No abstract

Calcium ions (Ca2+) play a complex role in orchestrating diverse cellular processes, including cell death and survival. To trigger signaling cascades, intracellular Ca2+ is shuffled between the cytoplasm and the major Ca2+ stores, the endoplasmic reticulum (ER), the mitochondria, and the lysosomes. A key role in the control of Ca2+ signals is attributed to the inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), the main Ca2+-release channels in the ER. IP3Rs can transfer Ca2+ to the mitochondria, thereby not only stimulating core metabolic pathways but also increasing apoptosis sensitivity and inhibiting basal autophagy. On the other hand, IP3-induced Ca2+ release enhances autophagy flux by providing cytosolic Ca2+ required to execute autophagy upon various cellular stresses, including nutrient starvation, chemical mechanistic target of rapamycin inhibition, or drug treatment. Similarly, IP3Rs are able to amplify Ca2+ signals from the lysosomes and, therefore, impact autophagic flux in response to lysosomal channels activation. Furthermore, indirect modulation of Ca2+ release through IP3Rs may also be achieved by controlling the sarco/endoplasmic reticulum Ca2+ ATPases Ca2+ pumps of the ER. Considering the complex role of autophagy in cancer development and progression as well as in response to anticancer therapies, it becomes clear that it is important to fully understand the role of the IP3R and its cellular context in this disease. In cancer cells addicted to ER–mitochondrial Ca2+ fueling, IP3R inhibition leads to cancer cell death via mechanisms involving enhanced autophagy or mitotic catastrophe. Moreover, IP3Rs are the targets of several oncogenes and tumor suppressors and the functional loss of these genes, as occurring in many cancer types, can result in modified Ca2+ transport to the mitochondria and in modulation of the level of autophagic flux. Similarly, IP3R-mediated upregulation of autophagy can protect some cancer cells against natural killer cells-induced killing. The involvement of IP3Rs in the regulation of both autophagy and apoptosis, therefore, directly impact cancer cell biology and contribute to the molecular basis of tumor pathology.

The first event of photosynthesis is the harvesting of solar energy by a large array of pigments. These pigments are coordinated to proteins that organize them to assure efficient excitation energy transfer. The protein plays an essential role in tuning the spectroscopic properties of the pigments, by determining their site energy and/or by favoring pigment-pigments interactions. Here we investigate how the protein modulates the pigment properties by using a single-point-mutation approach. We monitor changes in the low-energy absorption/emission band of Lhca4, which is well separated from the bulk absorption and thus represents an attractive model system. Moreover, it was recently shown that Lhca4 exists in at least two conformations, a dominating one emitting at 720 nm and a second one emitting at 685 nm (Kruger et al. PNAS 2011). Here we show that a single amino-acid substitution (from Asn to Gln, which are both chlorophyll-binding residues and only differ for one C-C bond), moves the equilibrium almost completely towards the 685-nm conformation. This indicates that small changes in the protein can have a large effect on the properties of the pigments. We show that His99, which was suggested to coordinate a red-absorbing chlorophyll (Melkozernov and Blankenship, JBC 2003), is not a chlorophyll ligand. We also show that single amino-acid substitutions nearby the chlorophylls allow to tune the emission spectrum of the pigments over a wide range of wavelengths and to modulate the excited-state lifetimes of the complex. These findings are discussed in the light of previously proposed non-photochemical quenching models.

Previous work revealed that intracellular Ca signals and the inositol 1,4,5-trisphosphate (IP) receptors (IPR) are essential to increase autophagic flux in response to mTOR inhibition, induced by either nutrient starvation or rapamycin treatment. Here, we investigated whether autophagy induced by resveratrol, a polyphenolic phytochemical reported to trigger autophagy in a non-canonical way, also requires IPRs and Ca signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells. Importantly, autophagy induced by resveratrol (80μM, 2h) was completely abolished in the presence of 10μM BAPTA-AM, an intracellular Ca-chelating agent. To elucidate the IPR's role in this process, we employed the recently established HEK 3KO cells lacking all three IPR isoforms. In contrast to the HEK293 wt cells and to HEK 3KO cells re-expressing IPR1, autophagic responses in HEK 3KO cells exposed to resveratrol were severely impaired. These altered autophagic responses could not be attributed to alterations in the mTOR/p70S6K pathway, since resveratrol-induced inhibition of S6 phosphorylation was not abrogated by chelating cytosolic Ca or by knocking out IPRs. Finally, we investigated whether resveratrol by itself induced Ca release. In permeabilized HeLa cells, resveratrol neither affected the sarco- and endoplasmic reticulum Ca ATPase (SERCA) activity nor the IP-induced Ca release nor the basal Ca leak from the ER. Also, prolonged (4 h) treatment with 100μM resveratrol did not affect subsequent IP-induced Ca release. However, in intact HeLa cells, although resveratrol did not elicit cytosolic Ca signals by itself, it acutely decreased the ER Ca-store content irrespective of the presence or absence of IPRs, leading to a dampened agonist-induced Ca signaling. In conclusion, these results reveal that IPRs and cytosolic Ca signaling are fundamentally important for driving autophagic flux, not only in response to mTOR inhibition but also in response to non-canonical autophagy inducers like resveratrol. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

