Timothy Schneider

Imaging Interorganelle Contacts and Local Calcium Dynamics at the ER-Mitochondrial Interface

The ER-mitochondrial junction provides a local calcium signaling domain that is critical for both matching energy production with demand and the control of apoptosis. Here, we visualize ER-mitochondrial contact sites and monitor the localized [Ca(2+)] changes ([Ca(2+)](ER-mt)) using drug-inducible fluorescent interorganelle linkers. We show that all mitochondria have contacts with the ER, but plasma membrane (PM)-mitochondrial contacts are less frequent because of interleaving ER stacks in both RBL-2H3 and H9c2 cells. Single mitochondria display discrete patches of ER contacts and show heterogeneity in the ER-mitochondrial Ca(2+) transfer. Pericam-tagged linkers revealed IP(3)-induced [Ca(2+)](ER-mt) signals that exceeded 9 microM and endured buffering bulk cytoplasmic [Ca(2+)] increases. Altering linker length to modify the space available for the Ca(2+) transfer machinery had a biphasic effect on [Ca(2+)](ER-mt) signals. These studies provide direct evidence for the existence of high-Ca(2+) microdomains between the ER and mitochondria and suggest an optimal gap width for efficient Ca(2+) transfer.

Mitofusin 2-Containing Mitochondrial-Reticular Microdomains Direct Rapid Cardiomyocyte Bioenergetic Responses Via Interorganelle Ca 2! Crosstalk

Rationale: Mitochondrial Ca2+ uptake is essential for the bioenergetic feedback response through stimulation of Krebs cycle dehydrogenases. Close association of mitochondria to the sarcoplasmic reticulum (SR) may explain efficient mitochondrial Ca2+ uptake despite low Ca2+ affinity of the mitochondrial Ca2+ uniporter. However, the existence of such mitochondrial Ca2+ microdomains and their functional role are presently unresolved. Mitofusin (Mfn) 1 and 2 mediate mitochondrial outer membrane fusion, whereas Mfn2 but not Mfn1 tethers endoplasmic reticulum to mitochondria in noncardiac cells. Objective: To elucidate roles for Mfn1 and 2 in SR-mitochondrial tethering, Ca2+ signaling, and bioenergetic regulation in cardiac myocytes. Methods and Results: Fruit fly heart tubes deficient of the Drosophila Mfn ortholog MARF had increased contraction-associated and caffeine-sensitive Ca2+ release, suggesting a role for Mfn in SR Ca2+ handling. Whereas cardiac-specific Mfn1 ablation had no effects on murine heart function or Ca2+ cycling, Mfn2 deficiency decreased cardiomyocyte SR-mitochondrial contact length by 30% and reduced the content of SR-associated proteins in mitochondria-associated membranes. This was associated with decreased mitochondrial Ca2+ uptake (despite unchanged mitochondrial membrane potential) but increased steady-state and caffeine-induced SR Ca2+ release. Accordingly, Ca2+-induced stimulation of Krebs cycle dehydrogenases during &bgr;-adrenergic stimulation was hampered in Mfn2-KO but not Mfn1-KO myocytes, evidenced by oxidation of the redox states of NAD(P)H/NAD(P)+ and FADH2/FAD. Conclusions: Physical tethering of SR and mitochondria via Mfn2 is essential for normal interorganelle Ca2+ signaling in the myocardium, consistent with a requirement for SR-mitochondrial Ca2+ signaling through microdomains in the cardiomyocyte bioenergetic feedback response to physiological stress.

