Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes

Phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by specific morphological and metabolic events which may result in the killing of internalized micro-organism. Hydrogen peroxide is produced in increased amounts during phagocytosis (17) and in combination with myeloperoxidase and halide ions constitute a potent, microbicidal mechanism (8,9,11). There can be direct iodination of micro-organisms (10), or alternatively, other intermediate reaction products, i.e. chloramines and aldehydes (21), can exert a microbicidal effect. The H2O2-peroxidase-halide system is presumed to operate within the phagocytic vacuole (12,18). Myeloperoxidase, present in the primary granules of PMN, enters the phagocytic vacuole during degranulation (1,4,7), and halide ions are probably derived from the extracellular medium or are present in the PMN (see 11, 18). For the operation of this system in intact cells, the presence of H2O2 in the phagocytic vacuole is necessary, and indeed this has been suggested by the work of several investigators (12, 18, 21). In the present investigation, the diaminobenzidine reaction of Graham and Karnovsky (5), modified to utilize endogenous myeloperoxidase and hydrogen peroxide, has been applied to actively phagocytizing PMN to demonstrate cytochemically the presence of H2O2 in the phagocytic vacuole.     

The KEEP ON GOING Protein of Arabidopsis Regulates Intracellular Protein Trafficking and Is Degraded during Fungal Infection

In plants, the trans-Golgi network and early endosomes (TGN/EE) function as the central junction for major endomembrane trafficking events, including endocytosis and secretion. Here, we demonstrate that the KEEP ON GOING (KEG) protein of Arabidopsis thaliana localizes to the TGN/EE and plays an essential role in multiple intracellular trafficking processes. Loss-of-function keg mutants exhibited severe defects in cell expansion, which correlated with defects in vacuole morphology. Confocal microscopy revealed that KEG is required for targeting of plasma membrane proteins to the vacuole. This targeting process appeared to be blocked at the step of multivesicular body (MVB) fusion with the vacuolar membrane as the MVB-associated small GTPase ARA6 was also blocked in vacuolar delivery. In addition, loss of KEG function blocked secretion of apoplastic defense proteins, indicating that KEG plays a role in plant immunity. Significantly, KEG was degraded specifically in cells infected by the fungus Golovinomyces cichoracearum, suggesting that this pathogen may target KEG to manipulate the host secretory system as a virulence strategy. Taking these results together, we conclude that KEG is a key component of TGN/EE that regulates multiple post-Golgi trafficking events in plants, including vacuole biogenesis, targeting of membrane-associated proteins to the vacuole, and secretion of apoplastic proteins.     

Protein storage vacuoles are transformed into lytic vacuoles in root meristematic cells of germinating seedlings by multiple, cell type-specific mechanisms

We have investigated the structural events associated with vacuole biogenesis in root tip cells of tobacco (Nicotiana tabacum) seedlings preserved by high-pressure freezing and freeze-substitution techniques. Our micrographs demonstrate that the lytic vacuoles (LVs) of root tip cells are derived from protein storage vacuoles (PSVs) by cell type-specific sets of transformation events. Analysis of the vacuole transformation pathways has been aided by the phytin-dependent black osmium staining of PSV luminal contents. In epidermal and outer cortex cells, the central LVs are formed by a process involving PSV fusion, storage protein degradation, and the gradual replacement of the PSV marker protein α-tonoplast intrinsic protein (TIP) with the LV marker protein γ-TIP. In contrast, in the inner cortex and vascular cylinder cells, the transformation events are more complex. During mobilization of the stored molecules, the PSV membranes collapse osmotically upon themselves, thereby squeezing the vacuolar contents into the remaining bulging vacuolar regions. The collapsed PSV membranes then differentiate into two domains: (1) vacuole "reinflation" domains that produce pre-LVs, and (2) multilamellar autophagosomal domains that are later engulfed by the pre-LVs. The multilamellar autophagosomal domains appear to originate from concentric sheets of PSV membranes that create compartments within which the cytoplasm begins to break down. Engulfment of the multilamellar autophagic vacuoles by the pre-LVs gives rise to the mature LVs. During pre-LV formation, the PSV marker α-TIP disappears and is replaced by the LV marker γ-TIP. These findings demonstrate that the central LVs of root cells arise from PSVs via cell type-specific transformation pathways.     

Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.     

