Benjamin Bonneau

Bcl-wav and the mitochondrial calcium uniporter drive gastrula morphogenesis in zebrafish

Bcl-2 proteins are acknowledged as key regulators of programmed cell death. However, increasing data suggest additional roles, including regulation of the cell cycle, metabolism and cytoskeletal dynamics. Here we report the discovery and characterization of a new Bcl-2-related multidomain apoptosis accelerator, Bcl-wav, found in fish and frogs. Genetic and molecular studies in zebrafish indicate that Bcl-wav and the recently identified mitochondrial calcium uniporter (MCU) contribute to the formation of the notochord axis by controlling blastomere convergence and extension movements during gastrulation. Furthermore, we found that Bcl-wav controls intracellular Ca(2+) trafficking by acting on the mitochondrial voltage-dependent anion channel, and possibly on MCU, with direct consequences on actin microfilament dynamics and blastomere migration guidance. Thus, from an evolutionary point of view, the original function of Bcl-2 proteins might have been to contribute in controlling the global positioning system of blastomeres during gastrulation, a critical step in metazoan development.

The Apoptotic Regulator Nrz Controls Cytoskeletal Dynamics via the Regulation of Ca2+ Trafficking in the Zebrafish Blastula

Bcl-2 family members are key regulators of apoptosis. Their involvement in other cellular processes has been so far overlooked. We have studied the role of the Bcl-2 homolog Nrz in the developing zebrafish. Nrz was found to be localized to the yolk syncytial layer, a region containing numerous mitochondria and ER membranes. Nrz knockdown resulted in developmental arrest before gastrulation, due to free Ca(2+) increase in the yolk cell, activating myosin light chain kinase, which led to premature contraction of actin-myosin cables in the margin and separation of the blastomeres from the yolk cell. In the yolk syncytial layer, Nrz appears to prevent the release of Ca(2+) from the endoplasmic reticulum by directly interacting with the IP3R1 Ca(2+) channel. Thus, the Bcl-2 family may participate in early development, not only by controlling apoptosis but also by acting on cytoskeletal dynamics and cell movements via Ca(2+) fluxes inside the embryo.

The Bcl-2 Homolog Nrz Inhibits Binding of IP3 to Its Receptor to Control Calcium Signaling During Zebrafish Epiboly

Phosphorylation of a Bcl-2–like protein allows calcium signals needed for the early stages of development. Nrz-IP3 Tug-of-War During early development of zebrafish, cells migrate over the yolk in a process known as epiboly. Epiboly requires Ca2+-dependent myosin contraction along actin filaments at the leading edge of the migratory epithelium. Growth factors stimulate production of inositol trisphosphate (IP3), which binds to and activates the IP3 receptor, a Ca2+ channel on the endoplasmic reticulum (ER). Bonneau et al. found that the Bcl-2–like protein Nrz was phosphorylated in zebrafish during early epiboly and that replacing endogenous Nrz with a mutant that could not be phosphorylated disrupted normal Ca2+ oscillations, actin assembly at the leading edge, and epiboly progression. In human cultured cells, wild-type Nrz, but not Nrz with phosphomimetic mutations, bound to the IP3 receptor, prevented its interaction with IP3, and blocked Ca2+ release from the ER. Thus, these data suggest that phosphorylation of Nrz during early epiboly enables IP3 to bind to its receptor and promote Ca2+ waves that are important for assembly of the actin-myosin ring required for cell migration. Members of the Bcl-2 protein family regulate mitochondrial membrane permeability and also localize to the endoplasmic reticulum where they control Ca2+ homeostasis by interacting with inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs). In zebrafish, Bcl-2–like 10 (Nrz) is required for Ca2+ signaling during epiboly and gastrulation. We characterized the mechanism by which Nrz controls IP3-mediated Ca2+ release during this process. We showed that Nrz was phosphorylated during early epiboly, and that in embryos in which Nrz was knocked down, reconstitution with Nrz bearing mutations designed to prevent its phosphorylation disrupted cyclic Ca2+ transients and the assembly of the actin-myosin ring and led to epiboly arrest. In cultured cells, wild-type Nrz, but not Nrz with phosphomimetic mutations, interacted with the IP3 binding domain of IP3R1, inhibited binding of IP3 to IP3R1, and prevented histamine-induced increases in cytosolic Ca2+. Collectively, these data suggest that Nrz phosphorylation is necessary for the generation of IP3-mediated Ca2+ transients and the formation of circumferential actin-myosin cables required for epiboly. Thus, in addition to their role in apoptosis, by tightly regulating Ca2+ signaling, Bcl-2 family members participate in the cellular events associated with early vertebrate development, including cytoskeletal dynamics and cell movement.

