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Article ; Online: The Role of Phospholipase D in Regulated Exocytosis.

Rogasevskaia, Tatiana P / Coorssen, Jens R

2015 Volume 290, Issue 48, Page(s) 28683–28696

Abstract: There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca(2+)-triggered steps of regulated exocytosis. To quantitatively address functional and ... ...

Abstract	There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca(2+)-triggered steps of regulated exocytosis. To quantitatively address functional and molecular aspects of the involvement of phospholipase D-derived phosphatidic acid in regulated exocytosis, we used an array of phospholipase D inhibitors for ex vivo and in vitro treatments of sea urchin eggs and isolated cortices and cortical vesicles, respectively, to study late steps of exocytosis, including docking/priming and fusion. The experiments with fluorescent phosphatidylcholine reveal a low level of phospholipase D activity associated with cortical vesicles but a significantly higher activity on the plasma membrane. The effects of phospholipase D activity and its product phosphatidic acid on the Ca(2+) sensitivity and rate of fusion correlate with modulatory upstream roles in docking and priming rather than to direct effects on fusion per se.
MeSH term(s)	Animals ; Calcium/metabolism ; Exocytosis/physiology ; Membrane Fusion/physiology ; Oocytes/cytology ; Oocytes/enzymology ; Phosphatidic Acids/metabolism ; Phospholipase D/metabolism ; Strongylocentrotus purpuratus/cytology ; Strongylocentrotus purpuratus/enzymology
Chemical Substances	Phosphatidic Acids ; Phospholipase D (EC 3.1.4.4) ; Calcium (SY7Q814VUP)
Language	English
Publishing date	2015-10-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M115.681429
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Article ; Online: Structure-function relationships of K

Jalloul, Ali H / Szerencsei, Robert T / Rogasevskaia, Tatiana P / Schnetkamp, Paul P M

Cell calcium

2019 Volume 86, Page(s) 102153

Abstract: ... ...

Abstract	K
MeSH term(s)	Amino Acid Sequence ; Amino Acid Substitution ; Animals ; Humans ; Models, Molecular ; Protein Transport ; Sodium-Calcium Exchanger/chemistry ; Sodium-Calcium Exchanger/metabolism ; Structure-Activity Relationship
Chemical Substances	Sodium-Calcium Exchanger
Language	English
Publishing date	2019-12-30
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	757687-0
ISSN	1532-1991 ; 0143-4160
ISSN (online)	1532-1991
ISSN	0143-4160
DOI	10.1016/j.ceca.2019.102153
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Article ; Online: A new approach to the molecular analysis of docking, priming, and regulated membrane fusion.

Rogasevskaia, Tatiana P / Coorssen, Jens R

Journal of chemical biology

2011 Volume 4, Issue 3, Page(s) 117–136

Abstract: Studies using isolated sea urchin cortical vesicles have proven invaluable in dissecting mechanisms of Ca(2+)-triggered membrane fusion. However, only acute molecular manipulations are possible in vitro. Here, using selective pharmacological ... ...

Abstract	Studies using isolated sea urchin cortical vesicles have proven invaluable in dissecting mechanisms of Ca(2+)-triggered membrane fusion. However, only acute molecular manipulations are possible in vitro. Here, using selective pharmacological manipulations of sea urchin eggs ex vivo, we test the hypothesis that specific lipidic components of the membrane matrix selectively affect defined late stages of exocytosis, particularly the Ca(2+)-triggered steps of fast membrane fusion. Egg treatments with cholesterol-lowering drugs resulted in the inhibition of vesicle fusion. Exogenous cholesterol recovered fusion extent and efficiency in cholesterol-depleted membranes; α-tocopherol, a structurally dissimilar curvature analogue, selectively restored fusion extent. Inhibition of phospholipase C reduced vesicle phosphatidylethanolamine and suppressed both the extent and kinetics of fusion. Although phosphatidylinositol-3-kinase inhibition altered levels of polyphosphoinositide species and reduced all fusion parameters, sequestering polyphosphoinositides selectively inhibited fusion kinetics. Thus, cholesterol and phosphatidylethanolamine play direct roles in the fusion pathway, contributing negative curvature. Cholesterol also organizes the physiological fusion site, defining fusion efficiency. A selective influence of phosphatidylethanolamine on fusion kinetics sheds light on the local microdomain structure at the site of docking/fusion. Polyphosphoinositides have modulatory upstream roles in priming: alterations in specific polyphosphoinositides likely represent the terminal priming steps defining fully docked, release-ready vesicles. Thus, this pharmacological approach has the potential to be a robust high-throughput platform to identify molecular components of the physiological fusion machine critical to docking, priming, and triggered fusion.
Language	English
Publishing date	2011-02-08
Publishing country	Germany
Document type	Journal Article
ZDB-ID	2432741-4
ISSN	1864-6166 ; 1864-6158
ISSN (online)	1864-6166
ISSN	1864-6158
DOI	10.1007/s12154-011-0056-8
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Article ; Online: Cellular localization of the K

