Inheritance of the mammalian Golgi apparatus during the cell cycle

AbstractThe creation and propagation of the intricate Golgi architecture during the cell cycle poses a fascinating problem for biologists. Similar to the inheritance process for nuclear DNA, the inheritance of the Golgi apparatus consists of biogenesis (replication) and partitioning (mitosis/meiosis) phases, in which Golgi components must double in unit mass, then be appropriately divided between nascent daughter cells during cytokinesis. In this article we focus discussion on the recent advances in the area of Golgi inheritance, first outlining our current understanding of the behaviour of the Golgi apparatus during cell division, then concluding with a more conceptual discussion of the Golgi biogenesis problem. Throughout, we attempt to integrate ultrastructural and biochemical findings with more recent information obtained using live cell microscopy and morphological techniques

Segregation of COPI-rich and anterograde-cargo-rich domains in endoplasmic-reticulum-to-Golgi transport complexes

AbstractMembrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in the selective packaging of anterograde cargo into coated transport vesicles budding from the ER [1]. In mammalian cells, these vesicles coalesce to form tubulo-vesicular transport complexes (TCs), which shuttle anterograde cargo from the ER to the Golgi complex [2–4]. In contrast, COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER [1,5–7]. The binding of COPI to COPII-coated TCs [3,8,9], however, has led to the proposal that COPI binds to TCs and specifically packages recycling proteins into retrograde vesicles for return to the ER [3,9]. To test this hypothesis, we tracked fluorescently tagged COPI and anterograde-transport markers simultaneously in living cells. COPI predominated on TCs shuttling anterograde cargo to the Golgi complex and was rarely observed on structures moving in directions consistent with retrograde transport. Furthermore, a progressive segregation of COPI-rich domains and anterograde-cargo-rich domains was observed in the TCs. This segregation and the directed motility of COPI-containing TCs were inhibited by antibodies that blocked COPI function. These observations, which are consistent with previous biochemical data [2,9], suggest a role for COPI within TCs en route to the Golgi complex. By sequestering retrograde cargo in the anterograde-directed TCs, COPI couples the sorting of ER recycling proteins [10] to the transport of anterograde cargo

A Na,K-ATPase–Fodrin–Actin Membrane Cytoskeleton Complex is Required for Endothelial Fenestra Biogenesis

Fenestrae are transcellular plasma membrane pores that mediate blood–tissue exchange in specialised vascular endothelia. The composition and biogenesis of the fenestra remain enigmatic. We isolated and characterised the protein composition of large patches of fenestrated plasma membrane, termed sieve plates. Loss-of-function experiments demonstrated that two components of the sieve plate, moesin and annexin II, were positive and negative regulators of fenestra formation, respectively. Biochemical analyses showed that moesin is involved in the formation of an actin–fodrin submembrane cytoskeleton that was essential for fenestra formation. The link between the fodrin cytoskeleton and the plasma membrane involved the fenestral pore protein PV-1 and Na,K-ATPase, which is a key regulator of signalling during fenestra formation both in vitro and in vivo. These findings provide a conceptual framework for fenestra biogenesis, linking the dynamic changes in plasma membrane remodelling to the formation of a submembrane cytoskeletal signalling complex

A Na,K-ATPase–Fodrin–Actin Membrane Cytoskeleton Complex is Required for Endothelial Fenestra Biogenesis

Fenestrae are transcellular plasma membrane pores that mediate blood–tissue exchange in specialised vascular endothelia. The composition and biogenesis of the fenestra remain enigmatic. We isolated and characterised the protein composition of large patches of fenestrated plasma membrane, termed sieve plates. Loss-of-function experiments demonstrated that two components of the sieve plate, moesin and annexin II, were positive and negative regulators of fenestra formation, respectively. Biochemical analyses showed that moesin is involved in the formation of an actin–fodrin submembrane cytoskeleton that was essential for fenestra formation. The link between the fodrin cytoskeleton and the plasma membrane involved the fenestral pore protein PV-1 and Na,K-ATPase, which is a key regulator of signalling during fenestra formation both in vitro and in vivo. These findings provide a conceptual framework for fenestra biogenesis, linking the dynamic changes in plasma membrane remodelling to the formation of a submembrane cytoskeletal signalling complex