TIF1γ requires sumoylation to exert its repressive activity on TGFβ signaling.

Summary TIF1&ggr;, a new regulator of TGF&bgr; signaling, inhibits the Smad4-mediated TGF&bgr; response by interaction with Smad2/3 or ubiquitylation of Smad4. We have shown that TIF1&ggr; participates in TGF&bgr; signaling as a negative regulator of Smad4 during the TGF&bgr;-induced epithelial-to-mesenchymal transition (EMT) in mammary epithelial cells, and during terminal differentiation of mammary alveolar epithelial cells and lactation. We demonstrate here that TIF1&ggr; is sumoylated and interacts with Ubc9, the only known SUMO-conjugating enzyme. Four functional sumoylation sites lie within the middle domain of TIF1&ggr;, the Smad interaction domain. We show that a sumoylation-defective TIF1&ggr; mutant significantly reduces TIF1&ggr; inhibition of Smad complexes and that of the Smad-mediated TGF&bgr; transcriptional response. Moreover, chromatin immunoprecipitation experiments indicate that TIF1&ggr; sumoylation is required to limit Smad4 binding on the PAI-1 TGF&bgr; target gene promoter. Ectopic expression of TIF1&ggr; in mammary epithelial cells inhibits TGF&bgr;-induced EMT, an effect relieved by expression of non-sumoylated TIF1&ggr;. Taken together, our results identify a new TGF&bgr; regulatory layer, whereby sumoylation strengthens the TIF1&ggr; repressive action on canonical TGF&bgr; signaling.

Remodeling of Ca 2+ signaling in cancer: Regulation of inositol 1,4,5-trisphosphate receptors through oncogenes and tumor suppressors

The calcium ion (Ca2+) is a ubiquitous intracellular signaling molecule that regulates diverse physiological and pathological processes, including cancer. Increasing evidence indicates that oncogenes and tumor suppressors regulate the Ca2+ transport systems. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are IP3-activated Ca2+ release channels located on the endoplasmic reticulum (ER). They play pivotal roles in the regulation of cell death and survival by controlling Ca2+ transfer from the ER to mitochondria through mitochondria-associated ER membranes (MAMs). Optimal levels of Ca2+ mobilization to mitochondria are necessary for mitochondrial bioenergetics, whereas excessive Ca2+ flux into mitochondria causes loss of mitochondrial membrane integrity and apoptotic cell death. In addition to well-known functions on outer mitochondrial membranes, B-cell lymphoma 2 (Bcl-2) family proteins are localized on the ER and regulate IP3Rs to control Ca2+ transfer into mitochondria. Another regulatory protein of IP3R, IP3R-binding protein released with IP3 (IRBIT), cooperates with or counteracts the Bcl-2 family member depending on cellular states. Furthermore, several oncogenes and tumor suppressors, including Akt, K-Ras, phosphatase and tensin homolog (PTEN), promyelocytic leukemia protein (PML), BRCA1, and BRCA1 associated protein 1 (BAP1), are localized on the ER or at MAMs and negatively or positively regulate apoptotic cell death through interactions with IP3Rs and regulation of Ca2+ dynamics. The remodeling of Ca2+ signaling by oncogenes and tumor suppressors that interact with IP3Rs has fundamental roles in the pathology of cancers.

Cytoskeleton dynamics in early zebrafish development: A matter of phosphorylation?

