SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition

Contact sites are discrete areas of organelle proximity that coordinate essential physiological processes across membranes, including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy. However, tools to easily image inter-organelle proximity over a range of distances in living cells and in vivo are lacking. Here we report a split-GFP-based contact site sensor (SPLICS) engineered to fluoresce when organelles are in proximity. Two SPLICS versions efficiently measured narrow (8\u201310 nm) and wide (40\u201350 nm) juxtapositions between endoplasmic reticulum and mitochondria, documenting the existence of at least two types of contact sites in human cells. Narrow and wide ER\u2013mitochondria contact sites responded differently to starvation, ER stress, mitochondrial shape modifications, and changes in the levels of modulators of ER\u2013mitochondria juxtaposition. SPLICS detected contact sites in soma and axons of D. rerio Rohon Beard (RB) sensory neurons in vivo, extending its use to analyses of organelle juxtaposition in the whole anim

Preparation of Diazoalkane Complexes of Ruthenium and Their Cyclization Reactions with Alkenes and Alkynes

The diazoalkane complexes [Ru(η5 -C5H5)- (N2CAr1Ar2)(PPh3)(L)]BPh4 (1−5: Ar1 = Ar2 = Ph (a), Ar1 = Ph and Ar2 = p-tolyl (b), Ar1Ar2 = C12H8 (c), Ar1 = Ph and Ar2 = PhCO (d); L = PPh3 (1), P(OMe)3 (2), P(OEt)3 (3), PPh(OEt)2 (4), But NC (5)) were prepared by allowing the chloro compounds RuCl(η5 -C5H5)(PPh3)(L) to react with the diazoalkanes Ar1Ar2CN2 in ethanol. Treatment of complexes 1−5 with ethylene (CH2CH2) under mild conditions (1 atm, room temperature) led not only to the η2 -ethylene complexes [Ru(η5 -C5H5)(η2 -CH2CH2)(PPh3)(L)]BPh4 (10−14) but also to dipolar (3 + 2) cycloaddition, affording the 4,5-dihydro-3H-pyrazole derivatives [Ru(η5 -C5H5){η1 -N NC(Ar1Ar2)CH2CH2}(PPh3)(L)]BPh4 (6−9). Acrylonitrile (CH2C(H)CN) reacted with diazoalkane complexes 2 and 3 to give the 1H-pyrazoline derivatives [Ru(η5 -C5H5){η1 -NC(CN)CH2C(Ar1Ar2)NH}(PPh3)(L)]BPh4 (19, 20). However, reactions with propylene (CH2C(H)CH3), maleic anhydride (ma, CHCHCO(O)CO) and dimethyl maleate (dmm, CH3OCOCHCHOCOCH3) led to the η2 -alkene complexes [Ru(η5 -C5H5)(η2 -R1CHCHR2)(PPh3)(L)]BPh4 (17−22). Treatment of the diazoalkane complexes 1 and 2 with acetylene CHCH under mild conditions (1 atm, room temperature) led to dipolar cycloaddition, affording the 3H-pyrazole complexes [Ru(η5 -C5H5){η1 -NNC(Ar1Ar2)CHCH}(PPh3) {P(OMe)3}]BPh4 (24), whereas reactions with the terminal alkynes PhCCH and But CCH gave the vinylidene derivatives [Ru(η5 -C5H5){CC(H)R}(PPh3){P(OMe)3}]BPh4 (25, 26). The alkyl propiolates HCCCOOR1 (R1 = Me, Et) also reacted with complexes 2 to give the 3H-pyrazole complexes [Ru(η5 -C5H5){η1 -NNC(Ar1Ar2)C(COOR1)CH}(PPh3)- {P(OMe)3}]BPh4 (27, 28). The complexes were characterized by spectroscopy and by X-ray crystal structure determinations of [Ru(η5 -C5H5){η1 -NC(CN)CH2C(Ph)(p-tolyl)NH}(PPh3){P(OMe)3}]BPh4 (19b), [Ru(η5 -C5H5){η2 -CHCHCO(O)CO}- (PPh3){P(OMe)3}]BPh4 (21), and [Ru(η5 -C5H5){η1 -NNC(C12H8)CHCH}(PPh3){P(OMe)3}]BPh4 (24c)

Expression profile of the pore-forming subunits \u3b11A and \u3b11D in the fetal bovine hypothalamus: a mammal with a long gestation.

