Localization of Autocrine Motility Factor Receptor to Caveolae and Clathrin-independent Internalization of Its Ligand to Smooth Endoplasmic Reticulum

Autocrine motility factor receptor (AMF-R) is a cell surface receptor that is also localized to a smooth subdomain of the endoplasmic reticulum, the AMF-R tubule. By postembedding immunoelectron microscopy, AMF-R concentrates within smooth plasmalemmal vesicles or caveolae in both NIH-3T3 fibroblasts and HeLa cells. By confocal microscopy, cell surface AMF-R labeled by the addition of anti-AMF-R antibody to viable cells at 4°C exhibits partial colocalization with caveolin, confirming the localization of cell surface AMF-R to caveolae. Labeling of cell surface AMF-R by either anti-AMF-R antibody or biotinylated AMF (bAMF) exhibits extensive colocalization and after a pulse of 1–2 h at 37°C, bAMF accumulates in densely labeled perinuclear structures as well as fainter tubular structures that colocalize with AMF-R tubules. After a subsequent 2- to 4-h chase, bAMF is localized predominantly to AMF-R tubules. Cytoplasmic acidification, blocking clathrin-mediated endocytosis, results in the essentially exclusive distribution of internalized bAMF to AMF-R tubules. By confocal microscopy, the tubular structures labeled by internalized bAMF show complete colocalization with AMF-R tubules. bAMF internalized in the presence of a 10-fold excess of unlabeled AMF labels perinuclear punctate structures, which are therefore the product of fluid phase endocytosis, but does not label AMF-R tubules, demonstrating that bAMF targeting to AMF-R tubules occurs via a receptor-mediated pathway. By electron microscopy, bAMF internalized for 10 min is located to cell surface caveolae and after 30 min is present within smooth and rough endoplasmic reticulum tubules. AMF-R is therefore internalized via a receptor-mediated clathrin-independent pathway to smooth ER. The steady state localization of AMF-R to caveolae implicates these cell surface invaginations in AMF-R endocytosis.     

Spatial associations between actin filaments, endoplasmic reticula, mitochondria and fungal hyphae in symbiotic cells of orchid protocorms

The relationships between endoplasmic reticula (ER), mitochondria, and actin filaments (Afs) were observed in uncolonized and colonized cells of symbiotic protocorms ofSpiranthes sinensis (Orchidaceae) germinated in the presence of the fungus,Ceratobasidium cornigerum. Mitochondria and ER were observed by transmission electron microscopy, and with the fluorescent probe DiOC₆ (3) (3,3′-dihexyloxacarbocyanine) combined with confocal laser scanning microscopy (CLSM). An indirect immunofluorescence method using CLSM and an indirect, pre-embedding immunogold method at the ultrastructural level were used for observation of Afs. In uncolonized cells, cortical ER showed a polygonal pattern and ER formed a network throughout the cytoplasm. In the cortex, a smooth face of ER contacted the plasma membrane. Mitochondria were associated with ER. Afs were in close proximity to ER, mitochondria and amyloplasts. Colonized cells retained cortical ER, and a smooth face of ER was also closely associated with the perifungal membrane. ER and mitochondria were present in the cytoplasmic channels bridging between the central peloton and the peripheral cytoplasm. This distribution of ER and mitochondria during fungal colonization and senescence coincided with that of Afs. The changes in the arrays of Afs accompanying symbiotic fungal colonization and senescence occurred concomitantly with the changes in ER.     

Regulation of cytosolic free calcium concentration by intrasynaptic mitochondria.

