Confocal-microscopic study of skeletal muscle fibre membrane organelles during Zenker's (spreading) necrosis

The changes of T-system and cellular acidic organelles during spreading (Zenker's) necrosis of frog skeletal muscle fibres have been investigated using laser confocal microscopy and several vital fluorescent dyes acridine orange, RH 414, DiOC6(3), rhodamine 123, fluorescein dextran. The formation of numerous vacuoles as a result of local T-system swelling is most characteristic for initial steps of Zenker's necrosis. Vacuoles can attain tens microns in length. They are located both near nuclear poles and between myofibres. Vacuoles maintain connections with the extracellular space up to the moment of contraction knot rejection, and under definite conditions (glycerol influx to fibre) vacuoles are reversible. They deform nuclei and sarcoplasmic reticulum cisternae. Cellular acidic organelles, accumulating acridine orange (lysosomes, late endosomes, Golgi apparatus cisternae) are situated in direct vicinity with normal and vacuolated T-system. The increase in acidic organelles number and size occur during the pathological process development, and tendency to vacuoles clusterization may be seen. Vacuolation of T-system during necrosis is not followed by vacuole content acidification. The role of cellular acidic organelles and of T-system vacuolation in the development of different muscle pathological changes is discussed.     

Confocal microscopy study of membrane organelles of the skeletal muscle fiber in the process of Zenker’s (spreading) necrosis

Using laser confocal microscopy and some vital fluorescent dyes (acridine orange, RH 414, DiOC₆(3), rhodamine 123, fluorescein dextran), changes of the T-system and cellular acidic organelles were studied during spreading (Zenker’s) necrosis of isolated frog skeletal muscle fibers. The most characteristic of the initial stages of development of Zenker’s necrosis is the formation of numerous vacuoles as a result of local T-system swellings. The vacuole length can reach tens of micrometers. They are located both near nuclear poles and between myofibrils. Until the moment of contraction knot separation, the vacuoles preserve their connections with normal T-tubules and under certain conditions (glycerol influx to the fiber) are reversible. The vacuoles deform nuclei and cisternae of the sarcoplasmic reticulum. Acidic cell organelles accumulating acridine orange (lysosomes, late endosomes, trans-Golgi cisternae) are located in the immediate vicinity both of normal and of vacuolated T-tubules. In the course of the development of the pathological process, the size and number of acidic organelles increases and they tend to be clustered. Vacuolation of the T-system during necrosis was not accompanied by vacuole content acidification. At late stages of necrosis, alterations of nuclei and sarcoplasmic reticulum were observed. The role of cellular acidic organelles and of the T-system vacuolation in development of various muscle pathologies is discussed.     

Regulation of stress responses by intracellular vesicle trafficking?

Being grounded to one place, plants are constantly exposed to unexpected changes in the surrounding environment. Often, the changes in environmental conditions can be very rapid, compelling the plants to continuously monitor the outside environment and to adjust their metabolism to new conditions. Many of the primary environmental stresses ensue the development of a secondary oxidative stress, resulting in tissue damage and necrosis. The acclimation process almost invariably involves changes in the pattern of expressed proteins and other molecules. This necessitates the removal of the existing molecules from their compartments and the delivery of new compounds to their target organelles. The trafficking of macromolecules is performed by a bi-directional intracellular vesicle trafficking system that delivers newly synthesized molecules to organelles and retrieves material from the organelles to cytosolic compartments, such as vacuoles or lysosomes. The plasma membrane is among the organelles that are most exposed to oxidative stress damage and therefore must be constantly recycled. Here I propose that, by adjusting the rate of trafficking to and from the plasma membrane, the cells can regulate the stress outcome. Since the vesicle trafficking is closely linked to general signal transduction pathways, such as the phosphoinositide kinase pathway, and is influenced by major plant hormones, such as abscisic acid and auxin, the vesicle trafficking machinery holds the potential to regulate the plant responses to different environmental stresses.     

