Mitochondria cannot be generated de novo; they must grow, replicate their genome, and divide in order to be inherited to each daughter cell during mitosis. Mitochondrial division is a structural challenge that requires a massive remodeling of membrane morphology 1–3. Although division factors differ across organisms, the need for multiple constriction steps and a dynamin-related protein (Drp1, Dnm1 in yeast) has been conserved 4–6. In mammalian cells, mitochondrial division has been shown to proceed with at least two sequential constriction steps: 1. endoplasmic reticulum (ER) and actin collaborate to generate constrictions suitable for Drp1 assembly; 2. Drp1 further constricts membranes until fission occurs 2,7–9. However, in vitro experiments argue that Drp1 does not have the dynamic range to complete membrane fission per se 7. In contrast to Drp1, the neuronal-specific classical Dynamin-1 (Dyn1) has been shown to assemble on narrower lipid profiles and facilitates spontaneous membrane fission upon GTP hydrolysis 10,11. Here we discovered that the ubiquitously-expressed classical Dynamin-2 (Dyn2) is a fundamental component of the mitochondrial division machinery. A combination of live-cell and electron microscopy reveals that Dyn2 works in concert with Drp1 to orchestrate sequential constriction events leading up to division. Our work underscores the biophysical limitations of Drp1 and positions Dyn2, which has intrinsic membrane fission properties, at the final step of mitochondrial division.

The endoplasmic reticulum (ER) has a remarkably complex structure, composed of a single bilayer that forms the nuclear envelope, along with a network of sheets and dynamic tubules. Our understanding of the biological significance of the complex architecture of the ER has improved dramatically in the last few years. The identification of proteins and forces required for maintaining ER shape, as well as more advanced imaging techniques, has allowed the relationship between ER shape and function to come into focus. These studies have also revealed unexpected new functions of the ER and novel ER domains regulating alterations in ER dynamics. The importance of ER structure has become evident as recent research has identified diseases linked to mutations in ER-shaping proteins. In this review, we discuss what is known about the maintenance of ER architecture, the relationship between ER structure and function, and diseases associated with defects in ER structure.

Tethered interactions between the endoplasmic reticulum (ER) and other membrane-bound organelles allow for efficient transfer of ions and/or macromolecules and provide a platform for organelle fission. Here, we describe an unconventional interface between membraneless ribonucleoprotein granules, such as processing bodies (P-bodies, or PBs) and stress granules, and the ER membrane. We found that PBs are tethered at molecular distances to the ER in human cells in a tunable fashion. ER-PB contact and PB biogenesis were modulated by altering PB composition, ER shape, or ER translational capacity. Furthermore, ER contact sites defined the position where PB and stress granule fission occurs. We thus suggest that the ER plays a fundamental role in regulating the assembly and disassembly of membraneless organelles.

We report unexpected nongenomic functions of signal transducer and activator of transcription (STAT) 5 species in the cytoplasm aimed at preserving the structure and function of the Golgi apparatus and rough endoplasmic reticulum (ER) in vascular cells. Immunoimaging and green fluorescent protein-tagged-STAT5a protein localization studies showed the constitutive association of nonphosphorylated STAT5a, and to a lesser extent STAT5b, with the Golgi apparatus and of STAT5a with centrosomes in human pulmonary arterial endothelial and smooth muscle cells. Acute knockdown of STAT5a/b species using small interfering RNAs (siRNAs), including in the presence of an mRNA synthesis inhibitor (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole), produced a dramatic phenotype within 1 day, consisting of dilatation and fragmentation of Golgi cisternae, a marked tubule-to-cyst change in the ER, increased accumulation of reticulon-4 (RTN4)/Nogo-B and atlastin-3 (ATL3) at cyst-zone boundaries, cystic separation of the outer and inner nuclear membranes, accompanied by scalloped/lunate distortion of the nucleus, with accumulation of RTN4 on convex sides of distorted nuclei. These cells showed inhibition of vesicular stomatitis virus G protein glycoprotein trafficking, mitochondrial fragmentation, and reduced mitochondrial function. STAT5a/b(-/-) mouse embryo fibroblasts also showed altered ER/Golgi dynamics. RTN4 knockdown using siRNA did not affect development of the cystic phenotype; ATL3 siRNA led to effacement of cyst-zone boundaries. In magnetic-bead cross-immunopanning assays, ATL3 bound both STAT5a and STAT5b. Remarkably, this novel cystic ER/lunate nucleus phenotype was characteristic of vascular cells in arterial lesions of idiopathic pulmonary hypertension, an unrelentingly fatal human disease. These data provide evidence of a STAT-family protein regulating the structure of a cytoplasmic organelle and implicate this mechanism in the pathogenesis of a human disease.

Although reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), its consequences on organellar structure and function within vascular cells is largely unexplored. We investigated the effect of reduced NO on the structure of the Golgi apparatus as assayed by giantin or GM130 immunofluorescence in human pulmonary arterial endothelial (HPAECs) and smooth muscle (HPASMCs) cells, bovine PAECs, and human EA.hy926 endothelial cells. Golgi structure was also investigated in cells in tissue sections of pulmonary vascular lesions in idiopathic PAH (IPAH) and in macaques infected with a chimeric simian immunodeficiency virus containing the human immunodeficiency virus (HIV)-nef gene (SHIV-nef) with subcellular three-dimensional (3D) immunoimaging. Compounds with NO scavenging activity including 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), methylene blue, N-acetylcysteine, and hemoglobin markedly fragmented the Golgi in all cell types evaluated as did monocrotaline pyrrole, while LY-83583, sildenafil, fasudil, Y-27632, Tiron, Tempol, or H(2)O(2) did not. Golgi fragmentation by NO scavengers was inhibited by diethylamine NONOate, was evident in HPAECs after selective knockdown of endothelial nitric oxide synthase using small interfering RNA (siRNA), was independent of microtubule organization, required the GTPase dynamin 2, and was accompanied by depletion of α-soluble N-ethylmaleimide-sensitive factor (NSF) acceptor protein (α-SNAP) from Golgi membranes and codispersal of the SNAP receptor (SNARE) Vti1a with giantin. Golgi fragmentation was confirmed in endothelial and smooth muscle cells in pulmonary arterial lesions in IPAH and the SHIV-nef-infected macaque with subcellular 3D immunoimaging. In SHIV-nef-infected macaques Golgi fragmentation was observed in cells containing HIV-nef-bearing endosomes. The observed Golgi fragmentation suggests that NO plays a significant role in modulating global protein trafficking patterns that contribute to changes in the cell surface landscape and functional signaling in vascular cells.