By the use of digitonin permeabilized presynaptic nerve terminals (synaptosomes), we have found that intrasynaptic mitochondria, when studied "in situ," i.e., surrounded by their cytosolic environment, are able to buffer calcium in a range of calcium concentrations close to those usually present in the cytosol of resting synaptosomes. Adenine nucleotides and polyamines, which are usually lost during isolation of mitochondria, greatly improve the calcium-sequestering activity of mitochondria in permeabilized synaptosomes. The hypothesis that the mitochondria contributes to calcium homeostasis at low resting cytosolic free calcium concentration ([Ca2+]i) in synaptosomes has been tested; it has been found that in fact this is the case. Intrasynaptic mitochondria actively accumulates calcium at [Ca2+]i around 10(-7) M, and this activity is necessary for the regulation of [Ca2+]i. When compared with other membrane-limited calcium pools, it was found that depending on external concentration the calcium pool mobilized from mitochondria is similar or even greater than the IP3- or caffeine-sensitive calcium pools. In summary, the results presented argue in favor of a more prominent role of mitochondria in regulating [Ca2+]i in presynaptic nerve terminals, a role that should be reconsidered for other cellular types in light of the present evidence.     

Red blood cells of the lizards Ameiva ameiva (Squamata, Teiidae) display multiple mechanisms to control cytosolic calcium

We have previously reported that lizard red blood cells control their cytosolic calcium concentration by sequestering calcium ions in pools, which could be discharged by thapsigargin, by the Na+/H+ ionophore, monensin, by the K+/H+ ionophore, nigericin and by the proton pump inhibitor, bafilomycin A1 [1]. We have now demonstrated, with the aid of confocal microscopy, the presence in these cells of organelles, which accumulate the dye acridine orange and are thus by inference the sites of proton pools. We have found, moreover, that monensin, nigericin and bafilomycin all act to discharge these pools. We further show that calcium release ensues when the calcium ionophore, ionomycin, is added after thapsigargin and monensin; this implies the existence of a third pool, besides the acidic pool and the Endoplasmic Reticulum (ER), which participates in calcium homeostasis. The ER calcium pool can de discharged by the addition of the second messenger, IP3, and we present evidence, based on confocal microscopy, that the IP3 receptors are located in or close to the nucleus.     

Complex calcium oscillations and the role of mitochondria and cytosolic proteins

Intracellular calcium oscillations, which are oscillatory changes of cytosolic calcium concentration in response to agonist stimulation, are experimentally well observed in various living cells. Simple calcium oscillations represent the most common pattern and many mathematical models have been published to describe this type of oscillation. On the other hand, relatively few theoretical studies have been proposed to give an explanation of complex intracellular calcium oscillations, such as bursting and chaos. In this paper, we develop a new possible mechanism for complex calcium oscillations based on the interplay between three calcium stores in the cell: the endoplasmic reticulum (ER), mitochondria and cytosolic proteins. The majority ( approximately 80%) of calcium released from the ER is first very quickly sequestered by mitochondria. Afterwards, a much slower release of calcium from the mitochondria serves as the calcium supply for the intermediate calcium exchanges between the ER and the cytosolic proteins causing bursting calcium oscillations. Depending on the permeability of the ER channels and on the kinetic properties of calcium binding to the cytosolic proteins, different patterns of complex calcium oscillations appear. With our model, we are able to explain simple calcium oscillations, bursting and chaos. Chaos is also observed for calcium oscillations in the bursting mode.     

Mitochondria determine the sequential propagation of the calcium macrodomains revealed by the super-resolution calcium lantern imaging

Despite the wide application of super-resolution(SR) microscopy in biological studies of cells, the technology is rarely used to monitor functional changes in live cells. By combining fast spinning disc-confocal structured illumination microscopy(SD-SIM)with loading of cytosolic fluorescent Ca~(2+) indicators, we have developed an SR method for visualization of regional Ca~(2+) dynamics and related cellular organelle morphology and dynamics, termed SR calcium lantern imaging. In COS-7 cells stimulated with ATP, we have identified various calcium macrodomains characterized by different types of Ca~(2+) release from endoplasmic reticulum(ER) stores. Finally, we demonstrated various roles of mitochondria in mediating calcium signals from different sources; while mitochondria can globally potentiate the Ca~(2+) entry associated with store release, mitochondria also locally control Ca~(2+) release from the neighboring ER stores and assist in their refilling processes.     

