scholarly journals ClCd and ClCf act redundantly at the TGN/EE and prevent acidification of the Golgi stack

2021 ◽  
Author(s):  
Stefan Scholl ◽  
Stefan Hilmer ◽  
Melanie Krebs ◽  
Karin Schumacher
Keyword(s):  
Chloride Channel ◽  
Male Gametophyte ◽  
Anion Transport ◽  
Early Endosome ◽  
Ph Measurements ◽  
Golgi Stack ◽  
Temporal Aspects ◽  
Spatio Temporal ◽  
Golgi Network

The trans-Golgi network/early endosome (TGN/EE) serves as the central hub in which exo- and endocytic trafficking pathways converge and specificity of cargo routing needs to be achieved. Acidification is a hallmark of the TGN/EE and is maintained by the vacuolar H+-ATPase (V-ATPase) with support of proton-coupled antiporters. We show here that ClCd and ClCf, two distantly related members of the Arabidopsis chloride channel (ClC)-family co-localize in the TGN/EE, act redundantly, and are essential for male gametophyte development. Combining an inducible knock-down approach and in vivo pH-measurements, we show here that reduced ClC-activity does not affect pH in the TGN/EE but causes hyperacidification of trans-Golgi cisternae. Taken together, our results show that ClC-mediated anion transport into the TGN/EE is essential and affects spatio-temporal aspects of TGN/EE-maturation as well as its functional separation from the Golgi stack.

scholarly journals CLCd and CLCf act redundantly at the TGN/EE and prevent acidification of the Golgi stack

2021 ◽  
Author(s):  
Stefan Scholl ◽  
Stefan Hillmer ◽  
Melanie Krebs ◽  
Karin Schumacher
Keyword(s):  
Male Gametophyte ◽  
Anion Transport ◽  
Early Endosome ◽  
Ph Measurements ◽  
Golgi Stack ◽  
Temporal Aspects ◽  
In Trans ◽  
Spatio Temporal ◽  
Golgi Network

The trans-Golgi network/early endosome (TGN/EE) serves as the central hub in which exo- and endocytic trafficking pathways converge and specificity of cargo routing needs to be achieved. Acidification is a hallmark of the TGN/EE and is maintained by the vacuolar H+-ATPase (V-ATPase) with support of proton-coupled antiporters. We show here that CLCd and CLCf, two distantly related members of the Arabidopsis chloride channel (CLC)-family that co-localize in the TGN/EE act redundantly and are essential for male gametophyte development. Combining an inducible knock-down approach and in vivo pH-measurements, we show here that reduced CLC-activity does not affect pH in the TGN/EE but causes accumulation of the V-ATPase in trans-Golgi cisternae leading to their hyper-acidification. Taken together, our results show that CLC-mediated anion transport into the TGN/EE is essential and affects spatio-temporal aspects of TGN/EE-maturation as well as its functional separation from the Golgi stack.

Retrograde trafficking of both Golgi complex and TGN markers to the ER induced by nordihydroguaiaretic acid and cyclofenil diphenol

1998 ◽  
Vol 111 (7) ◽  
pp. 951-965 ◽  
Author(s):  
D. Drecktrah ◽  
P. de Figueiredo ◽  
R.M. Mason ◽  
W.J. Brown
Keyword(s):  
Golgi Complex ◽  
Membrane Trafficking ◽  
Molecular Mechanisms ◽  
Nordihydroguaiaretic Acid ◽  
Retrograde Transport ◽  
Lipoxygenase Inhibitor ◽  
Golgi Stack ◽  
Golgi Network ◽  
In Vivo Effects

Previous studies have shown that the Golgi stack and the trans-Golgi network (TGN) may play a role in capturing escaped resident endoplasmic reticulum (ER) proteins, and directing their retrograde transport back to that organelle. Whether this retrograde movement represents a highly specific or more generalized membrane trafficking pathway is unclear. To better understand both the retrograde and anterograde trafficking pathways of the secretory apparatus, we examined more closely the in vivo effects of two structurally unrelated compounds, the potent lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), and the non-steroidal estrogen cyclofenil diphenol (CFD), both of which are known to inhibit secretion. In the presence of these compounds, transport of vesicular stomatitis virus G membrane glycoprotein from the ER to the Golgi complex, and from the TGN to the cell surface, was inhibited potently and rapidly. Surprisingly, we found that NDGA and CFD stimulated the rapid, but not concomitant, retrograde movement of both Golgi stack and TGN membrane proteins back to the ER until both organelles were morphologically absent from cells. Both NDGA- and CFD-stimulated TGN and Golgi retrograde membrane trafficking were inhibited by microtubule depolymerizing agents and energy poisons. Removal of NDGA and CFD resulted in the complete, but not concomitant, reformation of both Golgi stacks and their closely associated TGN compartments. These studies suggest that NDGA and CFD unmask a generalized bulk recycling pathway to the ER for both Golgi and TGN membranes and, further, that NDGA and CFD are useful for investigating the molecular mechanisms that control the formation and maintenance of both the Golgi stack proper and the TGN.

scholarly journals SMAP2, a Novel ARF GTPase-activating Protein, Interacts with Clathrin and Clathrin Assembly Protein and Functions on the AP-1–positive Early Endosome/Trans-Golgi Network

2006 ◽  
Vol 17 (6) ◽  
pp. 2592-2603 ◽  
Author(s):  
Waka Natsume ◽  
Kenji Tanabe ◽  
Shunsuke Kon ◽  
Naomi Yoshida ◽  
Toshio Watanabe ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Adaptor Proteins ◽  
Early Endosome ◽  
Dependent Manner ◽  
Transferrin Receptors ◽  
Gtpase Activating Protein ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Golgi Network

We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1–dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.