程序性坏死(Necroptosis)的分子机制

程序性坏死(Necroptosis)是一种不同于凋亡及传统坏死的细胞程序性死亡方式, 可由肿瘤坏死因子受体(Tumor necrosis factor receptor, TNFR)或模式识别受体(Pattern recognition receptor, PRR)调控启动。受体相互作用蛋白(Receptor-interacting protein, RIP)1和3是启动necroptosis的两个关键蛋白, necroptosis启动后需要一系列分子传递和执行死亡信号, 如多核苷酸二磷酸-核糖聚合酶-1(Poly(ADP-ribose) polymerase, PARP-1)、活性氧簇(Reactive oxygen species, ROS)、Ca²⁺等, 这些分子破坏线粒体及其他细胞器, 最终使细胞在缺乏天冬氨酸半胱氨酸蛋白酶(Caspase)的情况下死亡。Necroptosis细胞可将损伤相关模式分子(Damage-associated molecular patterns, DAMPs)暴露到细胞外, 被吞噬细胞识别并清除。文章对启动necroptosis的受体分子、传递执行细胞坏死的重要分子和坏死细胞的清除过程进行了概述。     

The vacuolar H+ -ATPase mediates intracellular acidification required for neurodegeneration in C. elegans

Numerous studies implicate necrotic cell death in devastating human pathologies such as stroke and neurodegenerative diseases. Investigations in both nematodes and mammals converge to implicate specific calpain and aspartyl proteases in the execution of necrotic cell death. It is believed that these proteases become activated under conditions that inflict necrotic cell death. However, the factors that modulate necrosis and govern the erroneous activation of these otherwise benign enzymes are largely unknown. Here we show that the function of the vacuolar H(+)-ATPase, a pump that acidifies lysosomes and other intracellular organelles, is essential for necrotic cell death in C. elegans. Cytoplasmic pH drops in dying cells. Intracellular acidification requires the vacuolar H(+)-ATPase, whereas alkalization of endosomal and lysosomal compartments by weak bases protects against necrosis. In addition, we show that vacuolar H(+)-ATPase activity is required downstream of cytoplasmic calcium overload during necrosis. Thus, intracellular pH is an important modulator of necrosis in C. elegans. We propose that vacuolar H(+)-ATPase activity is required to establish necrosis-promoting, acidic intracellular conditions that augment the function of executioner aspartyl proteases in dying cells. Similar mechanisms may contribute to necrotic cell death that follows extreme acidosis-for example, during stroke-in humans.     

Compromising the plasma membrane as a secondary target in photodynamic therapy-induced necrosis

Photodynamic therapy (PDT) is a non-invasive treatment widely applied to different cancers. The goal of PDT is the photo-induced destruction of cancer cells by the activation of different cell death mechanisms, including apoptosis and/or necrosis. Recent efforts focusing on understanding the mechanisms of cell death activated by PDT find that it depends on the type of photosensitizer (PS), targeted organelles, and nature of the light used. It is generally accepted that very short incubation times are required to direct the PS to the plasma membrane (PM), while longer periods result in the accumulation of the PS in internal compartments such as the endoplasmic reticulum or mitochondria. Glycosylation of the PS targets cancer via saccharide receptors on the cell surface, and is generally assumed that these compounds rapidly internalize and accumulate, e.g. in the endoplasmic reticulum. Herein we demonstrate that a minor fraction of a glycosylated chlorin compound residing at the PM of cancer cells can activate necrosis upon illumination by compromising the PM independently of the length of the incubation period. The results presented here show that the PM can also be targeted by glycosylated PS designed to accumulate in internal organelles. PS activation to induce necrosis by compromising the plasma membrane has the benefits of fast cell death and shorter irradiation times. The findings described here expand our understanding of the cellular damage induced by phototherapies, presenting the possibility of activating another cell death mechanism based on the incubation time and type of light used.     

Morphological homeostasis by autophagy

With cellular organelles coming in all shapes and sizes, the principle ‘form follows function’ is readily discernible through the cytologist’s lens. Architecturally, one might ask whether there is feedback in this organization. Does a cell ‘know’ when it has constructed membrane into the stacks of the Golgi, the cisternae of the mitochondria or the tubules of the endoplasmic reticulum? Proofreading can occur in vivo as both errors in nucleic acids and misfolds in proteins are recognized by the cell. Are there analogous systems which maintain/regulate the architectural integrity of organelles? Our recent paper entitled “Generation of cubic membranes from controlled homotypic interactions of membrane proteins in the endoplasmic reticulum” suggests that autophagy may play such a role.     

