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Fluorescent Method Images Cellular Phosphatidic Acid Synthesis

By MedImaging International staff writers
Posted on 12 Oct 2016

Image: A fluorescence image of a HeLa cell with PA labeled with a green fluorescent dye (Photo courtesy of Timothy Bumpus / Cornell).

A new study describes how an innovative chemoenzymatic strategy can be used to image cellular synthesis sites of the potent lipid secondary messenger phosphatidic acid (PA).

Developed at Cornell University (Cornell; Ithaca, NY, USA), the new method is based on a two-step process, which involves first attaching an ethanol-like molecule to phospholipase D (PLD), an enzyme that facilitates that process by catalyzing phospholipid head-group exchange with alkynols to generate alkyne-labeled PA analogues within cells. A second azide–alkyne cycloaddition reaction that is catalyzed by copper is then used to attach a fluorescent tag to PA when it is produced.

The fluorophore tagging allowed the researchers to image PA at the moment and location where it is created using fluorescence microscopy and quantification by high performance liquid chromatography (HPLC). The researchers were thus able to reveal the intracellular sites of PLD-mediated PA synthesis. Future applications include dissecting PA-dependent signaling pathways, imaging PLD activity in disease, and remodeling intracellular membranes with new functionality. The study was published on September 16, 2016, in Angewandte Chemie.

“Our imaging approach enables us to directly visualize in cells the locations where the lipid is produced; we believe that where PA is produced has tremendous impact on subsequent downstream biology that occurs,” said senior author Jeremy Baskin, of the department of chemistry and chemical biology at the Cornell Weill Institute for Cell and Molecular Biology. “Also, from an engineering perspective, the click reaction with PLD may allow bioengineers to attach other components onto PA to remodel or re-engineer membranes inside of cells and change their properties and functions.”

Phosphatidic acid is the acid form of phosphatidate, a part of common phospholipids, which are major constituents of cell membranes. PA fulfills three major functions: it is the precursor for the biosynthesis of many other lipids; it’s physical properties influence membrane curvature; and it acts as a signaling lipid, recruiting cytosolic proteins to appropriate membranes.

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67 (27.894973754883): select jos_linkedin.link from jos_linkedin where cat=10

68 (36.001205444336): SELECT fid FROM jos_deeppockets WHERE id=294766394 AND published='1' union SELECT fid FROM jos_deeppockets_archive WHERE id=294766394 AND published='1' LIMIT 1

69 (30.994415283203): SELECT id,name,sponsor_text FROM jos_deeppockets dp left outer join jos_channel_sponsor cs on dp.id=cs.fid WHERE cat=10 AND TYPE='file_folder' AND published=1 AND id=215 ORDER BY `ordering`

70 (246.04797363281): SELECT fid FROM jos_deeppockets WHERE cat=10 AND id=294766394 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

71 (208.13941955566): SELECT fid FROM jos_deeppockets_archive WHERE cat=10 AND id=294766394 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

72 (36.954879760742): SELECT id,name,title FROM jos_deeppockets WHERE cat=10 AND TYPE='file_folder' AND published=1 AND id != 294743300 AND id != 294743301 AND id != 294743302 AND id != 294743303 AND id != 294743304 AND id != 294743305 AND id != 294743306 AND id != 294743307 AND id != 294743308 AND id != 294743309 AND id != 294743310 AND metakey != 'aacc' ORDER BY `ordering`