Cytochrome c Release upon Fas Receptor Activation Depends on Translocation of Full-length Bid and the Induction of the Mitochondrial Permeability Transition

In Jurkat cells Bid was cleaved upon activation of the Fas receptor with an anti-Fas antibody. The caspase-8 inhibitor benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F (IETD) prevented the cleavage of Bid and the loss of viability. The nuclear enzyme poly(ADP-ribose)polymerase (PARP) was also cleaved upon the activation of caspases, and IETD similarly prevented PARP cleavage. The PARP inhibitor 3-aminobenzamide (3-AB) restored the cell killing in the presence of IETD, an effect that occurred without restoration of the cleavage of Bid or PARP. In the presence of 3-AB and IETD, translocation occurred of full-length Bid to the mitochondria. The induction of the mitochondrial permeability transition (MPT) was documented by the cyclosporin A (CyA) sensitivity of the release of cytochrome c, the release of malate dehydrogenase from the mitochondrial matrix, the loss of the mitochondrial membrane potential, and the pronounced swelling of these organelles, as assessed by electron microscopy. In addition to preventing all evidence of the MPT, CyA prevented the loss of cell viability, without effect on the cleavage of either Bid or PARP. The prevention of PARP cleavage by inhibition of caspase-3 resulted in a 10-fold activation of the enzyme and a resultant depletion of NAD and ATP. The PARP inhibitor 3-AB prevented the loss of NAD and ATP. Depletion of ATP by metabolic inhibitors similarly prevented the cell killing. It is concluded that the cleaving of PARP in Fas-mediated apoptosis allowed expression of an energy-dependent cell death program that included the translocation of full-length Bid to the mitochondria with induction of the MPT.

Evidence for vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes.

Plasmodium falciparum malaria parasites actively remodel the host cell cytosol and plasma membrane during the erythrocytic cycle. The focus of this investigation was to characterize intra-parasitic and -erythrocytic secretory pathways. Electron-dense vesicles, similar in appearance to mammalian secretory vesicles were detected in proximity to smooth tubo-vesicular elements at the periphery of the parasite cytoplasm in mature parasites by transmission electron microscopy. Vesicles (60-100 nm diameter), which appeared to be coated, were visualized on the erythrocytic side of the parasite vacuolar membrane and in the erythrocyte cytosol. The vesicles seemed to bind to and fuse with the erythrocyte membrane, giving rise to cup-shaped electron-dense structures, which might be intermediates in knob structure formation. Treatment of mature parasites with aluminum tetrafluoride, an activator of GTP-binding proteins, resulted in the accumulation of the vesicles with an electron-dense limiting membrane in the erythrocyte cytosol into multiple vesicle strings. These vesicle complexes were often associated with and closely abutted the erythrocyte membrane, but were apparently prevented from fusing by the aluminum fluoride treatment. The parasite proteins PfEMP1 and PfEMP3 were found by immunoelectron microscopy to be associated with these vesicles, suggesting they are responsible for transporting these proteins to the erythrocyte membrane.

Quantification of calcium signal transmission from sarco-endoplasmic reticulum to the mitochondria

1 Recent studies have shown that ryanodine and IP3 receptor (RyR/IP3R)‐mediated cytosolic Ca2+ signals propagate to the mitochondria, initiating chains of events vital in the regulation of different cellular functions. However, the fraction of released Ca2+ utilized by the mitochondria during these processes has not been quantified. 2 To measure the amount of Ca2+ taken up by the mitochondria, we used a novel approach that involves simultaneous fluorescence imaging of mitochondrial and cytosolic [Ca2+] in permeabilized H9c2 myotubes and RBL‐2H3 mast cells. Communication between sarco‐endoplasmic reticulum (SR/ER) and mitochondria is maintained in these permeabilized cells, as evidenced by the large RyR/IP3R‐driven mitochondrial matrix [Ca2+] and NAD(P)H signals and also by preservation of the morphology of the SR/ER‐mitochondrial junctions. 3 Ca2+ was released from the SR/ER by addition of saturating caffeine or IP3 and subsequently thapsigargin (Tg), an inhibitor of SR/ER Ca2+ pumps. The amount of Ca2+ transmitted to the mitochondria was determined by measuring increases of global [Ca2+] in the incubation medium (cytosolic [Ca2+] ([Ca2+]c)). Mitochondrial Ca2+ uptake was calculated from the difference between [Ca2+]c responses recorded in the absence and presence of uncoupler or from [Ca2+]c elevations evoked by uncoupler or ionophore applied after complete Ca2+ mobilization from the SR/ER. [Ca2+]c increases were calibrated by adding Ca2+ pulses to the permeabilized cells. 4 In H9c2 cells, caffeine induced partial mobilization of SR Ca2+ and mitochondria accumulated 26% of the released Ca2+. Sequential application of caffeine and Tg elicited complete discharge of SR Ca2+ without further increase in mitochondrial Ca2+ uptake. In RBL‐2H3 mast cells, IP3 by itself elicited complete discharge of the ER Ca2+ store and the increase of the ionophore‐releasable mitochondrial Ca2+ content reached 50% of the Ca2+ amount mobilized by IP3+ Tg. Thus, RyR/IP3R direct a substantial fraction of released Ca2+ to the mitochondria.

