1
SUPPLEMENTARY MATERIAL
Acknowledgments
Supported by the UK Medical Research Council (Research grant #G0500180) and a Centre Award from the Stanley Medical Research Institute. We thank Akira
Sawa for his contributions.

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Supplementary Methods and Materials
Cell Culture and Transfection with V5-tagged Leu607 or Phe607 DISC-1.
SH-SY5Y cells (European Collection of Cell Cultures, Porton Down, UK) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Sigma, Poole, UK) supplemented with 10% foetal calf serum (Sigma), 2mM L-glutamine (Sigma) and 1% non-essential amino acids (Sigma). Cells were maintained in a humidified incubator at 37°C and 5% CO
2
. Constructs containing either V5tagged Leu607 or Phe607 DISC-1 were transiently transfected into cells using
Turbofect (Fermentas, York, UK) in accordance with manufacturer’s instructions.
For western blotting and [ 3 H] noradrenaline neurotransmitter release assays, cells were sub-cultured into six-well culture plates at a density of 2.5 x 10 5 cells per well. 72 hours after plating, 2 µg per well of plasmid was mixed with transfection reagent and applied to 3 wells, with transfection reagent alone applied to the remaining 3 wells as experimental controls. Cells were grown for a further 24 hours before utilization. Each experimental run consisted of cells at a different passage number in which two culture plates per variant were run in parallel, one plate for the neurotransmitter assay, with cells from the other being harvested for western blotting studies.
For co-localization studies, undifferentiated SH-SY5Y cells were plated at a density of 5 x 10 5 cells per chamber into a 4 well chamber slide (VWR
International, Lutterworth, UK). After 24 hours, 0.5µg per well of plasmid was mixed with transfection reagent and constructs containing either variant applied to 2 wells, with transfection reagent alone applied to the remaining 2 wells as experimental controls.

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DISC-1 and PCM1 Co-localization Studies
24 hours after transfection, SH-SY5Y cells were fixed with methanol at -20°C for
6 min, given 2 X 10 min washes with phosphate buffered saline (PBS) and blocked for 1 hour at RT with 10% normal goat serum (Sigma) diluted in PBS containing 0.1% Triton X-100 (PBS-T) and 2% bovine serum albumin (BSA;
Sigma). Cells were then incubated for 1 hour at RT with primary antibodies diluted in PBS-T containing 1% normal goat serum and 2% BSA (rabbit antipericentriolar material-1 (PCM-1) at 1:200, Santa Cruz, Heidelberg, Germany; mouse anti V5 at 1:1000, Sigma). After 3 X 10 min washes in PBS, cells were incubated for 1 hour at RT with secondary antibodies (goat anti-rabbit Alexa
Fluor 568, goat anti mouse Alexa Fluor 488, both at 1:2000; Invitrogen) diluted in PBS-T containing 1% normal goat serum and 2% BSA. Cells were given 3 X10 min washes in PBS, dipped into distilled water, and cover slipped using
Vectashield (Vector Laboratories Ltd, Peterborough, UK). Cell staining was examined using a Nikon Eclipse 3600 microscope and images captured using a
MCID Elite version 7.0 image analysis system (Interfocus, Haverhill, UK). PCM1 centrosomal immunoreactive area was measured in a total of 60 cells for each variant and ~90 untransfected cells (in three separate experiments).
Neurotransmitter and western blotting assays were performed in 9 replicate experiments.
Western blotting
Immediately before cell harvesting, to each 10 ml of suspension buffer (0.1M
NaCl, 0.01M Tris-HCl, 0.001M EDTA, 1% sodium dodecyl sulphate) one complete mini protease inhibitor cocktail tablet (Roche Diagnostics Ltd, Burgess Hill, UK) was added. Cells were harvested using 200 µl per well of 0.25% Trypsin-EDTA

4
(Sigma). After incubating at 37°C for 2 min, to each well 800 µl of PBS was added, and the cells transferred to 1 ml eppendorf tubes. Cells were pelleted by centrifuging at 12,000g for 5 min, and the cell pellet resuspended in 100 µl of suspension buffer. Samples were then boiled for 10 min, centrifuged at 12,000g for 10 min and the supernatant collected. The protein content of each sample was assessed using the Bradford assay (Sigma).
Pilot studies established that loading 2 µg of protein was in the linear range of detection for all proteins examined. Immediately before loading samples were mixed with 5X SDS gel-loading buffer (0.5M Tris-HCl, 10% sodium dodecyl sulphate, 50% glycerol, 5% β-mercaptoethanol, 0.1% bromophenol blue) and boiled for 10 min. Samples were fractionated by electrophoresis through a 12% polyacrylamide gel together with precision plus protein standards (Bio-Rad,
Hemel Hempstead, UK). Separated proteins were transferred onto polyvinyl difluoride (PVDF) membranes using an iBlot dry blotting system (Program 3, 7 min; Invitrogen, Paisely, UK).
All washes and incubations were performed with PBS containing 0.1% Tween-20
(PBS-Tw). Non-specific binding sites were blocked by incubating membranes for
1 hour at RT with 2% non-fat milk. For verification of transfection with V5tagged Phe607- and Leu607 DISC-1, blots were then incubated with horseradish peroxidase (HRP) labelled anti-V5 (1:5000, Invitrogen) in 5% non-fat milk for 2 hours at RT, after which they were given 3 X 15 min washed in PBS-
Tw, and developed using an ECL-plus western blotting detection system (GE
Healthcare, Amersham, UK). For western blotting of tyrosinated and detyrosinated alpha-tubulin, blots were incubated for 1 hour at RT with primary

