ATPase activity of DFCP1 controls selective autophagy.

Cellular homeostasis is governed by removal of damaged organelles and protein aggregates by selective autophagy mediated by cargo adaptors such as p62/SQSTM1. Autophagosomes can assemble in specialized cup-shaped regions of the endoplasmic reticulum (ER) known as omegasomes, which are characterized by the presence of the ER protein DFCP1/ZFYVE1. The function of DFCP1 is unknown, as are the mechanisms of omegasome formation and constriction. Here, we demonstrate that DFCP1 is an ATPase that is activated by membrane binding and dimerizes in an ATP-dependent fashion. Whereas depletion of DFCP1 has a minor effect on bulk autophagic flux, DFCP1 is required to maintain the autophagic flux of p62 under both fed and starved conditions, and this is dependent on its ability to bind and hydrolyse ATP. While DFCP1 mutants defective in ATP binding or hydrolysis localize to forming omegasomes, these omegasomes fail to constrict properly in a size-dependent manner. Consequently, the release of nascent autophagosomes from large omegasomes is markedly delayed. While knockout of DFCP1 does not affect bulk autophagy, it inhibits selective autophagy, including aggrephagy, mitophagy and micronucleophagy. We conclude that DFCP1 mediates ATPase-driven constriction of large omegasomes to release autophagosomes for selective autophagy.

The phosphoinositide coincidence detector Phafin2 promotes macropinocytosis by coordinating actin organisation at forming macropinosomes.

Uptake of large volumes of extracellular fluid by actin-dependent macropinocytosis has an important role in infection, immunity and cancer development. A key question is how actin assembly and disassembly are coordinated around macropinosomes to allow them to form and subsequently pass through the dense actin network underlying the plasma membrane to move towards the cell center for maturation. Here we show that the PH and FYVE domain protein Phafin2 is recruited transiently to newly-formed macropinosomes by a mechanism that involves coincidence detection of PtdIns3P and PtdIns4P. Phafin2 also interacts with actin via its PH domain, and recruitment of Phafin2 coincides with actin reorganization around nascent macropinosomes. Moreover, forced relocalization of Phafin2 to the plasma membrane causes rearrangement of the subcortical actin cytoskeleton. Depletion of Phafin2 inhibits macropinosome internalization and maturation and prevents KRAS-transformed cancer cells from utilizing extracellular protein as an amino acid source. We conclude that Phafin2 promotes macropinocytosis by controlling timely delamination of actin from nascent macropinosomes for their navigation through the dense subcortical actin network.

Cosegregation of asymmetric features during cell division.

Cellular asymmetry plays a major role in the ageing and evolution of multicellular organisms. However, it remains unknown how the cell distinguishes 'old' from 'new' and whether asymmetry is an attribute of highly specialized cells or a feature inherent in all cells. Here, we investigate the segregation of three asymmetric features: old and new DNA, the spindle pole body (SPB, the centrosome analogue) and the old and new cell ends, using a simple unicellular eukaryote, Schizosaccharomyces pombe. To our knowledge, this is the first study exploring three asymmetric features in the same cells. We show that of the three chromosomes of S. pombe, chromosome I containing the new parental strand, preferentially segregated to the cells inheriting the old cell end. Furthermore, the new SPB also preferentially segregated to the cells inheriting the old end. Our results suggest that the ability to distinguish 'old' from 'new' and to segregate DNA asymmetrically are inherent features even in simple unicellular eukaryotes.

The GAS6-AXL signaling pathway triggers actin remodeling that drives membrane ruffling, macropinocytosis, and cancer-cell invasion.

AXL, a member of the TAM (TYRO3, AXL, MER) receptor tyrosine kinase family, and its ligand, GAS6, are implicated in oncogenesis and metastasis of many cancer types. However, the exact cellular processes activated by GAS6-AXL remain largely unexplored. Here, we identified an interactome of AXL and revealed its associations with proteins regulating actin dynamics. Consistently, GAS6-mediated AXL activation triggered actin remodeling manifested by peripheral membrane ruffling and circular dorsal ruffles (CDRs). This further promoted macropinocytosis that mediated the internalization of GAS6-AXL complexes and sustained survival of glioblastoma cells grown under glutamine-deprived conditions. GAS6-induced CDRs contributed to focal adhesion turnover, cell spreading, and elongation. Consequently, AXL activation by GAS6 drove invasion of cancer cells in a spheroid model. All these processes required the kinase activity of AXL, but not TYRO3, and downstream activation of PI3K and RAC1. We propose that GAS6-AXL signaling induces multiple actin-driven cytoskeletal rearrangements that contribute to cancer-cell invasion.