Steady-state localization of a medial-Golgi glycosyltransferase involves transit through the trans-Golgi network

2001 ◽  
Vol 358 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Andrew S. OPAT ◽  
Fiona HOUGHTON ◽  
Paul A. GLEESON
Keyword(s):  
Steady State ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Retrograde Transport ◽  
Asymmetric Distribution ◽  
Transport Pathways ◽  
Golgi Stack ◽  
Trans Golgi Network ◽  
Golgi Network

The steady-state localization of medial-Golgi enzymes is likely to involve retrograde transport pathways; however, the trafficking of these resident enzymes through the Golgi stack is unclear. To investigate if the medial-Golgi enzyme β-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI) is transported to the late Golgi, a modified GlcNAc-TI bearing an N-glycan site on the C-terminus was constructed. The modified GlcNAc-TI was demonstrated to be functionally active in vivo, and was localized to the Golgi stack of transfected cells. In stable Chinese-hamster ovary (CHO) cell clones, the N-glycosylated GlcNAc-TI carried sialylated complex N-glycan chains. Pulse-chase studies showed that the majority of GlcNAc-TI was sialylated within 60min of synthesis. Treatment of transfected CHO cells with Brefeldin A resulted in the glycosylated GlcNAc-TI bearing endo-β-N-acetylglucosaminidase H resistant chains; however, the sialylation of glycosylated GlcNAc-TI was dramatically reduced. These data imply that, in CHO cells, newly synthesized GlcNAc-TI is transported rapidly through the Golgi stack to the trans-Golgi network, suggesting that GlcNAc-TI continuously recycles from the late Golgi. Furthermore, this data suggests that retrograde transport pathways play an important role in establishing the asymmetric distribution of GlcNAc-TI within the Golgi stack.

scholarly journals Actin and Microtubules Differently Contribute to Vacuolar Targeting Specificity during the Export from the ER

Membranes ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 299
Author(s):  
Monica De Caroli ◽  
Fabrizio Barozzi ◽  
Luciana Renna ◽  
Gabriella Piro ◽  
Gian-Pietro Di Sansebastiano
Keyword(s):  
Spatial Targeting ◽  
Traffic Regulation ◽  
Emergent Features ◽  
Vacuolar Sorting ◽  
Er Export ◽  
Golgi Network ◽  
Relationship Of ◽  
The Relationship ◽  
Fine Tune

Plants rely on both actin and microtubule cytoskeletons to fine-tune sorting and spatial targeting of membranes during cell growth and stress adaptation. Considerable advances have been made in recent years in the comprehension of the relationship between the trans-Golgi network/early endosome (TGN/EE) and cytoskeletons, but studies have mainly focused on the transport to and from the plasma membrane. We address here the relationship of the cytoskeleton with different endoplasmic reticulum (ER) export mechanisms toward vacuoles. These emergent features of the plant endomembrane traffic are explored with an in vivo approach, providing clues on the traffic regulation at different levels beyond known proteins’ functions and interactions. We show how traffic of vacuolar markers, characterized by different vacuolar sorting determinants, diverges at the export from the ER, clearly involving different components of the cytoskeleton.

scholarly journals Machine learning estimation of tissue optical properties

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Brett H. Hokr ◽  
Joel N. Bixler
Keyword(s):  
Neural Network ◽  
Optical Properties ◽  
Biological Tissues ◽  
Homogeneous Layer ◽  
In Vivo Measurement ◽  
Tissue Optical Properties ◽  
The Neural Network ◽  
Spatio Temporal ◽  
Fully Connected

AbstractDynamic, in vivo measurement of the optical properties of biological tissues is still an elusive and critically important problem. Here we develop a technique for inverting a Monte Carlo simulation to extract tissue optical properties from the statistical moments of the spatio-temporal response of the tissue by training a 5-layer fully connected neural network. We demonstrate the accuracy of the method across a very wide parameter space on a single homogeneous layer tissue model and demonstrate that the method is insensitive to parameter selection of the neural network model itself. Finally, we propose an experimental setup capable of measuring the required information in real time in an in vivo environment and demonstrate proof-of-concept level experimental results.

scholarly journals Synaptotagmin IV is necessary for the maturation of secretory granules in PC12 cells

2006 ◽  
Vol 173 (2) ◽  
pp. 241-251 ◽  
Author(s):  
Malika Ahras ◽  
Grant P. Otto ◽  
Sharon A. Tooze
Keyword(s):  
Pc12 Cells ◽  
Secretory Granules ◽  
Granule Formation ◽  
Synaptotagmin Iv ◽  
Prohormone Convertase 2 ◽  
Granule Maturation ◽  
Golgi Network ◽  
Interfering Rna

In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA–mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation.

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