Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells.

Synaptophysin is a major transmembrane glycoprotein of a type of small vesicle with an electron-translucent content (SET vesicles), including the approximately 50-nm presynaptic vesicles in neuronal cells, and of similar, somewhat larger (< or = approximately 90 nm) vesicles (SLMV) in neuroendocrine (NE) cells. When certain epithelial non-NE cells, such as human hepatocellular carcinoma PLC cells, were cDNA transfected to synthesize synaptophysin, the new molecules appeared in specific SET vesicles. As this was in contrast to other reports that only NE cells were able to sort synaptophysin away from other plasma membrane proteins into presynaptic- or SLMV-type vesicles, we have further characterized the vesicles containing synaptophysin in transfected PLC cells. Using fractionation and immunoisolation techniques, we have separated different kinds of vesicles, and we have identified a distinct type of synaptophysin-rich, small (30-90-nm) vesicle that contains little, if any, protein of the constitutive secretory pathway marker hepatitis B surface antigen, of the fluid phase endocytosis marker HRP, and of the plasma membrane recycling endosomal marker transferrin receptor. In addition, we have found variously sized vesicles that contained both synaptophysin and transferrin receptor. A corresponding result was also obtained by direct visualization, using double-label immunofluorescence microscopy for the endocytotic markers and synaptophysin in confocal laser scan microscopy and in double-immunogold label electron microscopy. We conclude that diverse non-NE cells of epithelial nature are able to enrich the "foreign" molecule synaptophysin in a category of SET vesicles that are morphologically indistinguishable from SLMV of NE cells, including one type of vesicle in which synaptophysin is sorted away from endosomal marker proteins. Possible mechanisms of this sorting are discussed.

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Rab7-harboring vesicles are carriers of the transferrin receptor through the biosynthetic secretory pathway

Science Advances ◽

10.1126/sciadv.aba7803 ◽

2021 ◽

Vol 7 (2) ◽

pp. eaba7803

Author(s):

Maika S. Deffieu ◽

Ieva Cesonyte ◽

François Delalande ◽

Gaelle Boncompain ◽

Cristina Dorobantu ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Transport ◽

Transferrin Receptor ◽

Intracellular Trafficking ◽

Secretory Pathway ◽

Gene Editing ◽

The Other ◽

Secretory Vesicles ◽

Important Intermediate ◽

Intermediate Compartment

The biosynthetic secretory pathway is particularly challenging to investigate as it is underrepresented compared to the abundance of the other intracellular trafficking routes. Here, we combined the retention using selective hook (RUSH) to a CRISPR-Cas9 gene editing approach (eRUSH) and identified Rab7-harboring vesicles as an important intermediate compartment of the Golgi–to–plasma membrane transport of neosynthesized transferrin receptor (TfR). These vesicles did not exhibit degradative properties and were not associated to Rab6A-harboring vesicles. Rab7A was transiently associated to neosynthetic TfR-containing post–Golgi vesicles but dissociated before fusion with the plasma membrane. Together, our study reveals a role for Rab7 in the biosynthetic secretory pathway of the TfR, highlighting the diversity of the secretory vesicles’ nature.

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CD133 Expression in the Nucleus Is Associated with Endometrial Carcinoma Staging and Tumor Angioinvasion

Journal of Clinical Medicine ◽

10.3390/jcm10102144 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2144

Author(s):

Milosz Pietrus ◽

Kazimierz Pitynski ◽

Marcin Waligora ◽

Katarzyna Milian-Ciesielska ◽

Monika Bialon ◽

...

Keyword(s):

Endometrial Cancer ◽

Plasma Membrane ◽

Tumor Progression ◽

Endometrial Carcinoma ◽

Molecular Level ◽

Risk Group ◽

High Risk Group ◽

Cd133 Expression ◽

Transmembrane Glycoprotein ◽

The Impact

Background: (1) Endometrial cancer is one of the most common cancers affecting women, with a growing incidence. To better understand the different behaviors associated with endometrial cancer, it is necessary to understand the changes that occur at a molecular level. CD133 is one of the factors that regulate tumor progression, which is primarily known as the transmembrane glycoprotein associated with tumor progression or cancer stem cells. The aim of our study was to assess the impact of subcellular CD133 expression on the clinical course of endometrial cancer. (2) Methods: CD133 expression in the plasma membrane, nucleus, and cytoplasm was assessed by immunohistochemical staining in a group of 64 patients with endometrial cancer representing FIGO I-IV stages, grades 1–3 and accounting for tumor angioinvasion. (3) Results: Nuclear localization of CD133 expression was increased in FIGO IB-IV stages compared to FIGO IA. Furthermore, CD133 expression in the nucleus and plasma membrane is positively and negatively associated with a higher grade of endometrial cancer and angioinvasion, respectively. (4) Conclusions: Our findings suggest that positive nuclear CD133 expression in the tumor may be related to a less favorable prognosis of endometrial carcinoma patients and has emerged as a useful biomarker of a high-risk group.

