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Molecular Biology of the Cell May 2022Granule-plasma membrane docking and fusion can only occur when proteins that enable these reactions are present at the granule-plasma membrane contact. Thus, the...
Granule-plasma membrane docking and fusion can only occur when proteins that enable these reactions are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may influence docking and membrane fusion. We measured the mobility of vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field produced by total internal reflection was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, enabling the measurement of diffusion coefficients. We found that VAMP2-EGFP and Syt7-EGFP are mobile with a diffusion coefficient of ∼3 × 10 cm/s. Syt1-EGFP mobility was below the detection limit. Utilizing these diffusion parameters, we estimated the time required for these proteins to arrive at docking and nascent fusion sites to be many tens of milliseconds. Our analyses raise the possibility that the diffusion characteristics of VAMP2 and Syt proteins could be a factor that influences the rate of exocytosis.
Topics: Calcium; Chromaffin Cells; Chromaffin Granules; Exocytosis; Membrane Fusion; Synaptotagmin I; Vesicle-Associated Membrane Protein 2
PubMed: 34851717
DOI: 10.1091/mbc.E21-10-0494 -
Pflugers Archiv : European Journal of... Jan 2018The chromaffin cells (CCs) of the adrenal medulla play a key role in the control of circulating catecholamines to adapt our body function to stressful conditions. A huge...
The chromaffin cells (CCs) of the adrenal medulla play a key role in the control of circulating catecholamines to adapt our body function to stressful conditions. A huge research effort over the last 35 years has converted these cells into the Escherichia coli of neurobiology. CCs have been the testing bench for the development of patch-clamp and amperometric recording techniques and helped clarify most of the known molecular mechanisms that regulate cell excitability, Ca signals associated with secretion, and the molecular apparatus that regulates vesicle fusion. This special issue provides a state-of-the-art on the many well-known and unsolved questions related to the molecular processes at the basis of CC function. The issue is also the occasion to highlight the seminal work of Antonio G. García (Emeritus Professor at UAM, Madrid) who greatly contributed to the advancement of our present knowledge on CC physiology and pharmacology. All the contributors of the present issue are distinguished scientists who are either staff members, external collaborators, or friends of Prof. García.
Topics: Adrenal Medulla; Animals; Chromaffin Granules; Humans; Signal Transduction
PubMed: 29110079
DOI: 10.1007/s00424-017-2082-z -
Pflugers Archiv : European Journal of... Jan 2018Many of the molecular players in the stimulus-secretion chain are similarly active in neurosecretion and catecholamine release. Therefore, studying chromaffin cells... (Review)
Review
Many of the molecular players in the stimulus-secretion chain are similarly active in neurosecretion and catecholamine release. Therefore, studying chromaffin cells uncovered many details of the processes of docking, priming, and exocytosis of vesicles. However, morphological specializations at synapses, called active zones (AZs), confer extra speed of response and another layer of control to the fast release of vesicles by action potentials. Work at the Calyx of Held, a glutamatergic nerve terminal, has shown that in addition to such rapidly released vesicles, there is a pool of "Slow Vesicles," which are held to be perfectly release-competent, but lack a final step of tight interaction with the AZ. It is argued here that such "Slow Vesicles" have many properties in common with chromaffin granules. The added complexity in the AZ-dependent regulation of "Fast Vesicles" can lead to misinterpretation of data on neurosecretion. Therefore, the study of Slow Vesicles and of chromaffin granules may provide a clearer picture of the early steps in the highly regulated process of neurosecretion.
Topics: Animals; Chromaffin Granules; Humans; Neurosecretion; Synaptic Transmission
PubMed: 28801866
DOI: 10.1007/s00424-017-2051-6 -
Folia Biologica 2011Chromogranin A (CgA) is a hydrophilic acidic one-chain peptide containing 439 amino acids, preceded by NH2-terminal 18-amino-acid signal peptide; the complete... (Review)
Review
Chromogranin A (CgA) is a hydrophilic acidic one-chain peptide containing 439 amino acids, preceded by NH2-terminal 18-amino-acid signal peptide; the complete pre-chromogranin A molecule thus encompasses 457 amino acids. It is a member of the chromogranin family that comprises several proteins. The CgA gene is a single-copy gene localized in the locus 14q32. Chromogranin A is produced by endocrine and neuroendocrine cells. The largest amount of CgA occurs in chromaffin granules of adrenal medulla and in the dense-core vesicles of sympathetic nerves. Its biological functions have not been completely elucidated, but it is known that it acts as a precursor of many biologically active peptides generated by cleavage at specific sites. It is the major soluble protein co-stored and co-released along with resident catecholamines and polypeptide hormones or cell-specific neurotransmitters. Because of its widespread distribution in neuroendocrine tissue, it can be used both as immunohistochemical marker and serum marker of neuroendocrine tumours. CgA has been used as a rather reliable tumour marker because its level is significantly increased in neuroendocrine tumours and changes of its level reflect the tumour response to therapy or tumour recurrence.
