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 [Ca2+] changes ([Ca2+]ER-mt) using drug-inducible fluorescent interorganelle linkers. We show that all mitochondria have contacts with the ER but plasma membrane-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 Ca2+ transfer. Pericam-tagged linkers revealed IP3-induced [Ca2+]ER-mt signals that exceeded 9μM and endured buffering bulk cytoplasmic [Ca2+] increases. Altering linker length to modify the space available for the Ca2+ transfer machinery had a biphasic effect on [Ca2+]ER-mt signals. These studies provide direct evidence for the existence of high Ca2+ microdomains between the ER and mitochondria, and suggest an optimal gap width for efficient Ca2+ transfer.

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, while Mfn2, but not Mfn1, tethers endoplasmic reticulum to mitochondria in non-cardiac 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. While 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 β-adrenergic stimulation was hampered in Mfn2-, 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 inter-organelle 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.

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.

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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. 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. 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. 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.

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Hemoglobin degradation during the asexual cycle of Plasmodium falciparum is an obligate process for parasite development and survival. It is established that hemoglobin is transported from the host erythrocyte to the parasite digestive vacuole (DV), but this biological process is not well characterized. Three-dimensional reconstructions made from serial thin-section electron micrographs of untreated, trophozoite-stage P. falciparum-infected erythrocytes (IRBC) or IRBC treated with different pharmacological agents provide new insight into the organization and regulation of the hemoglobin transport pathway. Hemoglobin internalization commences with the formation of cytostomes from localized, electron-dense collars at the interface of the parasite plasma and parasitophorous vacuolar membranes. The cytostomal collar does not function as a site of vesicle fission but rather serves to stabilize the maturing cytostome. We provide the first evidence that hemoglobin transport to the DV uses an actin-myosin motor system. Short-lived, hemoglobin-filled vesicles form from the distal end of the cytostomes through actin and dynamin-mediated processes. Results obtained with IRBC treated with N-ethylmaleimide (NEM) suggest that fusion of hemoglobin-containing vesicles with the DV may involve a soluble NEM-sensitive factor attachment protein receptor-dependent mechanism. In this report, we identify new key components of the hemoglobin transport pathway and provide a detailed characterization of its morphological organization and regulation. Malaria is a devastating disease that infects Ͼ300 million people each year, resulting in Ͼ1 million deaths, with the protozoan species Plasmodium falciparum being responsible for the majority of these deaths (1). The morbidity and mortality associated with the disease are largely the result of the parasite's asexual intraerythrocytic cycle (2). P. falciparum digests host cell hemoglobin to support parasite growth and asexual replication during the intraerythrocytic stage (3, 4). In order for the parasite to survive within the erythrocyte host, it degrades approximately 80% of the erythrocyte hemoglobin (5), with the majority of this digestion occurring during the trophozoite stage (18 to 32 h postinvasion) (3). The bulk of hemoglobin degradation occurs via a semiordered process by proteases contained within the parasite's digestive vacuole (DV) (6).The transport of hemoglobin from the host erythrocyte cytosol to the parasite DV is a long-known but poorly understood biological process. The internalization of hemoglobin is thought to occur through an unusual structure, the cytostome. A cytostome is defined as a localized invagination of the parasite's outer membranes (the parasitophorous vacuolar membrane [PVM] and the parasite plasma membrane [PPM]), with a submembranous electron-dense collar associated with the neck of the cytostome (7, 8). It has traditionally been assumed that hemoglobin transport commences with the cytostome pinching off at the neck to form a double-membrane, hemoglobin-f...

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