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Fig mosaic emaravirus p4 protein is involved in cell-to-cell movement

Journal of General Virology ◽

10.1099/vir.0.047860-0 ◽

2013 ◽

Vol 94 (3) ◽

pp. 682-686 ◽

Cited By ~ 27

Author(s):

Kazuya Ishikawa ◽

Kensaku Maejima ◽

Ken Komatsu ◽

Osamu Netsu ◽

Takuya Keima ◽

...

Keyword(s):

Plasma Membrane ◽

Laser Scanning ◽

Movement Protein ◽

Rna Virus ◽

Cell Movement ◽

Plant Cells ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Series Of Experiments ◽

Fig Mosaic Virus

Fig mosaic virus (FMV), a member of the newly formed genus Emaravirus, is a segmented negative-strand RNA virus. Each of the six genomic FMV segments contains a single ORF: that of RNA4 encodes the protein p4. FMV-p4 is presumed to be the movement protein (MP) of the virus; however, direct experimental evidence for this is lacking. We assessed the intercellular distribution of FMV-p4 in plant cells by confocal laser scanning microscopy and we found that FMV-p4 was localized to plasmodesmata and to the plasma membrane accompanied by tubule-like structures. A series of experiments designed to examine the movement functions revealed that FMV-p4 has the capacity to complement viral cell-to-cell movement, prompt GFP diffusion between cells, and spread by itself to neighbouring cells. Altogether, our findings demonstrated that FMV-p4 shares several properties with other viral MPs and plays an important role in cell-to-cell movement.

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Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

Biochemical Journal ◽

10.1042/bj3300853 ◽

1998 ◽

Vol 330 (2) ◽

pp. 853-860 ◽

Cited By ~ 44

Author(s):

N. J. Silvia MORENO ◽

Li ZHONG ◽

Hong-Gang LU ◽

Wanderley DE SOUZA ◽

Marlene BENCHIMOL

Keyword(s):

Plasma Membrane ◽

Toxoplasma Gondii ◽

Laser Scanning ◽

Membrane Surface ◽

Surface Proteins ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Dependent Manner ◽

Cytoplasmic Ph ◽

Bafilomycin A1

Cytoplasmic pH (pHi) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Their mean baseline pHi (7.07±0.06; n = 5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3- but was significantly decreased in a dose-dependent manner by low concentrations of N,Nʹ-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A1. Bafilomycin A1 also inhibited the recovery of tachyzoite pHi after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A1 produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H+-ATPases. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pHi homoeostasis in these cells.

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pH-dependent Cargo Sorting from the Golgi

Journal of Biological Chemistry ◽

10.1074/jbc.m110.197889 ◽

2011 ◽

Vol 286 (12) ◽

pp. 10058-10065 ◽

Cited By ~ 34

Author(s):

Chunjuan Huang ◽

Amy Chang

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Atpase Activity ◽

Secretory Pathway ◽

Ph Dependence ◽

Glucose Deprivation ◽

Ph Homeostasis ◽

Plasma Membrane Proteins ◽

Cytosolic Ph ◽

Cargo Sorting

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.

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Membrane traffic between secretory compartments is differentially affected during mitosis.

Cell Regulation ◽

10.1091/mbc.1.5.415 ◽

1990 ◽

Vol 1 (5) ◽

pp. 415-424 ◽

Cited By ~ 25

Author(s):

T Kreiner ◽

H P Moore

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Secretory Pathway ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Membrane Traffic ◽

Human Growth ◽

Secretory Proteins ◽

Viral Membrane ◽

Mitotic Cells

Membrane traffic has been shown to be regulated during cell division. In particular, with the use of viral membrane proteins as markers, endoplasmic reticulum (ER)-to-Golgi transport in mitotic cells has been shown to be essentially blocked. However, the effect of mitosis on other steps in the secretory pathway is less clear, because an early block makes examination of following steps difficult. Here, we report studies on the functional characteristics of secretory pathways in mitotic mammalian tissue culture cells by the use of a variety of markers. Chinese hamster ovary cells were transfected with cDNAs encoding secretory proteins. Consistent with earlier results following viral membrane proteins, we found that the overall secretory pathway is nonfunctional in mitotic cells, and a major block to secretion is at the step between ER and Golgi: the overall rate of secretion of human growth hormone is reduced at least 10-fold in mitotic cells, and export of truncated vesicular stomatitis virus G protein from the ER is inhibited to about the same extent, as judged by acquisition of endoglycosidase H resistance. To ascertain the integrity of transport from the trans-Golgi to plasma membrane, we followed the secretion of sulfated glycosaminoglycan (GAG) chains, which are synthesized in the Golgi and thus are not subject to the earlier ER-to-Golgi block. GAG chains are valid markers for the pathway taken by constitutive secretory proteins; both protein secretion and GAG chain secretion are sensitive to treatment with n-ethyl-maleimide and monensin and are blocked at 19 degrees C. We found that the extent of GAG-chain secretion is not altered during mitosis, although the initial rate of secretion is reduced about twofold in mitotic compared with interphase cells. Thus, during mitosis, transport from the trans-Golgi to plasma membrane is much less hindered than ER-to-Golgi traffic. We conclude that transport steps are not affected to the same extent during mitosis.