JIP4 is recruited by the phosphoinositide-binding protein Phafin2 to promote recycling tubules on macropinosomes.

Macropinocytosis allows cells to take up extracellular material in a non-selective manner into large vesicles called macropinosomes. After internalization, macropinosomes acquire phosphatidylinositol 3-phosphate (PtdIns3P) on their limiting membrane as they mature into endosomal-like vesicles. The molecular mechanisms that underlie recycling of membranes and transmembrane proteins from these macropinosomes still need to be defined. Here, we report that JIP4 (officially known as SPAG9), a protein previously described to bind to microtubule motors, is recruited to tubulating subdomains on macropinosomes by the PtdIns3P-binding protein Phafin2 (officially known as PLEKHF2). These JIP4-positive tubulating subdomains on macropinosomes contain F-actin, the retromer recycling complex and the retromer cargo VAMP3. Disruption of the JIP4-Phafin2 interaction, deletion of Phafin2 or inhibition of PtdIns3P production by VPS34 impairs JIP4 recruitment to macropinosomes. Whereas knockout of JIP4 suppresses tubulation, its overexpression enhances tubulation from macropinosomes. JIP4-knockout cells display increased retention of macropinocytic cargo in both early and late macropinosomes. Collectively, these data identify JIP4 and Phafin2 as components of a tubular recycling pathway that operates from macropinosomes. This article has an associated First Person interview with the first author of the paper.

Dual Targeting of BRAF and mTOR Signaling in Melanoma Cells with Pyridinyl Imidazole Compounds.

BRAF inhibitors can delay the progression of metastatic melanoma, but resistance usually emerges, leading to relapse. Drugs simultaneously targeting two or more pathways essential for cancer growth could slow or prevent the development of resistant clones. Here, we identified pyridinyl imidazole compounds SB202190, SB203580, and SB590885 as dual inhibitors of critical proliferative pathways in human melanoma cells bearing the V600E activating mutation of BRAF kinase. We found that the drugs simultaneously disrupt the BRAF V600E-driven extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in melanoma cells. Pyridinyl imidazole compounds directly inhibit BRAF V600E kinase. Moreover, they interfere with the endolysosomal compartment, promoting the accumulation of large acidic vacuole-like vesicles and dynamic changes in mTOR signaling. A transient increase in mTORC1 activity is followed by the enrichment of the Ragulator complex protein p18/LAMTOR1 at contact sites of large vesicles and delocalization of mTOR from the lysosomes. The induced disruption of the endolysosomal pathway not only disrupts mTORC1 signaling, but also renders melanoma cells sensitive to endoplasmic reticulum (ER) stress. Our findings identify new activities of pharmacologically relevant small molecule compounds and provide a biological rationale for the development of anti-melanoma therapeutics based on the pyridinyl imidazole core.

WDFY2 restrains matrix metalloproteinase secretion and cell invasion by controlling VAMP3-dependent recycling.

Cancer cells secrete matrix metalloproteinases to remodel the extracellular matrix, which enables them to overcome tissue barriers and form metastases. The membrane-bound matrix metalloproteinase MT1-MMP (MMP14) is internalized by endocytosis and recycled in endosomal compartments. It is largely unknown how endosomal sorting and recycling of MT1-MMP are controlled. Here, we show that the endosomal protein WDFY2 controls the recycling of MT1-MMP. WDFY2 localizes to endosomal tubules by binding to membranes enriched in phosphatidylinositol 3-phosphate (PtdIns3P). We identify the v-SNARE VAMP3 as an interaction partner of WDFY2. WDFY2 knockout causes a strong redistribution of VAMP3 into small vesicles near the plasma membrane. This is accompanied by increased, VAMP3-dependent secretion of MT1-MMP, enhanced degradation of extracellular matrix, and increased cell invasion. WDFY2 is frequently lost in metastatic cancers, most predominantly in ovarian and prostate cancer. We propose that WDFY2 acts as a tumor suppressor by serving as a gatekeeper for VAMP3 recycling.

