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DISC-1 Leu607Phe alleles differentially affect centrosomal PCM1
localization and neurotransmitter release
Eastwood et al.
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.
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
. 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 =
*> noradrenaline neurotrans&itter release assays-
cells were sub+cultured into si?+well culture plates at a density o% 3. ? !0
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
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
'&&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/;$- !2 sodiu& dodecyl sulphate# one co&plete
&ini protease inhibitor coc)tail tablet (Roche /iagnostics 4td- Eurgess *ill- UK#
was added. Cells were harCested using 300 @l per well o% 0.32 ;rypsin+E/;$
(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
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+!- =
noradrenaline neurotrans&itter release was e?a&ined using an established
;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
- !.3&M K*
- !&M MgCl
0.3&M ascorbic acid- 0.3&M pargyline and
:&M /+glucose#. Cells were then loaded with =
*> 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
. $%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
>#. 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
>#- adherent cells
were lysed and detached %ro& the culture plate by the addition o% d*
collected (re&aining noradrenaline#. ;he Kuantities o% released and re&aining
*> noradrenaline was deter&ined by liKuid scintillation counting- and e?pressed
as a percentage o% total =
*> 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#.
better characterise which &echanis& &ay underlie =
*> noradrenaline release at
and !00&M =K
> under our e?peri&ental conditions- pilot studies were
conducted. !0@& desipra&ine has been shown to inhibit the noradrenaline
transporter in both directions-
and was applied during post incubation washes
and during release.
$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
Su##lementary /esults and Discussion
$lthough release at &M =K
> was una%%ected by !0@& desipra&ine- release
sti&ulated by !00 &M =K
> was reduced to &M =K
> leCels (Supple&entary
<igure !#- indicating that release at high =K
>in our assay conditions is due to
reCersal o% the noradrenaline transporter. ;he %inding that at &M =K
noradrenaline release was una%%ected by !0@& desipra&ine indicates that
reCersal o% the noradrenaline transporter is unli)ely to be the &echanis&
underlying this %or& o% release- and instead re%lects spontaneous Cesicle %usion.
Microtu)ules and syna#tic function
/ata %ro& seCeral sources suggest that &icrotubules play a role in synaptic
%or&ation- &aintenance and %unction. <or e?a&ple- &icrotubules haCe been
de&onstrated to be i&portant in dendritic spine deCelop&ent and &orphology-
whilst growth cone turning-
a?on path %inding-
and a?on branching
are all dependent on interactions between &icrotubules and &icro%ila&ents.
<urther&ore- &icrotubules %unction as rails along which &olecular &otors
including )inesin super%a&ily proteins and dyneins transport intracellular cargoes
such as &RG$s- protein co&ple?es and organelles-
including precursors o%
synaptic Cesicles to a?on ter&inals.
'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.
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Su##lementary 0i"ure 1' =
*> noradrenaline release at !00&M =K
attenuated by incubation with !0@M desipra&ine. (9alues are &ean M S/- nQ5#.
] 100mM [K
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