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Investigation of the role of microtubules in protein secretion from lactating mouse mammary epithelial cells

Journal of Cell Science ◽

10.1242/jcs.102.2.239 ◽

1992 ◽

Vol 102 (2) ◽

pp. 239-247 ◽

Cited By ~ 2

Author(s):

M.E. Rennison ◽

S.E. Handel ◽

C.J. Wilde ◽

R.D. Burgoyne

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Protein Secretion ◽

Secretory Pathway ◽

Mammary Epithelial Cells ◽

Early Stage ◽

Major Effect ◽

Vesicle Transport ◽

Mammary Epithelial ◽

Mammary Cells

Disruption of microtubules has been shown to reduce protein secretion from lactating mammary epithelial cells. To investigate the involvement of microtubules in the secretory pathway in these cells we have examined the effect of nocodazole on protein secretion from mammary epithelial cells derived from the lactating mouse. Mouse mammary cells have extensive microtubule networks and 85% of their tubulin was in a polymeric form. Treatment with 1 micrograms/ml nocodazole converted most of the tubulin into a soluble form. In a continuous labelling protocol it was found that nocodazole did not interfere with protein synthesis but over a 5 h period secretion was markedly inhibited. To determine whether the inhibition was at the level of early or late stages of the secretory pathway mammary cells were pulse-labelled for 1 h to label protein throughout the secretory pathway before nocodazole treatment. When secretion was subsequently assayed it was found to be slower and only partially inhibited. These findings suggest that the major effect of nocodazole is on an early stage of the secretory pathway and that microtubules normally facilitate vesicle transport to the plasma membrane. An involvement of microtubules in vesicle transport to the plasma membrane is consistent with an observed accumulation of casein vesicles in nocodazole-treated cells. Exocytosis stimulated by the calcium ionophore ionomycin was unaffected by nocodazole treatment. We conclude from these results that the major effect of nocodazole is at an early stage of the secretory pathway, one possible target being casein vesicle biogenesis in the trans-Golgi network.

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Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

Eukaryotic Cell ◽

10.1128/ec.00042-12 ◽

2012 ◽

Vol 11 (5) ◽

pp. 590-600 ◽

Cited By ~ 6

Author(s):

Fabien Lefèbvre ◽

Valérie Prouzet-Mauléon ◽

Michel Hugues ◽

Marc Crouzet ◽

Aurélie Vieillemard ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Membrane Trafficking ◽

Cell Cycle Progression ◽

Secretory Pathway ◽

Rho Gtpase ◽

Secretory Vesicles ◽

Gtpase Activating Protein ◽

Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

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The Effects of Surface Roughness of Composite Resin on Biofilm Formation of Streptococcus mutans in the Presence of Saliva

Operative Dentistry ◽

10.2341/11-371-l ◽

2012 ◽

Vol 37 (5) ◽

pp. 532-539 ◽

Cited By ~ 36

Author(s):

JW Park ◽

CW Song ◽

JH Jung ◽

SJ Ahn ◽

JL Ferracane

Keyword(s):

Surface Roughness ◽

Silicon Carbide ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Composite Resin ◽

Resin Composite ◽

Fluid Phase ◽

Confocal Laser ◽

Carbohydrate Source ◽

Effect Of Surface

SUMMARY The purpose of this study was to investigate the effects of surface roughness of resin composite on biofilm formation of Streptococcus mutans in the presence of saliva. To provide uniform surface roughness on composites, disks were prepared by curing composite against 400-grit silicon carbide paper (SR400), 800-grit silicon carbide paper (SR800), or a glass slide (SRGlass). The surface roughness was examined using confocal laser microscopy. For biofilm formation, S. mutans was grown for 24 hours with each disk in a biofilm medium with either glucose or sucrose in the presence of fluid-phase or surface-adsorbed saliva. The adherent bacteria were quantified via enumeration of the total viable counts of bacteria. Biofilms were examined using scanning electron microscopy. This study showed that SR400 had deeper and larger, but fewer depressions than SR800. Compared to SRGlass and SR800, biofilm formation was significantly increased on SR400. In addition, the differences in the effect of surface roughness on the amount of biofilm formation were not significantly influenced by either the presence of saliva or the carbohydrate source. Considering that similar differences in surface roughness were observed between SR400 and SR800 and between SR800 and SRGlass, this study suggests that surface topography (size and depth of depressions) may play a more important role than surface roughness in biofilm formation of S. mutans.

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Retrograde Transport of Golgi-localized Proteins to the ER

The Journal of Cell Biology ◽

10.1083/jcb.140.1.1 ◽

1998 ◽

Vol 140 (1) ◽

pp. 1-15 ◽

Cited By ~ 153

Author(s):

Nelson B. Cole ◽

Jan Ellenberg ◽

Jia Song ◽

Diane DiEuliis ◽

Jennifer Lippincott-Schwartz

Keyword(s):

Plasma Membrane ◽

Golgi Complex ◽

Fusion Proteins ◽

Secretory Pathway ◽

Molecular Mechanisms ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Nonpermissive Temperature ◽

Viral Glycoprotein ◽

Lumenal Domain

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.

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