Whorls Of Endoplasmic Reticulum Research Articles

Role of BcSfb3, the subunit of COPII vesicles, in fungal development and pathogenicity, ER-phagy and autophagy in the gray mold fungus Botrytis cinerea

Cytoplasmic coat protein complex II (COPII) plays a multifunctional role in the transport of newly synthesized proteins, autophagosome formation, and endoplasmic reticulum (ER)—ER-phagy. However, the molecular mechanisms of the COPII subunit in ER-phagy in plant pathogens remain unknown. Here, we identified the subunit of COPII vesicles (BcSfb3) and explored the importance of BcSfb3 in Botrytis cinerea. BcSfb3 deletion affected vegetative growth, conidiation, conidial morphology, and plasma membrane integrity. We confirmed that the increase in infectious hyphal growth was delayed in the ΔBcSfb3 mutant, reducing its pathogenicity in the host plant. Furthermore, the ΔBcSfb3 mutant was sensitive to ER stress, which caused massive ER expansion and induced the formation of ER whorls that were taken up into the vacuole. Further examination demonstrated that BcSfb3 deletion caused ER stress initiated by unfolded protein response, and which led to the promotion of ER-phagy and autophagy that participate in sclerotia formation. In conclusion, these results demonstrate that BcSfb3 plays an important role in fungal development, pathogenesis, ER-phagy and autophagy in B. cinerea.

EIF2α-mediated integrated stress response links multiple intracellular signaling pathways to reprogram vascular smooth muscle cell fate in carotid artery plaque

BackgroundCarotid arterial atherosclerotic stenosis is a well-recognized pathological basis of ischemic stroke; however, its underlying molecular mechanisms remain unknown. Vascular smooth muscle cells (VSMCs) play fundamental roles in the initiation and progression of atherosclerosis. Organelle dynamics have been reported to affect atherosclerosis development. However, the association between organelle dynamics and various cellular stresses in atherosclerotic progression remain ambiguous. MethodsIn this study, we conducted transcriptomics and bioinformatics analyses of stable and vulnerable carotid plaques. Primary VSMCs were isolated from carotid plaques and subjected to histopathological staining to determine their expression profiles. Endoplasmic reticulum (ER), mitochondria, and lysosome dynamics were observed in primary VSMCs and VSMC cell lines using live-cell imaging. Moreover, the mechanisms underlying disordered organelle dynamics were investigated using comprehensive biological approaches. ResultsER whorls, a representative structural change under ER stress, are prominent dynamic reconstructions of VSMCs between vulnerable and stable plaques, followed by fragmented mitochondria and enlarged lysosomes, suggesting mitochondrial stress and lysosomal defects, respectively. Induction of mitochondrial stress alleviated ER stress and autophagy in an eukaryotic translation initiation factor (eIF)-2α-dependent manner. Furthermore, the effects of eIF2α on ER stress, mitochondrial stress, and lysosomal defects were validated using clinical samples. ConclusionOur results indicate that morphological and functional changes in VSMC organelles, especially in ER whorls, can be used as reliable biomarkers for atherosclerotic progression. Moreover, eIF2α plays an important role in integrating multiple stress-signaling pathways to determine the behavior and fate of VSMCs.

ER whorls triggered by GhCNX6 were discovered involved in drought stress on cotton

Drought stress is the main abiotic stress causing damage to cotton growth and severe yield loss. Under environmental stress conditions, the endoplasmic reticulum (ER) folding environment is disturbed, resulting in the accumulation of unfolded/misfolded proteins, causing ER stress and triggering the unfolded protein response (UPR). Analysis of drought RNA-sequencing data revealed a Gh_D09G2402.1 ( GhCNX ) gene involved in ER protein processing. Through the observation of the ultrastructure of the ER under drought stress in cotton, the characteristic structure of the endoplasmic reticulum whorl was found. Virus-induced gene silencing (VIGS) mediated GhCNX6 silenced cotton plants were found highly sensitive to drought stress. Arabidopsis thaliana plants overexpressing GhCNX6 showed stronger tolerance to drought stress, indicating that the GhCNX6 gene is involved in the action mechanism of cotton drought stress and enhances the tolerance to drought stress. Drought stress leads to ER stress response, and the resulting ER whorl and CNX gene play a role in the ER protein processing pathway, thereby regulating the mechanism of cotton response to drought stress. This study will help to further explore the mechanism of cotton drought resistance. • Endoplasmic reticulum whorls found in cotton under drought stress. • First preliminary verification of GhCNX6 positively responding to drought stress. • The regulatory mechanism of GhCNX6 and ER whorls under drought stress.

