scholarly journals HID-1 is required for homotypic fusion of immature secretory granules during maturation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Wen Du ◽  
Maoge Zhou ◽  
Wei Zhao ◽  
Dongwan Cheng ◽  
Lifen Wang ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Molecular Mechanisms ◽  
Secretory Granules ◽  
Three Dimensional ◽  
Conditional Knockout ◽  
Active Peptides ◽  
Early Maturation ◽  
Proinsulin Processing ◽  
Granule Maturation ◽  
Golgi Network

Secretory granules, also known as dense core vesicles, are generated at the trans-Golgi network and undergo several maturation steps, including homotypic fusion of immature secretory granules (ISGs) and processing of prehormones to yield active peptides. The molecular mechanisms governing secretory granule maturation are largely unknown. Here, we investigate a highly conserved protein named HID-1 in a mouse model. A conditional knockout of HID-1 in pancreatic β cells leads to glucose intolerance and a remarkable increase in the serum proinsulin/insulin ratio caused by defective proinsulin processing. Large volume three-dimensional electron microscopy and immunofluorescence imaging reveal that ISGs are much more abundant in the absence of HID-1. We further demonstrate that HID-1 deficiency prevented secretory granule maturation by blocking homotypic fusion of immature secretory granules. Our data identify a novel player during the early maturation of immature secretory granules.

scholarly journals A novel function for Rab1 and Rab11 during secretory granule maturation

2021 ◽  
Author(s):  
Sarah D. Neuman ◽  
Annika R. Lee ◽  
Jane E. Selegue ◽  
Amy T. Cavanagh ◽  
Arash Bashirullah
Keyword(s):  
Salivary Glands ◽  
Secretory Granule ◽  
Secretory Granules ◽  
Granule Size ◽  
Dependent Manner ◽  
Rab Gtpases ◽  
Cargo Proteins ◽  
Granule Maturation ◽  
Golgi Network ◽  
Granule Growth

Regulated exocytosis is an essential process whereby specific cargo proteins are secreted in a stimulus-dependent manner. Cargo-containing secretory granules are synthesized in the trans-Golgi Network (TGN); after budding from the TGN, granules undergo modifications, including an increase in size. These changes occur during a poorly understood process called secretory granule maturation. Here we leverage the Drosophila larval salivary glands as a model to characterize a novel role for Rab GTPases during granule maturation. We find that secretory granules increase in size ∼300-fold between biogenesis and release, and loss of Rab1 or Rab11 reduces granule size. Surprisingly, we find that Rab1 and Rab11 localize to secretory granule membranes. Rab11 associates with granule membranes throughout maturation, and Rab11 recruits Rab1. In turn, Rab1 associates specifically with immature granules and drives granule growth. In addition to roles in granule growth, both Rab1 and Rab11 appear to have additional functions during exocytosis; Rab11 function is necessary for exocytosis, while the presence of Rab1 on immature granules may prevent precocious exocytosis. Overall, these results highlight a new role for Rab GTPases in secretory granule maturation.

scholarly journals Inside the Insulin Secretory Granule

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 515
Author(s):  
Mark Germanos ◽  
Andy Gao ◽  
Matthew Taper ◽  
Belinda Yau ◽  
Melkam A. Kebede
Keyword(s):  
Secretory Granules ◽  
Β Cells ◽  
Β Cell ◽  
Proinsulin Processing ◽  
Membrane Bound ◽  
Golgi Network ◽  
Insulin Secretory Granule ◽  
Insulin Storage ◽  
Cellular Stimulation

The pancreatic β-cell is purpose-built for the production and secretion of insulin, the only hormone that can remove glucose from the bloodstream. Insulin is kept inside miniature membrane-bound storage compartments known as secretory granules (SGs), and these specialized organelles can readily fuse with the plasma membrane upon cellular stimulation to release insulin. Insulin is synthesized in the endoplasmic reticulum (ER) as a biologically inactive precursor, proinsulin, along with several other proteins that will also become members of the insulin SG. Their coordinated synthesis enables synchronized transit through the ER and Golgi apparatus for congregation at the trans-Golgi network, the initiating site of SG biogenesis. Here, proinsulin and its constituents enter the SG where conditions are optimized for proinsulin processing into insulin and subsequent insulin storage. A healthy β-cell is continually generating SGs to supply insulin in vast excess to what is secreted. Conversely, in type 2 diabetes (T2D), the inability of failing β-cells to secrete may be due to the limited biosynthesis of new insulin. Factors that drive the formation and maturation of SGs and thus the production of insulin are therefore critical for systemic glucose control. Here, we detail the formative hours of the insulin SG from the luminal perspective. We do this by mapping the journey of individual members of the SG as they contribute to its genesis.