The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria

The unique endoplasmic reticulum (ER) subdomain termed the mitochondria-associated ER membrane (MAM) engages the physical connection between the ER and the mitochondrial outer membrane and plays a role in regulating IP(3) receptor-mediated Ca(2+) influx and the phospholipid transport between the two organelles. The MAM contains certain signaling and membrane-tethering proteins but also lipids including cholesterol. The biophysical role of lipids at the MAM, specifically in the physical interaction between the MAM of the ER and mitochondria, remains not totally clarified. Here we employed the in vitro membrane association assay to investigate the role of cholesterol in the association between MAMs and mitochondria. The purified MAMs and mitochondria were mixed in vitro in a test tube and then the physical association of the two subcellular organelles was quantified indirectly by measuring the presence of the MAM-specific protein sigma-1 receptors in the mitochondria fraction. Purified MAMs contained free cholesterol approximately 7 times higher than that in microsomes. We found that depletion of cholesterol in MAMs with methyl-β-cyclodextrin (MβC) significantly increases the association between MAMs and mitochondria, whereas MβC saturated with cholesterol does not change the association. (14)C-Serine pulse-labeling demonstrated that the treatment of living cells with MβC decreases the level of de novo synthesized (14)C-phosphatidylserine (PtSer) and concomitantly increases greatly the synthesis of (14)C-phosphatidylethanolamine (PtEt). Apparently, cholesterol depletion increased the PtSer transport from MAMs to mitochondria. Our findings suggest that cholesterol is an important substrate in regulating the association between MAMs of the ER and mitochondria.     

Structural and functional features and significance of the physical linkage between ER and mitochondria

The role of mitochondria in cell metabolism and survival is controlled by calcium signals that are commonly transmitted at the close associations between mitochondria and endoplasmic reticulum (ER). However, the physical linkage of the ER-mitochondria interface and its relevance for cell function remains elusive. We show by electron tomography that ER and mitochondria are adjoined by tethers that are approximately 10 nm at the smooth ER and approximately 25 nm at the rough ER. Limited proteolysis separates ER from mitochondria, whereas expression of a short "synthetic linker" (<5 nm) leads to tightening of the associations. Although normal connections are necessary and sufficient for proper propagation of ER-derived calcium signals to the mitochondria, tightened connections, synthetic or naturally observed under apoptosis-inducing conditions, make mitochondria prone to Ca2+ overloading and ensuing permeability transition. These results reveal an unexpected dependence of cell function and survival on the maintenance of proper spacing between the ER and mitochondria.     

Targeting of the c-Abl tyrosine kinase to mitochondria in endoplasmic reticulum stress-induced apoptosis

The ubiquitously expressed c-Abl tyrosine kinase localizes to the nucleus and cytoplasm. Using confocal microscopy, we demonstrated that c-Abl colocalizes with the endoplasmic reticulum (ER)-associated protein grp78. Expression of c-Abl in the ER was confirmed by immunoelectron microscopy. Subcellular fractionation studies further indicate that over 20% of cellular c-Abl is detectable in the ER. The results also demonstrate that induction of ER stress with calcium ionophore A23187, brefeldin A, or tunicamycin is associated with translocation of ER-associated c-Abl to mitochondria. In concert with targeting of c-Abl to mitochondria, cytochrome c is released in the response to ER stress by a c-Abl-dependent mechanism, and ER stress-induced apoptosis is attenuated in c-Abl-deficient cells. These findings indicate that c-Abl is involved in signaling from the ER to mitochondria and thereby the apoptotic response to ER stress.     

