Michal Jarnik

Distribution of ESCRT Machinery at HIV Assembly Sites Reveals Virus Scaffolding of ESCRT Subunits

Viral ESCRT The ESCRT (Endosomal Sorting Complex Required for Transport) protein complex plays a role in budding into multivesicular bodies, cytokinesis, and HIV budding, but the details of how the ESCRTs facilitate viral budding are unclear. Now, using high-resolution light and electron microscopical imaging techniques, Van Engelenburg et al. (p. 653, published online 16 January) dissect the role for ESCRT proteins in HIV budding. The findings suggest that the ESCRT machinery required for the scission of HIV particles from infected cells is located within the core of the virus particle and not, as might have been expected based on previous work, on the cellular side of the membrane scission event involved in viral budding. ESCRT-III proteins scaffold within assembling HIV particles to mediate viral membrane abscission. The human immunodeficiency virus (HIV) hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus release from infected cells. The nanoscale organization of ESCRT machinery necessary for mediating viral abscission is unclear. Here, we applied three-dimensional superresolution microscopy and correlative electron microscopy to delineate the organization of ESCRT components at HIV assembly sites. We observed ESCRT subunits localized within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by plasma membrane association and selective remodeling of ESCRT subunits.

BORC, a Multisubunit Complex that Regulates Lysosome Positioning

The positioning of lysosomes within the cytoplasm is emerging as a critical determinant of many lysosomal functions. Here we report the identification of a multisubunit complex named BORC that regulates lysosome positioning. BORC comprises eight subunits, some of which are shared with the BLOC-1 complex involved in the biogenesis of lysosome-related organelles, and the others of which are products of previously uncharacterized open reading frames. BORC associates peripherally with the lysosomal membrane, where it functions to recruit the small GTPase Arl8. This initiates a chain of interactions that promotes the kinesin-dependent movement of lysosomes toward the plus ends of microtubules in the peripheral cytoplasm. Interference with BORC or other components of this pathway results in collapse of the lysosomal population into the pericentriolar region. In turn, this causes reduced cell spreading and migration, highlighting the importance of BORC-dependent centrifugal transport for non-degradative functions of lysosomes.

Signal-Mediated, AP-1/Clathrin-Dependent Sorting of Transmembrane Receptors to the Somatodendritic Domain of Hippocampal Neurons

Plasma membranes of the somatodendritic and axonal domains of neurons are known to have different protein compositions, but the molecular mechanisms that determine this polarized protein distribution remain poorly understood. Herein we show that somatodendritic sorting of various transmembrane receptors in rat hippocampal neurons is mediated by recognition of signals within the cytosolic domains of the proteins by the μ1A subunit of the adaptor protein-1 (AP-1) complex. This complex, in conjunction with clathrin, functions in the neuronal soma to exclude somatodendritic proteins from axonal transport carriers. Perturbation of this process affects dendritic spine morphology and decreases the number of synapses. These findings highlight the primary recognition event that underlies somatodendritic sorting and contribute to the evolving view of AP-1 as a global regulator of cell polarity.

ARF induces autophagy by virtue of interaction with Bcl-xl.

The ARF tumor suppressor controls a well-described p53/Mdm2-dependent oncogenic stress checkpoint. In addition, ARF has recently been shown to localize to mitochondria, and to induce autophagy; however, this has never before been demonstrated for endogenous ARF, and the molecular basis for this activity of ARF has not been elucidated. Using an unbiased mass spectrometry-based approach, we show that mitochondrial ARF interacts with the Bcl2 family member Bcl-xl, which normally protects cells from autophagy by inhibiting the Beclin-1/Vps34 complex, which is essential for autophagy. We find that increased expression of ARF decreases Beclin-1/Bcl-xl complexes in cells, thereby providing a basis for ARF-induced autophagy. Our data also indicate that silencing p53 leads to high levels of ARF and increased autophagy, thereby providing a possible basis for the finding by others that p53 inhibits autophagy. The combined data support the premise that ARF induces autophagy in a p53-independent manner in part by virtue of its interaction with Bcl-xl.

The ARF tumor suppressor can promote the progression of some tumors

p14/p19ARF (ARF) is a tumor suppressor gene that is frequently mutated in human cancer. ARF has multiple tumor suppressor functions, some of which are mediated by signaling to p53. Surprisingly, a significant fraction of human tumors retain persistently high levels of ARF, suggesting that ARF may possess a prosurvival function. We show that ARF protein is markedly up-regulated in cells exposed to nutrient starvation. Cells with silenced ARF show reduced autophagy and reduced viability when placed under conditions of starvation. We show for the first time that ARF silencing can limit the progression of some tumors, such as lymphoma, but not others, such as E1A/Ras-induced tumors. Specifically, myc-driven lymphomas with mutant p53 tend to overexpress ARF; we show that silencing ARF in these tumors greatly impedes their progression. These data are the first to show that ARF can act in a p53-independent manner to promote the progression of some tumors.

Coordinated elevation of mitochondrial oxidative phosphorylation and autophagy help drive hepatocyte polarization

Cell polarization requires increased cellular energy and metabolic output, but how these energetic demands are met by polarizing cells is unclear. To address these issues, we investigated the roles of mitochondrial bioenergetics and autophagy during cell polarization of hepatocytes cultured in a collagen sandwich system. We found that as the hepatocytes begin to polarize, they use oxidative phosphorylation to raise their ATP levels, and this energy production is required for polarization. After the cells are polarized, the hepatocytes shift to become more dependent on glycolysis to produce ATP. Along with this central reliance on oxidative phosphorylation as the main source of ATP production in polarizing cultures, several other metabolic processes are reprogrammed during the time course of polarization. As the cells polarize, mitochondria elongate and mitochondrial membrane potential increases. In addition, lipid droplet abundance decreases over time. These findings suggest that polarizing cells are reliant on fatty acid oxidation, which is supported by pharmacologic inhibition of β-oxidation by etomoxir. Finally, autophagy is up-regulated during cell polarization, with inhibition of autophagy retarding cell polarization. Taken together, our results describe a metabolic shift involving a number of coordinated metabolic pathways that ultimately serve to increase energy production during cell polarization.

Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo.

Liver kinase B1 (LKB1) and its downstream effector AMP‐activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich‐cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver‐specific (albumin‐Cre) LKB1 knockout mice (LKB1−/−) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1−/− livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6‐carboxyfluorescein diacetate (6‐CFDA), which is processed in hepatocytes into its fluorescent derivative 6‐carboxyfluorescein (6‐CF) and secreted into the canaliculi. In LKB1−/− mice, 6‐CF remained largely in hepatocytes, canalicular secretion was delayed, and 6‐CF appeared in the blood. To test whether 6‐CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6‐CFDA. In wild‐type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1−/− mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. Conclusion: LKB1 plays a critical role in regulating the maintenance of TJ and paracellular permeability, which may explain how various drugs, chemicals, and metabolic states that inhibit the LKB1/AMPK pathway result in cholestasis. (Hepatology 2016;64:1317‐1329)

LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.

Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation.

TORC1 regulators Iml1/GATOR1 and GATOR2 control meiotic entry and oocyte development in Drosophila

Significance The target of rapamycin complex 1 (TORC1) promotes cell growth and anabolic metabolism. In yeast, entry into meiosis is contingent on the down-regulation of TORC1 activity by the increased minichromosome loss 1/GTPase-activating proteins toward Rags 1 (Iml1/GATOR1) complex in response to amino acid starvation. Here we define the developmental requirements for the TORC1 regulators Iml1/GATOR1 and GATOR2 during Drosophila oogenesis. We demonstrate that, as is observed in yeast, the Iml1/GATOR1 complex down-regulates TORC1 activity to facilitate the mitotic/meiotic transition in Drosophila ovarian cysts. Later in oogenesis, components of the GATOR2 complex oppose the activity of GATOR1 to enable a rise in TORC1 activity that drives oocyte development and growth. Thus, a conserved nutrient stress pathway has been incorporated into a developmental program that regulates meiotic progression in Drosophila. In single-cell eukaryotes the pathways that monitor nutrient availability are central to initiating the meiotic program and gametogenesis. In Saccharomyces cerevisiae an essential step in the transition to the meiotic cycle is the down-regulation of the nutrient-sensitive target of rapamycin complex 1 (TORC1) by the increased minichromosome loss 1/ GTPase-activating proteins toward Rags 1 (Iml1/GATOR1) complex in response to amino acid starvation. How metabolic inputs influence early meiotic progression and gametogenesis remains poorly understood in metazoans. Here we define opposing functions for the TORC1 regulatory complexes Iml1/GATOR1 and GATOR2 during Drosophila oogenesis. We demonstrate that, as is observed in yeast, the Iml1/GATOR1 complex inhibits TORC1 activity to slow cellular metabolism and drive the mitotic/meiotic transition in developing ovarian cysts. In iml1 germline depletions, ovarian cysts undergo an extra mitotic division before meiotic entry. The TORC1 inhibitor rapamycin can suppress this extra mitotic division. Thus, high TORC1 activity delays the mitotic/meiotic transition. Conversely, mutations in Tor, which encodes the catalytic subunit of the TORC1 complex, result in premature meiotic entry. Later in oogenesis, the GATOR2 components Mio and Seh1 are required to oppose Iml1/GATOR1 activity to prevent the constitutive inhibition of TORC1 and a block to oocyte growth and development. To our knowledge, these studies represent the first examination of the regulatory relationship between the Iml1/GATOR1 and GATOR2 complexes within the context of a multicellular organism. Our data imply that the central role of the Iml1/GATOR1 complex in the regulation of TORC1 activity in the early meiotic cycle has been conserved from single cell to multicellular organisms.

The GATOR2 Component Wdr24 Regulates TORC1 Activity and Lysosome Function

TORC1 is a master regulator of metabolism in eukaryotes that responds to multiple upstream signaling pathways. The GATOR complex is a newly defined upstream regulator of TORC1 that contains two sub-complexes, GATOR1, which inhibits TORC1 activity in response to amino acid starvation and GATOR2, which opposes the activity of GATOR1. While the GATOR1 complex has been implicated in a wide array of human pathologies including cancer and hereditary forms of epilepsy, the in vivo relevance of the GATOR2 complex remains poorly understood in metazoans. Here we define the in vivo role of the GATOR2 component Wdr24 in Drosophila. Using a combination of genetic, biochemical, and cell biological techniques we demonstrate that Wdr24 has both TORC1 dependent and independent functions in the regulation of cellular metabolism. Through the characterization of a null allele, we show that Wdr24 is a critical effector of the GATOR2 complex that promotes the robust activation of TORC1 and cellular growth in a broad array of Drosophila tissues. Additionally, epistasis analysis between wdr24 and genes that encode components of the GATOR1 complex revealed that Wdr24 has a second critical function, the TORC1 independent regulation of lysosome dynamics and autophagic flux. Notably, we find that two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. These findings represent a surprising and previously unrecognized function of GATOR2 complex components in the regulation of lysosomes. Consistent with our findings in Drosophila, through the characterization of a wdr24-/- knockout HeLa cell line we determined that Wdr24 promotes lysosome acidification and autophagic flux in mammalian cells. Taken together our data support the model that Wdr24 is a key effector of the GATOR2 complex, required for both TORC1 activation and the TORC1 independent regulation of lysosomes.