ESCRT machinery mediates selective microautophagy of endoplasmic reticulum in yeast

ER‐phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER‐phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro‐ER‐phagy has been discovered, the molecules and mechanisms mediating micro‐ER‐phagy remain unknown. Here, we first show that micro‐ER‐phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1‐Spo7 phosphatase complex and the ESCRT machinery as key components for micro‐ER‐phagy. Third, we demonstrate that macro‐ and micro‐ER‐phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that the ESCRT machinery directly functions in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro‐ER‐phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis.

Ultrastructural features of the early secretory pathway in Trichoderma reesei.

We have systematically analysed the ultrastructure of the early secretory pathway in the Trichoderma reesei hyphae in the wild-type QM6a, cellulase-overexpressing Rut-C30 strain and a Rut-C30 transformant BV47 overexpressing a recombinant BiP1-VenusYFP fusion protein with an endoplasmic reticulum (ER) retention signal. The hyphae were studied after 24h of growth using transmission electron microscopy, confocal microscopy and quantitative stereological techniques. All three strains exhibited different spatial organisation of the ER at 24h in both a cellulase-inducing medium and a minimal medium containing glycerol as a carbon source (non-cellulase-inducing medium). The wild-type displayed a number of ER subdomains including parallel tubular/cisternal ER, ER whorls, ER-isolation membrane complexes with abundant autophagy vacuoles and dense bodies. Rut-C30 and its transformant BV47 overexpressing the BiP1-VenusYFP fusion protein also contained parallel tubular/cisternal ER, but no ER whorls; also, there were very few autophagy vacuoles and an increasing amount of punctate bodies where particularly the recombinant BiP1-VenusYFP fusion protein was localised. The early presence of distinct strain-specific features such as the dominance of ER whorls in the wild type and tub/cis ER in Rut-C30 suggests that these are inherent traits and not solely a result of cellular response mechanisms by the high secreting mutant to protein overload.

ER-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery

ABSTRACTSelective autophagy of damaged or redundant organelles is an important mechanism for maintaining cell homeostasis. We found previously that endoplasmic reticulum (ER) stress in the yeast Saccharomyces cerevisiae causes massive ER expansion and triggers the formation of large ER whorls. Here, we show that stress-induced ER whorls are selectively taken up into the vacuole, the yeast lysosome, by a process termed ER-phagy. Import into the vacuole does not involve autophagosomes but occurs through invagination of the vacuolar membrane, indicating that ER-phagy is topologically equivalent to microautophagy. Even so, ER-phagy requires neither the core autophagy machinery nor several other proteins specifically implicated in microautophagy. Thus, autophagy of ER whorls represents a distinct type of organelle-selective autophagy. Finally, we provide evidence that ER-phagy degrades excess ER membrane, suggesting that it contributes to cell homeostasis by controlling organelle size.

KMS1 and KMS2, two plant endoplasmic reticulum proteins involved in the early secretory pathway

We have identified two endoplasmic reticulum (ER)-associated Arabidopsis proteins, KMS1 and KMS2, which are conserved among most species. Fluorescent protein fusions of KMS1 localised to the ER in plant cells, and over-expression induced the formation of a membrane structure, identified as ER whorls by electron microscopy. Hydrophobicity analysis suggested that KMS1 and KMS2 are integral membrane proteins bearing six transmembrane domains. Membrane protein topology was assessed by a redox-based topology assay (ReTA) with redox-sensitive GFP and confirmed by a protease protection assay. A major loop domain between transmembrane domains 2 and 3, plus the N- and C-termini were found on the cytosolic side of the ER. A C-terminal di(tri)-lysine motif is involved in retrieval of KMS1 and deletion led to a reduction of the GFP-KMS1 signal in the ER. Over-expression of KMS1/KMS2 truncations perturbed ER and Golgi morphology and similar effects were also seen when KMS1/KMS2 were knocked-down by RNA interference. Microscopy and biochemical experiments suggested that expression of KMS1/KMS2 truncations inhibited ER to Golgi protein transport.

