The view of organelles and how they operate together has changed dramatically over the last two decades. The textbook view of organelles was that they operated largely independently and were ...connected by vesicular trafficking and the diffusion of signals through the cytoplasm. We now know that all organelles make functional close contacts with one another, often called membrane contact sites. The study of these sites has moved to center stage in cell biology as it has become clear that they play critical roles in healthy and developing cells and during cell stress and disease states. Contact sites have important roles in intracellular signaling, lipid metabolism, motor-protein-mediated membrane dynamics, organelle division, and organelle biogenesis. Here, we summarize the major conceptual changes that have occurred in cell biology as we have come to appreciate how contact sites integrate the activities of organelles.
Membrane contact sites between organelles act as hubs for lipid transport, calcium signaling, lipid droplet biogenesis, and organelle dynamics. This review discusses the conceptual changes in cell biology that have occurred, with the examination of how membrane contact sites modulate organelle function and integrate their operations.
Generation of mitochondrial signals is believed to be important in the commitment to apoptosis, but the mechanisms coordinating the output of individual mitochondria remain elusive. We show that in ...cardiac myotubes exposed to apoptotic agents, Ca2+ spikes initiate depolarization of mitochondria in discrete subcellular regions, and these mitochondria initiate slow waves of depolarization and Ca2+ release propagating through the cell. Traveling mitochondrial waves are prevented by Bcl‐xL, involve permeability transition pore (PTP) opening, and yield cytochrome c release, caspase activation and nuclear apoptosis. Mito chondrial Ca2+ uptake is critical for wave propagation, and mitochondria at the origin of waves take up Ca2+ particularly effectively, providing a mechanism that may underlie selection of the initiation sites. Thus, apoptotic agents transform the mitochondria into an excitable state by sensitizing PTP to Ca2+. Expansion of the local excitation by mitochondrial waves propagating through the whole cell can be especially important in activation of the apoptotic machinery in large cells.
Functional states of mitochondria are often reflected in characteristic mitochondrial morphology. One of the most fundamental stress conditions, hypoxia-reoxygenation has been known to cause impaired ...mitochondrial function accompanied by structural abnormalities, but the underlying mechanisms need further investigation. Here, we monitored bioenergetics and mitochondrial fusion-fission in real time to determine how changes in mitochondrial dynamics contribute to structural abnormalities during hypoxia-reoxygenation. Hypoxia-reoxygenation resulted in the appearance of shorter mitochondria and a decrease in fusion activity. This fusion inhibition was a result of impaired ATP synthesis rather than Opa1 cleavage. A striking feature that appeared during hypoxia in glucose-free and during reoxygenation in glucose-containing medium was the formation of donut-shaped (toroidal) mitochondria. Donut formation was triggered by opening of the permeability transition pore or K(+) channels, which in turn caused mitochondrial swelling and partial detachment from the cytoskeleton. This then favored anomalous fusion events (autofusion and fusion at several sites among 2-3 mitochondria) to produce the characteristic donuts. Donuts effectively tolerate matrix volume increases and give rise to offspring that can regain ΔΨ(m). Thus, the metabolic stress during hypoxia-reoxygenation alters mitochondrial morphology by inducing distinct patterns of mitochondrial dynamics, which includes processes that could aid mitochondrial adaptation and functional recovery.
In many cell types, IP
3 and ryanodine receptor (IP
3R/RyR)-mediated Ca
2+ mobilization from the sarcoendoplasmic reticulum (ER/SR) results in an elevation of mitochondrial matrix Ca
2+. Although ...delivery of the released Ca
2+ to the mitochondria has been established as a fundamental signaling process, the molecular mechanism underlying mitochondrial Ca
2+ uptake remains a challenge for future studies. The Ca
2+ uptake can be divided into the following three steps: (1) Ca
2+ movement from the IP
3R/RyR to the outer mitochondrial membrane (OMM); (2) Ca
2+ transport through the OMM; and (3) Ca
2+ transport through the inner mitochondrial membrane (IMM). Evidence has been presented that Ca
2+ delivery to the OMM is facilitated by a local coupling between closely apposed regions of the ER/SR and mitochondria. Recent studies of the dynamic changes in mitochondrial morphology and visualization of the subcellular pattern of the calcium signal provide important clues to the organization of the ER/SR–mitochondrial interface. Interestingly, key steps of phospholipid synthesis and transfer to the mitochondria have also been confined to subdomains of the ER tightly associated with the mitochondria, referred as mitochondria-associated membranes (MAMs). Through the OMM, the voltage-dependent anion channels (VDAC, porin) have been thought to permit free passage of ions and other small molecules. However, recent studies suggest that the VDAC may represent a regulated step in Ca
2+ transport from IP
3R/RyR to the IMM. A novel proposal regarding the IMM Ca
2+ uptake site is a mitochondrial RyR that would mediate rapid Ca
2+ uptake by mitochondria in excitable cells. An overview of the progress in these directions is described in the present paper.
One half million patients suffer from colorectal cancer in industrialized nations, yet this disease exhibits a low incidence in underdeveloped countries. This geographic imbalance suggests an ...environmental contribution to the resistance of endemic populations to intestinal neoplasia. A common epidemiological characteristic of these colon cancer-spared regions is the prevalence of enterotoxigenic bacteria associated with diarrheal disease. Here, a bacterial heat-stable enterotoxin was demonstrated to suppress colon cancer cell proliferation by a guanylyl cyclase C-mediated signaling cascade. The heat-stable enterotoxin suppressed proliferation by increasing intracellular cGMP, an effect mimicked by the cell-permeant analog 8-br-cGMP. The antiproliferative effects of the enterotoxin and 8-br-cGMP were reversed by L-cis-diltiazem, a cyclic nucleotide-gated channel inhibitor, as well as by removal of extracellular Ca2+, or chelation of intracellular Ca2+. In fact, both the enterotoxin and 8-br-cGMP induced an L-cis-diltiazem-sensitive conductance, promoting Ca2+ influx and inhibition of DNA synthesis in colon cancer cells. Induction of this previously unrecognized antiproliferative signaling pathway by bacterial enterotoxin could contribute to the resistance of endemic populations to intestinal neoplasia, and offers a paradigm for targeted prevention and therapy of primary and metastatic colorectal cancer.