73 (48.160552978516): SELECT show_live FROM jos_com_show_setting LIMIT 1

74 (29.087066650391): SELECT show_live FROM jos_com_show_setting LIMIT 1

75 (26.941299438477): SELECT show_live FROM jos_com_show_setting LIMIT 1

76 (27.894973754883): SELECT show_live FROM jos_com_show_setting LIMIT 1

77 (26.941299438477): SELECT show_live FROM jos_com_show_setting LIMIT 1

78 (27.894973754883): SELECT show_live FROM jos_com_show_setting LIMIT 1

79 (28.133392333984): SELECT show_live FROM jos_com_show_setting LIMIT 1

80 (28.133392333984): SELECT show_live FROM jos_com_show_setting LIMIT 1

81 (27.894973754883): SELECT show_live FROM jos_com_show_setting LIMIT 1

82 (26.941299438477): SELECT show_live FROM jos_com_show_setting LIMIT 1

83 (26.941299438477): SELECT show_live FROM jos_com_show_setting LIMIT 1

84 (26.941299438477): SELECT show_live FROM jos_com_show_setting LIMIT 1

85 (37.908554077148): SELECT show_live FROM jos_com_show_setting LIMIT 1

86 (31.948089599609): SELECT show_live FROM jos_com_show_setting LIMIT 1

87 (41.007995605469): SELECT fid FROM jos_deeppockets WHERE cat=10 AND id=294766394 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

88 (29.087066650391): SELECT fid FROM jos_deeppockets_archive WHERE cat=10 AND id=294766394 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

89 (30.994415283203): SELECT id,name,title FROM jos_deeppockets WHERE cat=10 AND TYPE='file_folder' AND published=1 AND id != 294743300 AND id != 294743301 AND id != 294743302 AND id != 294743303 AND id != 294743304 AND id != 294743305 AND id != 294743306 AND id != 294743307 AND id != 294743308 AND id != 294743309 AND id != 294743310 AND metakey != 'aacc' ORDER BY `ordering`

90 (44.822692871094): SELECT show_live FROM jos_com_show_setting LIMIT 1

91 (30.040740966797): SELECT show_live FROM jos_com_show_setting LIMIT 1

92 (28.133392333984): SELECT show_live FROM jos_com_show_setting LIMIT 1

93 (28.133392333984): SELECT show_live FROM jos_com_show_setting LIMIT 1

94 (51.021575927734): SELECT show_live FROM jos_com_show_setting LIMIT 1

95 (29.087066650391): SELECT show_live FROM jos_com_show_setting LIMIT 1

96 (51.975250244141): SELECT show_live FROM jos_com_show_setting LIMIT 1

97 (30.040740966797): SELECT show_live FROM jos_com_show_setting LIMIT 1

98 (27.894973754883): SELECT show_live FROM jos_com_show_setting LIMIT 1

99 (27.894973754883): SELECT show_live FROM jos_com_show_setting LIMIT 1

100 (26.941299438477): SELECT show_live FROM jos_com_show_setting LIMIT 1

101 (27.894973754883): SELECT show_live FROM jos_com_show_setting LIMIT 1

102 (26.941299438477): SELECT show_live FROM jos_com_show_setting LIMIT 1

103 (26.941299438477): SELECT show_live FROM jos_com_show_setting LIMIT 1

104 (66.041946411133): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

105 (50.067901611328): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

106 (48.160552978516): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.cat=10 AND dp.id=294766394 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

107 (45.061111450195): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets_archive as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.cat=10 AND dp.id=294766394 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

108 (72.002410888672): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

109 (44.822692871094): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

110 (46.014785766602): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.id=294766394 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

111 (458.95576477051): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets_archive as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.id=294766394 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

112 (536.91864013672): SELECT dp.name, dp.id, dp2.name as channel, ex.name as author_name, ex.specialization, ex.images FROM jos_deeppockets as dp LEFT JOIN jos_experts as ex ON ex.id = dp.author LEFT JOIN jos_deeppockets as dp2 ON dp2.id = dp.fid WHERE dp.author != 0 AND dp.cat = 10 AND dp.published = 1 AND ( (dp.fid = 294743300) OR (dp.fid = 294743301) OR (dp.fid = 294743302) OR (dp.fid = 294743303) OR (dp.fid = 294743304) OR (dp.fid = 294743305) OR (dp.fid = 294743306) OR (dp.fid = 294743307) OR (dp.fid = 294743308) OR (dp.fid = 294743309) OR (dp.fid = 294743310) ) ORDER BY RAND() LIMIT 1