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.

scholarly journals Secretory granule protein chromogranin B (CHGB) forms an anion channel in membranes

2018 ◽  
Vol 1 (5) ◽  
pp. e201800139 ◽  
Author(s):  
Gaya P Yadav ◽  
Hui Zheng ◽  
Qing Yang ◽  
Lauren G Douma ◽  
Linda B Bloom ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Chloride Channels ◽  
Molecular Mechanisms ◽  
Single Channel ◽  
Secretory Granules ◽  
Anion Channel ◽  
Local Concentration ◽  
Chromogranin B ◽  
Fast Kinetics ◽  
Granule Protein

Regulated secretion is an intracellular pathway that is highly conserved from protists to humans. Granin family proteins were proposed to participate in the biogenesis, maturation and release of secretory granules in this pathway. However, the exact molecular mechanisms underlying the intracellular functions of the granin family proteins remain unclear. Here, we show that chromogranin B (CHGB), a secretory granule protein, inserts itself into membrane and forms a chloride-conducting channel. CHGB interacts strongly with phospholipid membranes through two amphipathic α helices. At a high local concentration, CHGB insertion in membrane causes significant bilayer remodeling, producing protein-coated nanoparticles and nanotubules. Fast kinetics and high cooperativity for anion efflux from CHGB vesicles suggest that CHGB tetramerizes to form a functional channel with a single-channel conductance of ∼125 pS (150/150 mM Cl−). The CHGB channel is sensitive to an anion channel blocker and exhibits higher anion selectivity than the other six known families of Cl−channels. Our data suggest that the CHGB subfamily of granin proteins forms a new family of organelle chloride channels.

scholarly journals A novel function for Rab proteins during maturation of secretory granules

2021 ◽  
Author(s):  
Sarah D Neuman ◽  
Annika R Lee ◽  
Jane E Selegue ◽  
Amy T Cavanagh ◽  
Arash Bashirullah
Keyword(s):  
Salivary Glands ◽  
Secretory Granule ◽  
Secretory Granules ◽  
Secretory Cells ◽  
Rab Proteins ◽  
Dependent Manner ◽  
Rab Gtpases ◽  
Cargo Proteins ◽  
Granule Maturation ◽  
Granule Growth

Regulated exocytosis is an essential process whereby professional secretory cells synthesize and secrete specific cargo proteins in a stimulus-dependent manner. Cargo-containing secretory granules are synthesized in the trans-Golgi Network (TGN); after budding from the TGN, granules undergo many modifications, including a dramatic increase in size. These changes occur during a poorly understood process called secretory granule maturation. Here we leverage the professional secretory cells of the Drosophila larval salivary glands as a model system to characterize a novel and unexpected role for Rab GTPases during secretory granule maturation. We find that secretory granules in the larval salivary glands increase in size ~300-fold between biogenesis and release, and loss of Rab1 or Rab11 dramatically reduces granule size. Surprisingly, we find that Rab1 and Rab11 protein localize to secretory granule membranes. Rab11 associates with granule membranes throughout the maturation process, and Rab11 is required for recruitment of Rab1. In turn, Rab1 associates specifically with immature secretory granules and drives granule growth. In addition to their roles in granule growth, both Rab1 and Rab11 appear to have additional roles during exocytosis; Rab11 function is necessary for exocytosis, while the presence of Rab1 on immature granules may prevent precocious exocytosis. Overall, these results highlight a new and unexpected role for Rab GTPases in secretory granule maturation.

scholarly journals Small disulfide loops in peptide hormones mediate self-aggregation and secretory granule sorting

