Materials and Reagents

Proteins: PSD-95, IRSp53, Shank3, GKAP, and Homer3 [see Feng et al. (2022) for protein production details]
Rabbit skeletal muscle actin (Cytoskeleton, Inc., catalog number: AKL99), rhodamine actin from rabbit skeletal muscle (Cytoskeleton, Inc., catalog number: AR05)
Lipid components:
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (Avanti, catalog number: 850457P)
1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-4’,5’-bisphosphate) [18:1 PI (4,5) P2] (Avanti, catalog number: 850155P)
1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl] (DGS-NTA) (Avanti, catalog number: 790404P)
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (PEG5000PE) (Avanti, catalog number: 880230P)
Fluorophores:
Alexa Fluor^TM 647 NHS ester (Thermo Fisher, catalog number: A20006)
Cy3^® NHS ester (AAT Bioquest, catalog number: 271)
Alexa Fluor^TM 488 C₅ maleimide (Thermo Fisher, catalog number: A10254)
DiO perchlorate (AAT Bioquest, catalog number: 22066)
Chambered cover glass (Lab-tek^®, catalog number: 155409)
Amber glass vials (Thermo Fisher, catalog number: B7800-1-9A)
Hellmanex III (Helma Analytics^TM, catalog number: Z805939)
Gastight syringes [Agilent, catalog numbers: 5190-1471 (2 μL), 5190-1483 (10 μL), 5190-1493 (25 μL), and 5190-1507 (100 μL)]
Chloroform (Scharlab, product code: 10289473)
NaOH (Scharlab, catalog number: SO04251000)
Sodium cholate (Sigma, catalog number: 27029)
BSA (Goldbio, catalog number: A-421-500)
ATP (Sigma, product number: A6144)
MgCl₂(VWR Chemicals BDH^®, catalog number: BDH9244)
NaH₂PO₄ (Millipore, catalog number: 567545)
KH₂PO₄ (Sigma, catalog number: P0662)
NaCl (Santa Cruz Biotechnology^®, catalog number: SC-203274)
KCl (VWR Chemicals BDH^®, catalog number: 26764)
Tris-HCl (Goldbio, catalog number: T-400-5)
DL-Dithiothreitol (DTT) (Sigma, catalog number: D0632)
CaCl₂ (Sigma, catalog number: C4901)
HiTrap desalting column (Cytiva, catalog number: 89501-384)
PBS buffer (see Recipes)
Reaction buffer (see Recipes)
G-buffer (see Recipes)

Equipment

Water bath (Shel lab, model number: 1201-2E)
Oven incubator (Binder GmbH, art number: 9010-0002)
High-speed centrifuge (Eppendorf, catalog number: 540600097)
AKTApurifier (GE Healthcare, USA)
Zeiss LSM 800 microscope (Zeiss)
Nanodrop

Software

FIJI (ImageJ)

Procedure

Preparation of small unilamellar vesicles (SUV)
1. Warm up individual lipid stocks (dissolved in chloroform) to room temperature.
2. Use glass syringe (see Materials and Methods for details of syringes used here) to prepare lipid mixture with the following composition: 95.9% POPC, 2% DGS-NTA (Ni), 2% PI (4,5) P2, and 0.1% PEG5000PE.
  Mix the following components in a glass vial:
  POPC (13.16 mM stock) 7.1 μL
  DGS-NTA (Ni) (4.74 mM stock) 0.42 μL
  PIP2 (500 μM stock) 3.9 μL
  PEG5000PE (170 μM stock) 0.57 μL
  Note that the above lipid quantities are calculated to coat a single cover glass well (i.e., 75 μg total).
3. Dry the lipid mix under a stream of nitrogen gas with rotation. You will see multiple white layers adhered to the wall of the vial after the lipids are dried (Figure 1). Note that 1% DiO perchlorate dye is included to label the lipids for a better visualization in Figure 1.
  
  Figure 1. Image showing dried lipid layers adhered to the wall of a glass vial.
  1% DiO dye is included to label the lipids for better demonstration.
4. Continue to dry the lipids under vacuum for at least 1 h to ensure removal of residual chloroform.
5. Resuspend the lipid mixture with vigorous pipetting and vortexing in ≤300 μL (the resuspension volume/the column injection volume is determined by the column resolution) of PBS buffer supplemented with 1% v/v sodium cholate. Lipids should be completely transparent once fully dissolved in detergent-containing buffer.
6. Subject the dissolved lipid mixture to a HiTrap desalting column equilibrated with the detergent-free PBS buffer. Use an FPLC system and pool fractions with detected UV absorbance at 280 nm, followed by dilution to the desired volume with PBS buffer. Add 150 μL of lipid solution to coat each well glass to generate SLBs.