Targeting autophagy for drug-induced hepatotoxicity

Autophagy is a lysosomal degradation pathway for bulk cytosolic proteins and damaged organelles, and is well known to act as a cell survival mechanism. Acetaminophen (APAP) overdose can cause liver injury in animals and humans by inducing necrosis due to mitochondrial damage. We recently found that pharmacological induction of autophagy by rapamycin protects against, whereas pharmacological suppression of autophagy by chloroquine exacerbates, APAP-induced liver injury in mice. Autophagy is induced to remove APAP-induced damaged mitochondria and thus attenuates APAP-induced hepatocyte necrosis. To our surprise, we found that liver-specific Atg5 knockout mice are not more susceptible, but are resistant to APAP-induced liver injury due to compensatory effects. Our work suggests that pharmacological modulation of autophagy is a novel therapeutic approach to ameliorate APAP-induced liver injury. Moreover, our work also suggests that caution needs to be exercised when using genetic autophagy gene knockout mice for pathophysiological studies.     

Neurohormonal regulation of calcium in the cell

Neurohormone C (NC) is a glycopeptide isolated from bovine hypothalamus, which inhibits Ca-calmodulin (CaM)-dependent cAMP and cGMP phosphodiesterase (PDE) and is a regulator of Ca in the cell. Distribution of [⁴⁵Ca]CaCl₂ in the mitochondria and reticulum (SR) of heart and brain mitochondria and changes of Ca-binding proteins in these organelles under NC influence have been studied in the myocardium before and after isoproterenol-induced necrosis. Intraperitoneal administration of 80–100 mU of PDE inhibitory activity of NC to rats did not cause any noticeable changes in the protein content of intracellular organelles, but altered the affinity of certain proteins to⁴⁵Ca²⁺. This property of NC was especially noticable after isoproterenol necrosis. Necrotic injury of the myocardium induced Ca²⁺ storage in the mitochondria and SR of brain, and decreased the Ca²⁺ concentration in myocardial mitochondria. NC injection to the animals with necrosis was followed by Ca²⁺ release from all the studied organelles.     

Lysosomal labilization

The lysosomal compartment is the place for cellular degradation of endocytosed and autophagocytosed material and a center for normal turnover of organelles as well as most long-lived proteins. Lysosomes were long considered stable structures that broke and released their many hydrolytic enzymes only following necrotic cell death. It is now realized that lysosomes instead are quite vulnerable, although in a heterogeneous way. Their exposure to a number of events, such as oxidative stress, lysosomotropic detergents and aldhydes, as well as overexpression of the p53 protein, causes time-and-dose-dependent lysosomal rupture that is followed by apoptosis or necrosis. Partial lysosomal rupture has often been found to be an early upstream event in apoptosis, while necrosis results from fulminant lysosomal rupture. Consequently, factors influencing the stability of lysosomes, for instance their content of labile and redox-active iron, seem to be essential for the survival of cells.     

Death receptor ligation triggers membrane scrambling between Golgi and mitochondria

Subcellular organelles such as mitochondria, endoplasmic reticulum (ER) and the Golgi complex are involved in the progression of the cell death programme. We report here that soon after ligation of Fas (CD95/Apo1) in type II cells, elements of the Golgi complex intermix with mitochondria. This mixing follows centrifugal dispersal of secretory membranes and reflects a global alteration of membrane traffic. Activation of apical caspases is instrumental for promoting the dispersal of secretory organelles, since caspase inhibition blocks the outward movement of Golgi-related endomembranes and reduces their mixing with mitochondria. Caspase inhibition also blocks the FasL-induced secretion of intracellular proteases from lysosomal compartments, outlining a novel aspect of death receptor signalling via apical caspases. Thus, our work unveils that Fas ligand-mediated apoptosis induces scrambling of mitochondrial and secretory organelles via a global alteration of membrane traffic that is modulated by apical caspases.     

Condensed,Microtubule-coating Thin Organelles for Orthogonal Translation in Mammalian Cells

Membraneless organelles are capable of selectively performing complex tasks in living cells despite dynamically exchanging with their surroundings. This is an exquisite example how self-organization of proteins and RNAs can lead to more complex functionalities in living systems. Importantly, the absence of a membrane boundary can enable easier access to larger macromolecular complexes that can be challenging to be transported across a membrane. We previously formed orthogonally translating designer membraneless organelles by combining phase separation with kinesin motor proteins to highly enrich engineered translational factors in large organelles. We also showed that even submicron thick designer organelles can be formed, by mounting them onto membranes, which, presumable assisted by 2D condensation, leads to thin film-like condensates. In this study we show that orthogonal translation can also be built with fiber-like appearing organelles. Here, the microtubule-end binding protein EB1 was used to form fiber-like OT organelles along the microtubule cytoskeleton that perform highly selective and efficient orthogonal translation. We also show an improved simplified design of OT organelles. Together this extends OT organelle technology and demonstrates that the microtubule cytoskeleton is a powerful platform for advanced synthetic organelle engineering.     

