scholarly journals Dynamics of Immature Secretory Granules: Role of Cytoskeletal Elements during Transport, Cortical Restriction, and F-Actin-dependent Tethering

2001 ◽  
Vol 12 (5) ◽  
pp. 1353-1365 ◽  
Author(s):  
Rüdiger Rudolf ◽  
Thorsten Salm ◽  
Amin Rustom ◽  
Hans-Hermann Gerdes
Keyword(s):  
Secretory Granules ◽  
Late Phase ◽  
Cell Cortex ◽  
Cell Periphery ◽  
Dependent Manner ◽  
Chromogranin B ◽  
Cooperative Action ◽  
Cytoskeletal Elements ◽  
Golgi Network

Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.

scholarly journals Expression of Regulated Secretory Proteins Is Sufficient to Generate Granule-like Structures in Constitutively Secreting Cells

2004 ◽  
Vol 279 (19) ◽  
pp. 20242-20249 ◽  
Author(s):  
Nicole Beuret ◽  
Hansruedi Stettler ◽  
Anja Renold ◽  
Jonas Rutishauser ◽  
Martin Spiess
Keyword(s):  
Protease Inhibitor ◽  
Secretory Granules ◽  
Secretory Proteins ◽  
Ionophore A23187 ◽  
Cell Periphery ◽  
Chromogranin B ◽  
Granule Formation ◽  
Trans Golgi Network ◽  
Secretogranin Ii ◽  
Golgi Network

The formation of secretory granules and regulated secretion are generally assumed to occur only in specialized endocrine, neuronal, or exocrine cells. We discovered that regulated secretory proteins such as the hormone precursors pro-vasopressin, pro-oxytocin, and pro-opiomelanocortin, as well as the granins secretogranin II and chromogranin B but not the constitutive secretory protein α1-protease inhibitor, accumulate in granular structures at the Golgi and in the cell periphery in transfected COS-1 fibroblast cells. The accumulations were observed in 30–70% of the transfected cells expressing the pro-hormones and for virtually all of the cells expressing the granins. Similar structures were also generated in other cell lines believed to be lacking a regulated secretory pathway. The accumulations resembled secretory granules morphologically in immunofluorescence and electron microscopy. They were devoid of markers of the endoplasmic reticulum, endosomes, and lysosomes but in part stained positive for the trans-Golgi network marker TGN46, consistent with their formation at the trans-Golgi network. When different regulated proteins were coexpressed, they were frequently found in the same granules, whereas α1-protease inhibitor could not be detected in accumulations formed by secretogranin II, demonstrating segregation of regulated from constitutive secretory proteins. In pulse-chase experiments, significant intracellular storage of secretogranin II and chromogranin B was observed and secretion of retained secretogranin II was stimulated with the calcium ionophore A23187. The results suggest that expression of regulated cargo proteins is sufficient to generate structures that resemble secretory granules in the background of constitutively secreting cells, supporting earlier proposals on the mechanism of granule formation.

scholarly journals Decoding the temporal nature of brain GR activity in the NFκB signal transition leading to depressive-like behavior

2021 ◽  
Author(s):  
Young-Min Han ◽  
Min Sun Kim ◽  
Juyeong Jo ◽  
Daiha Shin ◽  
Seung-Hae Kwon ◽  
...  
Keyword(s):  
Inhibitory Action ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Late Phase ◽  
Fine Tuning ◽  
Dependent Manner ◽  
Middle Phase ◽  
Temporal Shift

AbstractThe fine-tuning of neuroinflammation is crucial for brain homeostasis as well as its immune response. The transcription factor, nuclear factor-κ-B (NFκB) is a key inflammatory player that is antagonized via anti-inflammatory actions exerted by the glucocorticoid receptor (GR). However, technical limitations have restricted our understanding of how GR is involved in the dynamics of NFκB in vivo. In this study, we used an improved lentiviral-based reporter to elucidate the time course of NFκB and GR activities during behavioral changes from sickness to depression induced by a systemic lipopolysaccharide challenge. The trajectory of NFκB activity established a behavioral basis for the NFκB signal transition involved in three phases, sickness-early-phase, normal-middle-phase, and depressive-like-late-phase. The temporal shift in brain GR activity was differentially involved in the transition of NFκB signals during the normal and depressive-like phases. The middle-phase GR effectively inhibited NFκB in a glucocorticoid-dependent manner, but the late-phase GR had no inhibitory action. Furthermore, we revealed the cryptic role of basal GR activity in the early NFκB signal transition, as evidenced by the fact that blocking GR activity with RU486 led to early depressive-like episodes through the emergence of the brain NFκB activity. These results highlight the inhibitory action of GR on NFκB by the basal and activated hypothalamic-pituitary-adrenal (HPA)-axis during body-to-brain inflammatory spread, providing clues about molecular mechanisms underlying systemic inflammation caused by such as COVID-19 infection, leading to depression.

