Myosin VI is associated with secretory granules and is present in the nucleus in adrenal medulla chromaffin cells.

Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments. Here, MVI, but not myosins IB or IIB, was detected in chromaffin granules isolated from bovine medulla and found to be tightly associated with the granule apical surface. MVI also localized to secretory granules within rat pheochromocytoma PC12 cells as well as to the Golgi apparatus, endoplasmic reticulum and clathrin-coated pits. Notably, it was also found in the nucleus. RT-PCR revealed that MVI splice variants with a large insert (LI), characteristic of polarized cells, were barely detectable in PC12 cells, whereas variants with a small insert (SI) were the major isoforms. The presented data indicate that MVI in adrenal medulla cells is engaged in secretory vesicle trafficking within the cytoplasm and possibly also involved in transport within the nucleus.

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Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells.

The Journal of Cell Biology ◽

10.1083/jcb.127.5.1419 ◽

1994 ◽

Vol 127 (5) ◽

pp. 1419-1433 ◽

Cited By ~ 97

Author(s):

Y Liu ◽

E S Schweitzer ◽

M J Nirenberg ◽

V M Pickel ◽

C J Evans ◽

...

Keyword(s):

Pc12 Cells ◽

Gradient Centrifugation ◽

Secretory Vesicle ◽

Dense Core ◽

Gamma Aminobutyric Acid ◽

Dense Core Vesicles ◽

Protein Marker ◽

Preferential Localization ◽

Large Dense Core Vesicles ◽

Pheochromocytoma Pc12

Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH-terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs.

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Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures.

Journal of Histochemistry & Cytochemistry ◽

10.1177/44.12.8985140 ◽

1996 ◽

Vol 44 (12) ◽

pp. 1481-1488 ◽

Cited By ~ 50

Author(s):

V A Tanner ◽

T Ploug ◽

J H Tao-Cheng

Keyword(s):

Subcellular Localization ◽

Pc12 Cells ◽

Cell Cultures ◽

Transmembrane Protein ◽

Secretory Granules ◽

Secretory Vesicle ◽

Secretory Vesicles ◽

Experimental Conditions ◽

Gold Probe ◽

20 Nm

We demonstrated the subcellular localization of SV2, a transmembrane protein associated with neuroendocrine secretory vesicles, in NGF-treated PC12 cells by preembedding EM immunocytochemistry (ICC), using a small gold probe followed by silver enhancement. The use of a multiwell chamber slide substantially improved the efficiency of the preembedding EM ICC procedures for cell cultures. The advantages and related caveats of this method are discussed. SV2 was distinctly localized on dusters of synaptic vesicles and large dense-cored vesicles (LDCV). The distribution of SV2 on these two types of secretory vesicles was compared quantitatively to that of another secretory vesicle-associated transmembrane protein, synaptophysin. In cultures under similar experimental conditions, the ratio of SV2 vs synaptophysin ICC staining on synaptic vesicle dusters was about 1:1, whereas it was about 9:1 on LDCV membranes. Furthermore, whereas SV2 is localized on the membranes of the LDCVs, chromogranin A, an acidic protein in secretory granules, is clearly in the core of the LDCVs. This is the first demonstration of these two antigens in such dose (approximately 20 nm) yet distinct compartments within a single organelle.

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Rab27A and its effector MyRIP link secretory granules to F-actin and control their motion towards release sites

The Journal of Cell Biology ◽

10.1083/jcb.200302157 ◽

2003 ◽

Vol 163 (3) ◽

pp. 559-570 ◽

Cited By ~ 109

Author(s):

Claire Desnos ◽

Jean-Sébastien Schonn ◽

Sébastien Huet ◽

Viet Samuel Tran ◽

Aziz El-Amraoui ◽

...

Keyword(s):

Pc12 Cells ◽

Evanescent Wave ◽

Chromaffin Cells ◽

Secretory Granules ◽

Adrenal Chromaffin Cells ◽

Actin Cortex ◽

Myosin Va ◽

Myosin Viia ◽

Adrenal Chromaffin ◽

And Control

The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose that Rab27A and MyRIP link SGs to F-actin and control their motion toward release sites through the actin cortex.