Critical role for Piccolo in synaptic vesicle retrieval.

Loss of function of the active zone protein Piccolo has recently been linked to a disease, Pontocerebellar Hypoplasia type 3, which causes brain atrophy. Here, we address how Piccolo inactivation in rat neurons adversely affects synaptic function and thus may contribute to neuronal loss. Our analysis shows that Piccolo is critical for the recycling and maintenance of synaptic vesicles. We find that boutons lacking Piccolo have deficits in the Rab5/EEA1 dependent formation of early endosomes and thus the recycling of SVs. Mechanistically, impaired Rab5 function was caused by reduced synaptic recruitment of Pra1, known to interact selectively with the zinc finger domains of Piccolo. Importantly, over-expression of GTPase deficient Rab5 or the Znf1 domain of Piccolo restores the size and recycling of SV pools. These data provide a molecular link between the active zone and endosome sorting at synapses providing hints to how Piccolo contributes to developmental and psychiatric disorders.

Simultaneous defeat of MCF7 and MDA-MB-231 resistances by a hypericin PDT-tamoxifen hybrid therapy.

Currently the greatest challenge in oncology is the lack of homogeneity of the lesions where different cell components respond differently to treatment. There is growing consensus that monotherapies are insufficient to eradicate the disease and there is an unmet need for more potent combinatorial treatments. We have previously shown that hypericin photodynamic therapy (HYP-PDT) triggers electron transport chain (ETC) inhibition in cell mitochondria. We have also shown that tamoxifen (TAM) enhances cytotoxicity in cells with high respiration, when combined with ETC inhibitors. Herein we introduce a synergistic treatment based on TAM chemotherapy and HYP-PDT. We tested this novel combinatorial treatment (HYPERTAM) in two metabolically different breast cancer cell lines, the triple-negative MDA-MB-231 and the estrogen-receptor-positive MCF7, the former being quite sensitive to HYP-PDT while the latter very responsive to TAM treatment. In addition, we investigated the mode of death, effect of lipid peroxidation, and the effect on cell metabolism. The results were quite astounding. HYPERTAM exhibited over 90% cytotoxicity in both cell lines. This cytotoxicity was in the form of both necrosis and autophagy, while high levels of lipid peroxidation were observed in both cell lines. We, consequently, translated our research to an in vivo pilot study encompassing the MDA-MB-231 and MCF7 tumor models in NOD SCID-γ immunocompromised mice. Both treatment cohorts responded very positively to HYPERTRAM, which significantly prolonged mice survival. HYPERTAM is a potent, synergistic modality, which may lay the foundations for a novel, composite anticancer treatment, effective in diverse tumor types.

Concerted ESCRT and clathrin recruitment waves define the timing and morphology of intraluminal vesicle formation.

The endosomal sorting complex required for transport (ESCRT) machinery mediates cargo sorting, membrane deformation and membrane scission on the surface of endosomes, generating intraluminal vesicles (ILVs) to degrade signaling receptors. By live-cell imaging of individual endosomes in human cells, we find that ESCRT proteins are recruited in a repetitive pattern: ESCRT-0 and -I show a gradual and linear recruitment and dissociation, whereas ESCRT-III and its regulatory ATPase VPS4 display fast and transient dynamics. Electron microscopy shows that ILVs are formed consecutively, starting immediately after endocytic uptake of cargo proteins and correlating with the repeated ESCRT recruitment waves, unraveling the timing of ILV formation. Clathrin, recruited by ESCRT-0, is required for timely ESCRT-0 dissociation, efficient ILV formation, correct ILV size and cargo degradation. Thus, cargo sorting and ILV formation occur by concerted, coordinated and repetitive recruitment waves of individual ESCRT subcomplexes and are controlled by clathrin.

Neutrophils induce macrophage anti-inflammatory reprogramming by suppressing NF-κB activation.

