scholarly journals The EF-Hand Ca2+-binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca2+-binding Requirements

2004 ◽  
Vol 15 (2) ◽  
pp. 481-496 ◽  
Author(s):  
Josefa Andrade ◽  
Hu Zhao ◽  
Brian Titus ◽  
Sandra Timm Pearce ◽  
Margarida Barroso
Keyword(s):  
Endoplasmic Reticulum ◽  
Conformational Changes ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Network Formation ◽  
Binding Protein ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Independent Manner ◽  
Ef Hand

We have reported that p22, an N-myristoylated EF-hand Ca2+-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca2+, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca2+-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based “bulk microinjection” assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca2+-binding p22 mutant, which is unable to undergo Ca2+-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca2+-independent manner and affects ER network assembly in a Ca2+-dependent manner.

scholarly journals Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.

1991 ◽  
Vol 115 (1) ◽  
pp. 31-43 ◽  
Author(s):  
H Plutner ◽  
A D Cox ◽  
S Pind ◽  
R Khosravi-Far ◽  
J R Bourne ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Essential Role ◽  
Secretory Pathway ◽  
Binding Protein ◽  
Vesicular Transport ◽  
Initial Step ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Golgi Compartment

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.

scholarly journals The Actomyosin Ring Recruits Early Secretory Compartments to the Division Site in Fission Yeast

2008 ◽  
Vol 19 (3) ◽  
pp. 1125-1138 ◽  
Author(s):  
Aleksandar Vjestica ◽  
Xin-Zi Tang ◽  
Snezhana Oliferenko
Keyword(s):  
Endoplasmic Reticulum ◽  
Fission Yeast ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Physical Force ◽  
Daughter Cells ◽  
Division Site ◽  
Ring Assembly ◽  
Membrane Barrier ◽  
Ring Constriction

The ultimate goal of cytokinesis is to establish a membrane barrier between daughter cells. The fission yeast Schizosaccharomyces pombe utilizes an actomyosin-based division ring that is thought to provide physical force for the plasma membrane invagination. Ring constriction occurs concomitantly with the assembly of a division septum that is eventually cleaved. Membrane trafficking events such as targeting of secretory vesicles to the division site require a functional actomyosin ring suggesting that it serves as a spatial landmark. However, the extent of polarization of the secretion apparatus to the division site is presently unknown. We performed a survey of dynamics of several fluorophore-tagged proteins that served as markers for various compartments of the secretory pathway. These included markers for the endoplasmic reticulum, the COPII sites, and the early and late Golgi. The secretion machinery exhibited a marked polarization to the division site. Specifically, we observed an enrichment of the transitional endoplasmic reticulum (tER) accompanied by Golgi cisternae biogenesis. These processes required actomyosin ring assembly and the function of the EFC-domain protein Cdc15p. Cdc15p overexpression was sufficient to induce tER polarization in interphase. Thus, fission yeast polarizes its entire secretory machinery to the cell division site by utilizing molecular cues provided by the actomyosin ring.

scholarly journals Acyl-CoA-binding protein (ACBP) localizes to the endoplasmic reticulum and Golgi in a ligand-dependent manner in mammalian cells

2008 ◽  
Vol 410 (3) ◽  
pp. 463-472 ◽  
Author(s):  
Jesper S. Hansen ◽  
Nils J. Færgeman ◽  
Birthe B. Kragelund ◽  
Jens Knudsen
Keyword(s):  
Endoplasmic Reticulum ◽  
Fatty Acid ◽  
Ligand Binding ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Living Cells ◽  
Dependent Manner ◽  
Fold Reduction ◽  
Epithelial Cell Lines ◽  
Mammary Gland Epithelial Cell

In the present study, we microinjected fluorescently labelled liver bovine ACBP (acyl-CoA-binding protein) [FACI-50 (fluorescent acyl-CoA indicator-50)] into HeLa and BMGE (bovine mammary gland epithelial) cell lines to characterize the localization and dynamics of ACBP in living cells. Results showed that ACBP targeted to the ER (endoplasmic reticulum) and Golgi in a ligand-binding-dependent manner. A variant Y28F/K32A-FACI-50, which is unable to bind acyl-CoA, did no longer show association with the ER and became segregated from the Golgi, as analysed by intensity correlation calculations. Depletion of fatty acids from cells by addition of FAFBSA (fatty-acid-free BSA) significantly decreased FACI-50 association with the Golgi, whereas fatty acid overloading increased Golgi association, strongly supporting that ACBP associates with the Golgi in a ligand-dependent manner. FRAP (fluorescence recovery after photobleaching) showed that the fatty-acid-induced targeting of FACI-50 to the Golgi resulted in a 5-fold reduction in FACI-50 mobility. We suggest that ACBP is targeted to the ER and Golgi in a ligand-binding-dependent manner in living cells and propose that ACBP may be involved in vesicular trafficking.