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Doc2 is not associated with known regulated exocytotic or endosomal compartments in adrenal chromaffin cells

Biochemical Journal ◽

10.1042/bj3410179 ◽

1999 ◽

Vol 341 (1) ◽

pp. 179-183 ◽

Cited By ~ 1

Author(s):

Nathalie CHARVIN ◽

Geoff WILLIAMS ◽

Robert D. BURGOYNE

Keyword(s):

Pc12 Cells ◽

Adrenal Medulla ◽

Chromaffin Cells ◽

Subcellular Fractionation ◽

Neuroendocrine Cells ◽

Dense Core ◽

Dense Core Granules ◽

Adrenal Chromaffin ◽

Golgi Network ◽

General Component

Doc2 is a C2-domain-containing protein that is highly expressed in the nervous system and has a constitutively expressed isoform. It has been implicated as a potential Ca2+ sensor in regulated exocytosis, and has been suggested to be associated with synaptic vesicles. To examine whether Doc2 is associated with synaptic-like microvesicles (SLMVs) or dense-core granules in neuroendocrine cells, we examined the distribution of Doc2 in subcellular fractionation of chromaffin cells of the adrenal medulla and in PC12 cells. Doc2 did not co-distribute with SLMVs from either cell type, but did appear to co-distribute with dense-core granules from PC12 cells. In contrast, it was not associated with the dense-core granules during subcellular fractionation of the adrenal medulla, and nor did it appear to be associated with endosomes, cis-Golgi or the trans-Golgi network. In contrast, Doc2 co-distributed under all conditions with a mitochondrial marker. We conclude that Doc2 is not a general component of regulated secretory vesicles, but may instead be associated with mitochondria.

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The polymeric immunoglobulin receptor accumulates in specialized endosomes but not synaptic vesicles within the neurites of transfected neuroendocrine PC12 cells.

The Journal of Cell Biology ◽

10.1083/jcb.127.6.1603 ◽

1994 ◽

Vol 127 (6) ◽

pp. 1603-1616 ◽

Cited By ~ 17

Author(s):

F Bonzelius ◽

G A Herman ◽

M H Cardone ◽

K E Mostov ◽

R B Kelly

Keyword(s):

Epithelial Cells ◽

Synaptic Vesicle ◽

Pc12 Cells ◽

Cell Body ◽

Synaptic Vesicles ◽

Secretory Granules ◽

Density Lipoprotein ◽

Polymeric Immunoglobulin Receptor ◽

Apical Surface ◽

Immunoglobulin Receptor

We have expressed in neuroendocrine PC12 cells the polymeric immunoglobulin receptor (pIgR), which is normally targeted from the basolateral to the apical surface of epithelial cells. In the presence of nerve growth factor, PC12 cells extend neurites which contain synaptic vesicle-like structures and regulated secretory granules. By immunofluorescence microscopy, pIgR, like the synaptic vesicle protein synaptophysin, accumulates in both the cell body and the neurites. On the other hand, the transferrin receptor, which normally recycles at the basolateral surface in epithelial cells, and the cation-independent mannose 6-phosphate receptor, a marker of late endosomes, are largely restricted to the cell body. pIgR internalizes ligand into endosomes within the cell body and the neurites, while uptake of ligand by the low density lipoprotein receptor occurs primarily into endosomes within the cell body. We conclude that transport of membrane proteins to PC12 neurites as well as to specialized endosomes within these processes is selective and appears to be governed by similar mechanisms that dictate sorting in epithelial cells. Additionally, two types of endosomes can be identified in polarized PC12 cells by the differential uptake of ligand, a housekeeping type in the cell bodies and a specialized endosome in the neurites. Recent findings suggest that specialized axonal endosomes in neurons are likely to give rise to synaptic vesicles (Mundigl, O., M. Matteoli, L. Daniell, A. Thomas-Reetz, A. Metcalf, R. Jahn, and P. De Camilli. 1993. J. Cell Biol. 122:1207-1221). Although pIgR reaches the specialized endosomes in the neurites of PC12 cells, we find by subcellular fractionation that under a variety of conditions it is efficiently excluded from synaptic vesicle-like structures as well as from secretory granules.

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The GTPase Rab3a is associated with large dense core vesicles in bovine chromaffin cells and rat PC12 cells

Journal of Cell Science ◽

10.1242/jcs.108.4.1639 ◽

1995 ◽

Vol 108 (4) ◽

pp. 1639-1649 ◽

Cited By ~ 5

Author(s):

F. Darchen ◽

J. Senyshyn ◽

W.H. Brondyk ◽

D.J. Taatjes ◽

R.W. Holz ◽

...