scholarly journals Exophilin8 transiently clusters insulin granules at the actin-rich cell cortex prior to exocytosis

2011 ◽  
Vol 22 (10) ◽  
pp. 1716-1726 ◽  
Author(s):  
Kouichi Mizuno ◽  
José S. Ramalho ◽  
Tetsuro Izumi
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Small Gtpase ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Insulin Granules ◽  
Myosin Va ◽  
Exogenous Expression ◽  
Mutation Analyses

Exophilin8/MyRIP/Slac2-c is an effector protein of the small GTPase Rab27a and is specifically localized on retinal melanosomes and secretory granules. We investigated the role of exophilin8 in insulin granule trafficking. Exogenous expression of exophilin8 in pancreatic β cells or their cell line, MIN6, polarized (exophilin8-positive) insulin granules at the cell corners, where both cortical actin and the microtubule plus-end–binding protein, EB1, were present. Mutation analyses indicated that the ability of exophilin8 to act as a linker between Rab27a and myosin Va is essential for its granule-clustering activity. Moreover, exophilin8 and exophilin8-associated insulin granules were markedly stable and immobile. Total internal reflection fluorescence microscopy indicated that exophilin8 restricts the motion of insulin granules at a region deeper than that where another Rab27a effector, granuphilin, accumulates docked granules directly attached to the plasma membrane. However, the exophilin8-induced immobility of insulin granules was eliminated upon secretagogue stimulation and did not inhibit evoked exocytosis. Furthermore, exophilin8 depletion prevents insulin granules from being transported close to the plasma membrane and inhibits their fusion. These findings indicate that exophilin8 transiently traps insulin granules into the cortical actin network close to the microtubule plus-ends and supplies them for release during the stimulation.

scholarly journals Syntaxin-6 SNARE Involvement in Secretory and Endocytic Pathways of Cultured Pancreatic β-Cells

2004 ◽  
Vol 15 (4) ◽  
pp. 1690-1701 ◽  
Author(s):  
Regina Kuliawat ◽  
Elena Kalinina ◽  
Jason Bock ◽  
Lloyd Fricker ◽  
Timothy E. McGraw ◽  
...  
Keyword(s):  
Secretory Granules ◽  
Expression System ◽  
Secretory Cells ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Rate Limiting Step ◽  
Protein Receptor ◽  
Golgi Network ◽  
Rate Limiting

In pancreatic β-cells, the syntaxin 6 (Syn6) soluble N-ethylmaleimide-sensitive factor attachment protein receptor is distributed in the trans-Golgi network (TGN) (with spillover into immature secretory granules) and endosomes. A possible Syn6 requirement has been suggested in secretory granule biogenesis, but the role of Syn6 in live regulated secretory cells remains unexplored. We have created an ecdysone-inducible gene expression system in the INS-1 β-cell line and find that induced expression of a membrane-anchorless, cytosolic Syn6 (called Syn6t), but not full-length Syn6, causes a prominent defect in endosomal delivery to lysosomes, and the TGN, in these cells. The defect occurs downstream of the endosomal branchpoint involved in transferrin recycling, and upstream of the steady-state distribution of mannose 6-phosphate receptors. By contrast, neither acquisition of stimulus competence nor the ultimate size of β-granules is affected. Biosynthetic effects of dominant-interfering Syn6 seem limited to slowed intragranular processing to insulin (achieving normal levels within 2 h) and minor perturbation of sorting of newly synthesized lysosomal proenzymes. We conclude that expression of the Syn6t mutant slows a rate-limiting step in endosomal maturation but provides only modest and potentially indirect interference with regulated and constitutive secretory pathways, and in TGN sorting of lysosomal enzymes.

182 INTERACTION OF MICROTUBULES AND MICROFILAMENTS WITH DISTRIBUTION OF MITOCHONDRIA IN TWO-CELL HAMSTER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 207
Author(s):  
K. Kabashima ◽  
M. Matsuzaki ◽  
H. Suzuki
Keyword(s):  
Cell Membrane ◽  
Peripheral Region ◽  
Cytochalasin D ◽  
Microtubule Assembly ◽  
Cell Cortex ◽  
Dependent Manner ◽  
Perinuclear Region ◽  
Intermediate Region ◽  
Mitochondrial Distribution