Sequential SNARE disassembly and GATE-16–GOS-28 complex assembly mediated by distinct NSF activities drives Golgi membrane fusion

Characterization of mammalian NSF (G274E) and Drosophila NSF (comatose) mutants revealed an evolutionarily conserved NSF activity distinct from ATPase-dependent SNARE disassembly that was essential for Golgi membrane fusion. Analysis of mammalian NSF function during cell-free assembly of Golgi cisternae from mitotic Golgi fragments revealed that NSF disassembles Golgi SNAREs during mitotic Golgi fragmentation. A subsequent ATPase-independent NSF activity restricted to the reassembly phase is essential for membrane fusion. NSF/α-SNAP catalyze the binding of GATE-16 to GOS-28, a Golgi v-SNARE, in a manner that requires ATP but not ATP hydrolysis. GATE-16 is essential for NSF-driven Golgi reassembly and precludes GOS-28 from binding to its cognate t-SNARE, syntaxin-5. We suggest that this occurs at the inception of Golgi reassembly to protect the v-SNARE and regulate SNARE function

Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

Splicing of the human vascular endothelial growth factor-A (VEGF-A) gene has been reported to generate angiogenic (VEGFxxx) and anti-angiogenic (VEGFxxxb) isoforms. Corresponding VEGFxxxb isoforms have also been reported in rat and mouse. We examined VEGFxxxb expression in mouse fibrosarcoma cell lines expressing all or individual VEGF isoforms (VEGF120, 164 or 188), grown in vitro and in vivo, and compared results with those from normal mouse and human tissues. Importantly, genetic construction of VEGF164 and VEGF188 expressing fibrosarcomas, in which exon 7 is fused to the conventional exon 8, precludes VEGFxxxb splicing from occurring. Thus, these two fibrosarcoma cell lines provided endogenous negative controls. Using RT-PCR we show that primers designed to simultaneously amplify VEGFxxx and VEGFxxxb isoforms amplified only VEGFxxx variants in both species. Moreover, only VEGFxxx species were generated when mouse podocytes were treated with TGFβ-1, a reported activator of VEGFxxxb splice selection in human podocytes. A VEGF164/120 heteroduplex species was identified as a PCR artefact, specifically in mouse. VEGFxxxb isoform-specific PCR did amplify putative VEGFxxxb species in mouse and human tissues, but unexpectedly also in VEGF188 and VEGF164 fibrosarcoma cells and tumours, where splicing to produce true VEGFxxxb isoforms cannot occur. Moreover, these products were only consistently generated using reverse primers spanning more than 5 bases across the 8b/7 or 8b/5 splice junctions. Primer annealing to VEGFxxx transcripts and amplification of exon 8b primer ‘tails’ explained the artefactual generation of VEGFxxxb products, since the same products were generated when the PCR reactions were performed with cDNA from VEGF164/VEGF188 ‘knock-in’ vectors used in the generation of single VEGF isoform-expressing transgenic mice from which the fibrosarcoma lines were developed. Collectively, our results highlight important pitfalls in data interpretation associated with detecting VEGFxxxb isoforms using current methods, and demonstrate that anti-angiogenic isoforms are not commonly expressed in mouse or human tissues

TEG® and RapidTEG® are unreliable for detecting warfarin-coagulopathy: a prospective cohort study