FYVE1 Is Essential for Vacuole Biogenesis and Intracellular Trafficking in Arabidopsis

The plant vacuole is a central organelle that is involved in various biological processes throughout the plant life cycle. Elucidating the mechanism of vacuole biogenesis and maintenance is thus the basis for our understanding of these processes. Proper formation of the vacuole has been shown to depend on the intracellular membrane trafficking pathway. Although several mutants with altered vacuole morphology have been characterized in the past, the molecular basis for plant vacuole biogenesis has yet to be fully elucidated. With the aim to identify key factors that are essential for vacuole biogenesis, we performed a forward genetics screen in Arabidopsis (Arabidopsis thaliana) and isolated mutants with altered vacuole morphology. The vacuolar fusion defective1 (vfd1) mutant shows seedling lethality and defects in central vacuole formation. VFD1 encodes a Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing protein, FYVE1, that has been implicated in intracellular trafficking. FYVE1 localizes on late endosomes and interacts with Src homology-3 domain-containing proteins. Mutants of FYVE1 are defective in ubiquitin-mediated protein degradation, vacuolar transport, and autophagy. Altogether, our results show that FYVE1 is essential for plant growth and development and place FYVE1 as a key regulator of intracellular trafficking and vacuole biogenesis.The plant vacuole is the largest organelle in a plant cell in which proteins, metabolites, and ions can be stored or sequestered. The vacuole is essential for plant development and growth and is directly or indirectly involved in various biotic and abiotic stress responses (Zhang et al., 2014). The vacuole is also the central organelle for degradation of endocytic and autophagic protein substrates through the activity of vacuolar proteases. In both degradation pathways, substrates are transported to the vacuole by intracellular membrane trafficking. In endocytic degradation, plasma membrane-localized proteins are targeted to the vacuole for degradation by endosomes (Reyes et al., 2011). This process is important, among others, to control the abundance of plasma membrane receptors and thus downstream signaling events. Autophagic degradation is mainly involved in nutrient recycling. During this process, cytosolic proteins and organelles are either selectively or nonselectively transported by double membrane autophagosomes to the vacuole to be degraded (Liu and Bassham, 2012). Vacuolar transport defines an intracellular transport pathway by which de novo synthesized proteins or metabolic compounds are carried to the vacuole by vesicle transport (Drakakaki and Dandekar, 2013).In yeast (Saccharomyces cerevisiae), forward genetic screens aimed at finding mutants with defective vacuolar transport or vacuolar morphology have identified more than 30 VACUOLAR PROTEIN SORTING (VPS) and VACUOLAR MORPHOLOGY (VAM) genes (Banta et al., 1988; Raymond et al., 1992; Wada and Anraku, 1992). Closer analyses have shown that many of these mutants have defects both in protein sorting and in vacuole biogenesis, suggesting a close link between these processes. vps and vam mutants were classified into six mutant classes according to their phenotypes. The strategic success of these screens has been confirmed when later studies revealed that many of the genes categorized in the same mutant class were coding for subunits of the same protein complexes. Among them were complexes important for membrane transport and fusion events, such as the endosomal sorting complex required for transport (ESCRT)-I to ESCRT-III (Henne et al., 2011) or the homotypic fusion and vacuole protein sorting (HOPS) complex (Balderhaar and Ungermann, 2013).Sequence homologs of most yeast VPS genes can be found in the Arabidopsis (Arabidopsis thaliana) genome (Sanderfoot and Raikhel, 2003; Bassham et al., 2008), and some of them were reported to be involved in intracellular trafficking as well as vacuole biogenesis. For example, the Arabidopsis vacuoleless (vcl)/vps16 mutant is embryo lethal and lacks lytic vacuoles (Rojo et al., 2001). VPS16 is a subunit of the HOPS complex, suggesting that membrane fusion events mediated by VCL/VPS16 are also important for plant vacuole biogenesis. Several other Arabidopsis vps mutants were also shown to have altered vacuole morphology at the mature embryo stage (Shimada et al., 2006; Sanmartín et al., 2007; Ebine et al., 2008, 2014; Yamazaki et al., 2008; Zouhar et al., 2009; Shahriari et al., 2010), showing that there is a conserved mechanism regulating vacuolar transport and vacuole biogenesis. However, in contrast to yeast, in which mutants without vacuole or severe biogenesis defects are viable, plant vacuoles seem to be essential for plant development.We have previously shown that defects in the deubiquitinating enzyme (DUB) ASSOCIATED MOLECULE WITH THE Src homology-3 DOMAIN OF STAM3 (AMSH3) also lead to a severe vacuole biogenesis defect (Isono et al., 2010). AMSH homologs do not exist in budding yeast but are conserved in animals and plants. Our previous studies have shown that AMSH3 can directly interact with ESCRT-III subunits (Katsiarimpa et al., 2013). ESCRT-III is a multiprotein complex that is essential for multivesicular body (MVB) sorting (Winter and Hauser, 2006) and hence for plant growth and development (Haas et al., 2007; Spitzer et al., 2009; Katsiarimpa et al., 2011; Cai et al., 2014). AMSH proteins regulate intracellular trafficking events, including endocytic degradation, vacuolar transport, and autophagic degradation through its interaction with ESCRT-III (Isono et al., 2010; Katsiarimpa et al., 2011, 2013, 2014). Prior to our characterization of the amsh3 mutant, AMSH proteins had not been implicated in vacuole biogenesis. Thus, we reasoned that there might be additional, yet unidentified, factors important for regulating vacuole biogenesis in plants. Further, we reasoned that other mutants with a defect in vacuole biogenesis, analogous to amsh3, might also exhibit seedling lethality.Thus, with the goal to identify and characterize these factors, we carried out a two-step mutant screen. We first selected seedling lethal mutants from an ethyl methansulfonate (EMS)-mutagenized population and then examined the vacuole morphology in these mutants. The isolated mutants were designated vacuolar fusion defective (vfd). vfd1 is affected in the expression of a functional Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing FYVE1 protein. FYVE1 was originally identified in silico as one of 16 FYVE domain-containing proteins in Arabidopsis with no apparent homologs in yeast and mammals (van Leeuwen et al., 2004). FYVE domains bind phosphatidylinositol 3-P, a phospholipid that is a major constituent of endosomal membranes. Hence, FYVE domain-containing proteins are implicated in intracellular trafficking (van Leeuwen et al., 2004; Wywial and Singh, 2010). In a previous work, we have shown that a null mutant of FYVE1, fyve1-1, is defective in IRON-REGULATED TRANSPORTER1 (IRT1) polarization and that FYVE1 is essential for plant growth and development (Barberon et al., 2014). A very recent publication describing the same mutant has shown that FYVE1/FYVE domain protein required for endosomal sorting1 (FREE1) is also important for the early and late endosomal trafficking events (Gao et al., 2014). In this study, we show that FYVE1 is also regulating ubiquitin-dependent membrane protein degradation, vacuolar transport, autophagy, and vacuole biogenesis. Altogether, our results point toward FYVE1 being a key component of the intracellular trafficking machinery in plants.     

