaptin5 interactors: FIP200, a subunit of the ULK1 autophagy initiator complex, and ATG16L1, a central component of the E3-like enzyme in LC3 lipidation. Autophagy of early endosomes damaged by chloroquine or monensin treatment requires Rabaptin5 and particularly a short sequence motif that binds to the WD domain of ATG16L1. Rabaptin5 and its interaction with ATG16L1 further contributes to the autophagic elimination of Salmonella enterica early after infection, when it resides in phagosomes with early endosomal characteristics. Our results demonstrate a novel function of Rabaptin5 in quality control of early endosomes in the selective targeting of autophagy to damaged early endosomes and phagosomes.

PLIN4-myopathy is a recently identified autosomal dominant muscular disorder caused by the coding 99 bp repeat expansion in PLIN4, presenting with distal or proximal weakness. Here, we report one family and one sporadic case of adult-onset PLIN4-associated limb-girdle weakness, whose diagnoses were achieved by a comprehensive genetic analysis workup. We provided additional evidence that the combination of subsarcolemmal/cytoplasmic ubiquitin/p62 positive deposits and rimmed vacuoles could serve as a strong indicator of PLIN4-myopathy. Moreover, we found novel myopathological features that were ultrastructural subsarcolemmal filamentous materials and membrane-bound granulofilamentous inclusions formed by the co-deposition of disrupted lipid droplets and p62 protein aggregates.

The genus Anaplasma consists of tick-transmitted obligate intracellular bacteria that invade white or red blood cells to cause debilitating and potentially fatal infections. A. phagocytophilum, a human and veterinary pathogen, infects neutrophils to cause granulocytic anaplasmosis. A. marginale invades bovine erythrocytes. Evidence suggests that both species may also infect endothelial cells in vivo. In mammalian and arthropod host cells, A. phagocytophilum and A. marginale reside in host cell derived pathogen-occupied vacuoles (POVs). While it was recently demonstrated that the A. phagocytophilum-occupied vacuole (ApV) intercepts membrane traffic from the trans-Golgi network, it is unclear if it or the A. marginale-occupied vacuole (AmV) interacts with other secretory organelles. Here, we demonstrate that the ApV and AmV extensively interact with the host endoplasmic reticulum (ER) in endothelial, myeloid, and/or tick cells. ER lumen markers, calreticulin, and protein disulfide isomerase, and the ER membrane marker, derlin-1, were pronouncedly recruited to the peripheries of both POVs. ApV association with the ER initiated early and continued throughout the infection cycle. Both the ApV and AmV interacted with the rough ER and smooth ER. However, only derlin-1-positive rough ER derived vesicles were delivered into the ApV lumen where they localized with intravacuolar bacteria. Transmission electron microscopy identified multiple ER-POV membrane contact sites on the cytosolic faces of both species' vacuoles that corresponded to areas on the vacuoles' lumenal faces where intravacuolar Anaplasma organisms closely associated. A. phagocytophilum is known to hijack Rab10, a GTPase that regulates ER dynamics and morphology. Yet, ApV-ER interactions were unhindered in cells in which Rab10 had been knocked down, demonstrating that the GTPase is dispensable for the bacterium to parasitize the ER. These data establish the ApV and AmV as pathogen-host interfaces that directly engage the ER in vertebrate and invertebrate host cells and evidence the conservation of ER parasitism between two Anaplasma species.

In recent years, the function of different tumour suppressors in the regulation of macroautophagy has been studied. We show here that BRCA1, unlike other tumour suppressors, negatively regulates formation of autophagosomes and lysosomal mass under conditions of both basal and enhanced autophagy. In MCF-7 breast cancer cells, increased formation of autophagic vacuoles after inactivation of BRCA1 by siRNAs is associated with an increase in reactive oxygen species, such as superoxide anion and hydrogen peroxide. This allows one to propose an antioxidant function for BRCA1 and suggests that dysfunctional mitochondria and the generated reactive oxygen species excess could explain the increased macroautophagy observed in the absence of BRCA1. In addition, a quick decrease in BRCA1 levels occurs when MCF-7 cells are switched to a nutrient-poor environment that stimulates macroautophagy and that is also reminiscent of certain phases of tumour growth. Inhibition of BRCA1 synthesis has an important role in this reduction, while there are almost no changes in BRCA1 degradation by lysosomes and proteasomes. Therefore, BRCA1 produces macroautophagy inhibition by reducing the formation of autophagic vacuoles, and this, together with the other results presented here, shows new functional aspects of BRCA1 that could help to clarify the role of autophagy in cancer development.