The inositol 1,4,5-trisphosphate (IP) receptor (IPR) is a ubiquitously expressed Ca-release channel localized in the endoplasmic reticulum (ER). The intracellular Ca signals originating from the activation of the IPR regulate multiple cellular processes including the control of cell death versus cell survival via their action on apoptosis and autophagy. The exact role of the IPRs in these two processes does not only depend on their activity, which is modulated by the cytosolic composition (Ca, ATP, redox status, …) and by various types of regulatory proteins, including kinases and phosphatases as well as by a number of oncogenes and tumor suppressors, but also on their intracellular localization, especially at the ER-mitochondrial and ER-lysosomal interfaces. At these interfaces, Ca microdomains are formed, in which the Ca concentration is finely regulated by the different ER, mitochondrial and lysosomal Ca-transport systems and also depends on the functional and structural interactions existing between them. In this review, we therefore discuss the most recent insights in the role of Ca signaling in general, and of the IPR in particular, in the control of basal mitochondrial bioenergetics, apoptosis, and autophagy at the level of inter-organellar contact sites.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) to reduce protein load and restore homeostasis, including via induction of autophagy. We used the proline analogue l-azetidine-2-carboxylic acid (AZC) to induce ER stress, and assessed its effect on autophagy and Ca2+ homeostasis. Treatment with 5 mM AZC did not induce poly adenosine diphosphate ribose polymerase (PARP) cleavage while levels of binding immunoglobulin protein (BiP) and phosphorylated eukaryotic translation initiation factor 2α (eIF2α) increased and those of activating transcription factor 6 (ATF6) decreased, indicating activation of the protein kinase RNA-like ER kinase (PERK) and the ATF6 arms of the UPR but not of apoptosis. AZC treatment in combination with bafilomycin A1 (Baf A1) led to elevated levels of the lipidated form of the autophagy marker microtubule-associated protein light chain 3 (LC3), pointing to activation of autophagy. Using the specific PERK inhibitor AMG PERK 44, we could deduce that activation of the PERK branch is required for the AZC-induced lipidation of LC3. Moreover, both the levels of phospho-eIF2α and of lipidated LC3 were strongly reduced when cells were co-treated with the intracellular Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraaceticacid tetra(acetoxy-methyl) ester (BAPTA-AM) but not when co-treated with the Na+/K+ ATPase inhibitor ouabain, suggesting an essential role of Ca2+ in AZC-induced activation of the PERK arm of the UPR and LC3 lipidation. Finally, AZC did not trigger Ca2+ release from the ER though appeared to decrease the cytosolic Ca2+ rise induced by thapsigargin while also decreasing the time constant for Ca2+ clearance. The ER Ca2+ store content and mitochondrial Ca2+ uptake however remained unaffected.

Invadopodia are actin‐rich membrane protrusions which have been assigned essential roles in cancer cell invasiveness. Here, we investigate the roles of ezrin, radixin and moesin (ERM), three closely related F‐actin‐plasma membrane linker proteins implicated in metastasis, and their binding partner the Na+/H+ exchanger isoform 1 (NHE1), in the formation and function of invadopodial rosettes in MCF‐7 human breast cancer cells. ERM proteins colocalized strongly with NHE1 in invadopodia in MCF‐7 cells +/− constitutively active, N‐terminally truncated ErbB2 (ΔNErbB2). In contrast, the tumor suppressor Merlin which is more distantly related to the ERM proteins, did not. siRNA‐mediated knockdown of NHE1 increased the average number of invadopodia per cell in ΔNErbB2‐expressing cells but not in vector controls. Overexpression of either dominant negative (DN) or constitutively active (CA) ezrin decreased the number of invadopodia per cell, in vector control cells only. Average invadopodial rosette diameter was unaffected by knockdown of ezrin or NHE1, or by expression of DN‐ or CA‐ezrin. Thus, in MCF‐7 cells, NHE1 colocalizes strongly with ERM proteins in invadopodial rosettes and both NHE1 and ezrin appear to affect their formation. Studies are ongoing to determine the generality and roles of these interactions in cancer invasiveness.Funding: Danish Cancer Society, Danish Research Council