scholarly journals Targeting of P-selectin to two regulated secretory organelles in PC12 cells.

1996 ◽  
Vol 134 (5) ◽  
pp. 1229-1240 ◽  
Author(s):  
J P Norcott ◽  
R Solari ◽  
D F Cutler
Keyword(s):  
Amino Acids ◽  
Pc12 Cells ◽  
Transient Expression ◽  
Subcellular Fractionation ◽  
Cytoplasmic Domain ◽  
Dense Core ◽  
The Other ◽  
Lysosomal Targeting ◽  
Dense Core Granules ◽  
Carboxy Terminal

Targeting of P-selectin to the regulated secretory organelles (RSOs) of phaeochromocytoma PC12 cells has been investigated. By expressing from cDNA a chimera composed of HRP and P-selectin, and then following HRP activity through subcellular fractionation, we have discovered that P-selectin contains signals that target HRP to the synaptic-like microvesicles (SLMV) as well as the dense-core granules (DCGs) of these cells. Mutagenesis of the chimera followed by transient expression in PC12 cells shows that at least two different sequences within the carboxy-terminal cytoplasmic tail of P-selectin are necessary, but that neither is sufficient for trafficking to the SLMV. One of these sequences is centred on the 10 amino acids of the membrane-proximal C1 exon that is also implicated in lysosomal targeting. The other sequence needed for trafficking to the SLMV includes the last four amino acids of the protein. The same series of mutations have a different effect on DCG targeting, showing that traffic to the two different RSOs depends on different features within the cytoplasmic domain of P-selectin.

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

1999 ◽  
Vol 341 (1) ◽  
pp. 179-183 ◽  
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.

scholarly journals A Complex Web of Signal-dependent Trafficking Underlies the Triorganellar Distribution of P-Selectin in Neuroendocrine PC12 Cells

1999 ◽  
Vol 145 (7) ◽  
pp. 1419-1433 ◽  
Author(s):  
Anastasiya D. Blagoveshchenskaya ◽  
Eric W. Hewitt ◽  
Daniel F. Cutler
Keyword(s):  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Pc12 Cells ◽  
Brefeldin A ◽  
Dense Core ◽  
Concomitant Increase ◽  
Targeting Signal ◽  
Lysosomal Targeting ◽  
Dense Core Granules ◽  
Lumenal Domain

By analyzing the trafficking of HRP–P-selectin chimeras in which the lumenal domain of P-selectin was replaced with horseradish peroxidase, we determined the sequences needed for targeting to synaptic-like microvesicles (SLMV), dense core granules (DCG), and lysosomes in neuroendocrine PC12 cells. Within the cytoplasmic domain of P-selectin, Tyr777 is needed for the appearance of P-selectin in immature and mature DCG, as well as for targeting to SLMV. The latter destination also requires additional sequences (Leu768 and 786DPSP789) which are responsible for movement through endosomes en route to the SLMV. Leu768 also mediates transfer from early transferrin (Trn)-positive endosomes to the lysosomes; i.e., operates as a lysosomal targeting signal. Furthermore, SLMV targeting of HRP–P-selectin chimeras, but not the endogenous SLMV protein synaptophysin/p38, previously shown to be delivered to SLMV directly from the plasma membrane, is a Brefeldin A–sensitive process. Together, these data are consistent with a model of SLMV biogenesis which involves an endosomal intermediate in PC12 cells. In addition, we have discovered that impairment of SLMV or DCG targeting results in a concomitant increase in lysosomal delivery, illustrating the entwined relationships between routes leading to regulated secretory organelles (RSO) and to lysosomes.

scholarly journals Human T-Cell Leukemia Virus Type 1 Envelope-Mediated Syncytium Formation Can Be Activated in Resistant Mammalian Cell Lines by a Carboxy-Terminal Truncation of the Envelope Cytoplasmic Domain