Ascorbate free radical enhances vacuolization in onion root meristems

Abstract. Ascorbate free radical (AFR) induced cell elongation in merislems of Allium cepa roots by promoting a high vacuolization as shown by the increased vacuole volume, vacuole volume density, tonoplast surface and tonoplast surface density. Accordingly, both plasma membrane- and tonoplast-associated ATPases and vacuole soluble acid phosphatase of meristematic cells were also increased. Neither the other subcellular organelles nor cell proliferation appeared to be significantly affected. It is suggested that AFR may be involved in some plasma membrane events related to the initiation of plant cell elongation.     

液泡信号通路依赖的细胞程序性死亡研究进展

液泡是植物细胞专一性器官之一,具有多种功能,参与细胞内环境调节和细胞解毒等过程。研究表明,液泡在植物细胞程序性死亡（programmed cell death,PCD）中具有重要作用。在液泡介导的PCD过程中,液泡加工酶（vacuolar processing enzyme,VPE）的调控和激活是PCD的关键环节。着眼于液泡信号通路依赖的PCD,对相关细胞事件和分子调控机制进行了讨论,并对未来的研究方向作了展望,以期能推进PCD机制解明。     

缺血再灌后血管内皮空泡的形成与内皮的损伤

目的：在活体上探讨缺血再灌后血灌内上细胞损伤及白细胞、血小板与内皮之间粘附的变化。方法：用失血及与再回输血液造成缺血再灌流模型,在高倍显微镜下观察肠系膜微血管内皮损伤及血细胞粘附的变化。结果：缺血再灌后1－3h细静脉、集合毛细血管内出现白细胞、血小板的粘附,血管内皮水肿、管壁增厚,有的血管内皮细胞的胞浆形成圆丘形的空泡,空泡从血管内皮突入管胺、空泡直径10－30μm多出现的细动脉内,在同一根血管内可同时出现几个空泡,大的空泡几科占据血管腔的2／3。结论：缺血再灌后血管内皮水肿及空泡形成,显示内皮细胞的严重损伤。     