Ultrastructure of neuromelanin granules in dog substantia nigra at different ages

The Authors study through ultrastructural observations the sequence of processes concerning the appearance and the depositing of the neuromelanic pigment in the neurons of dog mesencephalic substantia nigra nucleus at different ages. The results are as follows: in the one year old dog, in a low percentage of lysosome-like oval organelles, few masses, strongly electron-dense, appear on the matrix. In the five year old dog, these masses are deposited and increased in quantity inside many more organelles. In the specimens extracted from older dogs, these organelles, characterized moreover by the presence of lipidic globules, merging in groups, became larger and more irregular. The Authors discuss the results obtained with the ultrastructural observation, compared with those obtained with the optical methods.     

Targeting autophagy for drug-induced hepatotoxicity

Autophagy is a lysosomal degradation pathway for bulk cytosolic proteins and damaged organelles, and is well known to act as a cell survival mechanism. Acetaminophen (APAP) overdose can cause liver injury in animals and humans by inducing necrosis due to mitochondrial damage. We recently found that pharmacological induction of autophagy by rapamycin protects against, whereas pharmacological suppression of autophagy by chloroquine exacerbates, APAP-induced liver injury in mice. Autophagy is induced to remove APAP-induced damaged mitochondria and thus attenuates APAP-induced hepatocyte necrosis. To our surprise, we found that liver-specific Atg5 knockout mice are not more susceptible, but are resistant to APAP-induced liver injury due to compensatory effects. Our work suggests that pharmacological modulation of autophagy is a novel therapeutic approach to ameliorate APAP-induced liver injury. Moreover, our work also suggests that caution needs to be exercised when using genetic autophagy gene knockout mice for pathophysiological studies.     

Programmed cell death and cell necrosis activity during hyperthermic stress-induced bleaching of the symbiotic sea anemone Aiptasia sp.

Different cell death pathways were investigated during bleaching in the sea anemone Aiptasia sp. in response to hyperthermic treatment. Using a suite of techniques, (haematoxylin and eosin staining of paraffin wax-embedded tissue sections, in-situ end labelling (ISEL) of fragmented DNA, agarose gel electrophoresis electron microscopy) both necrotic and programmed cell death (PCD) activity were indicated. After a treatment period of 4 days, the host endoderm tissues underwent necrotic cell death. This was indicated by widespread cellular degradation, dilation of cell cytoplasm and organelles, cell swelling and rupture, irregular pyknotic condensation of nuclear chromatin, and abundant cell debris. Host cell necrosis was associated with the release of zooxanthellae with a normal, healthy appearance into the coelenteron. Longer periods of hyperthermic treatment (7 days) were correlated with further animal cell degradation and the in-situ degradation of zooxanthellae remaining within the degraded endoderm. Within the same degraded endoderm tissue, the degradation of zooxanthellae resulted from two forms of cell death occurring simultaneously, which were identified as programmed cell death and cell necrosis. Programmed cell death of zooxanthellae was characterised by condensation of the cytoplasm and organelles, cell shrinkage, formation of accumulation bodies at the periphery of the cell wall, and DNA fragmentation. Cell necrosis of zooxanthellae was characterised by dilation of the cytoplasm and organelles, cell swelling and lysis, dispersion of cell component debris, and DNA fragmentation. The existence of a programmed cell death pathway within zooxanthellae is important to the understanding of coral bleaching events, raising interesting questions regarding the evolution of this process and the activation of the cellular trigger mechanisms involved.     

Self-protection and survival of arbovirus-infected mosquito cells

Arboviruses are serious pathogens for men but cause little damage to their arthropod vectors. We have studied how a mosquito cell line derived from one of the relevant vectors for arboviruses responds to Bunyamwera virus, a well-characterized arbovirus. Confocal, live cell microscopy and electron microscopy showed that Bunyamwera virus induces deep changes in mosquito cells. Early in infection these cells develop long projections and create new intercellular connections where cell organelles and viral proteins are detected. Live cell microscopy shows that these connections are developed before viral protein can be detected by immunofluorescence. Interestingly, their proliferation is accompanied by a progressive trapping of the nucleocapsid and RNA polymerase viral proteins into large cytoplasmic aggregates. A significant drop in the release of infectious virions then follows. Before that, numerous viruses assemble in peripheral Golgi stacks and they apparently exit the cells immediately since they do not accumulate intracellularly. This mechanism of assembly seems to cause little damage to the integrity of cell endomembranes. The characterization of the antiviral mechanisms operating in mosquito cells can be of great help in the fight against pathogenic arboviruses.     