Topics: Adrenal Medulla; Amino Acid Sequence; Animals; Biomarkers, Tumor; Chromaffin Granules; Chromogranin A; Chromosomes, Human, Pair 14; Endocrine Glands; Gene Dosage; Humans; Molecular Sequence Data; Neuroendocrine Tumors; Secretory Vesicles
PubMed: 22123459
DOI: No ID Found -
Journal of Neurochemistry Sep 2020Synaptotagmin-7 (Syt-7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin-1 (Syt-1). Despite a broad...
Synaptotagmin-7 (Syt-7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin-1 (Syt-1). Despite a broad appreciation for the importance of Syt-7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations-mouse chromaffin cells lacking endogenous Syt-7 (KO cells) and a reconstituted system employing cell-derived granules expressing either Syt-7 or Syt-1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt-7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt-7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt-1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt-7, granules expressing only Syt-7 or Syt-1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt-7 confers substantially greater calcium sensitivity to granule fusion than Syt-1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt-7 plays a central role in regulating secretory output from adrenal chromaffin cells.
Topics: Acetylcholine; Animals; Calcium Signaling; Cell Movement; Chromaffin Granules; Electrophysiological Phenomena; Exocytosis; Female; Kinetics; Male; Membrane Fusion; Mice; Mice, Inbred C57BL; Mice, Knockout; PC12 Cells; Rats; Receptors, Calcium-Sensing; SNARE Proteins; Subcellular Fractions; Synaptotagmin I; Synaptotagmins
PubMed: 32058590
DOI: 10.1111/jnc.14986 -
Journal of Neurochemistry Jun 2016The accumulation of neurotransmitters within secretory vesicles (SVs) far exceeds the theoretical tonic concentrations in the cytosol, a phenomenon that has captivated... (Review)
Review
The accumulation of neurotransmitters within secretory vesicles (SVs) far exceeds the theoretical tonic concentrations in the cytosol, a phenomenon that has captivated the attention of scientists for decades. For instance, chromaffin granules can accumulate close to molar concentrations of catecholamines, along with many other products like ATP, calcium, peptides, chromogranins, ascorbate, and other nucleotides. In this short review, we will summarize the interactions that are currently believed to occur between the elements that make up the vesicular cocktail in the acidic environment of SVs, and how they permit the accumulation of such high concentrations of certain components. In addition, we will examine how the vesicular cocktail regulates the exocytosis of neurotransmitters. In this review, we have highlighted the mechanisms that permit the storage of neurotransmitters and hormones inside secretory vesicles. We also have proposed a novel model based in the intravesicular interactions of the main components of this inner cocktail - catecholamines, ATP, and chromogranins - to allow the accumulation of near molar concentrations of transmitters in secretory vesicles. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).
Topics: Adenosine Triphosphate; Animals; Chromaffin Granules; Chromogranins; Exocytosis; Humans; Models, Biological; Neurotransmitter Agents; Secretory Vesicles
PubMed: 26990968
DOI: 10.1111/jnc.13609 -
Purinergic Signalling Apr 2024Chromaffin granules are secretory granules present in adrenal medulla chromaffin cells. They contain high contents of catecholamines and nucleotides and have been... (Review)
Review
Chromaffin granules are secretory granules present in adrenal medulla chromaffin cells. They contain high contents of catecholamines and nucleotides and have been regarded as a model system for the study of vesicular transmitter uptake and release. In 1988, Dr. María Teresa Miras Portugal, when studying catecholamine biosynthesis, detected a novel group of nucleotides, the diadenosine polyphosphates, in the adrenal chromaffin granules. Based on this finding, she unraveled the existence of diadenosine polyphosphate-mediated chemical transmission, leading to a paradigm shift in the field of purinergic signaling. She is also a pioneer in the studies on vesicular nucleotide storage. First, María Teresa and her group characterized nucleotide transport in chromaffin granules and synaptic vesicles using fluorescent nucleotide derivatives such as 1, N6-ethenoadenosine triphosphates. Then, they revealed the presence of a hypothetical vesicular nucleotide transporter with unique properties in terms of substrate specificity. In this article, we will describe her contributions to vesicular nucleotide storage and the foundations she laid for future studies.