Rogasevskaia, Tatiana P / Szerencsei, Robert T / Jalloul, Ali H / Visser, Frank / Winkfein, Robert J / Schnetkamp, Paul P M

Pigment cell & melanoma research

2018 Volume 32, Issue 1, Page(s) 55–67

Abstract: NCKX5 is a bidirectional ... ...

Abstract	NCKX5 is a bidirectional K
MeSH term(s)	Amino Acid Sequence ; Animals ; Autoantigens/metabolism ; Calcium/metabolism ; Cell Count ; HEK293 Cells ; Humans ; Membrane Glycoproteins/metabolism ; Membrane Proteins/metabolism ; Mice ; Mutation/genetics ; Pigmentation/drug effects ; Potassium/pharmacology ; Protein Structure, Secondary ; Protein Transport/drug effects ; Sodium-Calcium Exchanger/chemistry ; Sodium-Calcium Exchanger/metabolism ; Structure-Activity Relationship ; Zebrafish ; trans-Golgi Network/drug effects ; trans-Golgi Network/metabolism
Chemical Substances	Autoantigens ; Golgin subfamily A member 2 ; Membrane Glycoproteins ; Membrane Proteins ; Sodium-Calcium Exchanger ; TGOLN2 protein, human ; Potassium (RWP5GA015D) ; Calcium (SY7Q814VUP)
Language	English
Publishing date	2018-07-25
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2409570-9
ISSN	1755-148X ; 1600-0749 ; 0893-5785 ; 1755-1471
ISSN (online)	1755-148X ; 1600-0749
ISSN	0893-5785 ; 1755-1471
DOI	10.1111/pcmr.12723
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Article ; Online: A Functional Study of Mutations in K+-dependent Na+-Ca2+ Exchangers Associated with Amelogenesis Imperfecta and Non-syndromic Oculocutaneous Albinism.

Jalloul, Ali H / Rogasevskaia, Tatiana P / Szerencsei, Robert T / Schnetkamp, Paul P M

The Journal of biological chemistry

2016 Volume 291, Issue 25, Page(s) 13113–13123

Abstract: K(+)-dependent Na(+)/Ca(2+) exchangers belong to the solute carrier 24 (SLC24A1-5) gene family of membrane transporters. Five different gene products (NCKX1-5) have been identified in humans, which play key roles in biological processes including vision, ...

Abstract	K(+)-dependent Na(+)/Ca(2+) exchangers belong to the solute carrier 24 (SLC24A1-5) gene family of membrane transporters. Five different gene products (NCKX1-5) have been identified in humans, which play key roles in biological processes including vision, olfaction, and skin pigmentation. NCKXs are bi-directional membrane transporters that transport 1 Ca(2+)+K(+) ions in exchange for 4 Na(+) ions. Recent studies have linked mutations in the SLC24A4 (NCKX4) and SLC24A5 (NCKX5) genes to amylogenesis imperfecta (AI) and non-syndromic oculocutaneous albinism (OCA6), respectively. Here, we introduced mutations found in patients with AI and OCA6 into human SLC24A4 (NCKX4) cDNA leading to single residue substitutions in the mutant NCKX4 proteins. We measured NCKX-mediated Ca(2+) transport activity of WT and mutant NCKX4 proteins expressed in HEK293 cells. Three mutant NCKX4 cDNAs represent mutations found in the SCL24A4 gene and three represent mutations found in the SCL24A5 gene involving residues conserved between NCKX4 and NCKX5. Five mutant proteins had no observable NCKX activity, whereas one mutation resulted in a 78% reduction in transport activity. Total protein expression and trafficking to the plasma membrane (the latter with one exception) were not affected in the HEK293 cell expression system. We also analyzed two mutations in a Drosophila NCKX gene that have been reported to result in an increased susceptibility for seizures, and found that both resulted in mutant proteins with significantly reduced but observable NCKX activity. The data presented here support the genetic analyses that mutations in SLC24A4 and SLC24A5 are responsible for the phenotypic defects observed in human patients.
MeSH term(s)	Albinism, Oculocutaneous/genetics ; Amelogenesis Imperfecta/genetics ; Amino Acid Sequence ; Antiporters/genetics ; Antiporters/metabolism ; Calcium Signaling ; Genetic Association Studies ; HEK293 Cells ; Humans ; Molecular Sequence Data ; Mutation, Missense
Chemical Substances	Antiporters ; SLC24A4 protein, human ; SLC24A5 protein, human
Language	English
Publishing date	2016-04-25
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M116.728824
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Article ; Online: Anionic lipids in Ca(2+)-triggered fusion.