Different concentrations of ascorbyl-2-monophosphate-magnesium as dietary sources of vitamin C for seabass, Lates calcarifer

In this study, the effects of different concentrations of ascorbyl-2-monophosphate-magnesium (APM; 0, 30, 60, or 100 mg kg −1 diet) were tested in seabass for 6 weeks. Fish fed the diet without vitamin C supplementation showed various clinical signs such as fin erosion, erratic swimming behavior and spinal deformities. Several pathological changes were found in the gills and kidneys of fish fed a diet deficient in APM. The most remarkable feature in the gills was deformation of the outer epithelial cell layer, separation of the epithelial tissues of the secondary lamellae, and hyperplasia of the respiratory epithelium. An accumulation of red blood cells in the space between the outer and inner layer of epithelial cells of secondary lamellae was also observed. Changes in the kidneys were found in fish fed the APM-free diet; these changes included swelling, deformation, and fusion of mitochondria, as well as membrane whorls of endoplasmic reticulum. Seabass fed APM-containing diets did not show any of these abnormalities in the gills or proximal tubule cells of the kidney.

Characterization of a polydnavirus (PDV) and virus-like filamentous particle (VLFP) in the braconid wasp Cotesia congregata (Hymenoptera: Braconidae)

Two morphologically distinct virions were found to replicate in the female reproductive tract of wasps belonging to the species Cotesia congregata. Polydnaviruses (PDV) replicate in the ovaries of braconid and ichneumonid endoparasitic wasps and are injected into host insects during oviposition. Ultrastructural studies showed that in females of C. congregata, the polydnavirus initiated replication in specialized cells of the lower calyx following pupation, during the final phases of adult development, and replication and virus assembly continued throughout adult life. Viral envelopes initially were synthesized de novo at the periphery of the virogenic stroma, then multiple nucleocapsids were encapsidated; the maximum number seen per sectioned virion was 14. The extracellular virions in the calyx lumen measured >150 nm in diameter and had a tail. The PDVs did not replicate in cells composing the calyx wall. Replication also did not occur in a small subset of cells containing cytoplasmic PDVs, which appeared to be encased in whorls of endoplasmic reticulum. A virus-like filamentous particle (VLFP) was seen in the nucleus and cytoplasm of a few upper calyx cells that did not support PDV replication. This virus replicated significantly later than the PDV and contained only a single nucleocapsid per envelope. Only a few extracellular filamentous virions were seen in the calyx fluid. Those present had a double envelope and measured ca. 60 nm in diameter; their length was variable and sometimes exceeded 1 μm. Two viruses replicate in the reproductive system of C. congregata, as reported for other Cotesia species.

The use of primary cultures of adult rat hepatocytes to study induction of enzymes and DNA synthesis: effect of nafenopin and electroporation.

Primary cultures of adult rat hepatocytes maintained in a well-differentiated state, in a chemically defined medium containing 2% DMSO, have been utilized to study the effect of non-mutagenic hepatocarcinogens such as the peroxisome proliferator nafenopin. The parameters chosen in this in vitro system were those that paralleled the major in vivo effects of nafenopin on the liver, mainly: the proliferation of the endoplasmic reticulum and induction of cytochrome P-452, the proliferation of the peroxisome compartment and the induction of cyanide-insensitive beta-oxidation of fatty acids and the stimulation of liver growth as measured by the DNA synthetic activity of the hepatocytes. In this review, we also describe the morphology of hepatocyte cultures prepared from previously electroporated hepatocytes and the potential for the use of electroporation to introduce growth related genes into hepatocyte cells to study the mechanisms of hepatocyte growth at the molecular level. In addition we describe the formation of endoplasmic reticulum whorls in these cultures as a consequence of nafenopin treatment. 'Whorl formation' by hepatotrophic chemicals has been previously shown to occur in vivo; in this report, it is described for the first time in vitro.

Endoplasmic reticulum whorls as a source of membranes for early cytaster formation in parthenogenetically stimulated sea urchin eggs.

Sea urchin eggs exposed to a continuous hypertonic treatment rapidly form many concentric whorls of endoplasmic reticulum (ER) during the pre-activation period of the parthenogenetic development. These whorls, however, are only a temporary configurational alteration of ER which begin to break up just prior to egg activation. The conversion back to normal vesicles and lamellae occurs not only concurrently with the appearance of early cytastral areas, but also frequently in close association with the formation of these membranous areas. It is revealed here that membrane elements from disrupting whorls may become incorporated into adjacent, developing clear areas, early cytastral areas, and that this ER constitutes an initial major source of membranes for these early astral areas. Having previously suggested that the actual formation of ER whorls occurs in direct response to released intracellular calcium in hypertonic stressed eggs, the new findings, along with other related data and correlations, further suggest that whorl disruption and the formation of associated astral areas can be correlated with a corresponding decrease in the concentration of this released calcium in the cytoplasm.