A variety of intracellular bacteria modulate the host cytoskeleton to establish subcellular niches for replication. However, the role of intermediate filaments, which are crucial for mechanical strength and resilience of the cell, and in bacterial vacuole preservation remains unclear. Here, we show that Salmonella effector SopB reorganizes the vimentin network to form cage-like structures that surround Salmonella-containing vacuoles (SCVs). Genetic removal of vimentin markedly disrupts SCV organization, significantly reduces bacterial replication and cell death. Mechanistically, SopB uses its N-terminal Cdc42-binding domain to interact with and activate Cdc42 GTPase, which in turn recruits vimentin around SCVs. A high-content imaging-based screening identified that MEK1/2 inhibition led to vimentin dispersion. Our work therefore elucidates the signaling axis SopB-Cdc42-MEK1/2 as mobilizing host vimentin to maintain concrete SCVs and identifies a mechanism contributing to Salmonella replication. Importantly, Trametinib, a clinically-approved MEK1/2 inhibitor identified in the screen, displayed significant anti-infection efficacy against Salmonella both in vitro and in vivo, and may provide a therapeutic option for treating drug-tolerant salmonellosis.

Legionella pneumophila, a causative agent of Legionnaires' disease, is a facultative intracellular parasite of freshwater protozoa. Legionella pneumophila features a unique developmental network that involves several developmental forms including the infectious cyst forms. Reservoirs of L. pneumophila include natural and man-made freshwater systems; however, recent studies have shown that isolates of L. pneumophila can also be obtained directly from garden potting soil suggesting the presence of an additional reservoir. A previous study employing the metazoan Caenorhabditis elegans, a member of the Rhabditidae family of free-living soil nematodes, demonstrated that the intestinal lumen can be colonized with L. pneumophila. While both replicative forms and differentiated forms were observed in C. elegans, these morphologically distinct forms were initially observed to be restricted to the intestinal lumen. Using live DIC imaging coupled with focused transmission electron microscopy analyses, we report here that L. pneumophila is able to invade and establish Legionella-containing vacuoles (LCVs) in the intestinal cells. In addition, LCVs containing replicative and differentiated cyst forms were observed in the pseudocoelomic cavity and gonadal tissue of nematodes colonized with L. pneumophila. Furthermore, establishment of LCVs in the gonadal tissue was Dot/Icm dependent and required the presence of the endocytic factor RME-1 to gain access to maturing oocytes. Our findings are novel as this is the first report, to our knowledge, of extraintestinal LCVs containing L. pneumophila cyst forms in C. elegans tissues, highlighting the potential of soil-dwelling nematodes as an alternate environmental reservoir for L. pneumophila.

The obligate intracellular parasite Toxoplasma gondii replicates in an unusual process, described as internal budding. Multiple dausghter parasites are formed sequentially within a single mother cell, requiring replication and distribution of essential organelles such as micronemes. These organelles are thought to be formed de novo in the developing daughter cells. Using dual labelling of a microneme protein MIC2 and super-resolution microscopy, we show that micronemes are recycled from the mother to the forming daughter parasites using a highly dynamic F-actin network. While this recycling pathway is F-actin dependent, de novo synthesis of micronemes appears to be F-actin independent. The F-actin network connects individual parasites, supports long, multidirectional vesicular transport, and regulates transport, density and localisation of micronemal vesicles. The residual body acts as a storage and sorting station for these organelles. Our data describe an F-actin dependent mechanism in apicomplexans for transport and recycling of maternal organelles during intracellular development.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by motoneuron loss, for which there is currently no effective treatment. Statins, as inhibitors of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, are used as drugs for treatment for a variety of disease such as ischemic diseases, neurodegenerative diseases, cancer, and inflammation. However, our previous evidence has demonstrated that simvastatin leads to cytotoxicity in NSC34-hSOD1G93A cells by aggravating the impairment of autophagic flux, but the role of simvastatin in ALS model remains elusive. In present study, we reported that after simvastatin treatment, SOD1G93A mice showed early onset of the disease phenotype and shortened life span, with aggravated autophagic flux impairment and increased aggregation of SOD1 protein in spinal cord motoneurons (MNs) of SOD1G93A mice. In addition, simvastatin repressed the ability of Rab7 localization on the membrane by inhibiting isoprenoid synthesis, leading to impaired late stage of autophagic flux rather than initiation. This study suggested that simvastatin significantly worsened impairment of late autophagic flux, resulting in massive MNs death in spinal cord and accelerated disease progression of SOD1G93A mice. Together, these findings might imply a potential risk of clinic application of statins in ALS.