Rogasevskaia, Tatiana P / Churchward, Matthew A / Coorssen, Jens R

Cell calcium

2012 Volume 52, Issue 3-4, Page(s) 259–269

Abstract: Anionic lipids are native membrane components that have a profound impact on many cellular processes, including regulated exocytosis. Nonetheless, the full nature of their contribution to the fast, Ca(2+)-triggered fusion pathway remains poorly defined. ... ...

Abstract	Anionic lipids are native membrane components that have a profound impact on many cellular processes, including regulated exocytosis. Nonetheless, the full nature of their contribution to the fast, Ca(2+)-triggered fusion pathway remains poorly defined. Here we utilize the tightly coupled quantitative molecular and functional analyses enabled by the cortical vesicle model system to elucidate the roles of specific anionic lipids in the docking, priming and fusion steps of regulated release. Studies with cholesterol sulfate established that effectively localized anionic lipids could contribute to Ca(2+)-sensing and even bind Ca(2+) directly as effectors of necessary membrane rearrangements. The data thus support a role for phosphatidylserine in Ca(2+) sensing. In contrast, phosphatidylinositol would appear to serve regulatory functions in the physiological fusion machine, contributing to priming and thus the modulation and tuning of the fusion process. We note the complexities associated with establishing the specific roles of (anionic) lipids in the native fusion mechanism, including their localization and interactions with other critical components that also remain to be more clearly and quantitatively defined.
MeSH term(s)	Animals ; Anions/chemistry ; Calcium/metabolism ; Cholesterol/metabolism ; Exocytosis/physiology ; Kinetics ; Membrane Fusion/drug effects ; Neomycin/pharmacology ; Phosphatidylinositols/pharmacology ; Phosphatidylserines/pharmacology ; Secretory Vesicles/drug effects ; Secretory Vesicles/metabolism ; Strongylocentrotus purpuratus/metabolism
Chemical Substances	Anions ; Phosphatidylinositols ; Phosphatidylserines ; Neomycin (1404-04-2) ; Cholesterol (97C5T2UQ7J) ; Calcium (SY7Q814VUP)
Language	English
Publishing date	2012-09
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	757687-0
ISSN	1532-1991 ; 0143-4160
ISSN (online)	1532-1991
ISSN	0143-4160
DOI	10.1016/j.ceca.2012.03.006
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Article ; Online: Cholesterol-independent effects of methyl-β-cyclodextrin on chemical synapses.

Ormerod, Kiel G / Rogasevskaia, Tatiana P / Coorssen, Jens R / Mercier, A Joffre

PloS one

2012 Volume 7, Issue 5, Page(s) e36395

Abstract: ... impulse propagation and decreased EJP amplitude by 40% (P<0.05) in preparations from crayfish acclimatized ... acclimatized group was associated with a 49% reduction in quantal content (P<0.05). MβCD had no effect on input ... 90% reduction in cold, P<0.05; 50% reduction in warm, P<0.05). MβCD reduced cholesterol in isolated ...