Early and late molecular events of glucose-induced pexophagy in Pichia pastoris require Vac8

We have identified the Pichia pastoris Vac8 homolog, a 60-64 kDa armadillo repeat protein, and have examined the role of PpVac8 in the degradative pathways involving the yeast vacuole. We report here that PpVac8 is required for glucose-induced pexophagy, but not ethanol-induced pexophagy or starvation-induced autophagy. This has been demonstrated by the persistence of peroxisomal alcohol oxidase activity in mutants lacking PpVac8 during glucose adaptation. During glucose-induced micropexophagy, in the absence of PpVac8, the vacuole was invaginated with arm-like "segmented" extensions that almost completely surrounded the adjacent peroxisomes. Vac8-GFP was found at the vacuolar membrane and concentrated at the base of the arm-like protrusions that extend from the vacuole to sequester the peroxisomes. The localization of Vac8-GFP to the vacuolar membrane occurred independent of PpAtg1, PpAtg9 or PpAtg11. Mutagenesis of the palmitoylated cysteines to alanines or deletion of the myristoylation and palmitoylation sites of PpVac8 resulted in decreased protein stability, impaired vacuolar association and reduced degradation of peroxisomal alcohol oxidase. Deletion of the central armadillo repeat domains of the PpVac8 did not alter its association with the vacuolar membrane, but resulted in a non-functional protein that suppressed the formation of the arm-like extensions from the vacuole to engulf the peroxisomes. PpVac8 is essential for the trafficking of PpAtg11, but not PpAtg1 or PpAtg18, to the vacuole membrane. Together, our results support a role for PpVac8 in early (formation of sequestering membranes) and late (post-MIPA membrane fusion) molecular events of glucose-induced pexophagy.     

Yeast homotypic vacuole fusion requires the Ccz1-Mon1 complex during the tethering/docking stage

The function of the yeast lysosome/vacuole is critically linked with the morphology of the organelle. Accordingly, highly regulated processes control vacuolar fission and fusion events. Analysis of homotypic vacuole fusion demonstrated that vacuoles from strains defective in the CCZ1 and MON1 genes could not fuse. Morphological evidence suggested that these mutant vacuoles could not proceed to the tethering/docking stage. Ccz1 and Mon1 form a stable protein complex that binds the vacuole membrane. In the absence of the Ccz1-Mon1 complex, the integrity of vacuole SNARE pairing and the unpaired SNARE class C Vps/HOPS complex interaction were both impaired. The Ccz1-Mon1 complex colocalized with other fusion components on the vacuole as part of the cis-SNARE complex, and the association of the Ccz1-Mon1 complex with the vacuole appeared to be regulated by the class C Vps/HOPS complex proteins. Accordingly, we propose that the Ccz1-Mon1 complex is critical for the Ypt7-dependent tethering/docking stage leading to the formation of a trans-SNARE complex and subsequent vacuole fusion.     

How the vacuole ESCRTs its own proteins to their final destination

Lysosomes (vacuoles in yeast) are master regulators of metabolism and protein turnover, but how they degrade their own resident proteins is unclear. Recently, multiple models have been proposed explaining yeast vacuole protein sorting, but the role of the ESCRT pathway was unclear. In this JCB issue, work from Yang et al. (https://doi.org/10.1083/jcb.202012104) highlights how the ESCRT pathway localizes to the vacuole surface to execute protein sorting of its resident proteins.

