Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Summary

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Abstract

Microtubules are polymers of αβ-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Introduction

Microtubules are biopolymers that form structural scaffolds essential for multiple cellular processes, ranging from intracellular transport and organelle positioning to cell division and elongation. To execute these diverse functions, individual microtubules are organized into micron-sized arrays, such as mitotic spindles, ciliary axonemes, neuronal bundles, interphase arrays, and plant cortical arrays. A ubiquitous architectural motif found in these structures is a bundle of microtubules crosslinked along their lengths¹. An intriguing feature of several microtubule-based structures is the coexistence of bundled microtubules and non-crosslinked....

Protocol

1. Prepare reagents

1. Prepare buffers and reagents as outlined in Table 1 and Table 2. During the experiment, keep all solutions on ice, unless otherwise noted.

Solution	Components		Recommended Storage Duration	Notes
5X BRB80	400 mM K-PIPES, 5 mM MgCl2, 5 mM EGTA, pH 6.8 with KOH, filter sterilize

Results

The experiment described above was performed using 647 nm fluorophore-labeled biotinylated microtubules, 560 nm fluorophore-labeled non-biotinylated microtubules, and 560 nm fluorophore-labeled soluble tubulin mix. Microtubules were crosslinked by the crosslinker protein PRC1 (GFP-labeled). After surface-immobilized bundles and single microtubules were generated (step 5.11), the imaging chamber was mounted on a TIRF 100X 1.49 NA oil objective and viewed in the 560 nm and 647 nm fluorescence channels. Single microtubules .......

Discussion

The experiment described here significantly expands the scope and complexity of conventional microtubule reconstitution assays, which are traditionally performed on single microtubules or on one type of array. The current assay provides a method to simultaneously quantify and compare the regulatory MAP activity on two populations, namely, single microtubules and crosslinked bundles. Further, this assay allows for the examination of two types of bundles: those that are pre-formed from stable seeds before the initiation of.......

Materials

Name	Company	Catalog Number	Comments
(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox)	Sigma Aldrich	238813
1,4-piperazinediethanesulfonic acid (PIPES)	Sigma Aldrich	P6757
18x18 mm #1.5 coverslips	Electron Microscopy Sciences	63787
2-Mercaptoethanol (BME)	Sigma Aldrich	M-6250
24x60 mm #1.5 coverslips	Electron Microscopy Sciences	63793
405/488/560/647 nm Laser Quad Band	Chroma	TRF89901-NK
Acetone	Sigma Aldrich	320110
Adenosine 5'-triphosphate disodium salt hydrate (ATP)	Sigma Aldrich	A7699-5G
Avidin, NeutrAvidin® Biotin-binding Protein (Molecular Probes®)	Thermo Fischer Scientific	A2666
Bath sonicator: Branson 2800 Cleaner	Branson	CPX2800H
Beckman Coulter Polycarbonate Thickwall Tubes, 11 x 34 mm	Beckman-Coulter	343778
Beckman Coulter Polycarbonate Thickwall Tubes, 8 x 34 mm	Beckman-Coulter	343776
Biotin-PEG-SVA, MW 5,000	Laysan Bio	#Biotin-PEG-SVA-5000
Bovine Serum Albumin (BSA)	Sigma Aldrich	2905
Catalase	Sigma Aldrich	C40
Corning LSE Mini Microcentrifuge, AC100-240V	Corning	6670
Delicate Task Wipes	Kimtech	34120
Dithiothreitol (DTT)	GoldBio	DTT10
Emission filter	Chroma	ET610/75m
Ethanol (200-proof)	Decon Labs	2705
Ethylene glycol tetraacetic acid (EGTA)	Sigma Aldrich	3777
Glucose Oxidase	Sigma Aldrich	G2133
GMPCPP	Jena Bioscience	NU-405
Guanosine 5'-triphosphate sodium salt hydrate (GTP)	Sigma Aldrich	G8877
Hellmanex III detergent	Sigma Aldrich	Z805939
Immersion oil, Type A	Fisher Scientific	77010
Kappa-casein	Sigma Aldrich	C0406
Lanolin	Fisher Scientific	S25376
Lens Cleaning Tissue	ThorLabs	MC-5
Magnesium Chloride (MgCl2)	Sigma Aldrich	M9272
Methylcellulose	Sigma Aldrich	M0512
Microfuge 16 Benchtop Centrifuge	Beckman-Coulter	A46474
Microscope Slides, Diamond White Glass, 25 x 75mm, 90° Ground Edges, WHITE Frosted	Globe Scientific	1380-50W
mPEG-Succinimidyl Valerate, MW 5,000	Laysan Bio	#NH2-PEG-VA-5K
Optima™ Max-XP Tabletop Ultracentrifuge	Beckman-Coulter	393315
Paraffin	Fisher Scientific	P31-500
PELCO Reverse (self-closing), Fine Tweezers	Ted Pella	5377-NM
Petrolatum, White	Fisher Scientific	18-605-050
Plasma Cleaner, 115V	Harrick Plasma	PDC-001
Potassium Hydroxide (KOH)	Sigma Aldrich	221473
Sodium bicarbonate	Sigma Aldrich	S6014
Sucrose	Sigma Aldrich	S7903
Thermal-Lok 1-Position Dry Heat Bath	USA Scientific	2510-1101
Thermal-Lok Block for 1.5 and 2.0 mL Tubes	USA Scientific	2520-0000
Thermo Scientific™ Pierce™ Bond-Breaker™ TCEP Solution, Neutral pH; 500mM	Thermo Fischer Scientific	PI-77720
TIRF 100X NA 1.49 Oil Objective	Nikon	CFI Apochromat TIRF 100XC Oil
TIRF microscope	Nikon	Eclipse Ti
TLA 120.1 rotor	Beckman-Coulter	362224
TLA 120.2 rotor	Beckman-Coulter	357656
Tubulin protein (>99% pure): porcine brain	Cytoskeleton	T240
Tubulin Protein (Biotin): Porcine Brain	Cytoskeleton	T333P
Tubulin protein (fluorescent HiLyte 647): porcine brain	Cytoskeleton	TL670M
Tubulin protein (X-rhodamine): bovine brain	Cytoskeleton	TL620M
VECTABOND® Reagent, Tissue Section Adhesion	Vector Biolabs	SP-1800-7
VWR® Personal-Sized Incubator, 120V, 50/60Hz, 0.6A	VWR	97025-630