Abstract	The cholesterol chelating agent, methyl-β-cyclodextrin (MβCD), alters synaptic function in many systems. At crayfish neuromuscular junctions, MβCD is reported to reduce excitatory junctional potentials (EJPs) by impairing impulse propagation to synaptic terminals, and to have no postsynaptic effects. We examined the degree to which physiological effects of MβCD correlate with its ability to reduce cholesterol, and used thermal acclimatization as an alternative method to modify cholesterol levels. MβCD impaired impulse propagation and decreased EJP amplitude by 40% (P<0.05) in preparations from crayfish acclimatized to 14 °C but not from those acclimatized to 21 °C. The reduction in EJP amplitude in the cold-acclimatized group was associated with a 49% reduction in quantal content (P<0.05). MβCD had no effect on input resistance in muscle fibers but decreased sensitivity to the neurotransmitter L-glutamate in both warm- and cold-acclimatized groups. This effect was less pronounced and reversible in the warm-acclimatized group (90% reduction in cold, P<0.05; 50% reduction in warm, P<0.05). MβCD reduced cholesterol in isolated nerve and muscle from cold- and warm-acclimatized groups by comparable amounts (nerve: 29% cold, 25% warm; muscle: 20% cold, 18% warm; P<0.05). This effect was reversed by cholesterol loading, but only in the warm-acclimatized group. Thus, effects of MβCD on glutamate-sensitivity correlated with its ability to reduce cholesterol, but effects on impulse propagation and resulting EJP amplitude did not. Our results indicate that MβCD can affect both presynaptic and postsynaptic properties, and that some effects of MβCD are unrelated to cholesterol chelation.
MeSH term(s)	Animals ; Astacoidea/physiology ; Cholesterol/metabolism ; Glutamic Acid/metabolism ; Muscle Fibers, Skeletal/metabolism ; Neuromuscular Junction/metabolism ; Synaptic Potentials/drug effects ; Synaptic Potentials/physiology ; beta-Cyclodextrins/pharmacology
Chemical Substances	beta-Cyclodextrins ; methyl-beta-cyclodextrin ; Glutamic Acid (3KX376GY7L) ; Cholesterol (97C5T2UQ7J)
Language	English
Publishing date	2012-05-08
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0036395
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Article: Anionic lipids in Ca²⁺-triggered fusion

Rogasevskaia, Tatiana P / Churchward, Matthew A / Coorssen, Jens R

Cell calcium. 2012 , v. 52, no. 3-4

2012

Abstract: Anionic lipids are native membrane components that have a profound impact on many cellular processes, including regulated exocytosis. Nonetheless, the full nature of their contribution to the fast, Ca²⁺-triggered fusion pathway remains poorly defined. ... ...

Abstract	Anionic lipids are native membrane components that have a profound impact on many cellular processes, including regulated exocytosis. Nonetheless, the full nature of their contribution to the fast, Ca²⁺-triggered fusion pathway remains poorly defined. Here we utilize the tightly coupled quantitative molecular and functional analyses enabled by the cortical vesicle model system to elucidate the roles of specific anionic lipids in the docking, priming and fusion steps of regulated release. Studies with cholesterol sulfate established that effectively localized anionic lipids could contribute to Ca²⁺-sensing and even bind Ca²⁺ directly as effectors of necessary membrane rearrangements. The data thus support a role for phosphatidylserine in Ca²⁺ sensing. In contrast, phosphatidylinositol would appear to serve regulatory functions in the physiological fusion machine, contributing to priming and thus the modulation and tuning of the fusion process. We note the complexities associated with establishing the specific roles of (anionic) lipids in the native fusion mechanism, including their localization and interactions with other critical components that also remain to be more clearly and quantitatively defined.
Keywords	calcium ; cholesterol ; exocytosis ; phosphatidylserines
Language	English
Dates of publication	2012-09
Size	p. 259-269.
Publishing place	Elsevier India Pvt Ltd.
Document type	Article
ZDB-ID	757687-0
ISSN	1532-1991 ; 0143-4160
ISSN (online)	1532-1991
ISSN	0143-4160
DOI	10.1016/j.ceca.2012.03.006
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Identifying critical components of native Ca2+-triggered membrane fusion. Integrating studies of proteins and lipids.

Furber, Kendra L / Churchward, Matthew A / Rogasevskaia, Tatiana P / Coorssen, Jens R

Annals of the New York Academy of Sciences

2009 Volume 1152, Page(s) 121–134

Abstract: Ca(2+)-triggered membrane fusion is the defining step of exocytosis. Despite realization that the fusion machinery must include lipids and proteins working in concert, only of late has work in the field focused more equally on both these components. Here ...