Ceramide increases mitochondrial free calcium levels via caspase 8 and Bid: role in initiation of cell death

We investigated how the mitochondrial phase of ceramide-mediated cell death is initiated in nerve growth factor (NGF)-differentiated PC12 cells. We distinguished three independent effects of ceramide: free radical production; a transient increase in cytosolic free calcium; and a long-lasting increase in mitochondrial free calcium. Only the latter led to cell death, which could be prevented by buffering of mitochondrial calcium with the calcium binding protein calbindin D-28K ectopically expressed in mitochondria. We showed that mitochondrial calcium did not increase as a result of the increase in cytosolic free calcium levels. Rather, it appears to derive from the endoplasmic reticulum (ER) since dantrolene, which inhibits release of calcium from ER into cytosol through ryanodine receptors, prevented the increase in cytosolic free calcium but potentiated the increase in mitochondrial free calcium. This suggests that a transfer of calcium occurs directly, or very locally, between the two organelles. This transfer implicated activation of caspase 8 and cleavage of its substrate Bid, a previously unknown function of these cell death intermediaries. The increase in mitochondrial free calcium was also responsible for the release of cytochrome c into the cytosol, underlining the critical role it plays in ceramide-mediated cell death.     

Role of smooth muscle cells on endothelial cell cytosolic free calcium in porcine coronary arteries

We tested the hypothesis that the cytosolic free calcium concentration in endothelial cells is under the influence of the smooth muscle cells in the coronary circulation. In the left descending branch of porcine coronary arteries, cytosolic free calcium concentration ([Ca(2+)](i)) was estimated by determining the fluorescence ratio of two calcium probes, fluo 4 and fura red, in smooth muscle and endothelial cells using confocal microscopy. Acetylcholine and potassium, which act directly on smooth muscle cells to increase [Ca(2+)](i), were found to indirectly elevate [Ca(2+)](i) in endothelial cells; in primary cultures of endothelial cells, neither stimulus affected [Ca(2+)](i), yet substance P increased the fluorescence ratio twofold. In response to acetylcholine and potassium, isometric tension developed by arterial strips with intact endothelium was attenuated by up to 22% (P < 0.05) compared with strips without endothelium. These findings suggest that stimuli that increase smooth muscle [Ca(2+)](i) can indirectly influence endothelial cell function in porcine coronary arteries. Such a pathway for negative feedback can moderate vasoconstriction and diminish the potential for vasospasm in the coronary circulation.     

Localization of intracellular calcium release in cells injured by venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) and dependence of calcium mobilization on G-protein activation

Venom from the ectoparasitic wasp Nasonia vitripennis induces cellular injury that appears to involve the release of intracellular calcium stores via the activation of phospholipase C, and culminates in oncotic death. A linkage between release of intracellular Ca2+ and oncosis has not been clearly established and was the focus of this study. When BTI-TN-5B1-4 cells were treated with suramin, an uncoupler of G-proteins, venom-induced swelling and oncotic death were inhibited in a dose-dependent manner for at least 24 h. Suramin also blocked increases in free cytosolic [Ca2+], arguing that venom induces calcium mobilization through G-protein signaling pathways. Endoplasmic reticulum (ER) was predicted to be the source of intracellular calcium release, but labeling with the fluorescent probe ER-tracker revealed no indication of organelle swelling or loss of membrane integrity as would be expected if the Ca(2+)-ATPase pump was disabled by crude venom. Incubation of cell monolayers with calmodulin or nitrendipine, modulators of ER calcium release channels, neither attenuated nor augmented the effects of wasp venom. These results suggest that wasp venom stimulates calcium release from ER compartments distinct from RyRs, L-type Ca2+ channels, and the Ca(2+)-ATPase pump, or calcium is released from some other intracellular store. A reduction of mitochondrial membrane potential delta psi(m) appeared to precede a rise in cytosolic free Ca2+ as evidenced by fluorescent microscopy using the calcium-sensitive probe fluo-4 AM. This argues that the initial insult to the cell resulting from venom elicits a rapid loss of (delta psi(m)), followed by unregulated calcium efflux from mitochondria into the cytosol. Mobilization of calcium in this fashion could stimulate cAMP formation, and subsequently promote calcium release from NAADP-sensitive stores.     