During early development, mitochondrial distribution in the ooplasm is dramatically changed. In mice and pigs, it has been reported that translocation of mitochondria is mediated by microtubules, but not by microfilaments. However, the study using hamster embryos suggested that microfilaments played a role in the mitochondrial distribution. In this study, the role of microtubules and microfilaments on distribution of mitochondria was evaluated in 2-cell hamster embryos. Female golden hamsters were superovulated on the day of post-estrus discharge by PMSG in a weight-dependent manner [Mackiernan and Bavister 2000 Hum. Reprod. 15, 157–164], and mated with males in the evening 3 days later. Two-cell embryos were collected from the oviducts at 0900–1000 on Day 2 of pregnancy. Embryos were cultured in HECM-10 medium with or without nocodazole (a microtubule assembly inhibitor), and/or cytochalasin D (a microfilament assembly inhibitor) for 8–10 h in a humidified atmosphere of 10% CO2, 5% O2, and 85% N2 at 37.5°C. After incubation of embryos with or without inhibitors, mitochondria were stained with rhodamine 123. Some of them were stained for microtubules, microfilaments, and DNA, as reported previously (Suzuki et al. 1999 Biol. Reprod. 61, 521–526). In 2-cell hamster embryos (controls, n = 44), most mitochondria were accumulated at the perinuclear region, while little mitochondria were noted in the cell cortex. Microtubules were found around the nuclei, and distribution of the mitochondria was somewhat correlated with that of the microtubules. In contrast, microfilaments were located in a high density just under the cell membrane. After nocodazole treatment (n = 22), mitochondria had extended into the subcortical (intermediate) region in various degrees, where mitochondria were aggregated into large masses. After cytochalasin D treatment (n = 17), distributional density of mitochondria at the peripheral region was decreased, and mitochondria moved back around the nucleus. After treatment by both inhibitors (n = 21), a distribution pattern of mitochondria was almost similar to that observed after cytochalasin D treatment. The present results suggest that the distribution of mitochondria at the perinuclear region is mediated by microtubules, whereas the movement of mitochondria to the cell cortex is regulated by microfilaments. Microfilaments seemed to play a role as the anchor of mitochondria at the cell cortex.

scholarly journals Essential Role of the Disulfide-bonded Loop of Chromogranin B for Sorting to Secretory Granules Is Revealed by Expression of a Deletion Mutant in the Absence of Endogenous Granin Synthesis

1998 ◽  
Vol 140 (6) ◽  
pp. 1331-1346 ◽  
Author(s):  
Andreas Krömer ◽  
Michael M. Glombik ◽  
Wieland B. Huttner ◽  
Hans-Hermann Gerdes
Keyword(s):  
Deletion Mutant ◽  
Secretory Granules ◽  
Full Length ◽  
Structural Motif ◽  
Secretory Proteins ◽  
Sequence Motifs ◽  
Chromogranin B ◽  
Recombinant Vaccinia ◽  
Vaccinia Viruses

Sorting of regulated secretory proteins in the TGN to immature secretory granules (ISG) is thought to involve at least two steps: their selective aggregation and their interaction with membrane components destined to ISG. Here, we have investigated the sorting of chromogranin B (CgB), a member of the granin family present in the secretory granules of many endocrine cells and neurons. Specifically, we have studied the role of a candidate structural motif implicated in the sorting of CgB, the highly conserved NH2-terminal disulfide– bonded loop. Sorting to ISG of full-length human CgB and a deletion mutant of human CgB (Δcys-hCgB) lacking the 22–amino acid residues comprising the disulfide-bonded loop was compared in the rat neuroendocrine cell line PC12. Upon transfection, i.e., with ongoing synthesis of endogenous granins, the sorting of the deletion mutant was only slightly impaired compared to full-length CgB. To investigate whether this sorting was due to coaggregation of the deletion mutant with endogenous granins, we expressed human CgB using recombinant vaccinia viruses, under conditions in which the synthesis of endogenous granins in the infected PC12 cells was shut off. In these conditions, Δcys-hCgB, in contrast to full-length hCgB, was no longer sorted to ISG, but exited from the TGN in constitutive secretory vesicles. Coexpression of full-length hCgB together with Δcys-hCgB by double infection, using the respective recombinant vaccinia viruses, rescued the sorting of the deletion mutant to ISG. In conclusion, our data show that (a) the disulfide-bonded loop is essential for sorting of CgB to ISG and (b) the lack of this structural motif can be compensated by coexpression of loop-bearing CgB. Furthermore, comparison of the two expression systems, transfection and vaccinia virus–mediated expression, reveals that analyses under conditions in which host cell secretory protein synthesis is blocked greatly facilitate the identification of sequence motifs required for sorting of regulated secretory proteins to secretory granules.

