The Bcl‐2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro‐apoptotic proteins. Yet the mechanistic details of the Bax‐induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring‐like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super‐resolution data provide direct evidence in support of large Bax‐delineated pores in the mitochondrial outer membrane as being crucial for Bax‐mediated MOMP in cells.
The translocase of the mitochondrial outer membrane (TOM) complex is the main import pore for nuclear-encoded proteins into mitochondria, yet little is known about its spatial distribution within the outer membrane. Super-resolution stimulated emission depletion microscopy was used to determine quantitatively the nanoscale distribution of Tom20, a subunit of the TOM complex, in more than 1,000 cells. We demonstrate that Tom20 is located in clusters whose nanoscale distribution is finely adjusted to the cellular growth conditions as well as to the specific position of a cell within a microcolony. The density of the clusters correlates to the mitochondrial membrane potential. The distributions of clusters of Tom20 and of Tom22 follow an inner-cellular gradient from the perinuclear to the peripheral mitochondria. We conclude that the nanoscale distribution of the TOM complex is finely adjusted to the cellular conditions, resulting in distribution gradients both within single cells and between adjacent cells.M itochondria are essential organelles in eukaryotes, occupying a central role in cellular energy metabolism. The activity of mitochondria is adapted to changing cellular conditions: It has been suggested that short-term variations in energy demand may be compensated without modification of the mitochondrial enzyme content, but modulation of the mitochondrial protein content has been observed during long-term adaptations (1).In human cells, only 13 proteins are encoded by the mitochondrial genome; most mitochondrial proteins are synthesized as precursor proteins in the cytosol and are imported into the organelle. The central entry gate for almost all nuclear-encoded mitochondrial proteins is the translocase of the outer mitochondrial membrane (TOM complex) (for a detailed review see refs. 2-4). After passing through this complex, the precursor proteins follow different routes to their final destinations within the organelle. The TOM complex consists of the receptors Tom20, Tom22, and Tom70, the channel-forming protein Tom40, and several small, associated subunits. Tom20 is the initial recognition site for preproteins with presequences (5, 6) and transfers the preproteins to the central receptor, Tom22 (7, 8). From there, the precursors are inserted into the Tom40 channel.Although the components of the TOM complex and their molecular functions have been described in great detail, little is known about the distributions of the TOM complexes within the outer membrane, and even less is known about the spatial distributions of the complexes with respect to changing mitochondrial activities. The optical resolution in far-field fluorescence microscopy, arguably the most suitable approach for studying quantitatively the distribution of protein complexes in mitochondria of intact cells, is limited to ∼200 nm by diffraction (9). This resolution is not sufficient to resolve individual TOM complexes in mitochondria (10, 11). To overcome this problem, we used stimulated emission depletion (STED) super-resolution microscopy (...
Members of the miR‐200 family are critical gatekeepers of the epithelial state, restraining expression of pro‐mesenchymal genes that drive epithelial–mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR‐200c and another epithelial‐enriched miRNA, miR‐375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA‐binding protein Quaking (QKI). During EMT, QKI‐5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI‐5 is both necessary and sufficient to direct EMT‐associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial‐derived cancer types. Importantly, several actin cytoskeleton‐associated genes are directly targeted by both QKI and miR‐200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT. These findings demonstrate the existence of a miR‐200/miR‐375/QKI axis that impacts cancer‐associated epithelial cell plasticity through widespread control of alternative splicing.
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