Keyword(s):

Subcellular Localization ◽

Pc12 Cells ◽

Immunoelectron Microscopy ◽

Chromaffin Cells ◽

Secretory Granules ◽

Dense Core ◽

Dense Core Vesicles ◽

Large Dense Core Vesicles ◽

Bovine Chromaffin Cells ◽

Specific Association

Small GTPases of the rab family control intracellular vesicle traffic in eukaryotic cells. Although the molecular mechanisms underlying the activity of the Rab proteins have not been elucidated yet, it is known that the function of these proteins is dependent on their precise subcellular localization. It has been suggested that Rab3a, which is mainly expressed in neural and endocrine cells, might regulate exocytosis. Recently, direct experimental evidence supporting this hypothesis has been obtained. Consistent with such a role for Rab3a in regulated exocytosis was the previously reported specific association of Rab3a with synaptic vesicles and with secretory granules in adrenal chromaffin cells. Since the latter result, based on subcellular fractionation, has been controversial, we have re-investigated the subcellular localization of this GTP-binding protein by using a combination of morphological techniques. Bovine chromaffin cells were labelled with an affinity-purified polyclonal anti-Rab3a antibody and analyzed by confocal microcopy. Rab3a was found to colocalize partially with dopamine beta-hydroxylase, a chromaffin granule marker. In agreement with this observation, immunoelectron microscopy revealed a specific staining of chromaffin granules. In addition to large dense core vesicles, some small vesicles were labelled. To eliminate the possibility that the staining was due to a Rab3a-related protein, we investigated by immunoelectron microscopy the localization of an epitope-tagged Rab3a expressed in rat PC12 cells. Secretory granules were specifically labelled, whereas clear microvesicles were not. These results provide further evidence supporting a specific association of the GTPase Rab3a with large dense core secretory vesicles.

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miR-375 negatively regulates the synthesis and secretion of catecholamines by targeting Sp1 in rat adrenal medulla

AJP Cell Physiology ◽

10.1152/ajpcell.00345.2016 ◽

2017 ◽

Vol 312 (5) ◽

pp. C663-C672 ◽

Cited By ~ 8

Author(s):

Yedan Gai ◽

Jinglin Zhang ◽

Chao Wei ◽

Wei Cao ◽

Yan Cui ◽

...

Keyword(s):

Tyrosine Hydroxylase ◽

Adrenal Gland ◽

Pc12 Cells ◽

Adrenal Medulla ◽

Untranslated Region ◽

Chromaffin Cells ◽

Endocrine Gland ◽

Functional Studies ◽

Response To Stress

The adrenal gland is an important endocrine gland in balancing homeostasis and the response to stress by synthesizing and secreting catecholamines (CATs), and it has been confirmed that microRNA-375 (miR-375) is highly expressed in adrenal medulla. However, up to now there are few reports about the functions and related mechanisms in adrenal medulla. The present study was thus designed to study the roles and related mechanisms in rat adrenal medulla. Our results showed that miR-375 was specifically expressed in rat adrenal medulla chromaffin cells, and its expression was downregulated when rats were exposed to stress. The further functional studies demonstrated that the inhibition of endogenous miR-375 induced the secretion of CATs in primary rat medulla chromaffin cells and PC12 cells, whereas miR-375 overexpression resulted in a decline of CAT secretion. In addition, our results showed that miR-375 negatively regulated tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH) and mediated adrenomedullary CAT biosynthesis. These functions of miR-375 were accomplished by its binding to the 3′-untranslated region of Sp1, which was involved in the regulation of TH and DBH expressions. These novel findings suggest that miR-375 acts as a potent negative mediator in regulating the synthesis and secretion of CATs in the adrenal medulla during the maintenance of homeostasis under stress.

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Interaction of antimycin with cytochrome b -561: A study in secretory granules and in plasma membrane isolated from chromaffin cells of bovine adrenal medulla (1983) FEBS Letters 160, 153-158

FEBS Letters ◽

10.1016/0014-5793(83)81185-5 ◽

1983 ◽

Vol 163 (1) ◽

pp. 153-153

Keyword(s):

Plasma Membrane ◽

Cytochrome B ◽

Adrenal Medulla ◽

Chromaffin Cells ◽

Secretory Granules ◽

Bovine Adrenal Medulla

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Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension

Nature Communications ◽

10.1038/s41467-019-12855-6 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 10

Author(s):

Matteo Biancospino ◽

Gwen R. Buel ◽

Carlos A. Niño ◽

Elena Maspero ◽

Rossella Scotto di Perrotolo ◽

...

Keyword(s):

Mammalian Cells ◽

Coated Vesicle ◽

Apical Surface ◽

Myosin Vi ◽

Coated Pits ◽

Interacting Protein ◽

Vesicle Budding ◽

Polarized Epithelia ◽

Actin Motor ◽

Huntingtin Interacting Protein

Abstract Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.

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