2021 ◽  
Author(s):  
Jennifer Reck ◽  
Nicole Beuret ◽  
Erhan Demirci ◽  
Cristina Prescianotto-Baschong ◽  
Martin Spiess
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Granule ◽  
Secretory Granules ◽  
Peptide Hormones ◽  
Secreted Proteins ◽  
Reporter Protein ◽  
Granule Formation ◽  
Functional Amyloids ◽  
Golgi Network ◽  
Self Aggregation

ABSTRACTUnlike constitutively secreted proteins, peptide hormones are stored in densely packed secretory granules, before regulated release upon stimulation. Secretory granules are formed at the trans-Golgi network (TGN) by self-aggregation of prohormones as functional amyloids. The nonapeptide hormone vasopressin, which forms a small disulfide loop, was shown to be responsible for granule formation of its precursor in the TGN as well as for toxic fibrillar aggregation of unfolded mutants in the endoplasmic reticulum (ER). Several other hormone precursors also contain similar small disulfide loops suggesting their function as a general device to mediate aggregation for granule biogenesis. To test this hypothesis, we studied the capacity of small disulfide loops of different hormone precursors to mediate aggregation in the ER and the TGN. They indeed induced ER aggregation although to different extents in Neuro-2a and COS-1 cells. Fused to a constitutively secreted reporter protein, they also promoted sorting into secretory granules, enhanced stimulated secretion, and increased Lubrol insolubility in AtT20 cells. These results support the hypothesis that small disulfide loops act as novel signals for secretory granule biogenesis and sorting by self-aggregation.

scholarly journals Protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment.

1992 ◽  
Vol 118 (3) ◽  
pp. 521-529 ◽  
Author(s):  
R Kuliawat ◽  
P Arvan
Keyword(s):  
Pancreatic Islets ◽  
Secretory Pathway ◽  
Protein Targeting ◽  
Secretory Granules ◽  
Membrane Vesicles ◽  
Isolated Pancreatic Islets ◽  
C Peptide ◽  
Granule Maturation ◽  
Time Required ◽  
Golgi Network

We have suggested the existence of a novel "constitutive-like" secretory pathway in pancreatic islets, which preferentially conveys a fraction of newly synthesized C-peptide, insulin, and proinsulin, and is related to the presence of immature secretory granules (IGs). Regulated exocytosis of IGs results in an equimolar secretion of C-peptide and insulin; however an assay of the constitutive-like secretory pathway recently demonstrated that this route conveys newly synthesized C-peptide in molar excess of insulin (Arvan, P., R. Kuliawat, D. Prabakaran, A.-M. Zavacki, D. Elahi, S. Wang, and D. Pilkey. J. Biol. Chem. 266:14171-14174). We now use this assay to examine the kinetics of constitutive-like secretion. Though its duration is much shorter than the life of mature granules under physiologic conditions, constitutive-like secretion appears comparatively slow (t1/2 approximately equal to 1.5 h) compared with the rate of proinsulin traffic through the ER and Golgi stacks. We have examined whether this slow rate is coupled to the rate of IG exit from the trans-Golgi network (TGN). Escape from the 20 degrees C temperature block reveals a t1/2 less than or equal to 12 min from TGN exit to stimulated release of IGs; the time required for IG formation is too rapid to be rate limiting for constitutive-like secretion. Further, conditions are described in which constitutive-like secretion is blocked yet regulated discharge of IGs remains completely intact. Thus, constitutive-like secretion appears to represent an independent secretory pathway that is kinetically restricted to a specific granule maturation period. The data support a model in which passive sorting due to insulin crystallization results in enrichment of C-peptide in membrane vesicles that bud from IGs to initiate the constitutive-like secretory pathway.

scholarly journals Kalirin/Trio Rho Guanine Nucleotide Exchange Factors Regulate a Novel Step in Secretory Granule Maturation

2007 ◽  
Vol 18 (12) ◽  
pp. 4813-4825 ◽  
Author(s):  
Francesco Ferraro ◽  
Xin-Ming Ma ◽  
Jacqueline A. Sobota ◽  
Betty A. Eipper ◽  
Richard E. Mains
Keyword(s):  
Secretory Granule ◽  
Molecular Mechanisms ◽  
Secretory Granules ◽  
Guanine Nucleotide Exchange Factors ◽  
Neuroendocrine Cells ◽  
Secretory Cells ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