Apoptotic cells modulate the function of macrophages to control and resolve inflammation. Here, we show that neutrophils induce a rapid and sustained suppression of NF-κB signalling in the macrophage through a unique regulatory relationship which is independent of apoptosis. The reduction of macrophage NF-κB activation occurs through a blockade in transforming growth factor ß-activated kinase 1 (TAK1) and IKKß activation. As a consequence, NF-κB (p65) phosphorylation is reduced, its translocation to the nucleus is inhibited and NF-κB-mediated inflammatory cytokine transcription is suppressed. Gene Set Enrichment Analysis reveals that this suppression of NF-κB activation is not restricted to post-translational modifications of the canonical NF-κB pathway, but is also imprinted at the transcriptional level. Thus neutrophils exert a sustained anti-inflammatory phenotypic reprogramming of the macrophage, which is reflected by the sustained reduction in the release of pro- but not anti- inflammatory cytokines from the macrophage. Together, our findings identify a novel apoptosis-independent mechanism by which neutrophils regulate the mediator profile and reprogramming of monocytes/macrophages, representing an important nodal point for inflammatory control.

Acute Effect of Hypervolemic Hemodilution on Retrobulbar Hemodynamics in Anterior Ischemic Optic Neuropathy.

PURPOSE: Ischemic ocular disorders may be treated by hypervolemic hemodilution. The presumed therapeutic benefit is based on a volume effect and improved rheological factors. The aim was to investigate the acute effect of intravenous hydroxyethyl starch on retrobulbar hemodynamics in patients with nonarteritic anterior ischemic optic neuropathy (NAION). METHODS: 24 patients with acute NAION were included. Retrobulbar hemodynamics were measured using color Doppler imaging before and 15 min after intravenous infusion of 250 cc 10% hydroxyethyl starch (HES). Peak systolic velocity (PSV), end diastolic velocity (EDV), and Pourcelot's resistive index (RI) were measured in the ophthalmic artery (OA), central retinal artery (CRA), and short posterior ciliary arteries (PCAs). RESULTS: After infusion of HES blood flow velocities significantly increased in the CRA (PSV from 7.53 ± 2.33 to 8.32 ± 2.51 (p < 0.001); EDV from 2.16 ± 0.56 to 2.34 ± 0.55 (p < 0.05)) and in the PCAs (PSV from 7.18 ± 1.62 to 7.56 ± 1.55 (p < 0.01); EDV from 2.48 ± 0.55 to 2.66 ± 0.6 cm/sec (p < 0.01)). The RI of all retrobulbar vessels remained unaffected. Blood pressure and heart rate remained unchanged. CONCLUSIONS: Hypervolemic hemodilution has an acute effect on blood flow velocities in the CRA and PCAs in NAION patients. Increased blood flow in the arteries supplying the optic nerve head may lead to a better perfusion in NAION patients. This trial is registered with DRKS00012603.

ErbB3 interacts with Hrs and is sorted to lysosomes for degradation.

The ErbB family of receptor tyrosine kinases mediates activation of a wide network of signaling pathways. ErbB3 has weak kinase activity, but its six docking sites for the p85 subunit of phosphoinositide 3-kinase make it an important contributor to proliferative signaling. ErbB3 has a relatively short half-life but the exact mechanisms controlling its turnover are unclear as contradictory reports exist. ErbB-mediated signaling is, however, negatively regulated by endocytosis of the receptors, followed by either recycling or degradation. Our previous studies showed that ErbB3 can be endocytosed and degraded in the absence of its ligand heregulin. However, binding of heregulin increased the degradation rate. In the current study we have investigated in more detail the trafficking and degradation of ErbB3 in the presence or absence of heregulin. We report that ErbB3 is internalized by clathrin-mediated endocytosis both in the presence and absence of heregulin. Moreover, we show that both proteasomal and lysosomal activity regulate ErbB3 degradation. Although steady-state expression of ErbB3 is regulated by proteasomal activity to a large extent, probably linked to a previously identified ER-localized quantity control, the results indicate that internalization, both constitutive and ligand-induced, causes lysosomal degradation of ErbB3. Furthermore, we show that ErbB3 interacts with the ESCRT-0 subunit Hrs both in the presence and absence of heregulin. This indicates an ESCRT-mediated sorting of ErbB3 to late endosomes and lysosomes, and in line with this we show that impaired ESCRT function leads to an endosomal accumulation of ErbB3.

