TIRF Microscopy to Visualize Actin and Microtubule Coupling Dynamics

Abstract

Source: Henty-Ridilla, J.L., Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy. J. Vis. Exp. (2022)

This video describes TIRF, a total internal reflection microscopy-based technique to visualize actin and microtubule polymerization dynamics. The method allows the high-resolution imaging of actin and microtubule coupling dynamics in real-time, which is essential for understanding cellular crosstalks.

Protocol

1. Washing the coverslips NOTE: Wash (24 mm x 60 mm, #1.5) coverslips according to Smith et al., 2013. Arrange coverslips in a plastic slide mailer container. Submerge coverslips sequentially in the following solutions and sonicate for 30-60 min, rinsing with ddH2O 10 times in between each solution: ddH2O with one drop of dish soap; 0.1 M KOH. Store coverslips in 100% ethanol for up to 6 months. <stro…

Representative Results

Figure 1. Experimental schematics: flow chamber assembly to image acquisition. (A) Imaging chamber assembly. Top to bottom: IBIDI imaging chambers are taped along perfusion wells (denoted by arrow); the second (white) layer of tape backing (left on in the image shown to better orient users) is removed and Epoxy is applied at the edge of the perfusion chamber (arrow). Note: To more easily orient users where to pl…

Materials

1% BSA (w/v)	Fisher Scientific	BP1600-100	For this purpose (blocking TIRF chambers), BSA is resuspended in ddH20 and filtered through a 0.22 µm filter.
1× BRB80	Homemade		80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8 with KOH
80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8 with KOH	Sigma Aldrich Inc, St. Louis, MO	G2133-50KU	Combined with catalase, aliquot and store at -80 oC until use
100 µM tubulin	Cytoskeleton Inc, Denver, CO	T240	Homemade tubulins should be recycled before use to remove polymerization-incompetent tubulin (Hyman et al. (1992)29; Li and Moore (2020)30 ). Commercially available tubulins are often too dilute to recycle but function well if resuspended according to the manufacturer’s instructions and pre-cleared via ultracentrifugation (278,000 × g) for 60 min, before use.
100 mM ATP	Gold Biotechnology Inc, Olivette, MO	A-08	Resuspended in ddH20 (pH 7.5) and filter sterilized.
100 mM GTP	Fisher Scientific	AC226250010	Resuspended in 1× BRB80 (pH 6.8) and filter sterilized.
120-150 mW solid-state lasers	Leica Microsystems	11889151; 11889148
2 mg/mL catalase	Sigma Aldrich Inc, St. Louis, MO	C40-100	Combined with glucose oxidase, aliquot and store at -80 oC until use
2× TIRF buffer	Homemad		2× BRB80, 100 mM KCl, 20 mM DTT, 80 mM glucose, 0.5% (v/v) methylcellulose (4,000 cp); Note: 1 µL of 0.1M GTP and 1 µL of 0.1M ATP added separately to TIRF reactions to avoid repeated freeze-thaw cycles.
24 × 60 mm, #1.5 coverglass	Fisher Scientific, Waltham, MA	22-266882	Coverglass must be extensively washed before use (Smith et al. (2014)22 )
37 oC heatblock
37 oC water bath
5 mg/mL Streptavidin (600x stock)	Avantor, Philadelphia, PA	RLS000-01	Resuspended in Tris-HCl (pH 8.8); dilute the aliquot to 1× in HEK buffer on day of use
5 min Epoxy resin and hardener	Loctite, Rocky Hill, CT	1365736	Combined resin and hardener may take up to 30 min to cure.
50% biotinylated-GpCpp microtubule seeds	Cytoskeleton Inc; Homemade	T333P	(optional) GppCpp or Taxol stabilized microtubule seeds can more efficiently mediate microtubule polymerization. Taxol and GppCpp stabilize microtubules in different ways that can affect the microtubule lattice structure and the ability of certain regulatory proteins to bind to the stabilized portion of the microtubule. A method to make diverse kinds of microtubule seeds is outlined in Hyman et al. (1992).
70 C incubator
Actin mix stock	Homemade; this protocol		A 12.5 µM actin mix comprised of labeled (fluorescent and biotinylated) and unlabeled actin for up to six reactions. 2 µL of stock is used in the final TIRF reaction. The final concentration of actin used in each reaction is 0.5 µM (10% Alexa-647; 0.09% biotin-labeled).
Appropriate buffer controls	Homemade		Combination of buffers from all proteins being assessed
Biotin-PEG-silane (MW 3,400)	Laysan Bio Inc	biotin-PEG-SIL-3400	Dispensed into 2-5 mg aliquots, backfilled with nitrogen, parafilmed closed, and stored at -20 C with desiccant until use
Biotinylated actin	Cytoskeleton Inc; Homemade	AB07	Biotin-actin is made by labeling on lysine residues and thus assumed to be at least 100% labeled, but varies with different lots/preparations. Optimal biotinylated actin concentrations must be empirically determined for particular uses/experimental designs. Higher concentrations permit more efficient tracking, but may impede polymerization or interactions with regulatory proteins. Here, a small percentage (0.09% or 900 pM) biotinylated actin is present in the final TIRF reaction.
Dish soap	Dawn, Procter and Gamble, Cincinnati, OH		For unknown reasons, the blue version cleans coverslips more efficiently than other available colors.
Dry ice
Fluorescently labeled actin	Cytoskeleton Inc; Homemade	AR05	Homemade fluorescently labeled-actin is stored in G-buffer supplemented with 50% glycerol at -20 C (Spudich et al. (1971) 47; Liu et al. (2022)48 ). Fluorescently-labeled actin is dialyzed against G-buffer and precleared via ultracentrifugation for 60 min at 278,000 × g before use.
Fluorescently labeled tubulin	Cytoskeleton Inc	TL488M, TLA590M, TL670M	Resuspended in 20 µL 1× BRB80 (10 µM final concentration) and pre-cleared via ultracentrifugation (278,000 × g) for 60 min, before use.
G-buffer	Homemade		3 mM Tris-HCl (pH 8.0), 0.2 mM CaCl2, 0.5 mM DTT, 0.2 mM ATP
HEK Buffer	Homemade		20 mM HEPES (pH 7.5), 1 mM EDTA (pH 8.0), 50 mM KCl
Ice
Ice bucket
Imaging chamber	IBIDI, Fitchburg, WI	80666	Order chambers with no bottom to utilize different coverslip coatings
iXon Life 897 EMCCD camera	Andor, Belfast, Northern Ireland	8114137
LASX Premium microscope software	Leica Microsystems	8114137
Methylcellulose (4,000 cp)	Sigma Aldrich Inc	M0512
Microscope base equipped with TIRF module	Leica Microsystems, Wetzlar, Germany	11889146
mPEG-silane (MW 2,000)