This document provides supplementary methods and results for a study examining how Leu607Phe alleles of the DISC1 gene differentially affect centrosomal PCM1 localization and neurotransmitter release. It describes the cell culture and transfection methods used to express tagged Leu607 or Phe607 DISC1 variants. It also details the protocols for studying co-localization with PCM1, western blotting, and a noradrenaline release assay to analyze the effects on neurotransmitter levels. Statistical analyses are outlined and pilot studies suggest high KCl-stimulated noradrenaline release in the assay is due to reversal of the noradrenaline transporter.
This document provides supplementary methods and results for a study examining how Leu607Phe alleles of the DISC1 gene differentially affect centrosomal PCM1 localization and neurotransmitter release. It describes the cell culture and transfection methods used to express tagged Leu607 or Phe607 DISC1 variants. It also details the protocols for studying co-localization with PCM1, western blotting, and a noradrenaline release assay to analyze the effects on neurotransmitter levels. Statistical analyses are outlined and pilot studies suggest high KCl-stimulated noradrenaline release in the assay is due to reversal of the noradrenaline transporter.
This document provides supplementary methods and results for a study examining how Leu607Phe alleles of the DISC1 gene differentially affect centrosomal PCM1 localization and neurotransmitter release. It describes the cell culture and transfection methods used to express tagged Leu607 or Phe607 DISC1 variants. It also details the protocols for studying co-localization with PCM1, western blotting, and a noradrenaline release assay to analyze the effects on neurotransmitter levels. Statistical analyses are outlined and pilot studies suggest high KCl-stimulated noradrenaline release in the assay is due to reversal of the noradrenaline transporter.
localization and neurotransmitter release Eastwood et al. Acno!led"ments Supported by the UK Medical Research Council (Research grant #G000!"0# and a Centre $ward %ro& the Stanley Medical Research 'nstitute. (e than) $)ira Sawa %or his contributions. 1 Su##lementary Methods and Materials Cell Culture and $ransfection !ith %&-ta""ed Leu607 or Phe607 DISC-1' S*+S,, cells (European Collection o% Cell Cultures- .orton /own- UK# were cultured in /ulbecco0s Modi%ied Eagle Mediu& (/MEM1 Sig&a- .oole- UK# supple&ented with !02 %oetal cal% seru& (Sig&a#- 3&M 4+gluta&ine (Sig&a# and !2 non+essential a&ino acids (Sig&a#. Cells were &aintained in a hu&idi%ied incubator at 567C and 2 C8 3 . Constructs containing either 9+ tagged 4eu:06 or .he:06 /'SC+! were transiently trans%ected into cells using ;urbo%ect (<er&entas- ,or)- UK# in accordance with &anu%acturer0s instructions. <or western blotting and = 5 *> noradrenaline neurotrans&itter release assays- cells were sub+cultured into si?+well culture plates at a density o% 3. ? !0
cells per well. 63 hours a%ter plating- 3 @g per well o% plas&id was &i?ed with trans%ection reagent and applied to 5 wells- with trans%ection reagent alone applied to the re&aining 5 wells as e?peri&ental controls. Cells were grown %or a %urther 3A hours be%ore utiliBation. Each e?peri&ental run consisted o% cells at a di%%erent passage nu&ber in which two culture plates per Cariant were run in parallel- one plate %or the neurotrans&itter assay- with cells %ro& the other being harCested %or western blotting studies. <or co+localiBation studies- undi%%erentiated S*+S,, cells were plated at a density o% ? !0