Early morphogenic movements are an important feature of embryonic development in vertebrates. During zebrafish gastrulation, epiboly progression is driven by the coordinated remodeling of the YSL microtubule network and F-actin cables. We recently described the implication of Nrz, an anti-apoptotic Bcl-2 homolog, in the control of the YSL cytoskeleton dynamics. Nrz knock-down induces premature actin-myosin ring formation leading to margin constriction, epiboly arrest and embryo lethality. At the molecular level, the Nrz protein controls the actin-myosin dynamics through IP3R-dependent calcium levels variation. Here, we discuss these novel findings and propose a model in which reversible phosphorylation of the Nrz/IP3R complex modulates the permeability of the IP3R calcium channel and thus may explain the Nrz-dependent control of IP3R opening required for proper epiboly completion.

Bcl-2 proteins, cell migration and embryonic development: lessons from zebrafish.

B-cell lymphoma-2 (Bcl-2) was cloned 30 years ago and associated with B-cell follicular lymphoma. A number of Bcl-2 homologs were identified later on. Importantly, the Bcl-2 family was found to control the mitochondrial outer membrane permeabilization: a key step of the mitochondrial pathway of apoptosis.1 Bcl-2 homologs are evolutionarily conserved throughout metazoans and considered as the hallmarks of multicellularity. Genetic manipulation in nematodes and mice demonstrated that the bcl-2 family has a pivotal role in tissue homeostasis by controlling cell death; however, an increased number of in vitro studies have identified additional non-apoptotic functions, suggesting that Bcl-2 proteins are in fact multitask factors (Figure 1a).2 Figure 1 Non-apoptotic functions of the Bcl-2 family of proteins. (a) Simplified representation of Bcl-2 proteins functions. Bcl-2 proteins are multitask factors and linked to non-apoptotic functions. The top panel represents the different Ca2+-independent ... Besides their mitochondrial localization, Bcl-2 proteins are also found in the endoplasmic reticulum (ER). In fact, a number of them contribute to apoptosis regulation though the control of Ca2+ exchanges at the level of the ER/mitochondria interface. Indeed physical proximity between these organelles creates intracellular microdomains, considered as Ca2+ hotspots.3 Mitochondria constantly uptake Ca2+ to ensure their physiological functions; they are also able to rapidly uptake Ca2+ when massively released from the ER, acting as a genuine Ca2+ buffer. This fast accumulation may lead to mitochondrial Ca2+ overload and, depending on Ca2+ levels, the cells will undergo apoptosis or necrosis.4 Bcl-2 proteins control Ca2+ exchanges through direct interactions with ER Ca2+ channels and pumps including the Inositol 1,4,5-Trisphosphate receptor (IP3R), the Ca2+-ATPase (SERCA) pump, the ryanodine receptor, the Bax inhibitor-1 channel, as well as, the voltage-dependent anion channel (VDAC) at the mitochondria (reviewed in Bonneau et al.2). It was reported that overexpression of Bcl-2 may lead to a decrease of the ER Ca2+ load,5 and the ability of Bcl-2 proteins to regulate intracellular Ca2+ homeostasis was linked to non-apoptotic functions (Figure 1a).

The yolk cell of the zebrafish blastula harbors functional apoptosis machinery

We recently described the implication of the Bcl-2 related anti-apoptotic Nrz protein during early zebrafish development. Nrz knock-down induces calcium-dependent cytoskeleton remodeling leading to margin constriction and premature embryo lethality. In the YSL, nrz knock-down embryos exhibit some typical features of apoptosis such as mitochondrial transmembrane potential loss and cytochrome c release. However, downstream caspase-3 activation has not been detected so far. Here, we report that the YSL contains fully functional apoptotic machinery that can activate caspase-3 following zBax ectopic expression. Furthermore, we present evidence that caspase-3 activation is actually detectable in nrz knock-down embryos when premature margin constriction is prevented.

Control of programmed cell death during zebrafish embryonic development

Programmed cell death (PCD) is a conserved cellular process, which is essential during embryonic development, morphogenesis and tissue homeostasis. PCD participates in the elimination of unwanted or potentially harmful cells, and contributes in this way to the precise shaping of the developing embryo. In this review, the current knowledge related to the role of PCD during zebrafish development is described and an overview is provided about the main actors that induce, control and execute the apoptotic pathways during zebrafish development. Finally, we point out some important issues regarding the regulation of apoptosis during the early stages of zebrafish development.