This study describes the expression of the voltage operated calcium channels (VOCCs) subunits alpha 1A (typical of the P/Q family) and alpha 1D (of the L family) in the bovine hypothalamus. The expression of both P/Q and L families has been characterized in the brain of adult mammals. However, their distribution and expression during foetal neuronal differentiation have not yet been determined. The expression profile of the alpha 1A and alpha 1D pore-forming subunits was investigated during four embryonic stages in bovine foetuses. Our data suggest that the expressions of alpha 1A and alpha 1D are correlated during development, with an increase only in males that peaks on the last period of gestation. Bovine male hypothalami showed significantly higher alpha 1A and alpha 1D expression values in comparison to female ones during the whole developmental period. In the females, the expression profiles of both genes were constant during all the developmental time. Immunohistochemical studies confirmed the presence of the alpha 1A and alpha 1D protein subunits in foetal hypothalamic neurones starting from the third foetal stage. Our data provide new information on the hypothalamic expression of alpha 1A and alpha 1D subunits during development in a mammal with a long gestation period and a large and convoluted brain. (C) 2013 Published by Elsevier Ireland Ltd

Preparation of Diazoalkane Complexes of Ruthenium and Their Cyclization Reactions with Alkenes and Alkynes

The diazoalkane complexes [Ru­(η⁵-C₅H₅)­(N₂CAr1Ar2)­(PPh₃)­(L)]­BPh₄ (<b>1</b>–<b>5</b>: Ar1 = Ar2 = Ph (<b>a</b>), Ar1 = Ph and Ar2 = <i>p-</i>tolyl (<b>b</b>), Ar1Ar2 = C₁₂H₈ (<b>c</b>), Ar1 = Ph and Ar2 = PhCO (<b>d</b>); L = PPh₃ (<b>1</b>), P­(OMe)₃ (<b>2</b>), P­(OEt)₃ (<b>3</b>), PPh­(OEt)₂ (<b>4</b>), Bu^<i>t</i>NC (<b>5</b>)) were prepared by allowing the chloro compounds RuCl­(η⁵-C₅H₅)­(PPh₃)­(L) to react with the diazoalkanes Ar1Ar2CN₂ in ethanol. Treatment of complexes <b>1</b>–<b>5</b> with ethylene (CH₂CH₂) under mild conditions (1 atm, room temperature) led not only to the η²-ethylene complexes [Ru­(η⁵-C₅H₅)­(η²-CH₂CH₂)­(PPh₃)­(L)]­BPh₄ (<b>10</b>–<b>14</b>) but also to dipolar (3 + 2) cycloaddition, affording the 4,5-dihydro-3<i>H</i>-pyrazole derivatives [Ru­(η⁵-C₅H₅)­{η¹-NNC­(Ar1Ar2)­CH₂CH₂}­(PPh₃)­(L)]­BPh₄ (<b>6</b>–<b>9</b>). Acrylonitrile (CH₂C­(H)­CN) reacted with diazoalkane complexes <b>2</b> and <b>3</b> to give the 1<i>H</i>-pyrazoline derivatives [Ru­(η⁵-C₅H₅)­{η¹-NC­(CN)­CH₂C­(Ar1Ar2)NH}­(PPh₃)­(L)]­BPh₄ (<b>19</b>, <b>20</b>). However, reactions with propylene (CH₂C­(H)­CH₃), maleic anhydride (ma, CHCHCO­(O)CO) and dimethyl maleate (dmm, CH₃OCOCHCHOCOCH₃) led to the η²-alkene complexes [Ru­(η⁵-C₅H₅)­(η²-R1CHCHR2)­(PPh₃)­(L)]­BPh₄ (<b>17</b>–<b>22</b>). Treatment of the diazoalkane complexes <b>1</b> and <b>2</b> with acetylene CHCH under mild conditions (1 atm, room temperature) led to dipolar cycloaddition, affording the 3<i>H</i>-pyrazole complexes [Ru­(η⁵-C₅H₅)­{η¹-NNC­(Ar1Ar2)­CHCH}­(PPh₃)­{P­(OMe)₃}]­BPh₄ (<b>24</b>), whereas reactions with the terminal alkynes PhCCH and Bu^<i>t</i>CCH gave the vinylidene derivatives [Ru­(η⁵-C₅H₅)­{CC­(H)­R}­(PPh₃)­{P­(OMe)₃}]­BPh₄ (<b>25</b>,<b> 26</b>). The alkyl propiolates HCCCOOR1 (R1 = Me, Et) also reacted with complexes <b>2</b> to give the 3<i>H</i>-pyrazole complexes [Ru­(η⁵-C₅H₅)­{η¹-NNC­(Ar1Ar2)­C­(COOR1)CH}­(PPh₃)­{P­(OMe)₃}]­BPh₄ (<b>27</b>,<b> 28</b>). The complexes were characterized by spectroscopy and by X-ray crystal structure determinations of [Ru­(η⁵-C₅H₅)­{η¹-NC­(CN)­CH₂C­(Ph)­(<i>p</i>-tolyl)NH}­(PPh₃)­{P­(OMe)₃}]­BPh₄ (<b>19b</b>), [Ru­(η⁵-C₅H₅)­{η²-CHCHCO­(O)CO}­(PPh₃)­{P­(OMe)₃}]­BPh₄ (<b>21</b>), and [Ru­(η⁵-C₅H₅)­{η¹-NNC­(C₁₂H₈)­CHCH}­(PPh₃)­{P­(OMe)₃}]­BPh₄ (<b>24c</b>)