Lee J, Reich R, Xu F, Sehgal PB. Golgi, trafficking, and mitosis dysfunctions in pulmonary arterial endothelial cells exposed to monocrotaline pyrrole and NO scavenging. Am J Physiol Lung Cell Mol Physiol 297: L715-L728, 2009. First published July 31, 2009 doi:10.1152/ajplung.00086.2009.-Although the administration of monocrotaline (MCT) into experimental animals is in widespread use today in investigations of pulmonary arterial hypertension (PAH), the underlying cellular and subcellular mechanisms that culminate in vascular remodeling are incompletely understood. Bovine pulmonary arterial endothelial cells (PAECs) in culture exposed to monocrotaline pyrrole (MCTP) develop "megalocytosis" 18 -24 h later characterized by enlarged hyperploid cells with enlarged Golgi, mislocalization of endothelial nitric oxide synthase away from the plasma membrane, decreased cell-surface/caveolar nitric oxide (NO), and hypo-S-nitrosylation of caveolin-1, clathrin heavy chain, and N-ethylmaleimidesensitive factor. We investigated whether MCTP did in fact affect functional intracellular trafficking. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) and the NO donor diethylamine NONOate were used for comparison. Both MCTP and c-PTIO produced distinctive four-to fivefold enlarged PAECs within 24 -48 h with markedly enlarged/dispersed Golgi, as visualized by immunostaining for the Golgi tethers/matrix proteins giantin, GM130, and p115. Live-cell uptake of the Golgi marker C5 ceramide revealed a compact juxtanuclear Golgi in untreated PAECs, brightly labeled enlarged circumnuclear Golgi after MCTP, but minimally labeled Golgi elements after c-PTIO. These Golgi changes were reduced by NONOate. After an initial inhibition during the first day, both MCTP and c-PTIO markedly enhanced anterograde secretion of soluble cargo (exogenous vector-expressed recombinant horseradish peroxidase) over the next 4 days. Live-cell internalization assays using fluorescently tagged ligands showed that both MCTP and c-PTIO inhibited the retrograde uptake of acetylated low-density lipoprotein, transferrin, and cholera toxin B. Moreover, MCTP, and to a variable extent c-PTIO, reduced the cell-surface density of all receptors assayed (LDLR, TfnR, BMPR, Tie-2, and PECAM-1/ CD31). In an important distinction, c-PTIO enhanced mitosis in PAECs but MCTP inhibited mitosis, even that due to c-PTIO, despite markedly exaggerated Golgi dispersal. Taken together, these data define a broad-spectrum Golgi and subcellular trafficking dysfunction syndrome in endothelial cells exposed to MCTP or NO scavenging. vascular remodeling; megalocytosis; anterograde and retrograde trafficking; ␤-actin INGESTION OR ADMINISTRATION of pyrrolizidene alkaloids leads to vascular occlusive disease including pulmonary arterial hypertension (PAH) in cattle, horses, pigs, dogs, and even primates (reviewed in Refs. 20, 48 and citations therein). Over the last four decades the experimental administration of the pyrrolizidine alkaloid monocrotaline into r...

STAT5a/b species are well known as transcription factors that regulate nuclear gene expression. In a novel line of research in human pulmonary arterial endothelial cells (HPAECs), we previously observed that STAT5a associated with the Golgi apparatus and that siRNA-mediated knockdown of STAT5a/b led to the rapid development of a dramatic cystic change in the endoplasmic reticulum (ER) characterized by deposition along cyst membranes and tubule-to-cyst boundaries of the proteins reticulon-4 (RTN4; also called Nogo-B) and the ER-resident GTPase atlastin-3 (ATL3) and Golgi fragmentation. We now report that STAT5a can be observed in ER sheets in digitonin-permeabilized HPAECs and that anti-STAT5a cross- immunopanned ATL3 but not RTN4. Moreover, there was marked accumulation of the 63-kDa cytoskeleton-linking membrane protein and ER-spacer CLIMP63 (also called cytoskeleton-associated protein 4, CKAP4) and KDEL-mCherry within the cysts. That the STAT5a/b-siRNA-induced cystic ER phenotype developed in the presence of the transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) had suggested that the mechanism was independent of the transcription factor functions of STAT5a/b, i.e., was "nongenomic." We have now definitively tested the requirement for the nucleus in eliciting the STAT5a/b-siRNA-induced cystic ER phenotype. Enucleated HPAEC cytoplasts were prepared using adherent 35-mm cultures using the cytochalasin B-centrifugation method (typically yielding 65-75% enucleation). STAT5a/b siRNAs readily elicited the cystic ER phenotype including the marked luminal accumulation of CLIMP63 and Golgi fragmentation in the recovered HPAEC cytoplasts demonstrably lacking a nucleus. These studies provide unequivocal evidence using enucleated cytoplasts for a nongenomic mechanism(s) underlying the cystic change in ER structure elicited by STAT5a/b knockdown.

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.