Acanthamoeba castellanii medusavirus is a member of the phylum Nucleocytoviricota, also known as giant viruses, and has a unique strategy of infecting Acanthamoeba castellanii and replicating viral genes in the host nucleus. Here, we show time series changes in the intracellular morphology, including the nucleus, of host cells infected with four types of giant viruses, including medusavirus, using time-lapse phase-contrast microscopy and image analysis. We updated our phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) to use multiple microscopic images with different focus positions to allow a more detailed analysis of their intracellular structures. Image analysis using PKA3 revealed that as medusavirus infection progressed, the host nucleus increased in size and the number of vacuoles decreased. In addition, infected host cells are known to become smaller and rounder at later stages of infection, but here they were found to be larger than uninfected cells at earlier stages. These results suggested that the propagation mechanism of medusavirus includes the formation of empty virus particles in the host cytoplasm, packaging of the viral genome replicated in the host nucleus, and then the release of viral particles. IMPORTANCE In this study, we quantitatively revealed how long the increase in host cell size or the increase in host nucleus size occurs after infection with giant viruses, especially medusavirus. To understand the underlying mechanism, we performed image analysis and determined that the host cell size increased at approximately 6 h postinfection (hpi) and the host nucleus enlarged at approximately 22 hpi, pointing to the importance of biochemical experiments. In addition, we showed that the intracellular structures could be quantitatively analyzed using multiple phase-contrast microscopy images with different focus positions at the same time point. Hence, morphological analyses of intracellular structures using phase-contrast microscopy, which have wide applications in live-cell observations, may be useful in studying various organisms that infect or are symbiotic with A. castellanii.

Salmonella colonizes a vacuolar niche in host cells during infection. Maturation of the Salmonella-containing vacuole (SCV) involves the formation of phosphatidylinositol 3-phosphate (PI(3)P) on its outer leaflet. SopB, a bacterial virulence factor with phosphoinositide phosphatase activity, was proposed to generate PI(3)P by dephosphorylating PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3. Here, we examine the mechanism of PI(3)P formation during Salmonella infection. SopB is required to form PI(3,4)P2/PI(3,4,5)P3 at invasion ruffles and PI(3)P on nascent SCVs. However, we uncouple these events experimentally and reveal that SopB does not dephosphorylate PI(3,4)P2/PI(3,4,5)P3 to produce PI(3)P. Instead, the phosphatase activity of SopB is required for Rab5 recruitment to the SCV. Vps34, a PI3-kinase that associates with active Rab5, is responsible for PI(3)P formation on SCVs. Therefore, SopB mediates PI(3)P production on the SCV indirectly through recruitment of Rab5 and its effector Vps34. These findings reveal a link between phosphoinositide phosphatase activity and the recruitment of Rab5 to phagosomes.

In this study, using the immunofluorescent method, the immunopositive signals to ubiquitin and proteasomes in nucleoli of root meristematic cells of soybean seedlings have been observed. In fact, those signals were present exclusively in nucleolar vacuoles. No signals were observed in the nucleolar territory out of the nucleolar vacuoles or in the nucleoli without vacuoles. The ubiquitin-proteasome system (UPS) may act within the nucleoli of plants with high metabolic activities and may provide an additional level of regulation of intracellular proteolysis via compartment-specific activities of their components. It is suggested that the presence of the UPS solely in vacuolated nucleoli serves as a mechanism that enhances the speed of ribosome subunit production in very actively transcribing nucleoli. On the other hand, nucleolar vacuoles in a cell/nucleus could play additional roles associated with temporary sequestration or storage of some cellular factors, including components of the ubiquitin-proteasome system.

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.

Fine regulation of exocytosis and endocytosis plays a basic role in pollen tube growth. Excess plasma membrane secreted during pollen tube elongation is known to be retrieved by endocytosis and partially reused in secretory pathways through the Golgi apparatus. Dissection of endocytosis has enabled distinct degradation pathways to be identified in tobacco pollen tubes and has shown that microtubules influence the transport of plasma membrane internalized in the tip region to vacuoles. Here, we used different drugs affecting the polymerization state of microtubules together with SYP21, a marker of prevacuolar compartments, to characterize trafficking of prevacuolar compartments in Nicotiana tabacum pollen tubes. Ultrastructural and biochemical analysis showed that microtubules bind SYP21-positive microsomes. Transient transformation of pollen tubes with LAT52-YFP-SYP21 revealed that microtubules play a key role in the delivery of prevacuolar compartments to tubular vacuoles.