Lysosomes are key metabolic organelles that influence nutrient sensing, protein trafficking, lipid homeostasis, and catabolic metabolism (1). Because of their many roles, how lysosomes receive and degrade proteins has been a pervasive question in cell biology, and has driven the discovery of multiple trafficking pathways that deliver proteins from different regions of the cell to the lysosome for turnover. In budding yeast, the vacuole (functionally equivalent to the lysosome) is a superb model to dissect the mechanisms of endolysosomal trafficking. However, how the vacuole/lysosome senses and degrades its own resident proteins has remained mysterious. This is a critical question, since the vacuole/lysosome surface is home to many nutrient and lipid transporters, which must be selectively maintained or degraded in response metabolic cues to enable cell homeostasis.Recently, a flurry of papers were published with models explaining how the turnover of resident vacuole proteins is achieved. Two opposing models emerged (Fig. 1). In one, the ESCRT (endosomal sorting complexes required for transport) proteins, which are traditionally known to sort cargoes on the endosome surface into intralumenal vesicles that push into the endosome, were proposed to localize to the vacuole surface and execute a topologically similar protein sorting mechanism (albeit now on a different organelle; 2, 3, 4). However, another model called the intralumenal fragment (ILF) model proposed a radically different mechanism. Here, the homotypic fusion of the vacuole with itself could create a bubble-like fragment inside, containing vacuole surface proteins to be degraded. Critically, the formation of this fragment would be ESCRT-independent but require other membrane trafficking machinery like Rab7 (5, 6, 7). Paradoxically, both models were implicated in sorting the same vacuole proteins. Whether these two models were mutually exclusive, and how they related to one another, was unclear.Open in a separate windowFigure 1.Cartoon schematic of the models for resident vacuole protein turnover. Left: ESCRT-dependent sorting of vacuole proteins via recruitment of ESCRTs to ubiquitinated surface proteins, which are sorted into a vesicle that protrudes into the vacuole lumen and is degraded. Right: ILF pathway showing an ESCRT-independent selective sorting of proteins into a fragment, which via homotypic vacuole fusion is deposited into the vacuole lumen and then degraded.In a recent paper published by JCB, Yang and colleagues (8) shed new light on the mystery of how resident vacuole proteins are degraded and resolve several of the issues between these two contrasting models. To begin, Yang et al. used Western blotting and a microfluidics-based imaging system to monitor GFP-tagged vacuole proteins. Capitalizing on a tetracycline (TET)-OFF system that enabled them to essentially conduct pulse-chase assays, they could monitor each GFP-tagged protein and its turnover kinetics following treatment with rapamycin, which initiated the degradation process. They learned that some proteins like Zrt3-GFP (a zinc transporter) were first sorted into foci on the vacuole surface, then a short time later accumulated in the vacuole lumen, consistent with a sorting process that enabled Zrt3-GFP breakdown by vacuolar proteases. Indeed, Western blotting revealed free GFP accumulation over time as the Zrt3-GFP was degraded, indicating the fusion protein was being broken down in the vacuole lumen, leaving behind soluble GFP.Using this experimental setup, they next examined a vacuole protein previously reported to be a substrate of the ILF pathway, Fth1 (5). Fth1 had been proposed to be selectively sorted via the ILF pathway, whereas its binding partner Fet5 was not. However, Yang et al. were able to show that these proteins coimmunoprecipitated together, suggesting they exist in a complex and were unlikely to be separated. Indeed, deletion of Fth1 caused Fet5 to be trapped at the ER, consistent with it needing Fth1 for stability. Furthermore, time-lapse imaging indicated that Fth1 turnover was very slow, in contrast to earlier reports that Fth1 was constitutively turned over via the ILF pathway. Motivated by these observations, they next examined other ILF cargoes reported to be degraded by the ILF pathway following exposure of yeast to heat or the drug cycloheximide. Again capitalizing on their time-lapse imaging system and Western blotting, they failed to observe significant turnover of these proteins, in contrast to previous work.A key difference between the two models is dependence on the ESCRT pathway. To further dissect, Yang et al. used imaging to interrogate how loss of ESCRT machinery impacted vacuole protein dynamics and degradation. By monitoring vacuole proteins whose turnover was stimulated by rapamycin, they observed that yeast lacking the ESCRT component Vps4 failed to sort Zrt3 and other cargoes into the vacuole. This argued that their degradation was ESCRT dependent, and likely not through the ILF pathway. Serendipitously, time-lapse imaging experiments also detected occasional ILF-like structures within the vacuole. However, imaging revealed that ILF structures were rare, and often contained the protein Zrc1-mCherry, which is stable and not degraded. Collectively, this argued that ESCRT machinery is required for the turnover of several vacuole proteins, and that the ILF system is likely not the predominant mechanism of resident vacuole protein degradation.Finally, Yang et al. used their imaging platform to interrogate how ubiquitin and the ESCRTs influence vacuole protein turnover. They took advantage of an inducible degradation system called RapIDeg that uses an FK506-binding protein–FKBP Rapamycin-binding domain system to inducibly attach ubiquitin to a GFP-tagged vacuole protein by adding rapamycin. Strikingly, this revealed that within 10–30 min of ubiquitination, the vacuole protein was sorted into bright foci on the membrane surface that contained ESCRT machinery, strongly suggesting this protein sorting was ESCRT dependent.Collectively, the data argue that many vacuole proteins are degraded via a ubiquitin and ESCRT-dependent pathway that operates on the vacuole surface in a manner topologically similar to the formation of multi-vesicular bodies at endosomes (Fig. 1). In this model, ubiquitination of vacuole proteins attracts ESCRT machinery, which bend the membrane away from the cytoplasm to create a vesicle within the vacuole, which is subsequently degraded by vacuolar proteases. Although ILFs were observed in this and previous studies, these structures are rare, and appear to lack the ability to selectively sort proteins.It should be noted that this study and the earlier ILF pathway studies used very different methodologies. The ILF studies relied on purified vacuoles in a cell-free system (5, 7), whereas Yang et al. employed in vivo time-lapse imaging and blotting (8). Since purified vacuoles lack the full cellular ubiquitination machinery, it is feasible that ESCRT-independent membrane remodeling may occur in vitro. Relatedly, it is possible that the ILF system may sort other proteins not examined by Yang et al. However, Yang clearly establishes that several vacuole proteins are sorted via an ESCRT-dependent pathway that acts on the vacuole surface. Second, it is worth noting that ILF structures are observed in several studies of vacuole remodeling. Although likely not a major mechanism for protein sorting, these ILF structures may provide means for the vacuole to control its membrane composition and even size. Third, the work of Yang et al. adds to the continually growing list of roles for ESCRTs in cellular homeostasis. ESCRTs now have established roles at endosomes, the plasma membrane, the nuclear envelope, and yeast vacuole. This versatile machinery is continually used to execute its topologically unique membrane remodeling. This is important to understand, as experiments that perturb or block ESCRT function likely impact multiple cellular processes and have pleiotropic effects.Given these insights, questions still remain. Although Yang et al. shed light on how the yeast vacuole consumes its proteins, how this system relates to mammalian lysosomes and how distinct resident proteins are selectively degraded or retained on the surface during metabolic cues remains to be explored. It is also unclear whether the ESCRT pathway, which utilizes several distinct complexes, operates completely the same on vacuoles as it does at other organelles. How ESCRTs function on vacuoles/lysosomes is of growing importance since ESCRTs have also been shown to facilitate micro-autophagy, delivering substrates such as lipid droplets into the vacuole lumen (9). These and other questions will no doubt drive further studies of the amazing yeast vacuole and mammalian lysosome, and how they organize and govern the lives of their many resident proteins.     