113 (355.00526428223): SELECT dp.name, dp.id, dp2.name as channel, ex.name as author_name, ex.specialization, ex.images FROM jos_deeppockets as dp LEFT JOIN jos_experts as ex ON ex.id = dp.author LEFT JOIN jos_deeppockets as dp2 ON dp2.id = dp.fid WHERE dp.author != 0 AND dp.cat = 10 AND dp.published = 1 AND ( (dp.fid = 294743300) OR (dp.fid = 294743301) OR (dp.fid = 294743302) OR (dp.fid = 294743303) OR (dp.fid = 294743304) OR (dp.fid = 294743305) OR (dp.fid = 294743306) OR (dp.fid = 294743307) OR (dp.fid = 294743308) OR (dp.fid = 294743309) OR (dp.fid = 294743310) ) ORDER BY RAND() LIMIT 1

114 (152.1110534668): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

115 (70.095062255859): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

116 (99.897384643555): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

117 (50.067901611328): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

118 (51.021575927734): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

119 (42.200088500977): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

120 (54.836273193359): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

121 (45.061111450195): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

122 (313.04359436035): SELECT * FROM trending_articles WHERE `journal_id` = 10 AND `timestamp` <= NOW() + INTERVAL 7 DAY ORDER BY `order`

123 (!!!!! 18860.101699829): SELECT * FROM jos_deeppockets WHERE jos_deeppockets.cat = 10 AND jos_deeppockets.type = 'deeppockets_item' AND jos_deeppockets.published = 1 AND jos_deeppockets.created + INTERVAL 14 DAY >= NOW() ORDER BY jos_deeppockets.hits DESC LIMIT 7

124 (534.0576171875): SELECT fid FROM jos_deeppockets WHERE cat=10 AND id=294766394 AND type='deeppockets_item' AND published='1' union SELECT fid FROM jos_deeppockets_archive WHERE cat=10 AND id=294766394 AND type='deeppockets_item' AND published='1' LIMIT 1

125 (!!!!! 7090.0917053223): ( SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.video != '' AND dp.video_source != '' AND dp.cat = 10 AND dp.published = 1 AND dp.type != 'deeppockets_item_link' ORDER BY created DESC LIMIT 3 ) UNION ( SELECT dpa.id, dpa.name, dpa.title, dpa.video, dpa.video_source, dpa.created FROM jos_deeppockets_archive AS dpa WHERE dpa.video != '' AND dpa.video_source != '' AND dpa.cat = 10 AND dpa.published = 1 AND dpa.type != 'deeppockets_item_link' ORDER BY created DESC LIMIT 3 ) ORDER BY created DESC LIMIT 3

126 (82.015991210938): ( SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.video != '' AND dp.video_source != '' AND dp.cat = 10 AND dp.published = 1 AND dp.type != 'deeppockets_item_link' ORDER BY created DESC LIMIT 3 ) UNION ( SELECT dpa.id, dpa.name, dpa.title, dpa.video, dpa.video_source, dpa.created FROM jos_deeppockets_archive AS dpa WHERE dpa.video != '' AND dpa.video_source != '' AND dpa.cat = 10 AND dpa.published = 1 AND dpa.type != 'deeppockets_item_link' ORDER BY created DESC LIMIT 3 ) ORDER BY created DESC LIMIT 3

127 (47.206878662109): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.id = 294803337 LIMIT 1

128 (28.848648071289): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.id = 294803337 LIMIT 1

129 (46.014785766602): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.id = 294803276 LIMIT 1

130 (29.087066650391): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.id = 294803276 LIMIT 1

131 (368.11828613281): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets_archive AS dp WHERE dp.id = 294803276 LIMIT 1

132 (92.029571533203): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets_archive AS dp WHERE dp.id = 294803276 LIMIT 1

133 (34.093856811523): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.id = 294802958 LIMIT 1

134 (26.941299438477): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.id = 294802958 LIMIT 1