Amorphous areas in the cytoplasm of Dendrobium tepal cells

In Dendrobium flowers some tepal mesophyll cells showed cytoplasmic areas devoid of large organelles. Such amorphous areas comprised up to about 40% of the cross-section of a cell. The areas were not bound by a membrane. The origin of these areas is not known. We show data suggesting that they can be formed from vesicle-like organelles. The data imply that these organelles and other material become degraded inside the cytoplasm. This can be regarded as a form of autophagy. The amorphous areas became surrounded by small vacuoles, vesicles or double membranes. These seemed to merge and thereby sequester the areas. Degradation of the amorphous areas therefore seemed to involve macroautophagy.     

Accumulation of the amyloid-beta precursor protein in multivesicular body-like organelles.

It has been suggested that the successive proteolytic events leading to the production of the amyloid-beta protein from its precursor may take place at different intracellular locations. Using cultured human leptomeningeal smooth muscle cells and brain pericytes, we modulated the intracellular localization of the amyloid-beta precursor protein (APP) to study possible effects on its processing. By using immunofluorescence and immunoelectron microscopy we demonstrated that, under normal conditions, the APP is found in small intracellular vesicles, some of which were characterized as lysosomes. Both the cytokine interferon-gamma and the lysosomotropic drug chloroquine, but not the cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor-alpha (TNF-alpha), induced an accumulation of APP in newly formed multivesicular body-like organelles. The secretion of the amyloid-beta precursor protein was slightly reduced by interferon-gamma or chloroquine. Double-labeling and tracer molecule uptake experiments showed that the multivesicular body-like organelles were part of the endocytic pathway. Our findings suggest that the multivesicular body-like organelles function as an intermediate organelle in the intracellular trafficking of the APP. Accumulation of the APP in this organelle is reflected by its reduced secretion from the cell.     

BP-80 and homologs are concentrated on post-Golgi,probable lytic prevacuolar compartments

Prevacuolar compartments (PVCs) are membrane-bound organelles that mediate protein traffic between Golgi and vacuoles in the plant secretory pathway. Here we identify and define organelles as the lytic prevacuolar compartments in pea and tobacco cells using confocal immunofluorescence. We use five different antibodies specific for a vacuolar sorting receptor (VSR) BP-80 and its homologs to detect the location of VSR proteins. In addition, we use well-established Golgi-markers to identify Golgi organelles. We further compare VSR-labeled organelles to Golgi organelles so that the relative proportion of VSR proteins in Golgi vs. PVCs can be quantitated. More than 90% of the BP-80-marked organelles are separate from Golgi organelles; thus, BP-80 and its homologs are predominantly concentrated on the lytic PVCs. Additionally, organelles marked by anti-AtPep12p (AtSYP21p) and anti-AtELP antibodies are also largely separate from Golgi apparatus, whereas VSR and AtPep12p (AtSYP21p) were largely colocalized. We have thus demonstrated in plant cells that VSR proteins are predominantly present in the lytic PVCs and have provided additional markers for defining plant PVCs using confocal immunofluorescence. Additionally, our approach will provide a rapid comparison between markers to quantitate protein distribution among various organelles.     

Apoptosis, autophagy, and more

Cell death has been subdivided into the categories apoptosis (Type I), autophagic cell death (Type II), and necrosis (Type III). The boundary between Type I and II has never been completely clear and perhaps does not exist due to intrinsic factors among different cell types and the crosstalk among organelles within each type. Apoptosis can begin with autophagy, autophagy can end with apoptosis, and blockage of caspase activity can cause a cell to default to Type II cell death from Type I. Furthermore, autophagy is a normal physiological process active in both homeostasis (organelle turnover) and atrophy. “Autophagic cell death” may be interpreted as the process of autophagy that, unlike other situations, does not terminate before the cell collapses. Since switching among the alternative pathways to death is relatively common, interpretations based on knockouts or inhibitors, and therapies directed at controlling apoptosis must include these considerations.