2003 ◽  
Vol 77 (2) ◽  
pp. 963-969 ◽  
Author(s):  
Felix J. Kim ◽  
Nicolas Manel ◽  
Yvan Boublik ◽  
Jean-Luc Battini ◽  
Marc Sitbon
Keyword(s):  
Amino Acids ◽  
Cell Lines ◽  
Leukemia Virus ◽  
Syncytium Formation ◽  
Cytoplasmic Domain ◽  
Cell Leukemia ◽  
Cell Infection ◽  
Human T Cell ◽  
T Cell Leukemia Virus ◽  
Carboxy Terminal

ABSTRACT Human T-cell leukemia virus (HTLV) envelope (Env) glycoproteins induce fusion, leading to rampant syncytium formation in a broad range of cell lines. Here, we identified murine, hamster, canine, and porcine cell lines that are resistant to HTLV-1 Env-induced syncytium formation. This resistance was not due to the absence of functional receptors for HTLV Env, as these cells were susceptible to infection with HTLV Env-pseudotyped virions. As murine leukemia virus (MLV) Env and HTLV Env present close structural homologies (F. J. Kim, I. Seiliez, C. Denesvre, D. Lavillette, F. L. Cosset, and M. Sitbon, J. Biol. Chem. 275:23417-23420, 2000), and because activation of syncytium formation by MLV Env generally requires cleavage of the R peptide in the cytoplasmic domain of the Env transmembrane (TM) component, we assessed whether truncation of the cytoplasmic domain of HTLV Env would alleviate this resistance. Indeed, in all resistant cell lines, truncation of the last 8 amino acids of the HTLV Env cytoplasmic domain (HdC8) was sufficient to overcome resistance to HTLV Env-induced syncytium formation. Furthermore, HdC8-mediated cell-to-cell infection titers varied according to the target cell lines and could be significantly higher than that observed with HTLV Env on HeLa cells. These data indicate that a determinant located within the 8 carboxy-terminal cytoplasmic amino acids of TM plays a distinct role in HTLV Env-mediated cell-to-cell infection and syncytium formation.

scholarly journals Use of a β1 Integrin-deficient Human T Cell to Identify β1 Integrin Cytoplasmic Domain Sequences Critical for Integrin Function

1998 ◽  
Vol 9 (10) ◽  
pp. 2715-2727 ◽  
Author(s):  
Nadine C. Romzek ◽  
Estelle S. Harris ◽  
Cheryl L. Dell ◽  
Jeffrey Skronek ◽  
Elizabeth Hasse ◽  
...  
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cytoplasmic Domain ◽  
Integrin Subunit ◽  
Β1 Integrin ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Stimulation Of

T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in β1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the β1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human β1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the β1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the β1 integrin with the activating β1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the β1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of β1 integrin structure and function in human T cells.

scholarly journals Sorting during transport to the surface of PC12 cells: divergence of synaptic vesicle and secretory granule proteins.

1990 ◽  
Vol 110 (3) ◽  
pp. 721-730 ◽  
Author(s):  
D F Cutler ◽  
L P Cramer
Keyword(s):  
Synaptic Vesicle ◽  
Pc12 Cells ◽  
Synaptic Vesicles ◽  
Secretory Pathway ◽  
Secretory Protein ◽  
Dense Granule ◽  
Dense Core ◽  
Protein Markers ◽  
Granule Fraction ◽  
Dense Core Granules

PC12 cells, a cell line derived from a rat pheochromocytoma, have both regulated and constitutive secretory pathways. Regulated secretion occurs via large dense core granules, which are related to chromaffin granules and are abundant in these cells. In addition, PC12 cells also contain small electron-lucent vesicles, whose numbers increase in response to nerve growth factor and which may be related to cholinergic synaptic vesicles. These could characterize a second regulated secretory pathway. We have investigated the trafficking of protein markers for both these organelles. We have purified and characterized the large dense core granules from these cells using sequential velocity and equilibrium gradients. We demonstrate the copurification of the major PC12 soluble regulated secretory protein (secretogranin II) with this organelle. As a marker for the synaptic vesicle-like organelles in this system, we have used the integral membrane glycoprotein p38 or synaptophysin. We show that the p38-enriched fraction of PC12 cells comigrates with rat brain synaptic vesicles on an equilibrium gradient. We also demonstrate that p38 purifies away from the dense core granules; less than 5% of this protein is found in our dense granule fraction. Finally we show that p38 does not pass through the dense granule fraction in pulse-chase experiments. These results rule out the possibility of p38 reaching the small clear vesicles via mature dense granules and imply that these cells may have two independently derived regulated pathways.

