Preparation of Polarity-Marked Microtubules Using a Plus-End Capping DARPin

Materials and reagents

Reagents

1. Porcine brain tubulin, prepared as previously reported [35,36]

2. Purified tandem DARPin D1 protein [(D1)₂], expressed in E. coli and purified according to standard DARPin expression and purification protocols [37,38]. A detailed description of the (D1)₂ expression on a 500 mL scale and purification via metal affinity chromatography and size exclusion chromatography can be found here [14,32]. The bacterial (D1)₂ expression plasmid can be requested from A. Pluckthun (plueckthun@bioc.uzh.ch)

3. Fluorescently labeled porcine brain tubulin (i.e., AlexaFluor647 or Atto647-labeled) prepared as previously reported [21,35]

   Note: Fluorescence labeling ratio should be determined by Nanodrop prior to starting the protocol.

4. Optional for neutravidin-biotin-based immobilization in, e.g., TIRF microscopy assays: biotinylated porcine brain tubulin, prepared as previously reported [21,35]

5. Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) (Sigma-Aldrich, catalog number: P6757)

6. Magnesium chloride (MgCl₂), 1 M (Sigma-Aldrich, catalog number: 63069)

7. Ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889)

8. Guanosine-5'-[(α,β)-methyleno]triphosphate (GMPCPP), 10 mM (Jena bioscience, catalog number: NU-405)

9. High-quality purified water, e.g., Millipore Milli-Q

   
   

For gliding assay:

1. An active, directional microtubule motor protein; we had the minus-end-directed HSET motor protein available in the laboratory, prepared as previously published [39]. Many other microtubule motors with established polarity, such as the plus-end directed kinesin-1 or its motor-domain truncations, can be used instead

2. Poly(L-lysine)-polyethylene glycol (PLL-PEG) [SuSoS, catalog number: PLL(20)-g[3.5]- PEG(2)]

3. β-Mercaptoethanol (β-ME), 14.3 M (Sigma-Aldrich, catalog number: M6250)

4. β-casein (Sigma-Aldrich, catalog number: C6905)

5. ATP (Sigma-Aldrich, catalog number: A6419)

   
   

1. EGTA, 0.5 M, pH 8 (see Recipes)

2. BRB80, 5× stock (see Recipes)

3. BRB80, 1× (see Recipes)

   
   

For gliding assay:

1. PLL-PEG, 2 mg/mL (see Recipes)

2. β-casein, 25 mg/mL (see Recipes)

3. ATP, 100 mM, pH 7 (see Recipes)

4. Gliding buffer (see Recipes)

   
   

Recipes

Note: All solutions should be prepared using high-quality purified water, e.g., Millipore Milli-Q.

1. EGTA, 0.5 M, pH 8

   Add solid KOH to dissolve EGTA powder and adjust pH only by adding KOH, being careful not to overshoot; store at room temperature.

   
   

2. BRB80, 5× (50 mL)

   Reagent	Final concentration	Quantity or Volume
   PIPES	400 mM	6.06 g
   EGTA (0.5 M, pH 8.0)	5 mM	0.5 mL
   MgCl2 (1 M)	5 mM	0.25 mL
   Total		50 mL, pH 6.8

   Filter (0.22 μm pore size) and store for up to 4 weeks at 4 °C.

   Note: PIPES dissolves upon the addition of solid KOH. MgCl₂ and EGTA should be added only after PIPES is dissolved (requiring ~1.35 g of KOH for these amounts). The final pH should be adjusted only by addition of KOH, being careful not to overshoot, to control the total ionic strength of the solution.

   
   

3. BRB80, 1×

   Diluted from the above recipe on the day of use; keep on ice.

   
   

4. PLL-PEG, 2 mg/mL, in water

   Aliquot and store at -20 °C.

   
   

5. β-casein, 25 mg/mL, in BRB80

   Ultracentrifuge, aliquot, snap-freeze in liquid nitrogen, and store at -80 °C.

   
   

6. ATP, 100 mM, pH 7

   Filter, aliquot, and store at -80 °C.

   
   

7. Gliding buffer

   BRB80 with 5 mM β-ME, 1 mM ATP, 1 mg/mL β-casein; prepare on the day of the experiments and keep at room temperature.

   
   

Laboratory supplies

1. 1.5 mL centrifuge tubes (e.g., Eppendorf, catalog number: 0030120086)

2. Ice bucket

3. Pipettes

4. Pipette tips

   
   

For imaging/ gliding assay:

1. Cover glasses, thickness #1.5, 22 × 22 mm (VWR, catalog number: 630-1843)

2. Standard microscopy slides, 76 × 26 mm (e.g., VWR, catalog number: 631-1550P)

3. Double-sided adhesive fleece transparent tape, 10 m × 15 mm (Tesa^®, catalog number: 053380000001)

4. Filter paper 3 mm CHR, 46 cm × 57 cm (Whatman^®, catalog number: 3030-917)

5. Korasilon^TM Paste, medium viscosity (Sigma-Aldrich, catalog number: 769681-1EA)

6. Diamond scribing pen (e.g., Ted Pella, catalog number: 54468)

7. Forceps (e.g., Sigma-Aldrich, catalog number: Z680214-1EA)

Equipment

1. Microcentrifuge (e.g., Eppendorf, model: 5418 R)

2. Heat block set to 37 °C (e.g., Eppendorf, model: ThermoStat C)

   
   

For imaging/ gliding assay:

1. Fluorescence microscope (e.g., Nikon Instruments, model: E600)

Software and datasets

1. ImageJ/FIJI (e.g., ImageJ version 1.54, https://imagej.net/ij/index.html)

Procedure