Visualization of PML nuclear import complexes reveals FG-repeat nucleoporins at cargo retrieval sites.

Selective nuclear import in eukaryotic cells involves sequential interactions between nuclear import receptors and phenylalanine-glycine (FG)-repeat nucleoporins. Traditionally, binding of cargoes to import receptors is perceived as a nuclear pore complex independent event, while interactions between import complexes and nucleoporins are thought to take place at the nuclear pores. However, studies have shown that nucleoporins are mobile and not static within the nuclear pores, suggesting that they may become engaged in nuclear import before nuclear pore entry. Here we have studied post-mitotic nuclear import of the tumor suppressor protein PML. Since this protein forms nuclear compartments called PML bodies that persist during mitosis, the assembly of putative PML import complexes can be visualized on the surface of these protein aggregates as the cell progress from an import inactive state in mitosis to an import active state in G1. We show that these post-mitotic cytoplasmic PML bodies incorporate a multitude of peripheral nucleoporins, but not scaffold or nuclear basket nucleoporins, in a manner that depends on FG-repeats, the KPNB1 import receptor, and the PML nuclear localization signal. The study suggests that nucleoporins have the ability to target certain nuclear cargo proteins in a nuclear pore-uncoupled state, before nuclear pore entry.

Dynamic Escherichia coli SeqA complexes organize the newly replicated DNA at a considerable distance from the replisome.

The Escherichia coli SeqA protein binds to newly replicated, hemimethylated DNA behind replication forks and forms structures consisting of several hundred SeqA molecules bound to about 100 kb of DNA. It has been suggested that SeqA structures either direct the new sister DNA molecules away from each other or constitute a spacer that keeps the sisters together. We have developed an image analysis script that automatically measures the distance between neighboring foci in cells. Using this tool as well as direct stochastic optical reconstruction microscopy (dSTORM) we find that in cells with fluorescently tagged SeqA and replisome the sister SeqA structures were situated close together (less than about 30 nm apart) and relatively far from the replisome (on average 200-300 nm). The results support the idea that newly replicated sister molecules are kept together behind the fork and suggest the existence of a stretch of DNA between the replisome and SeqA which enjoys added stabilization. This could be important in facilitating DNA transactions such as recombination, mismatch repair and topoisomerase activity. In slowly growing cells without ongoing replication forks the SeqA protein was found to reside at the fully methylated origins prior to initiation of replication.

Epigenetic modification of the PD-1 (Pdcd1) promoter in effector CD4(+) T cells tolerized by peptide immunotherapy.

Clinically effective antigen-based immunotherapy must silence antigen-experienced effector T cells (Teff) driving ongoing immune pathology. Using CD4(+) autoimmune Teff cells, we demonstrate that peptide immunotherapy (PIT) is strictly dependent upon sustained T cell expression of the co-inhibitory molecule PD-1. We found high levels of 5-hydroxymethylcytosine (5hmC) at the PD-1 (Pdcd1) promoter of non-tolerant T cells. 5hmC was lost in response to PIT, with DNA hypomethylation of the promoter. We identified dynamic changes in expression of the genes encoding the Ten-Eleven-Translocation (TET) proteins that are associated with the oxidative conversion 5-methylcytosine and 5hmC, during cytosine demethylation. We describe a model whereby promoter demethylation requires the co-incident expression of permissive histone modifications at the Pdcd1 promoter together with TET availability. This combination was only seen in tolerant Teff cells following PIT, but not in Teff that transiently express PD-1. Epigenetic changes at the Pdcd1 locus therefore determine the tolerizing potential of TCR-ligation.

Promyelocytic leukemia bodies tether to early endosomes during mitosis.

During mitosis the nuclear envelope breaks down, leading to potential interactions between cytoplasmic and nuclear components. PML bodies are nuclear structures with tumor suppressor and antiviral functions. Early endosomes, on the other hand, are cytoplasmic vesicles involved in transport and growth factor signaling. Here we demonstrate that PML bodies form stable interactions with early endosomes immediately following entry into mitosis. The 2 compartments remain stably associated throughout mitosis and dissociate in the cytoplasm of newly divided daughter cells. We also show that a minor subset of PML bodies becomes anchored to the mitotic spindle poles during cell division. The study demonstrates a stable mitosis-specific interaction between a cytoplasmic and a nuclear compartment.