Targeting of hepatitis C virus core protein to mitochondria through a novel C-terminal localization motif

The hepatitis C virus (HCV) core protein represents the first 191 amino acids of the viral precursor polyprotein and is cotranslationally inserted into the membrane of the endoplasmic reticulum (ER). Processing at position 179 by a recently identified intramembrane signal peptide peptidase leads to the generation and potential cytosolic release of a 179-amino-acid matured form of the core protein. Using confocal microscopy, we observed that a fraction of the mature core protein colocalized with mitochondrial markers in core-expressing HeLa cells and in Huh-7 cells containing the full-length HCV replicon. Subcellular fractionation confirmed this observation and showed that the core protein associates with purified mitochondrial fractions devoid of ER contaminants. The core protein also fractionated with mitochondrion-associated membranes, a site of physical contact between the ER and mitochondria. Using immunoelectron microscopy and in vitro mitochondrial import assays, we showed that the core protein is located on the mitochondrial outer membrane. A stretch of 10 amino acids within the hydrophobic C terminus of the processed core protein conferred mitochondrial localization when it was fused to green fluorescent protein. The location of the core protein in the outer mitochondrial membrane suggests that it could modulate apoptosis or lipid transfer, both of which are associated with this subcellular compartment, during HCV infection.     

The ependymal secretion of the fetal and adult rat subcommissural organ. morphological aspects linked to the synthesis,storage and release of the secretory products

Different localizations of secretory material are noted in adult and fetal subcommissural organ (SCO) in light microscopy. At the electron microscope level, the secretory ependymocytes reveal frequent associations among mitochondria and ribosomes of the endoplasmic reticulum (ER). In the SCO ependymocytes of the adult rat, the relationship between mitochondria and ribosomes of the ER is observed in the subgolgian zone, the ER cisternal profiles are smooth except where they face the mitochondria. Here, a constant interval of 40-45 nm separates the ribosome-coated ER membrane from the external membrane of the mitochondria. This association evidences a functional cooperation between mitochondria and ER, at least in some phases of the synthesis of the organ's gliosecretory material. By contrast, in the fetus (17-21 fetal day), the synthetic apparatus displays an entirely granular ER. The secretory products are stored as flocculent material which fills the ER cisternae. In the apical zone of the ependymocytes, as the membrane of the dense secretory granules fuses with the apical plasmalemma, the granules release their contents into the ventricular cavity. A possible link between the releasing process and the coated vesicles is discussed.     

Localization of GRP78 to mitochondria under the unfolded protein response

The ubiquitously expressed molecular chaperone GRP78 (78 kDa glucose-regulated protein) generally localizes to the ER (endoplasmic reticulum). GRP78 is specifically induced in cells under the UPR (unfolded protein response), which can be elicited by treatments with calcium ionophore A23187 and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor TG (thapsigargin). By using confocal microscopy, we have demonstrated that GRP78 was concentrated in the perinuclear region and co-localized with the ER marker proteins, calnexin and PDI (protein disulphide-isomerase), in cells under normal growth conditions. However, treatments with A23187 and TG led to diminish its ER targeting, resulting in redirection into a cytoplasmic vesicular pattern, and overlapping with the mitochondrial marker MitoTracker. Cellular fractionation and protease digestion of isolated mitochondria from ER-stressed cells suggested that a significant portion of GRP78 is localized to the mitochondria and is protease-resistant. Localizations of GRP78 in ER and mitochondria were confirmed by using immunoelectron microscopy. In ER-stressed cells, GRP78 mainly localized within the mitochondria and decorated the mitochondrial membrane compartment. Submitochondrial fractionation studies indicated further that the mitochondria-resided GRP78 is mainly located in the intermembrane space, inner membrane and matrix, but is not associated with the outer membrane. Furthermore, radioactive labelling followed by subcellular fractionation showed that a significant portion of the newly synthesized GRP78 is localized to the mitochondria in cells under UPR. Taken together, our results indicate that, at least under certain circumstances, the ER-resided chaperone GRP78 can be retargeted to mitochondria and thereby may be involved in correlating UPR signalling between these two organelles.     

Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle

Autocrine motility factor (AMF) is secreted by tumor cells and is capable of stimulating the motility of the secreting cells. In addition to being expressed on the cell surface, its receptor, AMF-R, is found within a Triton X-100 extractable intracellular tubular compartment. AMF-R tubules can be distinguished by double immunofluorescence microscopy from endosomes labeled with the transferrin receptor, lysosomes labeled with LAMP-2, and the Golgi apparatus labeled with beta-COP. AMF-R can also be separated from a LAMP-2 containing lysosomal fraction by differential centrifugation of MDCK cells and is found within a 100,000 g membrane pellet. By electron microscopic immunocytochemistry, AMF-R is localized predominantly to smooth vesicular and tubular membranous organelles as well as to a lesser extent to the plasma membrane and rough endoplasmic reticulum. AMF-R tubules have a variable diameter of 50-250 nm and can acquire an elaborate branched morphology. By immunofluorescence microscopy, AMF-R tubules are clearly distinguished from the calnexin labeled rough endoplasmic reticulum and AMF-R tubule expression is stable to extended cycloheximide treatment. The AMF-R tubule is therefore not a biosynthetic subcompartment of the endoplasmic reticulum. The tubular morphology of the AMF-R tubule is modulated by both the actin and microtubule cytoskeletons. In a similar fashion to that described previously for the tubular lysosome and endoplasmic reticulum, the linear extension and peripheral cellular orientation of the AMF-R tubule are dependent on the integrity of the microtubule cytoskeleton. The AMF-R tubule may thus form part of a family of microtubule- associated tubular organelles.     

Ultrastructural relationship of the phagophore with surrounding organelles

Phagophore nucleates from a subdomain of the endoplasmic reticulum (ER) termed the omegasome and also makes contact with other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes during its formation. We have used serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) to image phagophore biogenesis in 3 dimensions and to determine the relationship between the phagophore and surrounding organelles at high resolution. ET was performed to confirm whether membrane contact sites (MCSs) are evident between the phagophore and those surrounding organelles. In addition to the known contacts with the ER, we identified MCSs between the phagophore and membranes from putative ER exit sites, late endosomes or lysosomes, the Golgi complex and mitochondria. We also show that one phagophore can have simultaneous MCSs with more than one organelle. Future membrane flux experiments are needed to determine whether membrane contacts also signify lipid translocation.     

The ependymal secretion of the fetal and adult rat subcommissural organ. morphological aspects linked to the synthesis, storage and release of the secretory products

Different localizations of secretory material are noted in adult and fetal subcommissural organ (SCO) in light microscopy. At the electron microscope level, the secretory ependymocytes reveal frequent associations among mitochondria and ribosomes of the endoplasmic reticulum (ER). In the SCO ependymocytes of the adult rat, the relationship between mitochondria and ribosomes of the ER is observed in the subgolgian zone, the ER cisternal profiles are smooth except where they face the mitochondria. Here, a constant interval of 40-45 nm separates the ribosome-coated ER membrane from the external membrane of the mitochondria. This association evidences a functional cooperation between mitochondria and ER, at least in some phases of the synthesis of the organ's gliosecretory material. By contrast, in the fetus (17-21 fetal day), the synthetic apparatus displays an entirely granular ER. The secretory products are stored as flocculent material which fills the ER cisternae. In the apical zone of the ependymocytes, as the membrane of the dense secretory granules fuses with the apical plasmalemma, the granules release their contents into the ventricular cavity. A possible link between the releasing process and the coated vesicles is discussed.