Programmed cell death of plant tracheary elements differentiating in vitro

Summary We used various microscopic and labeling techniques to examine events occurring during the programmed cell death (PCD) of plant tracheary elements (TEs) developing in vitro. TEs differentiating in vitro synthesize a secondary cell wall which is complex in composition and pattern at approximately 72 h after hormone manipulation. The timing of PCD events was established relative to this developmental marker. Cytoplasmic streaming continues throughout secondary wall synthesis, which takes 6 h to complete in a typical cell. Vital dye staining and ultrastructural analysis show that the vacuole and plasma membrane are intact during secondary cell wall synthesis, but the cytoplasm becomes less dense in appearance, most likely through the action of confined hydrolysis by small vacuoles which are seen throughout the cell at this time. The final, preeminent step of TE PCD is a rapid collapse of the vacuole occurring after completion of secondary cell wall synthesis. Vacuole collapse is an irreversible commitment to death which results in the immediate cessation of cytoplasmic streaming and leads to the complete degradation of cellular contents, which is probably accomplished by release of hydrolytic enzymes sequestered in the vacuole. This event represents a novel form of PCD. The degradation of nuclear DNA is detectable by TUNEL, an in situ labeling method, and appears to occur near or after vacuole collapse. Our observations indicate that the process of cellular degradation that produces the hollow TE cell corpse is an active and cell-autonomous process which is distinguishable morphologically and kinetically from necrosis. Although TE PCD does not resemble apoptosis morphologically, we describe the production of spherical protoplast fragments by cultured cells that resemble apoptotic bodies but which are not involved in TE PCD. We also present evidence that, unlike the hypersensitive response (HR), TE PCD does not involve an oxidative burst. While this evidence does not exclude a role for reactive oxygen intermediates in TE PCD, it does suggest TE PCD is mechanistically distinct from cell death during the HR.Abbreviations BA 6-benzylamino-purine - DAPI 4,6-diamidino-2-phenylindole diacetate - DCF 2,7-dichlorofluorescein diacetate - DPI diphenyleneiodonium - FDA fluorescein diacetate - HR hypersensitive response - NAA -naphthalene-acetic acid - PCD programmed cell death - ROI reactive oxygen intermediate - TE tracheary element - TUNEL TdT-mediated dUTP nick end labeling     