Abstract	Ca(2+)-triggered membrane fusion is the defining step of exocytosis. Despite realization that the fusion machinery must include lipids and proteins working in concert, only of late has work in the field focused more equally on both these components. Here we use isolated sea urchin egg cortical vesicles (CV), a stage-specific preparation of Ca(2+)-sensitive release-ready vesicles that enables the tight coupling of molecular and functional analyses necessary to dissect molecular mechanisms. The stalk-pore hypothesis proposes that bilayer merger proceeds rapidly via transient, high-negative curvature, intermediate membrane structures. Consistent with this, cholesterol, a major component of the CV membrane, contributes to a critical local negative curvature that supports formation of lipidic fusion intermediates. Following cholesterol depletion, structurally dissimilar lipids having intrinsic negative curvature greater than or equal to cholesterol recover the ability of CV to fuse but do not recover fusion efficiency (Ca(2+) sensitivity and kinetics). Conversely, cholesterol- and sphingomyelin-enriched microdomains regulate the efficiency of the fusion mechanism, presumably by contributing spatial and functional organization of other critical lipids and proteins at the fusion site. Critical proteins are thought to participate in Ca(2+) sensing, initiating membrane deformations, and facilitating fusion pore expansion. Capitalizing on a novel effect of the thiol-reactive reagent iodoacetamide (IA), potentiation of the Ca(2+) sensitivity and kinetics, a fluorescently tagged IA has been used to enhance fusion efficiency and simultaneously label the proteins involved. Isolation of cholesterol-enriched CV membrane fractions, using density gradient centrifugation, is being used to narrow the list of protein candidates potentially critical to the mechanism of fast Ca(2+)-triggered membrane fusion.
MeSH term(s)	Animals ; Calcium/metabolism ; Cholesterol/metabolism ; Lipid Metabolism ; Membrane Fusion ; Proteins/metabolism
Chemical Substances	Proteins ; Cholesterol (97C5T2UQ7J) ; Calcium (SY7Q814VUP)
Language	English
Publishing date	2009-01
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	211003-9
ISSN	1749-6632 ; 0077-8923
ISSN (online)	1749-6632
ISSN	0077-8923
DOI	10.1111/j.1749-6632.2008.03993.x
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Article ; Online: Cholesterol-independent effects of methyl-β-cyclodextrin on chemical synapses.

Kiel G Ormerod / Tatiana P Rogasevskaia / Jens R Coorssen / A Joffre Mercier

PLoS ONE, Vol 7, Iss 5, p e

2012 Volume 36395

Abstract	The cholesterol chelating agent, methyl-β-cyclodextrin (MβCD), alters synaptic function in many systems. At crayfish neuromuscular junctions, MβCD is reported to reduce excitatory junctional potentials (EJPs) by impairing impulse propagation to synaptic terminals, and to have no postsynaptic effects. We examined the degree to which physiological effects of MβCD correlate with its ability to reduce cholesterol, and used thermal acclimatization as an alternative method to modify cholesterol levels. MβCD impaired impulse propagation and decreased EJP amplitude by 40% (P<0.05) in preparations from crayfish acclimatized to 14 °C but not from those acclimatized to 21 °C. The reduction in EJP amplitude in the cold-acclimatized group was associated with a 49% reduction in quantal content (P<0.05). MβCD had no effect on input resistance in muscle fibers but decreased sensitivity to the neurotransmitter L-glutamate in both warm- and cold-acclimatized groups. This effect was less pronounced and reversible in the warm-acclimatized group (90% reduction in cold, P<0.05; 50% reduction in warm, P<0.05). MβCD reduced cholesterol in isolated nerve and muscle from cold- and warm-acclimatized groups by comparable amounts (nerve: 29% cold, 25% warm; muscle: 20% cold, 18% warm; P<0.05). This effect was reversed by cholesterol loading, but only in the warm-acclimatized group. Thus, effects of MβCD on glutamate-sensitivity correlated with its ability to reduce cholesterol, but effects on impulse propagation and resulting EJP amplitude did not. Our results indicate that MβCD can affect both presynaptic and postsynaptic properties, and that some effects of MβCD are unrelated to cholesterol chelation.
Keywords	Medicine ; R ; Science ; Q
Subject code	150
Language	English
Publishing date	2012-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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