5 antibodies (mouse anti-tryosinated alpha tubulin at 1:50,000,clone TUB-1A2,
Sigma; rabbit anti-detryosinated alpha tubulin at 1:10,000, Millipore, Watford,
UK) diluted in 1% BSA. After 3 X 15 min washes, blots were incubated for 1 hour at RT with secondary antibodies diluted in 2% non-fat milk (HRP labelled goat anti-mouse and HRP labelled goat anti-rabbit, both at 1:5000, Bio-Rad), and after 3 final washes in PBS-Tw, membranes were developed as above.
Membranes were placed against ECL film (Amersham Hyperfilm ECL, GE
Heathcare) for the following times (anti-V5: 3 min; anti-tryosinated alpha tubulin: 15 sec; anti-detyrosinated alpha tubulin: 2 min). All quantitative assays were performed in duplicate in nine replicate experiments, and tyrosinated and de-tyrosinated alpha tubulin protein was measured using an AlphaImager 3400
(Gentic Research Instruments Ltd, Braintree, UK) image analysis system.
[ 3 H] Noradrenaline Release Assay
24 hours after transfection with either V5-tagged Leu607 or Phe607 DISC-1,
[ 3 H] noradrenaline neurotransmitter release was examined using an established assay.
S1 The culture media was removed and cells were given two brief washes in Hepes buffered saline (HBS: 20mM Hepes, 140mM NaCl, 5mM KCl, 2.5mM
CaCl
2
, 1.2mM KH
2
PO
4
, 1mM MgCl
2,
0.2mM ascorbic acid, 0.2mM pargyline and
6mM D-glucose). Cells were then loaded with [ 3 H] noradrenaline (0.5 µCi/well;
Perkin Elmer, Beaconsfield, UK) diluted in HBS for 1 hour in a humidified incubator at 37°C and 5% CO
2
. After 4 X 15 min washes in HBS cells at room temperature (RT), cells were incubated with HBS for 5 min at RT and the perfusate collected (5mM [K + ]). Cells were then incubated for 5 min at RT with
HBS containing 100mM KCl (with the concentration of NaCl reduced to 40mM to maintain osmolarity). After collecting the perfusate (100mM [K + ]), adherent cells

6 were lysed and detached from the culture plate by the addition of dH
2
0 and collected (remaining noradrenaline). The quantities of released and remaining
[ 3 H] noradrenaline was determined by liquid scintillation counting, and expressed as a percentage of total [ 3 H] noradrenaline collected.
Neurotransmitter assays were performed in 9 replicate experiments.
Neurotransmitters can be released by both vesicular exocytosis and by membrane carriers responsible for their uptake (carrier mediated release).
S2 To better characterise which mechanism may underlie [ 3 H] noradrenaline release at
5 and 100mM [K + ] under our experimental conditions, pilot studies were conducted. 10µm desipramine has been shown to inhibit the noradrenaline transporter in both directions, S3 and was applied during post incubation washes and during release.
Statistical Analyses
All datasets met criteria for normality using the Kolomogorov-Smirnov onesample test, and parametric tests were therefore applied. Paired sample t-tests were used in the statistical analyses of noradrenaline release and the tyrosinated to detyrosinated alpha-tubulin western blot studies. For each variant and experimental condition, values obtained after transfection were compared with those from their own experimental control (untransfected cells). For the PCM1 immunoreactivity studies differences between the three groups (untransfected, phe607 DISC-1, Leu607 DISC-1) was assessed by analysis of variance with planned comparisons between groups explored using least significant difference
(LSD) test.

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Supplementary Results and Discussion
Neurotransmitter Assay
Although release at 5mM [K + ] was unaffected by 10µm desipramine, release stimulated by 100 mM [K + ] was reduced to 5mM [K + ] levels (Supplementary
Figure 1), indicating that release at high [K + ]in our assay conditions is due to reversal of the noradrenaline transporter. The finding that at 5mM [K + ] noradrenaline release was unaffected by 10µm desipramine indicates that reversal of the noradrenaline transporter is unlikely to be the mechanism underlying this form of release, and instead reflects spontaneous vesicle fusion.
S4
Microtubules and synaptic function
Data from several sources suggest that microtubules play a role in synaptic formation, maintenance and function. For example, microtubules have been demonstrated to be important in dendritic spine development and morphology, S5-6 whilst growth cone turning, S7-8 axon path finding, S9-10 and axon branching S11-12 are all dependent on interactions between microtubules and microfilaments. Furthermore, microtubules function as rails along which molecular motors including kinesin superfamily proteins and dyneins transport intracellular cargoes such as mRNAs, protein complexes and organelles, S13 including precursors of synaptic vesicles to axon terminals.
S14-16 In this respect, microtubules have been demonstrated to be important in the transport and function of receptors, both during synaptogenesis and in adult synaptic plasiticity.
S17-20

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Supplementary Figure 1. [ 3 H] noradrenaline release at 100mM [K + ] is attenuated by incubation with 10µM desipramine. (Values are mean + SD, n=3).
12.5
10.0
7.5
5.0
2.5
0.0
5mM [K
+
] 100mM [K
+
]