scholarly journals The Ca2+-binding Protein ALG-2 Is Recruited to Endoplasmic Reticulum Exit Sites by Sec31A and Stabilizes the Localization of Sec31A

2006 ◽  
Vol 17 (11) ◽  
pp. 4876-4887 ◽  
Author(s):  
Akinori Yamasaki ◽  
Katsuko Tani ◽  
Akitsugu Yamamoto ◽  
Naomi Kitamura ◽  
Masayuki Komada
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Rich Region ◽  
Linked Gene ◽  
Transport Vesicles ◽  
Er Exit Sites ◽  
A Cell

The formation of transport vesicles that bud from endoplasmic reticulum (ER) exit sites is dependent on the COPII coat made up of three components: the small GTPase Sar1, the Sec23/24 complex, and the Sec13/31 complex. Here, we provide evidence that apoptosis-linked gene 2 (ALG-2), a Ca2+-binding protein of unknown function, regulates the COPII function at ER exit sites in mammalian cells. ALG-2 bound to the Pro-rich region of Sec31A, a ubiquitously expressed mammalian orthologue of yeast Sec31, in a Ca2+-dependent manner and colocalized with Sec31A at ER exit sites. A Ca2+binding-deficient ALG-2 mutant, which did not bind Sec31A, lost the ability to localize to ER exit sites. Overexpression of the Pro-rich region of Sec31A or RNA interference-mediated Sec31A depletion also abolished the ALG-2 localization at these sites. In contrast, depletion of ALG-2 substantially reduced the level of Sec31A associated with the membrane at ER exit sites. Finally, treatment with a cell-permeable Ca2+chelator caused the mislocalization of ALG-2, which was accompanied by a reduced level of Sec31A at ER exit sites. We conclude that ALG-2 is recruited to ER exit sites via Ca2+-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.

scholarly journals C4b Binding Protein Acts as an Innate Immune Effector Against Influenza A Virus

2021 ◽  
Vol 11 ◽  
Author(s):  
Praveen M. Varghese ◽  
Valarmathy Murugaiah ◽  
Nazar Beirag ◽  
Nigel Temperton ◽  
Haseeb A. Khan ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Matrix Protein ◽  
Binding Protein ◽  
Fluid Phase ◽  
Factor H ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Independent Manner

C4b Binding Protein (C4BP) is a major fluid phase inhibitor of the classical and lectin pathways of the complement system. Complement inhibition is achieved by binding to and restricting the role of activated complement component C4b. C4BP functions as a co-factor for factor I in proteolytic inactivation of both soluble and cell surface-bound C4b, thus restricting the formation of the C3-convertase, C4b2a. C4BP also accelerates the natural decay/dissociation of the C3 convertase. This makes C4BP a prime target for exploitation by pathogens to escape complement attack, as seen in Streptococcus pyogenes or Flavivirus. Here, we examined whether C4BP can act on its own in a complement independent manner, against pathogens. C4BP bound H1N1 and H3N2 subtypes of Influenza A Virus (IAV) most likely via multiple sites in Complement Control Protein (CCP) 1-2, 4-5, and 7-8 domains of its α-chain. In addition, C4BP CCP1-2 bound H3N2 better than H1N1. C4BP bound three IAV envelope proteins: Haemagglutinin (~70 kDa), Neuraminidase (~55 kDa), and Matrix protein 1 (~25kDa). C4BP suppressed H1N1 subtype infection into the lung epithelial cell line, A549, while it promoted infection by H3N2 subtype. C4BP restricted viral entry for H1N1 but had the opposite effect on H3N2, as evident from experiments using pseudo-typed viral particles. C4BP downregulated mRNA levels of pro-inflammatory IFN-α, IL-12, and NFκB in the case of H1N1, while it promoted a pro-inflammatory immune response by upregulating IFN- α, TNF-α, RANTES, and IL-6 in the case of H3N2. We conclude that C4BP differentially modulates the efficacy of IAV entry, and hence, replication in a target cell in a strain-dependent manner, and acts as an entry inhibitor for H1N1. Thus, CCP containing complement proteins such as factor H and C4BP may have additional defense roles against IAV that do not rely on the regulation of complement activation.

Regulation of duodenal Ca2+ pump by calmodulin and vitamin D-dependent Ca2+-binding protein

1986 ◽  
Vol 251 (2) ◽  
pp. G223-G229
Author(s):  
W. E. Ghijsen ◽  
C. H. Van Os ◽  
C. W. Heizmann ◽  
H. Murer
Keyword(s):  
Vitamin D ◽  
Endoplasmic Reticulum ◽  
Binding Protein ◽  
Specific Effect ◽  
Dependent Manner ◽  
Calmodulin Antagonists ◽  
Concentration Dependent Manner ◽  
High Affinity ◽  
Vesicle Preparation ◽  
Duodenal Epithelium