Preparation of supported lipid bilayers (SLBs)
1. Wash the chambered cover glass (well area of 0.7 cm²) with 5% Hellmanex III overnight at room temperature and thoroughly rinse with Milli-Q water the next morning.
2. Wash the cover glass with 5 M NaOH for 1 h at 50 °C and then rinse thoroughly with Milli-Q water. Repeat this wash step three times.
3. To each cleaned cover glass, add 500 μL of PBS buffer for equilibration, take out, and discard.
4. Add 150 μL of SUVs to each cover glass well and incubate for 1 h at 42 °C to generate SLBs.
5. Wash the SLBs with 750 μL of reaction buffer three times to achieve 216-fold dilution. It should be noted that SLBs should not be exposed to the atmosphere to prevent oxidation.
6. Block the SLBs with 1 mg/mL BSA (stock concentration of 100 mg/mL) in the reaction buffer at room temperature for 30 min.
7. Add 2 μM 10% Alexa 647- or Cy3-labeled, His8-tagged PSD-95 protein to the SLBs and leave for incubation with SLBs for 1 h at room temperature.
8. Wash away the unbound His-PSD-95 with the reaction buffer three times (750 μL per wash).
9. Image using Zeiss LSM 800 microscope (Figure 2Ai).

Imaging membrane localization of PIP2 binder
1. Add 1 μM 10% Alexa 488–labeled IRSp53 protein to the SLBs and leave for incubation with SLBs for 1 h at room temperature.
2. Wash the SLBs with the reaction buffer three times (750 μL per wash) to remove any unbound IRSp53 protein.
3. Image using Zeiss LSM 800 microscope (Figure 2Aii).
  
  Figure 2. Postsynaptic density (PSD) condensation and actin polymerization on supported lipid bilayers (SLBs). (A) Fluorescence images showing uniform coating of Cy3-labeled, His8-tagged PSD-95 (i) or Alexa 488–labeled IRSp53 (ii) on SLBs before the addition of other PSD scaffold proteins. (B) Representative fluorescence images showing actin filaments assembled from PSD condensates on the SLB. An actin-binding-deficient mutant of IRSp53 (K4E) shows significantly diminished actin bundling to PSD condensates on the membrane. A line plot of the rhodamine-labeled actin intensities along the dashed line is presented. Figures are adapted from Feng et al. (2022) with permission.

Actin reconstitution and labeled actin preparation
1. Reconstitute lyophilized muscle actin protein to 10 mg/mL with 100 μL of deionized water and then dilute to 0.4 mg/mL in G-buffer supplemented with 0.2 mM ATP and 0.5 mM DTT.
2. After reconstitution, incubate actin on ice for 1 h to depolymerize actin oligomers that form during storage. Further centrifuge the actin resuspension at 14,000 × g for 15 min at 4 °C.
3. Transfer the supernatant to a new microcentrifuge tube and determine the total protein concentration with a NanoDrop.
4. Reconstitute lyophilized rhodamine muscle actin protein to 10 mg/mL with 2 μL of deionized water and then dilute to 2 mg/mL with G-buffer supplemented with 0.2 mM ATP and 1 mM DTT.
5. Leave actin solution on ice for 1 h to depolymerize actin oligomers.
6. Centrifuge the rhodamine actin resuspension at 14,000 × g for 15 min at 4 °C.
7. Transfer the top 90% of the supernatant to a new tube and determine the total protein concentration and percentage of rhodamine-labeled protein with a NanoDrop.

Imaging microcluster formation and actin polymerization
1. Premix 250 nM of Shank3, GKAP, Homer3, and IRSp53, as well as 1 μM of 10% rhodamine-labeled actin in reaction buffer supplemented with 1 mM ATP and 2 mM MgCl₂. The total solution volume in each chambered glass well is 150 μL.
2. Add the pre-mixture to the Alexa 647–labeled His8-PSD-95-bound SLBs and incubate for 20 min to allow microcluster formation and actin polymerization on the membrane.
3. Image using Zeiss LSM 800 microscope. Actin polymerization occurs within 20 min after mixture and reaches a plateau after 1 h. All data should be collected within 8 h after lipid coating started.

Data analysis

Analyzing actin recruitment with FIJI (ImageJ)

Open the captured image using FIJI.
To analyze actin enrichment within membrane localized clusters, draw a diagonal line across the image and measure the fluorescence intensity by Plot Profile. Note that it is essential to use identical imaging settings for direct comparison between different experiments.

Recipes

PBS buffer
10 mM NaH₂PO₄/1.8 mM KH₂PO₄, pH 7.4
137 mM NaCl
27 mM KCl

Reaction buffer
50 mM Tris-HCl, pH 8.0
100 mM NaCl
2 mM DTT

G-buffer
5 mM Tris-HCl, pH 8.0
0.2 mM CaCl₂
0.2 mM ATP

Acknowledgments

This work was supported by grants from the Ministry of Science and Technology of China (2019YFA0508402), the National Science Foundation of China (82188101), Research Grants Council (RGC) of Hong Kong (AoE-M09-12, 16104518 and 16101419), a Human Frontier Science Program research grant (RGP0020/2019) to M. Zhang, and an RGC General Research Fund (GRF) grant (16102120) to Z. Feng. The protocol was adapted from Feng et al. (2022).

Competing interests

The authors declare no competing interests.

References

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