Distinctive subcellular alterations induced by hypertonic stress in sea urchin eggs

Sea urchin eggs continuously exposed to a hypertonic solution were ultrastructurally examined for osmotic-stress induced alterations. No fertilization membranes formed during the treatment and the surface-cortex complexes remained unaltered from the unfertilized state. However, the osmotic stress did induce a number of subcellular changes. During the first 30 minutes of the treatment the eggs formed many endoplasmic reticulum whorls and compacted Golgi body aggregations. Both of these new formations can be correlated with rapid changes in intracellular calcium, known to occur in hypertonic stressed eggs. Aggregations of mitochondria could be observed at later stages; these aggregations can also be related to subcellular stress and possible changes in internal calcium concentrations. The various morphological transitions within the cytoplasm, along with the lack of a cortical reaction in these eggs, not only supports the idea that calcium is released during parthenogenetic activation, but also suggests that this free calcium originates from stores other than the stores that are involved during fertilization or simple artificial activation.

OBSERVATIONS ON THE CYTOLOGY OF THE DEVELOPING FEMALE GERM CELL IN THE POLYCHAETEHARMOTHOE IMBRICATA(L.)

Development of the oocytes of Harmothoe imbricata (L.) occurs whilst they remain attached to the segmental blood vessels of the fertile segments, each oocyte surrounded by the cytoplasm of follicle cells. During the early stages of oogenesis, only protogonia and ‘tangled-knot’ stage premeiotic oocytes are readily recognisable. Preliminary ultra-structural observations on vitellogenic oocytes show that the Golgi apparatus is responsible for the production of cortical granules, but the origin of the yolk granules is not known. Large whorls of endoplasmic reticulum and regions of presumed transfer of material from the nucleus to the cytoplasm are seen at various stages during oogenesis. Mature oocytes, in metaphase I of meiosis, are collected into the nephridia after a brief coelomic phase, and are soon spawned.

Freeze-etch study of the teleostean pituitary

The structure of membranes in the pituitary of several teleostean species was studied with the freeze-etching technique. Nuclear pore density is higher in hormone-secreting than in stellate cells, suggesting a higher rate of nucleo-cytoplasmic interactions in the secretory cells. The perinuclear endoplasmic reticulum (ER) in the MAS-secreting cells has a large number of fenestrae, whereas in the ER whorls of the prolactin-secreting cells the fenestration occurs only rarely. Three different types of membrane specializations are described, presumably related to exo-endocytotic events at the level of the plasmalemma.

Intermediate (10 nm) filaments in human malignant mesothelioma.

Human malignant mesotheliomas were studied by electron microscopy. Three main types of cells were seen--submesothelial epithelioid cells, epithelial lining cells and fibroblast-like cells. In submesothelial epithelioid cells prominent arrays of intermediate (10 nm) filaments were often seen attached to plasma membrane, mitochondria, nuclei and concentric whorls of rough endoplasmic reticulum. The other types of cell found in the tumors, epithelial lining cells and fibroblast-like cells, lacked such distinct filaments. The intermediate filaments were especially abundant in cells with extensive whorling of endoplasmic reticulum. The association of intermediate filaments with such deranged cytoplasmic organization suggests that they play a role in the altered behavior of malignant cells.

Ultrastructural changes in shoot apex cells of winter wheat seedlings during ice encasement

The decline in viability of cold-hardy Kharkov winter wheat (Triticum aestivum L.) seedlings during ice encasement at −1 °C was accompanied by characteristic ultrastructural changes. A dramatic increase in endoplasmic reticulum was observed within a few days. This proliferation of endoplasmic reticulum often resulted in the formation of an elaborate series of parallel membranes, either dispersed randomly throughout the cytoplasm or in the form of concentric whorls. However, the structural integrity of many cellular organelles was largely unaffected even by prolonged ice encasement resulting in death of the plants. In contrast, exposure of cold-hardy seedlings to near lethal, subfreezing temperature resulted in severe disorganization of cellular organelles. Ice encasement of nonhardened seedlings resulted in complete kill within 4 h. After 16 h ice encasement, occasional concentric whorls of endoplasmic reticulum and copious amounts of osmiophilic material were observed in the cytoplasm. Upon removal of the ice encasement stress, the accumulated endoplasmic reticulum disappeared rapidly during recovery at either2 or20 °C.