The molecular mechanisms involved in the maturation of secretory granules, organelles that store hormones and neuropeptides, are poorly understood. As granule content proteins are processed, the composition of granule membranes changes, yielding constitutive-like secretion of immature content proteins and producing secretagogue-responsive mature granules. Constitutive-like secretion was not previously recognized as a process subject to regulation. We show that Kalirin and Trio, homologous Rho guanine nucleotide exchange factors (GEFs), which interact with a secretory granule resident protein, modulate cargo secretion from immature granules. Some of the Kalirin and Trio isoforms expressed in neuroendocrine cells colocalize with immature granules. Overexpression of their N-terminal GEF domain (GEF1) enhances secretion from immature granules, depleting cells of secretory cargo in the absence of secretagogue. This response requires GEF1 activity and is mimicked by Kalirin/Trio substrates Rac1 and RhoG. Accordingly, selective pharmacological inhibition of endogenous GEF1 activity decreases secretagogue-independent release of hormone precursors, accumulating product peptide in mature secretory granules. Kalirin/Trio modulation of cargo secretion from immature granules provides secretory cells with an extra layer of control over the sets of peptides released. Control of this step enhances the range of physiological responses that can be elicited, whereas lack of control could have pathological consequences.

scholarly journals AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

2011 ◽  
Vol 22 (12) ◽  
pp. 2094-2105 ◽  
Author(s):  
Jason Burgess ◽  
Miluska Jauregui ◽  
Julie Tan ◽  
Janet Rollins ◽  
Sylvie Lallet ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Secretory Granules ◽  
Adaptor Protein ◽  
Fruit Fly ◽  
Biologically Active ◽  
Granule Formation ◽  
The Third ◽  
Larval Salivary Gland ◽  
Clathrin Adaptor ◽  
Golgi Network

 Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing “glue granules” that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1– and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules.

Structural Studies of Fibrinolysis by Electron and Light Microscopy

1999 ◽  
Vol 82 (08) ◽  
pp. 277-282 ◽  
Author(s):  
Yuri Veklich ◽  
Jean-Philippe Collet ◽  
Charles Francis ◽  
John W. Weisel
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Plasminogen Activator ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Degradation Products ◽  
Molecular Model ◽  
Three Dimensional ◽  
Tissue Type ◽  
Type Plasminogen Activator

IntroductionMuch is known about the fibrinolytic system that converts fibrin-bound plasminogen to the active protease, plasmin, using plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator. Plasmin then cleaves fibrin at specific sites and generates soluble fragments, many of which have been characterized, providing the basis for a molecular model of the polypeptide chain degradation.1-3 Soluble degradation products of fibrin have also been characterized by transmission electron microscopy, yielding a model for their structure.4 Moreover, high resolution, three-dimensional structures of certain fibrinogen fragments has provided a wealth of information that may be useful in understanding how various proteins bind to fibrin and the overall process of fibrinolysis (Doolittle, this volume).5,6 Both the rate of fibrinolysis and the structures of soluble derivatives are determined in part by the fibrin network structure itself. Furthermore, the activation of plasminogen by t-PA is accelerated by the conversion of fibrinogen to fibrin, and this reaction is also affected by the structure of the fibrin. For example, clots made of thin fibers have a decreased rate of conversion of plasminogen to plasmin by t-PA, and they generally are lysed more slowly than clots composed of thick fibers.7-9 Under other conditions, however, clots made of thin fibers may be lysed more rapidly.10 In addition, fibrin clots composed of abnormally thin fibers formed from certain dysfibrinogens display decreased plasminogen binding and a lower rate of fibrinolysis.11-13 Therefore, our increasing knowledge of various dysfibrinogenemias will aid our understanding of mechanisms of fibrinolysis (Matsuda, this volume).14,15 To account for these diverse observations and more fully understand the molecular basis of fibrinolysis, more knowledge of the physical changes in the fibrin matrix that precede solubilization is required. In this report, we summarize recent experiments utilizing transmission and scanning electron microscopy and confocal light microscopy to provide information about the structural changes occurring in polymerized fibrin during fibrinolysis. Many of the results of these experiments were unexpected and suggest some aspects of potential molecular mechanisms of fibrinolysis, which will also be described here.

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