Calcium signal transmission between endoplasmic reticulum (ER) and mitochondria is supported by a local Ca2+ control that operates between IP3receptor Ca2+release channels (IP3R) and mitochondrial ...Ca2+uptake sites, and displays functional similarities to synaptic transmission. Activation of IP3R by IP3is known to evoke quantal Ca2+mobilization that is associated with incremental elevations of mitochondrial matrix Ca2+ (Ca2+m). Here we report that activation of IP3R by adenophostin-A (AP) yields non-quantal Ca2+mobilization in mast cells. We also show that the AP-induced continuous Ca2+release causes relatively small Ca2+mresponses, in particular, the sustained phase of Ca2+release is not sensed by the mitochondria. Inhibition of ER Ca2+pumps by thapsigargin slightly increases IP3-induced Ca2+mresponses, but augments AP-induced Ca2+mresponses in a large extent. In adherent permeabilized cells exposed to elevated Ca2+, ER Ca2+uptake fails to affect global cytosolic Ca2+, but attenuates Ca2+mresponses. Moreover, almost every mitochondrion exhibits a region very close to ER Ca2+pumps visualized by BODIPY-FL-thapsigargin or SERCA antibody. Thus, at the ER-mitochondrial junctions, localized ER Ca2+uptake provides a mechanism to attenuate the mitochondrial response during continuous Ca2+release through the IP3R or during gradual Ca2+influx to the junction between ER and mitochondria.
Enhanced formation of reactive oxygen species (ROS), superoxide ( O2· -), and hydrogen peroxide ( H2 O2) may result in either apoptosis or other forms of cell death. Here, we studied the mechanisms ...underlying activation of the apoptotic machinery by ROS. Exposure of permeabilized HepG2 cells to O2· - elicited rapid and massive cytochrome c release (CCR), whereas H2 O2 failed to induce any release. Both O2· - and H2 O2 promoted activation of the mitochondrial permeability transition pore by Ca2+, but Ca2+-dependent pore opening was not required for O2· --induced CCR. Furthermore, O2· - alone evoked CCR without damage of the inner mitochondrial membrane barrier, as mitochondrial membrane potential was sustained in the presence of extramitochondrial ATP. Strikingly, pretreatment of the cells with drugs or an antibody, which block the voltage-dependent anion channel (VDAC), prevented O2· --induced CCR. Furthermore, VDAC-reconstituted liposomes permeated cytochrome c after O2· - exposure, and this release was prevented by VDAC blocker. The proapoptotic protein, Bak, was not detected in HepG2 cells and O2· --induced CCR did not depend on Bax translocation to mitochondria. O2· --induced CCR was followed by caspase activation and execution of apoptosis. Thus, O2· - triggers apoptosis via VDAC-dependent permeabilization of the mitochondrial outer membrane without apparent contribution of proapoptotic Bcl-2 family proteins.
The small leucine-rich proteoglycan decorin interacts with the epidermal growth factor receptor (EGFR) and triggers a signaling cascade that leads to elevation of endogenous p21 and growth ...suppression. We demonstrate that decorin causes a sustained down-regulation of the EGFR. Upon stable expression of decorin, the EGFR number is reduced by approximately 40%, without changes in EGFR expression. However, EGFR phosphorylation is nearly completely abolished. Concurrently, decorin attenuates the EGFR-mediated mobilization of intracellular calcium and blocks the growth of tumor xenografts by down-regulating the EGFR kinase in vivo. Thus, decorin acts as an autocrine and paracrine regulator of tumor growth and could be utilized as an effective anti-cancer agent.
Mitochondrial Ca(2+) uptake has long been considered crucial for meeting the fluctuating energy demands of cells in the heart and other tissues. Increases in mitochondrial matrix Ca(2+) drive ...mitochondrial ATP production via stimulation of Ca(2+)-sensitive dehydrogenases. Mitochondria-targeted sensors have revealed mitochondrial matrix Ca(2+) rises that closely follow the cytoplasmic Ca(2+) signals in many paradigms. Mitochondrial Ca(2+) uptake is mediated by the Ca(2+) uniporter (mtCU). Pharmacological manipulation of the mtCU is potentially key to understanding its physiological significance, but no specific, cell-permeable inhibitors were identified. In the past decade, as the molecular identity of the mtCU was brought to light, efforts have focused on genetic targeting. However, in the cells/animals that are able to survive impaired mtCU function, robust compensatory changes were found in the mtCU as well as other mechanisms. Thus, the discovery, through chemical library screens on normal and mtCU-deficient cells, of new small-molecule inhibitors with improved cell permeability and specificity might offer a better chance to test the relevance of mitochondrial Ca(2+) uptake. Success with the development of small molecule mtCU inhibitors is also expected to have clinical impact, considering the growing evidence for the role of mitochondrial Ca(2+) uptake in a variety of diseases, including heart attack, stroke and various neurodegenerative disorders. Here, we review the progress in pharmacological targeting of mtCU and illustrate the challenges in this field using data obtained with MCU-i11, a new small molecule inhibitor.
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