Cytochrome c-dependent activation of caspase-3 by tumor necrosis factor requires induction of the mitochondrial permeability transition.

The killing of L929 mouse fibroblasts by tumor necrosis factor-alpha (TNF-alpha) in the presence of 0.5 microg/ml actinomycin D (Act D) is prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A(2) inhibitor aristolochic acid (ArA). The MPT is accompanied by the release of cytochrome c from the mitochondria, caspase-8 and caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. The caspase-3 inhibitor z-Asp-Glu-Val-aspartic acid fluoromethyl-ketone (Z-DEVD-FMK) did not prevent the loss of viability or the redistribution of cytochrome c, but it did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. Inhibition of the MPT reduced the activation of caspase-8 to the level occurring with TNF-alpha alone (no ActD). The caspase-8 inhibitor z-Ile-Glu(OMe)-Thr-Asp(OMe) fluoromethylketone (Z-IETD-FMK) did not prevent the cell killing and decreased only slightly the translocation of Bid to the mitochondria. These data indicate that induction of the MTP by TNF-alpha causes a release of cytochrome c, caspase-3 activation with PARP cleavage and DNA fragmentation. The loss of viability is dependent on the MPT but independent of the activation of caspase-3. The activation of caspase-8 is not dependent on the MPT. There is no evidence linking this enzyme to the loss of viability. Thus, the killing of L929 fibroblasts by TNF-alpha can occur in the absence of either caspase-3 or caspase-8 activity. Alternatively, cell death can be prevented despite an activation of caspase-8.

CHARACTERIZATION OF MACROMOLECULAR TRANSPORT PATHWAYS IN MALARIA-INFECTED ERYTHROCYTES

We have previously provided evidence for a pathway in Plasmodium falciparum-infected erythrocytes, coined the parasitophorous duct pathway, which provides serum (macro)molecules direct access to intraerythrocytic parasites . The present study addresses the purity of the fluorescent macromolecules used to define the duct pathway and provides ultrastructural evidence for its presence. The fluorescent tracers used to characterize transport remain intact during their incubation with infected erythrocytes. Transport of macromolecules in the external medium or host cell cytosol to the intracellular parasites is shown to occur by two distinct pathways. Fluorescent dextrans in the erythrocyte cytosol are ingested by the parasite via a specialized organelle, the cytostome, and are transported to the parasite food vacuole. Transport through this pathway occurs throughout the asexual life cycle. By contrast, fluorescent dextrans in the external medium bypass the erythrocyte cytosol, and are internalized by the parasite by a process resembling fluid-phase endocytosis. Serial sections of mature parasites fixed and stained by various methods for transmission electron microscopy reveal areas of apparent membrane continuity between the erythrocyte membrane and the parasitophorous vacuolar membrane that surrounds the parasite, that could leave the parasites exposed to the external medium. Using carboxylate and amidine-modified fluorescent latex spheres and laser scanning confocal microscopy, macromolecules up to 50-70 nm in diameter are found to have direct access to intraerythrocytic parasites. This size exclusion is consistent with the dimensions of the parasitophorous duct pathway revealed by electron microscopy. This investigation reports for the first time the existence of two, distinct macromolecular transport pathways in malaria-infected erythrocytes.

Vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes

During the development of the asexual stage of the malaria parasite, Plasmodium falciparum, the composition, structure and function of the host cell membrane is dramatically altered, including the ability to adhere to vascular endothelium. Crucial to these changes is the transport of parasite proteins, which become associated with or inserted into the erythrocyte membrane. Protein and membrane targeting beyond the parasite plasma membrane must require unique pathways, given the parasites intracellular location within a parasitophorous vacuolar membrane and the lack of organelles and biosynthetic machinery in the host cell necessary to support a secretory system. It is not clear how these proteins cross the parasitophorous vacuolar membrane or how they traverse the erythrocyte cytosol to reach their final destinations. The identification of: (1) a P. falciparum homologue of the protein Sar1p, which is an essential component of the COPII-based secretory system in mammalian cells and yeast and (2) electron-dense, possibly coated, secretory vesicles bearing P. falciparum erythrocyte membrane protein 1 and P. falciparum erythrocyte membrane protein 3 in the host cell cytosol of P. falciparum infected erythrocytes recently provided the first direct evidence of a vesicle-mediated pathway for the trafficking of some parasite proteins to the erythrocyte membrane. The major advance in uncovering the parasite-induced secretory pathway was made by incubating infected erythrocytes with aluminium tetrafluoride, an activator of guanidine triphosphate-binding proteins, which resulted in the accumulation of the vesicles into multiple vesicle strings. These vesicle complexes were often associated with and closely abutted the erythrocyte membrane, but were apparently prevented from fusing by the aluminium fluoride treatment, making their capture by electron microscopy possible. It appears that malaria parasites export proteins into the host cell cytosol to support a vesicle-mediated protein trafficking pathway.

Delivery of the Malaria Virulence Protein PfEMP1 to the Erythrocyte Surface Requires Cholesterol-Rich Domains

ABSTRACT The particular virulence of the human malaria parasite Plasmodium falciparum derives from export of parasite-encoded proteins to the surface of the mature erythrocytes in which it resides. The mechanisms and machinery for the export of proteins to the erythrocyte membrane are largely unknown. In other eukaryotic cells, cholesterol-rich membrane microdomains or “rafts” have been shown to play an important role in the export of proteins to the cell surface. Our data suggest that depletion of cholesterol from the erythrocyte membrane with methyl-β-cyclodextrin significantly inhibits the delivery of the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). The trafficking defect appears to lie at the level of transfer of PfEMP1 from parasite-derived membranous structures within the infected erythrocyte cytoplasm, known as the Maurers clefts, to the erythrocyte membrane. Thus our data suggest that delivery of this key cytoadherence-mediating protein to the host erythrocyte membrane involves insertion of PfEMP1 at cholesterol-rich microdomains. GTP-dependent vesicle budding and fusion events are also involved in many trafficking processes. To determine whether GTP-dependent events are involved in PfEMP1 trafficking, we have incorporated non-membrane-permeating GTP analogs inside resealed erythrocytes. Although these nonhydrolyzable GTP analogs reduced erythrocyte invasion efficiency and partially retarded growth of the intracellular parasite, they appeared to have little direct effect on PfEMP1 trafficking.

A new model for hemoglobin ingestion and transport by the human malaria parasite Plasmodium falciparum

The current model for hemoglobin ingestion and transport by intraerythrocytic Plasmodium falciparum malaria parasites shares similarities with endocytosis. However, the model is largely hypothetical, and the mechanisms responsible for the ingestion and transport of host cell hemoglobin to the lysosome-like food vacuole (FV) of the parasite are poorly understood. Because actin dynamics play key roles in vesicle formation and transport in endocytosis, we used the actin-perturbing agents jasplakinolide and cytochalasin D to investigate the role of parasite actin in hemoglobin ingestion and transport to the FV. In addition, we tested the current hemoglobin trafficking model through extensive analysis of serial thin sections of parasitized erythrocytes (PE) by electron microscopy. We find that actin dynamics play multiple, important roles in the hemoglobin transport pathway, and that hemoglobin delivery to the FV via the cytostomes might be required for parasite survival. Evidence is provided for a new model, in which hemoglobin transport to the FV occurs by a vesicle-independent process.