MicroRNA-155 negatively affects blood-brain barrier function during neuroinflammation.

Blood-brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple sclerosis (MS) and stroke. However, the molecular mechanisms underlying neurovascular dysfunction during BBB breakdown remain elusive. MicroRNAs (miRNAs) have recently emerged as key regulators of pathogenic responses, although their role in central nervous system (CNS) microvascular disorders is largely unknown. We have identified miR-155 as a critical miRNA in neuroinflammation at the BBB. miR-155 is expressed at the neurovascular unit of individuals with MS and of mice with experimental autoimmune encephalomyelitis (EAE). In mice, loss of miR-155 reduced CNS extravasation of systemic tracers, both in EAE and in an acute systemic inflammation model induced by lipopolysaccharide. In cultured human brain endothelium, miR-155 was strongly and rapidly upregulated by inflammatory cytokines. miR-155 up-regulation mimicked cytokine-induced alterations in junctional organization and permeability, whereas inhibition of endogenous miR-155 partially prevented a cytokine-induced increase in permeability. Furthermore, miR-155 modulated brain endothelial barrier function by targeting not only cell-cell complex molecules such as annexin-2 and claudin-1, but also focal adhesion components such as DOCK-1 and syntenin-1. We propose that brain endothelial miR-155 is a negative regulator of BBB function that may constitute a novel therapeutic target for CNS neuroinflammatory disorders.

Monitoring phosphatidylinositol 3-phosphate in multivesicular endosome biogenesis.

The endocytic pathway comprises a variety of intracellular compartments that regulate sorting of internalized plasma membrane constituents as well as extracellular material. A major sorting station on this route is the early endosome, where internalized receptors destined for degradation are trafficked from the limiting membrane into the interior of the endosome by formation of intraluminal vesicles (ILVs). This invagination and budding process leads to the biogenesis of multivesicular endosomes (MVEs). The formation of ILVs depends on the sequential action of protein complexes that are partly recruited in a phosphatidylinositol 3-phosphate (PtdIns3P)-dependent manner. The underlying mechanisms of the biogenesis of MVEs are still not completely understood and it is therefore of great interest to study the sorting of PtdIns3P in this process. We are describing several methods to track these sorting events by both light and electron microscopy and combination of both methods.

Arteriovenous passage times and visual field progression in normal tension glaucoma.

PURPOSE: Fluorescein angiographic studies revealed prolonged arteriovenous passage (AVP) times and increased fluorescein filling defects in normal tension glaucoma (NTG) compared to healthy controls. The purpose of this study was to correlate baseline AVP and fluorescein filling defects with visual field progression in patients with NTG. PATIENTS AND METHODS: Patients with a follow-up period of at least 3 years and at least 4 visual field examinations were included in this retrospective study. Fluorescein angiography was performed at baseline using a confocal scanning laser ophthalmoscope (SLO, Rodenstock Instr.); fluorescein filling defects and AVP were measured by digital image analysis and dye dilution curves (25 Hz). Visual field progression was evaluated using regression analysis of the MD (Humphrey-Zeiss, SITA-24-2, MD progression per year (dB/year)). 72 patients with NTG were included, 44 patients in study 1 (fluorescein filling defects) and 28 patients in study 2 (AVP). RESULTS: In study 1 (mean follow-up 6.6 ± 1.9 years, 10 ± 5 visual field tests), MD progression per year (-0.51 ± 0.59 dB/year) was significantly correlated to the age (P = 0.04, r = -0.29) but not to fluorescein filling defects, IOP, or MD at baseline. In study 2 (mean follow-up 6.6 ± 2.2 years, 10 ± 5 visual field tests), MD progression per year (-0.45 ± 0.51 dB/year) was significantly correlated to AVP (P = 0.03, r = 0.39) but not to age, IOP, or MD at baseline. CONCLUSION: Longer AVP times at baseline are correlated to visual field progression in NTG. Impaired retinal blood flow seems to be an important factor for glaucoma progression.