scholarly journals pH-dependent processing of secretogranin II by the endopeptidase PC2 in isolated immature secretory granules

1997 ◽  
Vol 321 (1) ◽  
pp. 65-74 ◽  
Author(s):  
Sylvie URBÉ ◽  
Andrea S. DITTIÉ ◽  
Sharon A. TOOZE
Keyword(s):  
Membrane Fraction ◽  
Secretory Granules ◽  
Dependent Manner ◽  
Ph Indicator ◽  
Standard Curve ◽  
Ph Values ◽  
Secretogranin Ii ◽  
Ph Dependent ◽  
Golgi Network ◽  
Immature Secretory Granule

We have previously characterized the processing of secretogranin II (SgII) in PC12 cells that were stably transfected with the endopeptidase PC2. Here we show that processing of SgII can be observed in isolated immature secretory granules (ISGs) derived from this cell line in a temperature- and ATP-dependent manner. The stimulatory effect of ATP on processing can be attributed to the activation of the vacuolar H+-ATPase and a concomitant decrease in intragranular pH. The immature secretory granule therefore provides an adequate environment for correct processing of SgII by PC2. The rate of SgII processing was strongly dependent on the intragranular pH, suggesting that processing of SgII can be used as a pH indicator for the granule interior. A standard curve was prepared using SgII processing in ISGs equilibrated at a range of pH values. The extent of processing in ISGs incubated in the presence of ATP at physiological pH was compared with the standard curve, and the intragranular pH was determined. From these observations, we propose an intragranular pH of 6.3±0.1 for ISGs in a physiological buffer in the presence of ATP. Hence, the pH of ISGs seems to be similar to the pH of the trans-Golgi network (TGN) and is clearly higher than the pH of mature secretory granules (pH 5.0–5.5). Interestingly, no processing of SgII could be observed in a membrane fraction that is highly enriched in TGN under conditions for which processing was readily obtained in isolated ISGs.

scholarly journals Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network.

1991 ◽  
Vol 115 (6) ◽  
pp. 1505-1519 ◽  
Author(s):  
E Chanat ◽  
W B Huttner
Keyword(s):  
Secretory Granules ◽  
Hormonal Treatment ◽  
Neuroendocrine Cells ◽  
Secretory Proteins ◽  
Chromogranin B ◽  
Granule Formation ◽  
Trans Golgi Network ◽  
High Calcium ◽  
Golgi Network ◽  
Selective Aggregation

Regulated secretory proteins are thought to be sorted in the trans-Golgi network (TGN) via selective aggregation. The factors responsible for this aggregation are unknown. We show here that two widespread regulated secretory proteins, chromogranin B and secretogranin II (granins), remain in an aggregated state when TGN vesicles from neuroendocrine cells (PC12) are permeabilized at pH 6.4 in 1-10 mM calcium, conditions believed to exist in this compartment. Permeabilization of immature secretory granules under these conditions allowed the recovery of electron dense cores. The granin aggregates in the TGN largely excluded glycosaminoglycan chains which served as constitutively secreted bulk flow markers. The low pH, high calcium milieu was sufficient to induce granin aggregation in the RER. In the TGN of pituitary GH4C1 cells, the proportion of granins conserved as aggregates was higher upon hormonal treatment known to increase secretory granule formation. Our data suggest that a decrease in pH and an increase in calcium are sufficient to trigger the selective aggregation of the granins in the TGN, segregating them from constitutive secretory proteins.

scholarly journals Role of Microtubules in Fusion of Post-Golgi Vesicles to the Plasma Membrane

2003 ◽  
Vol 14 (4) ◽  
pp. 1558-1569 ◽  
Author(s):  
Jan Schmoranzer ◽  
Sanford M. Simon
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Actin Filaments ◽  
Total Internal Reflection ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Cell Periphery ◽  
Golgi Vesicles ◽  
Cytoskeletal Elements

Biosynthetic cargo is transported away from the Golgi in vesicles via microtubules. In the cell periphery the vesicles are believed to engage actin and then dock to fusion sites at the plasma membrane. Using dual-color total internal reflection fluorescence microscopy, we observed that microtubules extended within 100 nm of the plasma membrane and post-Golgi vesicles remained on microtubules up to the plasma membrane, even as fusion to the plasma membrane initiated. Disruption of microtubules eliminated the tubular shapes of the vesicles and altered the fusion events: vesicles required multiple fusions to deliver all of their membrane cargo to the plasma membrane. In contrast, the effects of disrupting actin on fusion behavior were subtle. We conclude that microtubules, rather than actin filaments, are the cytoskeletal elements on which post-Golgi vesicles are transported until they fuse to the plasma membrane.

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