The Ca2+ pump in rat duodenal epithelium is studied as ATP-dependent Ca2+ uptake in a vesicle preparation with a 9-fold purification in Na+-K+-ATPase activity and a 20-fold purification of Na+-K+-ATPase with respect to an endoplasmic reticulum marker. ATP-dependent Ca2+ uptake is reduced by 60% by digitonin treatment of the vesicles, whereas high-affinity Ca2+-ATPase is stimulated by the same treatment. Different methods to deplete membrane preparations of calmodulin have been used. In EDTA osmotically shocked vesicles, calmodulin stimulated ATP-dependent Ca2+ transport up to 100% in a Ca2+ concentration-dependent manner. The duodenal Ca2+ pump is inhibited by calmodulin antagonists only at low Ca2+ concentrations and in membranes not depleted from calmodulin. Vitamin D-dependent Ca2+-binding protein (Mr = 10,000) in concentrations up to 5 microM did not affect the rate of ATP-dependent Ca2+ transport, either in Ca2+-EGTA-buffered solutions or in EGTA-free solutions. In membrane preparations from vitamin D-deficient rats, the effects of calmodulin and of Ca2+-binding protein were identical to the vitamin D-repleted control preparations. This excludes a specific effect of Ca2+-binding protein and calmodulin in the vitamin D dependency of duodenal Ca2+-ATPase.

Econazole induces increases in free intracellular Ca2+ concentrations in human osteosarcoma cells

2005 ◽  
Vol 24 (9) ◽  
pp. 453-458 ◽  
Author(s):  
H-T Chang ◽  
C-S Liu ◽  
C-T Chou ◽  
C-H Hsieh ◽  
C-H Chang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Phospholipase C ◽  
Dependent Manner ◽  
Osteosarcoma Cells ◽  
Independent Manner ◽  
Concentration Dependent Manner ◽  
Human Osteosarcoma Cells ◽  
Intracellular Ca ◽  
In Vitro Effects

Econazole is an antifungal drug with different in vitro effects. However, econazole's effect on osteoblast like cells is unknown. In human MG63 osteosarcoma cells, the effect of econazole on intracellular Ca2+ concentrations ([Ca2+]i) was explored by using fura-2. At a concentration of 0.1 μM, econazole started to cause a rise in [Ca2+]i in a concentration-dependent manner. Econazole-induced [Ca2+]i rise was reduced by 74% by removal of extracellular Ca2+. The econazole-induced Ca2+ influx was mediated via a nimodipine-sensitive pathway. In Ca2+ free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ ATPase, caused a [Ca2+]i rise, after which the increasing effect of econazole on [Ca2+]i was abolished. Pretreatment of cells with econazole to deplete Ca2+ stores totally prevented thapsigargin from releasing Ca2+. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate dependent Ca2+ mobilizer)-induced, but not econazoleinduced, [Ca2+]i rise. Econazole inhibited 76% of thapsigargin-induced store-operated Ca2+ entry. These findings suggest that in MG63 osteosarcoma cells, econazole increases [Ca2+]i by stimulating Ca2+ influx and Ca2+ release from the endoplasmic reticulum via a phospholipase C-independent manner. In contrast, econazole acts as a potent blocker of store-operated Ca2+ entry.

scholarly journals Calcium-binding protein S100A6 interaction with VEGF receptors integrates signaling and trafficking pathways

2021 ◽  
Author(s):  
Sreenivasan Ponnambalam ◽  
Leyuan Bao ◽  
Gareth W Fearnley ◽  
Chi-Chuan Lin ◽  
Adam F Odell ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Calcium Binding ◽  
Mapk Pathway ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Mitogen Activated Protein Kinase ◽  
Tyrosine Kinase Domain ◽  
Dependent Manner ◽  
Endothelial Cell Function

The mammalian endothelium which lines all blood vessels responds to soluble factors which control vascular development and sprouting. Endothelial cells bind to vascular endothelial growth factor A via two different receptor tyrosine kinases (VEGFR1, VEGFR2) which regulate such cellular responses. The integration of VEGFR signal transduction and membrane trafficking is not well understood. Here, we used a yeast-based membrane protein screen to identify VEGFR-interacting factor(s) which modulate endothelial cell function. By screening a human endothelial cDNA library, we identified a calcium-binding protein, S100A6, which can interact with either VEGFR. We found that S100A6 binds in a calcium-dependent manner to either VEGFR1 or VEGFR2. S100A6 binding was mapped to the VEGFR2 tyrosine kinase domain. Depletion of S100A6 impacts on VEGF-A-regulated signaling through the canonical mitogen-activated protein kinase (MAPK) pathway. Furthermore, S100A6 depletion caused contrasting effects on biosynthetic VEGFR delivery to the plasma membrane. Co-distribution of S100A6 and VEGFRs on tubular profiles suggest the presence of transport carriers that facilitate VEGFR trafficking. We propose a mechanism whereby S100A6 acts as a calcium regulated switch which facilitates biosynthetic VEGFR trafficking from the TGN-to-plasma membrane. VEGFR-S100A6 interactions thus enable integration of signaling and trafficking pathways in controlling the endothelial response to VEGF-A.

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