Topics: Female; Humans; Nucleotides; Adenosine Triphosphate; Portugal; Catecholamines; Chromaffin Granules
PubMed: 36525101
DOI: 10.1007/s11302-022-09912-z -
Pflugers Archiv : European Journal of... Jan 2018Actin is one of the most ubiquitous protein playing fundamental roles in a variety of cellular processes. Since early in the 1980s, it was evident that filamentous actin... (Review)
Review
Actin is one of the most ubiquitous protein playing fundamental roles in a variety of cellular processes. Since early in the 1980s, it was evident that filamentous actin (F-actin) formed a peripheral cortical barrier that prevented vesicles to access secretory sites in chromaffin cells in culture. Later, around 2000, it was described that the F-actin structure accomplishes a dual role serving both vesicle transport and retentive purposes and undergoing dynamic transient changes during cell stimulation. The complex role of the F-actin cytoskeleton in neuroendocrine secretion was further evidenced when it has been proved to participate in the scaffold structure holding together the secretory machinery at active sites and participate in the generation of mechanical forces that drive the opening of the fusion pore, during the first decade of the present century. The complex vision of the multiple roles of F-actin in secretion we have acquired to date comes largely from studies performed on traditional 2D cultures of primary cells; however, recent evidences suggest that these may not accurately mimic the 3D in vivo environment, and thus, more work is now needed on adrenomedullary cells kept in a more "native" configuration to fully understand the role of F-actin in regulating chromaffin granule transport and secretion under physiological conditions.
Topics: Actin Cytoskeleton; Actins; Animals; Chromaffin Granules; Exocytosis; Humans; Secretory Pathway
PubMed: 28730385
DOI: 10.1007/s00424-017-2040-9 -
Journal of Anatomy Oct 1993More than 25 years have elapsed since R. E. Coupland made his classic observations on the ultrastructure of chromaffin granules, on the histochemical differentiation of... (Review)
Review
More than 25 years have elapsed since R. E. Coupland made his classic observations on the ultrastructure of chromaffin granules, on the histochemical differentiation of noradrenaline and adrenaline storage granules and on their release by exocytosis. This essay attempts to demonstrate that subsequent studies on the biochemistry of chromaffin granules have yielded analytical and functional data relevant for all large dense core vesicles of endocrine and nervous tissue.
Topics: Adrenal Medulla; Animals; Antigens; Chromaffin Granules; Chromogranins; Endocrine Glands; Exocytosis; Glycoproteins; Membrane Proteins; Models, Biological; Neurons; Neuropeptides
PubMed: 8300414
DOI: No ID Found -
Journal of Neurochemistry Jun 2016In addition to playing a fundamental structural role, the F-actin cytoskeleton in neuroendocrine chromaffin cells has a prominent influence on governing the molecular...
In addition to playing a fundamental structural role, the F-actin cytoskeleton in neuroendocrine chromaffin cells has a prominent influence on governing the molecular mechanism and regulating the secretory process. Performing such roles, the F-actin network might be essential to first transport, and later locate the cellular organelles participating in the secretory cycle. Chromaffin granules are transported from the internal cytosolic regions to the cell periphery along microtubular and F-actin structures. Once in the cortical region, they are embedded in the F-actin network where these vesicles experience restrictions in motility. Similarly, mitochondria transport is affected by both microtubule and F-actin inhibitors and suffers increasing motion restrictions when they are located in the cortical region. Therefore, the F-actin cortex is a key factor in defining the existence of two populations of cortical and perinuclear granules and mitochondria which could be distinguished by their different location and mobility. Interestingly, other important organelles for controlling intracellular calcium levels, such as the endoplasmic reticulum network, present clear differences in distribution and much lower mobility than chromaffin vesicles and mitochondria. Nevertheless, both mitochondria and the endoplasmic reticulum appear to distribute in the proximity of secretory sites to fulfill a pivotal role, forming triads with calcium channels ensuring the fine tuning of the secretory response. This review presents the contributions that provide the basis for our current view regarding the influence that F-actin has on the distribution of organelles participating in the release of catecholamines in chromaffin cells, and summarizes this knowledge in simple models. In chromaffin cells, organelles such as granules and mitochondria distribute forming cortical and perinuclear populations whereas others like the ER present homogenous distributions. In the present review we discuss the role of transport systems and the existence of an F-actin cortical structure as the main factors behind the formation of organelle subpopulations in this neuroendocrine cell model. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015). Cover image for this issue: doi: 10.1111/jnc.13322.
Topics: Actin Cytoskeleton; Actins; Animals; Chromaffin Cells; Chromaffin Granules; Humans; Organelles
PubMed: 26843469
DOI: 10.1111/jnc.13560