BACKGROUND: Thromboelastography® (TEG) utilizes kaolin, an intrinsic pathway activator, to assess clotting function. Recent published studies suggest that TEG results are commonly normal in patients receiving warfarin, despite an increased International Normalized Ratio (INR). Because RapidTEG™ includes tissue factor, an extrinsic pathway activator, as well as kaolin, we hypothesized that RapidTEG would be more sensitive in detecting a warfarin-effect. METHODS: Included in this prospective study were 22 consecutive patients undergoing elective cardioversion and receiving warfarin. Prior to cardioversion, blood was collected to assess INR, Prothrombin Time, TEG, and RapidTEG. RESULTS: INR Results: 2.8 ± 0.5 (1.6 to 4.2). Prothrombin Time Results: 19.1 ± 2.2 (13.9. to 24.3). TEG Results (Reference Range): R-Time: 8.3 ± 2.7 (2–8); K-Time: 2.1 ± 1.4 (1–3); Angle: 62.5 ± 10.3 (55–78); MA: 63.2 ± 10.3 (51–69); G: 9.4 ± 3.5 (4.6-10.9); R-Time within normal range: 10 (45.5%) with INR 2.9 ± 0.3; Correlation coefficients for INR and each of the 5 TEG variables were insignificant (P > 0.05). RapidTEG Results (Reference Range): ACT: 132 ± 58 (86–118); K-Time: 1.2 ± 0.5 (1–2); Angle: 75.4 ± 5.2 (64–80); MA: 63.4 ± 5.1 (52–71); G: 8.9 ± 2.0 (5.0-11.6); ACT within normal range: 9 (40.9%) with INR 2.7 ± 0.5; Correlation coefficients for INR and each of the 5 RapidTEG variables were insignificant (P > 0.05). CONCLUSIONS: TEG, using kaolin activation, and RapidTEG, with kaolin and tissue factor activation, were normal in a substantial percent of warfarin patients, despite an increased INR. The false-negative rate for detecting warfarin coagulopathy with either test is unacceptable. The lack of correlation between INR and all TEG and RapidTEG components further indicates that these methodologies are insensitive to warfarin effects. Findings suggest that intrinsic pathway activation may mitigate detection of an extrinsic pathway coagulopathy

Delta-like 4 is indispensable in thymic environment specific for T cell development

The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells

Mode of action uncovered for the specific reduction of methane emissions from ruminants by the small molecule 3-nitrooxypropanol

Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to acetate, propionate, butyrate, CO, and methane. Whereas the short fatty acids are absorbed and metabolized by the animals, the greenhouse gas methane escapes via eructation and breathing of the animals into the atmosphere. Along with the methane, up to 12% of the gross energy content of the feedstock is lost. Therefore, our recent report has raised interest in 3-nitrooxypropanol (3-NOP), which when added to the feed of ruminants in milligram amounts persistently reduces enteric methane emissions from livestock without apparent negative side effects [Hristov AN, et al. (2015) Proc Natl Acad Sci USA 112(34):10663-10668]. We now show with the aid of in silico, in vitro, and in vivo experiments that 3-NOP specifically targets methyl-coenzyme M reductase (MCR). The nickel enzyme, which is only active when its Ni ion is in the+1 oxidation state, catalyzes the methane-forming step in the rumen fermentation. Molecular docking suggested that 3-NOP preferably binds into the active site of MCR in a pose that places its reducible nitrate group in electron transfer distance to Ni(I). With purified MCR, we found that 3-NOP indeed inactivates MCR at micromolar concentrations by oxidation of its active site Ni(I). Concomitantly, the nitrate ester is reduced to nitrite, which also inactivates MCR at micromolar concentrations by oxidation of Ni(I). Using pure cultures, 3-NOP is demonstrated to inhibit growth of methanogenic archaea at concentrations that do not affect the growth of nonmethanogenic bacteria in the rumen.We thank Peter Livant (Auburn University), Elisabeth Jimenez (Consejo Superior de Investigaciones Cientificas), John Wallace (University of Aberdeen), and Jamie Newbold (Aberystwyth University) for the use of the gas chromatograph, for the in vitro culture work, and for providing stock pure cultures, respectively; Ulrich Ermler and the staff of the PXII beam line at Swiss Light Source (Villigen, Switzerland) for helping with the X-ray data collection; and David Rinaldo (Schrödinger, LLC) for molecular modeling support.Peer Reviewe