cells per cha&ber into a A well cha&ber slide (9(R 'nternational- 4utterworth- UK#. $%ter 3A hours- 0.@g per well o% plas&id was &i?ed with trans%ection reagent and constructs containing either Cariant applied to 3 wells- with trans%ection reagent alone applied to the re&aining 3 wells as e?peri&ental controls. 2 DISC-1 and PCM1 Co-localization Studies 3A hours a%ter trans%ection- S*+S,, cells were %i?ed with ðanol at +307C %or : &in- giCen 3 D !0 &in washes with phosphate bu%%ered saline (.ES# and bloc)ed %or ! hour at R; with !02 nor&al goat seru& (Sig&a# diluted in .ES containing 0.!2 ;riton D+!00 (.ES+;# and 32 boCine seru& albu&in (ES$1 Sig&a#. Cells were then incubated %or ! hour at R; with pri&ary antibodies diluted in .ES+; containing !2 nor&al goat seru& and 32 ES$ (rabbit anti+ pericentriolar &aterial+! (.CM+!# at !F300- Santa CruB- *eidelberg- Ger&any1 &ouse anti 9 at !F!000- Sig&a#. $%ter 5 D !0 &in washes in .ES- cells were incubated %or ! hour at R; with secondary antibodies (goat anti+rabbit $le?a <luor :"- goat anti &ouse $le?a <luor A""- both at !F30001 'nCitrogen# diluted in .ES+; containing !2 nor&al goat seru& and 32 ES$. Cells were giCen 5 D!0 &in washes in .ES- dipped into distilled water- and coCer slipped using 9ectashield (9ector 4aboratories 4td- .eterborough- UK#. Cell staining was e?a&ined using a Gi)on Eclipse 5:00 &icroscope and i&ages captured using a MC'/ Elite Cersion 6.0 i&age analysis syste& ('nter%ocus- *aCerhill- UK#. .CM! centroso&al i&&unoreactiCe area was &easured in a total o% :0 cells %or each Cariant and HI0 untrans%ected cells (in three separate e?peri&ents#. Geurotrans&itter and western blotting assays were per%or&ed in I replicate e?peri&ents. (estern )lottin" '&&ediately be%ore cell harCesting- to each !0 &l o% suspension bu%%er (0.!M GaCl- 0.0!M ;ris+*Cl- 0.00!M E/;$- !2 sodiu& dodecyl sulphate# one co&plete &ini protease inhibitor coc)tail tablet (Roche /iagnostics 4td- Eurgess *ill- UK# was added. Cells were harCested using 300 @l per well o% 0.32 ;rypsin+E/;$ 3 (Sig&a#. $%ter incubating at 567C %or 3 &in- to each well "00 @l o% .ES was added- and the cells trans%erred to ! &l eppendor% tubes. Cells were pelleted by centri%uging at !3-000g %or &in- and the cell pellet resuspended in !00 @l o% suspension bu%%er. Sa&ples were then boiled %or !0 &in- centri%uged at !3-000g %or !0 &in and the supernatant collected. ;he protein content o% each sa&ple was assessed using the Erad%ord assay (Sig&a#. .ilot studies established that loading 3 @g o% protein was in the linear range o% detection %or all proteins e?a&ined. '&&ediately be%ore loading sa&ples were &i?ed with D S/S gel+loading bu%%er (0.M ;ris+*Cl- !02 sodiu& dodecyl sulphate- 02 glycerol- 2 J+&ercaptoethanol- 0.!2 bro&ophenol blue# and boiled %or !0 &in. Sa&ples were %ractionated by electrophoresis through a !32 polyacryla&ide gel together with precision plus protein standards (Eio+Rad- *e&el *e&pstead- UK#. Separated proteins were trans%erred onto polyCinyl di%luoride (.9/<# &e&branes using an iElot dry blotting syste& (.rogra& 5- 6 &in1 'nCitrogen- .aisely- UK#. $ll washes and incubations were per%or&ed with .ES containing 0.!2 ;ween+30 (.ES+;w#. Gon+speci%ic binding sites were bloc)ed by incubating &e&branes %or ! hour at R; with 32 non+%at &il). <or Ceri%ication o% trans%ection with 9+ tagged .he:06+ and 4eu:06 /'SC+!- blots were then incubated with horse+ radish pero?idase (*R.# labelled anti+9 (!F000- 'nCitrogen# in 2 non+%at &il) %or 3 hours at R;- a%ter which they were giCen 5 D ! &in washed in .ES+;w- and deCeloped using an EC4+plus western blotting detection syste& (GE *ealthcare- $&ersha&- UK#. <or western blotting o% tyrosinated and detyrosinated alpha+ tubulin- blots were incubated %or ! hour at R; with pri&ary antibodies (&ouse 4 anti+tryosinated alpha tubulin at !F0-000-clone ;UE+!$3- Sig&a1 rabbit anti+ detryosinated alpha tubulin at !F!0-000- Millipore- (at%ord- UK# diluted in !2 ES$. $%ter 5 D ! &in washes- blots were incubated %or ! hour at R; with secondary antibodies diluted in 32 non+%at &il) (*R. labelled goat anti+&ouse and *R. labelled goat anti+rabbit- both at !F000- Eio+Rad#- and a%ter 5 %inal washes in .ES+;w- &e&branes were deCeloped as aboCe. Me&branes were placed against EC4 %il& ($&ersha& *yper%il& EC4- GE *eathcare# %or the %ollowing ti&es (anti+9F 5 &in1 anti+tryosinated alpha tubulinF ! sec1 anti+ detyrosinated alpha tubulinF 3 &in#. $ll KuantitatiCe assays were per%or&ed in duplicate in nine replicate e?peri&ents- and tyrosinated and de+tyrosinated alpha tubulin protein was &easured using an $lpha'&ager 5A00 (Gentic Research 'nstru&ents 4td- Eraintree- UK# i&age analysis syste&. * + ,- .oradrenaline /elease Assay 3A hours a%ter trans%ection with either 9+tagged 4eu:06 or .he:06 /'SC+!