Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Summary

Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends.

Abstract

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion.

Introduction

Microtubules are highly conserved cytoskeletal structures that are important for cellular architecture, cell motility, cell division, and intracellular transport¹. These dynamic polymers assemble from tubulin in the presence of GTP, and they switch spontaneously between growth and shortening². Microtubules are very thin (only 25 nm in diameter) therefore special optical techniques to enhance contrast should be used to observe microtubules with a light microscope. Previous work with these polymers examined their dynamic behavior using differential interference contrast (DIC)³. This and similar studies in vitro revealed that und....

Protocol

Required equipment: The experiments described below require a light microscope equipped for DIC and fluorescence imaging (Table 1). Bright field LED illumination can be used to significantly improve the detection of the coverslip-attached microtubule seeds³⁷, which are difficult to observe with a regular Halogen lamp. To control liquid flow in microscopy chambers, the solutions should be exchanged with a peristaltic pump capable of flow speeds from 10-100 μl/min. A syringe pump can also be used but care should be taken to avoid air bubbles that may form when the flow speed is changed abruptly. For handling protein-coated be....

Results

Protein tracking with depolymerizing microtubule ends. Yeast kinetochore component Dam1 is by far the best tip-tracker of the depolymerizing microtubule ends¹⁴. This 10-subunit complex labeled with GFP can be readily expressed and purified from bacterial cells^18,38, so we recommend using it as a positive control for the tip-tracking assay. A fluorescent protein that tracks with the depolymerizing end of a microtubule is seen as a bright fluorescent spot steadily moving towards the c.......

Discussion

Many single molecule assays nowadays routinely use specially treated coverslips to drastically reduce nonspecific protein sticking. The procedure we describe here is a modification of the original protocol developed in Howard lab³², and we find that silanizing the coverslips is well worth the effort even with DIC-based bead assays, which do not use fluorescence. Chambers assembled with such coverslips show much cleaner surfaces, and the results obtained in the presence of soluble microtubule-binding protein ar.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Table 1. Microscopy and other equipment.
Microscope	Zeiss Nikon	Axio Imager 2 Eclipse Ti	other microscope models capable of DIC and epifluorescence-imaging can be used
Objective	Zeiss Nikon	420490-9900-000 CFI Apo 100x Oil 1.49	100X, DIC, 1.3-1.49 NA
Objective heater	Bioptechs	150803, 150819-19
Fluorescent filter cube	Chroma	49004 or 49008 41017 or 49020	optimized for Rhodamine fluorescence  optimized for GFP fluorescence
Acquisition software	freeware MicroManager Molecular Devices	not applicable MetaMorph 7.5	http://valelab.ucsf.edu/~MM/MMwiki/ other software can be used to acquire images and for a particle tracking
EMCCD camera	Andor	iXon3, DU-897E-cs0-#BV	Highly sensitive EMCCD camera
Trapping laser	IPG Photonics	YLR-10-1064-LP	1,064 nm laser, 10 W
Fluorescence excitation lasers	Coherent, Inc. Coherent, Inc.	Sapphire 488 LP Sapphire 552 LP	excitation of green fluorophores excitation of red fluorophores
Plasma Cleaner	Harrick Plasma	PDC-001
Commercial flow chambers	Warner Instruments	RC-20 or RC-30
Perfusion pump	Cole Palmer Harvard Apparatus	Masterflex 77120-52 Pico Plus	Both pumps provide the required rate of liquid flow but a peristaltic pump may pulse at very slow speed. The flow with a syringe pump is more consistent for a wide range of rates but this pump has inertia.
Table 2. Microscopy chamber preparation.
Modified microscope slides for reusable chambers	Precision Glassblowing of Colorado	Custom order www.precisionglassblowing.com	Sonic slots in slides using schematics in Figure 1
Polyethylene tubing	Intramedic	427410	I.D. 0.58 mm, O.D. 0.965 mm; use these tubes to connect assembled chamber to the pump and waste container
Polyethylene tubing	Intramedic	427400	I.D. 0.28 mm, O.D. 0.61 mm; use these tubes to make the reusable chamber
Regular microscope slides	VWR	48312-003	Other similar slides can be used
Coverslips	VWR	48393-150, 48366-067	Other similar coverslips can be used
Silicon sealant	World Precision Instruments	KIT, SILICON SEALANT 5 MIN CURE
Epoxy glue	Loctite	83082
Cyanoacrylate adhesive	Scotch 3M	AD114	Or cyanoacrylate adhesive from other manufacturers
Table 3. Coverslips cleaning and coating.
Molecular Sieves, Grade 564	Macron	4490-04
Coverglass Staining Jar	Ted Pella, Inc.	21036
Coverslip Ceramic Holder	Thomas Scientific	8542e40
PlusOne Repel Silane	GE Healthcare Biosciences	17-1332-01
Pluronic F-127	Sigma-Aldrich	P2443
Anti-digoxigenin AB	Roche Applied Science	11093274910
Table 4. Preparation of seeds and segmented microtubules.
Tubulin	purified from cow brains Cytoskeleton, Inc	T238P	For purification protocols see 49–51 Unlabeled porcine tubulin
Labeled tubulin	Cytoskeleton, Inc Invitrogen Invitrogen	TL590M C1171 (Rhodamine) A-2952 (Digoxigenin)	Rhodamine-labeled porcine tubulin Tubulin can be labeled with any amine-reactive dye as in reference52.
GMPCPP	Jena Biosciences	NU-405	Aliquot and store at -70 °C
VALAP	Vaseline, lanolin, and paraffin at 1:1:2 by mass		see reference9