DRP1 interacts directly with BAX to induce its activation and apoptosis.

The apoptotic executioner protein BAX and the dynamin-like protein DRP1 co-localize at mitochondria during apoptosis to mediate mitochondrial permeabilization and fragmentation. However, the molecular basis and functional consequences of this interplay remain unknown. Here, we show that BAX and DRP1 physically interact, and that this interaction is enhanced during apoptosis. Complex formation between BAX and DRP1 occurs exclusively in the membrane environment and requires the BAX N-terminal region, but also involves several other BAX surfaces. Furthermore, the association between BAX and DRP1 enhances the membrane activity of both proteins. Forced dimerization of BAX and DRP1 triggers their activation and translocation to mitochondria, where they induce mitochondrial remodeling and permeabilization to cause apoptosis even in the absence of apoptotic triggers. Based on this, we propose that DRP1 can promote apoptosis by acting as noncanonical direct activator of BAX through physical contacts with its N-terminal region.

SnapShot: Functions of Endoplasmic Reticulum Membrane Contact Sites.

The endoplasmic reticulum (ER) consists of the nuclear envelope and a reticulated interconnected network of tubules and sheets. ER sheets are studded with ribosomes and provide the entryway for proteins into the secretory pathway. ER tubules move dynamically on microtubules and form membrane contact sites with other organelles, where membranes are tethered, but not fused. This Snapshot reviews key biological processes that take place at ER contact sites with the Golgi, endosomes, and mitochondria.

Systems-level effects of ectopic galectin-7 reconstitution in cervical cancer and its microenvironment.

BACKGROUND: Galectin-7 (Gal-7) is negatively regulated in cervical cancer, and appears to be a link between the apoptotic response triggered by cancer and the anti-tumoral activity of the immune system. Our understanding of how cervical cancer cells and their molecular networks adapt in response to the expression of Gal-7 remains limited. METHODS: Meta-analysis of Gal-7 expression was conducted in three cervical cancer cohort studies and TCGA. In silico prediction and bisulfite sequencing were performed to inquire epigenetic alterations. To study the effect of Gal-7 on cervical cancer, we ectopically re-expressed it in the HeLa and SiHa cervical cancer cell lines, and analyzed their transcriptome and SILAC-based proteome. We also examined the tumor and microenvironment host cell transcriptomes after xenotransplantation into immunocompromised mice. Differences between samples were assessed with the Kruskall-Wallis, Dunn's Multiple Comparison and T tests. Kaplan-Meier and log-rank tests were used to determine overall survival. RESULTS: Gal-7 was constantly downregulated in our meta-analysis (p < 0.0001). Tumors with combined high Gal-7 and low galectin-1 expression (p = 0.0001) presented significantly better prognoses (p = 0.005). In silico and bisulfite sequencing assays showed de novo methylation in the Gal-7 promoter and first intron. Cells re-expressing Gal-7 showed a high apoptosis ratio (p < 0.05) and their xenografts displayed strong growth retardation (p < 0.001). Multiple gene modules and transcriptional regulators were modulated in response to Gal-7 reconstitution, both in cervical cancer cells and their microenvironments (FDR < 0.05 %). Most of these genes and modules were associated with tissue morphogenesis, metabolism, transport, chemokine activity, and immune response. These functional modules could exert the same effects in vitro and in vivo, even despite different compositions between HeLa and SiHa samples. CONCLUSIONS: Gal-7 re-expression affects the regulation of molecular networks in cervical cancer that are involved in diverse cancer hallmarks, such as metabolism, growth control, invasion and evasion of apoptosis. The effect of Gal-7 extends to the microenvironment, where networks involved in its configuration and in immune surveillance are particularly affected.

Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores.

Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual-color single-molecule localization-based super-resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc-shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane.

Structural model of active Bax at the membrane.

Bax plays a central role in the mitochondrial pathway of apoptosis. Upon activation, cytosolic Bax monomers oligomerize on the surface of mitochondria and change conformation concertedly to punch holes into the outer membrane. The subsequent release of cytochrome c initiates cell death. However, the structure of membrane-inserted Bax and its mechanism of action remain largely unknown. Here, we propose a 3D model of active Bax at the membrane based on double electron-electron resonance (DEER) spectroscopy in liposomes and isolated mitochondria. We show that active Bax is organized at the membrane as assemblies of dimers. In addition to a stable dimerization domain, each monomer contains a more flexible piercing domain involved in interdimer interactions and pore formation. The most important structural change during Bax activation is the opening of the hairpin formed by helices 5 and 6, which adopts a clamp-like conformation central to the mechanism of mitochondrial permeabilization.