- = 5 *> noradrenaline neurotrans&itter release was e?a&ined using an established assay. S! ;he culture &edia was re&oCed and cells were giCen two brie% washes in *epes bu%%ered saline (*ESF 30&M *epes- !A0&M GaCl- &M KCl- 3.&M CaCl 3 - !.3&M K* 3 .8 A - !&M MgCl 3- 0.3&M ascorbic acid- 0.3&M pargyline and :&M /+glucose#. Cells were then loaded with = 5 *> noradrenaline (0. @CiLwell1 .er)in El&er- Eeacons%ield- UK# diluted in *ES %or ! hour in a hu&idi%ied incubator at 567C and 2 C8 3 . $%ter A D ! &in washes in *ES cells at roo& te&perature (R;#- cells were incubated with *ES %or &in at R; and the per%usate collected (&M =K M >#. Cells were then incubated %or &in at R; with *ES containing !00&M KCl
(with the concentration o% GaCl reduced to A0&M to &aintain os&olarity#. $%ter collecting the per%usate (!00&M =K M >#- adherent cells 5 were lysed and detached %ro& the culture plate by the addition o% d* 3 0 and collected (re&aining noradrenaline#. ;he Kuantities o% released and re&aining = 5 *> noradrenaline was deter&ined by liKuid scintillation counting- and e?pressed as a percentage o% total = 5 *> noradrenaline collected. Geurotrans&itter assays were per%or&ed in I replicate e?peri&ents. Geurotrans&itters can be released by both Cesicular e?ocytosis and by &e&brane carriers responsible %or their upta)e (carrier &ediated release#. S3 ;o better characterise which &echanis& &ay underlie = 5 *> noradrenaline release at and !00&M =K M > under our e?peri&ental conditions- pilot studies were conducted. !0@& desipra&ine has been shown to inhibit the noradrenaline transporter in both directions- S5 and was applied during post incubation washes and during release. Statistical Analyses $ll datasets &et criteria %or nor&ality using the Kolo&ogoroC+S&irnoC one+ sa&ple test- and para&etric tests were there%ore applied. .aired sa&ple t+tests were used in the statistical analyses o% noradrenaline release and the tyrosinated to detyrosinated alpha+tubulin western blot studies. <or each Cariant and e?peri&ental condition- Calues obtained a%ter trans%ection were co&pared with those %ro& their own e?peri&ental control (untrans%ected cells#. <or the .CM! i&&unoreactiCity studies di%%erences between the three groups (untrans%ected- phe:06 /'SC+!- 4eu:06 /'SC+!# was assessed by analysis o% Cariance with planned co&parisons between groups e?plored using least signi%icant di%%erence (4S/# test. 6 Su##lementary /esults and Discussion .eurotransmitter Assay $lthough release at &M =K M > was una%%ected by !0@& desipra&ine- release sti&ulated by !00 &M =K M > was reduced to &M =K M > leCels (Supple&entary <igure !#- indicating that release at high =K M >in our assay conditions is due to reCersal o% the noradrenaline transporter. ;he %inding that at &M =K M > noradrenaline release was una%%ected by !0@& desipra&ine indicates that reCersal o% the noradrenaline transporter is unli)ely to be the &echanis& underlying this %or& o% release- and instead re%lects spontaneous Cesicle %usion. SA
Microtu)ules and syna#tic function /ata %ro& seCeral sources suggest that &icrotubules play a role in synaptic %or&ation- &aintenance and %unction. <or e?a&ple- &icrotubules haCe been de&onstrated to be i&portant in dendritic spine deCelop&ent and &orphology- S+ : whilst growth cone turning- S6+" a?on path %inding- SI+!0 and a?on branching S!!+!3 are all dependent on interactions between &icrotubules and &icro%ila&ents. <urther&ore- &icrotubules %unction as rails along which &olecular &otors including )inesin super%a&ily proteins and dyneins transport intracellular cargoes such as &RG$s- protein co&ple?es and organelles- S!5 including precursors o% synaptic Cesicles to a?on ter&inals. S!A+!: 'n this respect- &icrotubules haCe been de&onstrated to be i&portant in the transport and %unction o% receptors- both during synaptogenesis and in adult synaptic plasiticity. S!6+30