Phagosome fusion vesicles of paramecium. I. Thin-section morphology

Ultrastructural studies of the digestive system of Paramecium caudatum focusing on the first 5 min of digestive-vacuole age reveal a set of vesicles, named phagosome fusion vesicles (PFVs), which fuse with the digestive vacuole just after the vacuoles are released from the cytopharynx and concomitant with vacuole acidification. Serial thin-sections of vacuoles labeled with horseradish peroxidase (HRP) and/or latex beads in pulse-chase studies were observed. PFVs, irregularly shaped, electron-translucent vesicles ranging from a small diameter to over 1 micro, are first seen in the region of the cytopharynx where they bind to the nascent vacuole membrane. Within 30 sec of vacuole release the PFVs fuse with the vacuole where they remain for a brief time connected to the vacuole by a narrow annulus. HRP-reaction product is found in vacuoles but not in PFVs before PFVs fuse with the vacuoles. After fusion with PFVs HRP is quickly inactivated. Tubular extensions of vacuole membrane then form between the fused PFVs. By 3 to 5 min both PFVs and tubules disappear from the vacuole surface and lysosomes appear in their place. We believe the tubules are pinched off as PFV membrane is being added to the vacuole. Microfilaments coat the membrane during all these dynamic events. Since the pH of the vacuole becomes acid during the first few minutes, we are now looking for a direct correlation between PFV fusion and acidification.     

Digestion in the peritrich ciliateOphrydium versatile

Summary Digestion in the peritrich ciliateOphrydium versatile O.F.M. involves a complex sequence of intracytotic and exocytotic membrane fusion and recycling events. Food particulates are concentrated in the lower cytopharynx which forms a fusiform-shaped food vacuole. Upon release from the cytopharynx, this food vacuole begins to condense, concentrating the food particulates. Excess membrane is removed intracytotically. These released membranes pieces form discoidal vesicles which are recycled to the base of the cytopharynx, thus providing additional membrane for subsequent food vacuole formation. In the condensed food vacuole, digestion proceeds; hydrolytic enzymes are delivered to the food vacuole via rough endoplasmic reticulum and/or by the cup-shaped coated vesicles (CSCV). As these vesicles fuse with the food vacuole, the food vacuole enlarges, digestion proceeds and an electron-dense membrane coat appears along the luminal surface of the food vacuole. Prior to defecation, the food vacuole undergoes a final condensation; irregularly-shaped, electron dense, single-membrane bound vesicles are cut-off intracytotically from the old food vacuole. These vesicles undergo condensation and invagination to form the cup-shaped coated vesicles (CSCV) which fuse with younger food vacuoles.     

The initial phase of cyst formation in Sarcocystis dispersa sarcosporidians

Six white mice were inoculated orally with Sarcocystis dispersa sporocysts and killed on days 10, 12 and 14 post inoculation (p. i.). Precyst merozoites were found in leukocytes and muscular cells on days 10 and 12 p. i. Young cysts containing only metrocytes were found on days 12 and 14 p. i. Precyst merozoites in muscular cells were situated freely, without any parasitophorous vacuole. The process of merozoite transformation into a primary metrocyte runs parallel with the process of sarcocyst wall formation.     

The effect of puromycin and cycloheximide on vacuole formation and exocytosis in Tetrahymena pyriformis GL-9

It has been shown that both puromycin and cycloheximide, at concentrations of 434 and 100 g/ml respectively, produce a marked inhibition of vacuole formation and exocytosis in Tetrahymena pyriformis GL-9. These effects were analysed in a quantitative manner. At the same time as these inhibitions occurred the incorporation of 1-C¹⁴ leucine into trichloroacetic acid precipitable material was inhibited by 90% and 100% respectively over a 40 min period. This inhibition of protein synthesis by cycloheximide occurred almost immediately, whereas the inhibition of vacuole formation and egestion was delayed. The results suggested that the latter processes were dependent upon a continuing supply of proteinaceous material, of which there was only a small store within the cell. Cycloheximide inhibited exocytosis completely under the conditions employed (with 100% inhibition of protein synthesis) whereas puromycin (with a 90% inhibition of protein synthesis) only inhibited it by about 50%. This suggested that the amount of newly synthesized protein required for the exocytic egestion process was very small in relation to the total cell requirement for protein synthesis. The entry of both inhibitors into the cell was by means other than vacuole formation. Puromycin appeared to have some effect on vacuole formation which was unconnected with protein synthesis. Microscopic observations of living cells indicated that oral apparatus function and endocytic vacuole formation were probably both affected by the inhibitors. Chloramphenicol, at 200 g/ml, had little effect on vacuole formation by starved cells with an exposure of an hour. The uptake of 1-C¹⁴ leucine from the growth medium was found to be a selective process, giving a concentration of about 2000 times into the cells over a 1 hr period. The results are discussed.     