Proliferation of lamellar whorls in arcuate neurons of the hypothalamus of male rats treated with estradiol benzoate or cyproterone acetate

The arcuate nucleus of the hypothalamus (AH) of male rats which had been treated either with estradiol benzoate (E2B) or cyproterone acetate (CPA) was examined ultrastructurally for the presence of whorls of endoplasmic reticulum. The incidence of whorl containing neurons (WCN) was 2-4 times higher in the AH of animals treated for 2-3 weeks with E2B or for 2 weeks with CPA than in the AH of oil treated controls. CPA is a powerful anti-androgen while E2B acts both peripherally and centrally to limit testosterone production. These findings, together with previous evidence that whorls proliferate in AH of male rats deprived of androgen by morphine treatment or castration, suggest that steroid feedback (androgen alone or both androgen and estrogen) plays an important role in AH whorl preliferation. The possibility that WCN may be LH-RH containing neurons is suggested by the close correspondence between the number and location of WCN within AH as determined in this study and the distribution of LH-RH containing cells reported by others.

Proliferation of lamellar whorls in arcuate neurons of the hypothalamus of castrated and morphine-treated male rats.

A comprehensive ultrastructural examination of one cross-sectional level (middle 1/3) of the arcuate nucleus of the hypothalamus (AH) of male rats several weeks after castration or after two weeks of morphine treatment confirmed a marked increased in lamellar whorls of endoplasmic reticulum in AH neurons in each group. A comparable incidence of AH whorls was not detected in rats treated with lactose, those treated for only 1--3 days with morphine, or in those given testosterone plus morphine for 2 weeks. It is postulated that the testosterone deficiency following either castration or chronic morphine treatment stimulated the observed increase in AH whorls. A close correspondence was noted between the distribution within the AH of neurons containing whorls and those reported by others to contain luteinizing hormone releasing hormone (LH-RH). The possibility that whorls may be a marker for hypothalamic neurons which play a role in the LH-RH regulatory system warrants further consideration.

Ultrastructural modifications of renal cells in the three-spined stickleback (Gasterosteus aculeatus L.) after castration.

The mechanisms of cellular autophagy have been studied in the renal epithelium of male sticklebacks (Gasterosteus aculeatus L.) following castration. Two main types of alteration are described at the level of the proximal tubule cells. At first, the formation of endoplasmic reticulum whorls is observed, followed immediately by the secretion of lacunar progranules and by nucleolar modifications. These alterations lead to a progressive decrease of the glandular activity of the cells. The other main effect of castration is a general involution of the secretory granules still present in the cells. The involutive processes which lead to the complete destruction of the secretory material and, sometimes, of entire proximal cells, are described. In the collecting tubules, a marked and rapid decrease in glandular activity occurs, followed by disappearance of the mucous secretions. Small dense granules, supposedly lysosomes, are present. The processes described also appear to be characteristic of the normal involution of the renal cells occurring at the end of the breeding period. The findings strongly suggest that the male sex hormone may exert a double control on the renal cells in the stickleback, at both the cytoplasmic and nucleolar levels.

Effects of testosterone, hydrocortisone and insulin on the fine structure of the epithelium of rat ventral prostate in organ culture.

The explants of the rat ventral prostate were cultured on a chemically defined medium and the effects of individual hormones viz. testosterone, hydrocortisone and insulin on the fine structure of the epithelial cells were investigated. The epithelial cells without hormones underwent regressive changes resembling those seen in vivo after castration. Large autophagic vacuoles, residual bodies and dilations of rough endoplasmic reticulum (ER) were noted on day 2 of the culture. Four days later the amount of rough ER was markedly decreased and no secretory vesicles were visible. The treatment of the explants with testosterone resulted in an apparent intensification of the morphological equivalents of secretion. Beside the well developed rough ER and Golgi complex, numerous large secretory vesicles with flocculent content were observed. These observations are compatible with the idea that testosterone promotes the formation and probably also the excretion of the secretion in the epithelial cells. Testosterone also prevented partially the appearance of the signs of cellular degeneration. The most characteristic feature of hydrocortisone-treated epithelial cells was the copious supply of rough ER. Of particular interest was the formation of endoplasmic reticulum whorls which could not be observed in control and testosterone-treated cells. Hydrocortisone was shown to promote the formation of juvenile secretory vesicles on the second day. Insulin delayed the onset of the degeneration of the epithelial cells. Marked degenerative changes were, however, noted on day 6. There was no specific ultrastructural characteristic that could be interpreted to be specific for insulin action in the epithelial cell.