135 (593.18542480469): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

136 (377.89344787598): SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

137 (252.00843811035): SELECT name FROM jos_deeppockets WHERE id=215

138 (39.815902709961): SELECT name FROM jos_deeppockets WHERE id=215

139 (544.07119750977): SELECT *, DATE_FORMAT(publish_up,'%d %b %Y') AS date_publish_up, DATE_FORMAT(publish_down,'%d %b %Y') AS date_publish_down FROM jos_events WHERE state=1 AND publish_up > NOW() and extra_info REGEXP '((;)|(^))215((;)|($))' AND catid IN (0,110) ORDER BY publish_up ASC LIMIT 3

140 (557.18421936035): SELECT *, DATE_FORMAT(publish_up,'%d %b %Y') AS date_publish_up, DATE_FORMAT(publish_down,'%d %b %Y') AS date_publish_down FROM jos_events WHERE state=1 AND publish_up > NOW() and extra_info REGEXP '((;)|(^))215((;)|($))' AND catid IN (0,110) ORDER BY publish_up ASC LIMIT 3

141 (76.055526733398): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

142 (51.021575927734): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

143 (!!!!! 8008.0032348633): TRUNCATE TABLE jos_trademed_module

144 (93.936920166016): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.cat=10 AND dp.id=294766394 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

145 (51.021575927734): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets_archive as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.cat=10 AND dp.id=294766394 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

146 (348.09112548828): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

147 (60.796737670898): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

148 (66.995620727539): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

149 (46.014785766602): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

150 (!!!!! 1103.1627655029): SELECT jos_item.* FROM jos_item INNER JOIN jos_number ON jos_item.numberID=jos_number.id WHERE 1=1 AND (jos_item.imgMini<>'') AND jos_item.type='L' AND jos_item.numberID= ( SELECT jos_number.id FROM jos_number WHERE jos_number.journalID IN (10) and jos_number.id in (select distinct jos_number.id from jos_number join jos_item on jos_item.numberid=jos_number.id where jos_item.type='P') ORDER BY (CONVERT(SUBSTRING(issue,1,2),SIGNED) + (CONVERT(SUBSTRING(issue,3,2),SIGNED)*100)) DESC LIMIT 0,1 ) ORDER BY RAND() DESC LIMIT 6

151 (64.13459777832): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

152 (46.968460083008): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

153 (36.001205444336): SELECT fid FROM jos_deeppockets WHERE cat=10 AND id=294766394 AND type='deeppockets_item' AND published='1' union SELECT fid FROM jos_deeppockets_archive WHERE cat=10 AND id=294766394 AND type='deeppockets_item' AND published='1' LIMIT 1

154 (34.093856811523): select distinct links.* from jos_textlinks links join jos_textlinks_placement placement on links.id=placement.link_id where journal_id=(10) and (channel_id=215 or channel_id is null)

155 (34.093856811523): select distinct links.* from jos_textlinks links join jos_textlinks_placement placement on links.id=placement.link_id where journal_id=(10) and (channel_id=215 or channel_id is null)

156 (48.87580871582): SELECT livesite FROM jos_dp_categories WHERE id=10 AND published='1' LIMIT 1

157 (83.92333984375): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

158 (58.889389038086): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294766394 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294766394 AND published='1' LIMIT 1

159 (37.908554077148): select jos_twitters.link from jos_twitters where cat=10

160 (30.994415283203): select jos_facebook.link from jos_facebook where cat=10

161 (31.948089599609): select jos_google_plus.link from jos_google_plus where cat=10

162 (29.802322387695): select jos_linkedin.link from jos_linkedin where cat=10

163 (282.04917907715): update jos_bannersmanager_banner set impmade=impmade+1 where id in(14202,14203)

164 (927.92510986328): insert into jos_banners_delta values (14202,'2025-06-22 11:58:48','216.73.216.120','view'),(14203,'2025-06-22 11:58:48','216.73.216.120','view')

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