Primary Lysosomes of the Ciliate Pseudomicrothorax dubius: Cytochemical Identification and Role in Phagocytosis*

SYNOPSIS. Filamentous cyanobacteria are ingested through the cytopharynx of the ciliate Pseudomicrothorax dubius. The cytopharynx is a complex of microtubules and microfilaments located in a highly vesiculated cytoplasm, the phagoplasm. Two types of membrane-bounded phagoplasmic vesicles can be distinguished by their differences in size, fine structure, and acid phosphatase (AcPase) content. One type has a homogeneous, electron-dense interior which is AcPase-positive. These vesicles are present in fed cells and in unfed cells devoid of food vacuoles, and thus appear to be primary lysosomes. During phagocytosis, exocytosis within the cytopharynx of the primary lysosomes results in the elaboration of a food vacuole. The vacuole grows by incorporation of lysosomal membrane; lysosomal hydrolases are liberated into the vacuole. Within less than 1 second of AcPase's entry into the food vacuole, it is detectable within the cyanobacterial cytoplasm, and within 5 seconds, destruction of the cyanobacterial filament is observed. It is hypothesized that the rapidity of hydrolase penetration of the cyanobacterial cell wall is the result of the action of molecules analogous to the “killing agents” of neutrophil leukocytes, which rapidly render bacterial envelopes permeable. AcPase, and presumably other hydrolases, are present in the cyanobacterial filament when filament destruction occurs; they thus appear implicated in this process. Hydrolases may activate an autodestruction mechanism in the cyanobacterium. Firm adherence of the food vacuole membrane to the cyanobacterial filament is demonstrated, and its role in phagocytosis is discussed.     

A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.     

Paramecium Phagosome Membrane: From Oral Region to Cytoproct and Back Again1

Ten years of research on digestive vacuoles (phagosomes) of Paramecium caudatum have revealed sequential changes both within the vacuole lumen as well as within the surrounding membrane. Four vacuole stages can be recognized by a combination of thin section and freeze-fracture ultrastructural features. Three sets of vesicles (discoidal vesicles, acidosomes, and lysosomes) fuse with the vacuole, each at a predetermined stage, to bring about these membrane and physiological changes. At various times membrane is removed as vesicles from the vacuole surface, which has the effect of regulating vacuole size. Membrane recycling, membrane replacement, and specific membrane to membrane recognition all appear to be operating during the digestive cycle. Details of these events are summarized in this address and a number of unanswered questions suggest areas for future research.     

Pore formation, polymerization, hemolytic and leukotoxic effects of a new Enterobacter cloacae toxin neutralized by antiserum

A new toxin of Enterobacter cloacae was purified and studied by SDS-PAGE electrophoresis with the purpose of investigating its ability to generate polymers and their molecular mass. Monomer of 13.3 kDa and structures of multimeric mass were detected. The toxin of 66 kDa was the most abundant form of toxin. This polymer and the monomer were selected to examine blood cells damage. Membrane pores caused by both toxin forms seemed to be of similar dimension (estimated in 3.6 nm by experiments with osmotic protectors) and were able to lyse erythrocytes and leukocytes. The results obtained indicate that polymerization and pore formation are involved in the molecular events that participate in the cytotoxic effects of E. cloacae toxin. Immunization of rabbits with 13.3kDa toxin generated antibody response capable of inhibiting oxidative stress as well as hemolytic and leukotoxic effects. Immunoblotting indicated that monomer and polymer reacted with antihemolysin serum. The importance of E. cloacae toxin "in vivo" was studied in animals by means of assays performed in peritoneum of rats, inoculated with the hemolytic strain (C1) and a non-hemolytic variant (C4). Both strains stimulated infiltration of leukocytes in peritoneum, but C1 caused cell death and lysis wheras assays with C4 maintained the viability of leukocytes even within 5 h after extraction of samples.