scholarly journals Targeting of synaptotagmin to neurite terminals in neuronally differentiated PC12 cells

2000 ◽  
Vol 113 (8) ◽  
pp. 1389-1404
Author(s):  
P.A. Krasnov ◽  
G. Enikolopov
Keyword(s):  
Amino Acids ◽  
Pc12 Cells ◽  
Neuronal Cell ◽  
Structural Element ◽  
Alanine Scanning Mutagenesis ◽  
Mutant Proteins ◽  
Pheochromocytoma Pc12 ◽  
Carboxy Terminal ◽  
Related Proteins ◽  
Point Mutagenesis

We have investigated structural elements that determine the accumulation of synaptotagmin, a major synaptic vesicle protein, in neurite terminals of neuronally differentiated neuroendocrine pheochromocytoma PC12 cells. We performed extensive deletion and point mutagenesis of rat synaptotagmin II, expressed mutant proteins in PC12 cells differentiated by nerve growth factor (NGF) and monitored their intracellular distribution by immunofluorescence. We found a structural element located at the carboxy-terminal domain of the synaptotagmin molecule, which is necessary for its accumulation at the terminal. Using alanine-scanning mutagenesis, we have identified two amino acids in this element, tryptophan W405 and leucine L408, that are critical for correct targeting of synaptotagmin II to neurite terminals. Changing either one of them to alanine prevents the accumulation of the protein at the terminals. These amino acids are evolutionarily conserved throughout the entire synaptotagmin family and also among synaptotagmin-related proteins, suggesting that different synaptotagmins may have similar mechanisms of targeting to neuronal cell terminals.

Requirements for the identification of dense-core granules

2004 ◽  
Vol 14 (1) ◽  
pp. 13-19 ◽  
Author(s):  
Jacopo Meldolesi ◽  
Evelina Chieregatti ◽  
Maria Luisa Malosio
Keyword(s):  
Dense Core ◽  
Dense Core Granules

Natural Vulcanization Accelerators in Hevea Latex

1948 ◽  
Vol 21 (4) ◽  
pp. 853-859
Author(s):  
R. F. A. Altman
Keyword(s):  
Amino Acids ◽  
Experimental Results ◽  
The Other ◽  
Active Nitrogen ◽  
Nitrogen Bases ◽  
Other Hand ◽  
Decomposition Processes ◽  
Volatile Products ◽  
Nitrogenous Substances ◽  
The One

Abstract As numerous investigators have shown, some of the nonrubber components of Hevea latex have a decided accelerating action on the process of vulcanization. A survey of the literature on this subject points to the validity of certain general facts. 1. Among the nonrubber components of latex which have been investigated, certain nitrogenous bases appear to be most important for accelerating the rate of vulcanization. 2. These nitrogen bases apparently occur partly naturally in fresh latex, and partly as the result of putrefaction, heating, and other decomposition processes. 3. The nitrogen bases naturally present in fresh latex at later stages have been identified by Altman to be trigonelline, stachhydrine, betonicine, choline, methylamine, trimethylamine, and ammonia. These bases are markedly active in vulcanization, as will be seen in the section on experimental results. 4. The nitrogenous substances formed by the decomposition processes have only partly been identified, on the one hand as tetra- and pentamethylene diamine and some amino acids, on the other hand as alkaloids, proline, diamino acids, etc. 5. It has been generally accepted that these nitrogenous substances are derived from the proteins of the latex. 6. Decomposition appears to be connected with the formation of a considerable amount of acids. 7. The production of volatile nitrogen bases as a rule accompanies the decomposition processes. These volatile products have not been identified. 8. The active nitrogen bases, either already formed or derived from complex nitrogenous substances, seem to be soluble in water but only slightly soluble in acetone.

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