Preparation of Diazoalkane Complexes of Ruthenium and Their Cyclization Reactions with Alkenes and Alkynes

The diazoalkane complexes [Ru­(η⁵-C₅H₅)­(N₂CAr1Ar2)­(PPh₃)­(L)]­BPh₄ (<b>1</b>–<b>5</b>: Ar1 = Ar2 = Ph (<b>a</b>), Ar1 = Ph and Ar2 = <i>p-</i>tolyl (<b>b</b>), Ar1Ar2 = C₁₂H₈ (<b>c</b>), Ar1 = Ph and Ar2 = PhCO (<b>d</b>); L = PPh₃ (<b>1</b>), P­(OMe)₃ (<b>2</b>), P­(OEt)₃ (<b>3</b>), PPh­(OEt)₂ (<b>4</b>), Bu^<i>t</i>NC (<b>5</b>)) were prepared by allowing the chloro compounds RuCl­(η⁵-C₅H₅)­(PPh₃)­(L) to react with the diazoalkanes Ar1Ar2CN₂ in ethanol. Treatment of complexes <b>1</b>–<b>5</b> with ethylene (CH₂CH₂) under mild conditions (1 atm, room temperature) led not only to the η²-ethylene complexes [Ru­(η⁵-C₅H₅)­(η²-CH₂CH₂)­(PPh₃)­(L)]­BPh₄ (<b>10</b>–<b>14</b>) but also to dipolar (3 + 2) cycloaddition, affording the 4,5-dihydro-3<i>H</i>-pyrazole derivatives [Ru­(η⁵-C₅H₅)­{η¹-NNC­(Ar1Ar2)­CH₂CH₂}­(PPh₃)­(L)]­BPh₄ (<b>6</b>–<b>9</b>). Acrylonitrile (CH₂C­(H)­CN) reacted with diazoalkane complexes <b>2</b> and <b>3</b> to give the 1<i>H</i>-pyrazoline derivatives [Ru­(η⁵-C₅H₅)­{η¹-NC­(CN)­CH₂C­(Ar1Ar2)NH}­(PPh₃)­(L)]­BPh₄ (<b>19</b>, <b>20</b>). However, reactions with propylene (CH₂C­(H)­CH₃), maleic anhydride (ma, CHCHCO­(O)CO) and dimethyl maleate (dmm, CH₃OCOCHCHOCOCH₃) led to the η²-alkene complexes [Ru­(η⁵-C₅H₅)­(η²-R1CHCHR2)­(PPh₃)­(L)]­BPh₄ (<b>17</b>–<b>22</b>). Treatment of the diazoalkane complexes <b>1</b> and <b>2</b> with acetylene CHCH under mild conditions (1 atm, room temperature) led to dipolar cycloaddition, affording the 3<i>H</i>-pyrazole complexes [Ru­(η⁵-C₅H₅)­{η¹-NNC­(Ar1Ar2)­CHCH}­(PPh₃)­{P­(OMe)₃}]­BPh₄ (<b>24</b>), whereas reactions with the terminal alkynes PhCCH and Bu^<i>t</i>CCH gave the vinylidene derivatives [Ru­(η⁵-C₅H₅)­{CC­(H)­R}­(PPh₃)­{P­(OMe)₃}]­BPh₄ (<b>25</b>,<b> 26</b>). The alkyl propiolates HCCCOOR1 (R1 = Me, Et) also reacted with complexes <b>2</b> to give the 3<i>H</i>-pyrazole complexes [Ru­(η⁵-C₅H₅)­{η¹-NNC­(Ar1Ar2)­C­(COOR1)CH}­(PPh₃)­{P­(OMe)₃}]­BPh₄ (<b>27</b>,<b> 28</b>). The complexes were characterized by spectroscopy and by X-ray crystal structure determinations of [Ru­(η⁵-C₅H₅)­{η¹-NC­(CN)­CH₂C­(Ph)­(<i>p</i>-tolyl)NH}­(PPh₃)­{P­(OMe)₃}]­BPh₄ (<b>19b</b>), [Ru­(η⁵-C₅H₅)­{η²-CHCHCO­(O)CO}­(PPh₃)­{P­(OMe)₃}]­BPh₄ (<b>21</b>), and [Ru­(η⁵-C₅H₅)­{η¹-NNC­(C₁₂H₈)­CHCH}­(PPh₃)­{P­(OMe)₃}]­BPh₄ (<b>24c</b>)