The authors would like to thank F. I. Ataullakhanov for helping to design and manufacture reusable flow chambers, N. Dashkevich, N. Gudimchuk and A. Korbalev for providing images for figures, N. Gudimchuk and P. Zakharov for developing a protocol and providing reagents to prepare digoxigenin-labeled microtubule seeds, A. Potapenko for help with text editing and other members of Grishchuk lab for tips and discussions. This work was supported in part by NIH grant GM R01-098389 and a pilot grant from Pennsylvania Muscle Institute to E.L.G., who is a Kimmel Scholar, by RFBR grants 12-04-00111-a, 13-04-40190-H and 13-04-40188-H, Russian Academy of Sciences Presidium Grants (Mechanisms of the Molecular Systems Integration and Molecular and Molecular Cell Biology programs) to F. I. Ataullakhanov, NIH grant GM R01 GM033787 to J.R. McIntosh, and a Dmitry Zimin Dynasty Foundation postdoctoral fellowship to V.A.V.

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 &#956;l/min. A syring...

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...

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 under typical experimental conditions, the microtubules undergo catastrophe and switch to the depolymerization only rarely, once every 5-15 min (this frequency is for 7-15 mM soluble tubulin concentration examined at 28-32&#160;&#176;C)4. Different techniques have thus been proposed to induce microtubule depolymerization in a controlled manner. Microtubule shortening can be triggered by washing away soluble tubulin5,6, cutting microtubules with a laser beam7, or using segmented microtubules8, as described here. Previous work using segmented microtubules, as well as stochastically switching polymers, has found that small intracellular cargos, such as chromosomes, vesicles, and protein-coated beads, can move at the ends of the shortening microtubules9-13. This phenomenon is thought to have a direct implication for chromosome motions in mitotic cells, and the underlying mechanisms are currently under active investigation14-16.
Recently, fluorescent-based techniques, including the total internal reflection fluorescence (TIRF) microscopy, have been employed to study motility with dynamic microtubule ends17-24. The advantage of this approach is that it allows examination of interactions between microtubules and microtubule-binding proteins in real time using proteins labeled with different fluorophores. Several protein complexes were found to move processively with elongating and/or shortening microtubule ends. They include the microtubule-associated proteins Dam110,12,18, Ska119, and XMAP21520, as well as kinesin motors Kif18A21,22, MCAK23 and CENP-E24. These proteins exhibit processive tip-tracking, which is fundamentally different from that of the classic tip-tracking proteins like EB125. Although EB1 molecules and the associated partners appear to remain stably associated with dynamic microtubule ends, the individual molecules remain bound to the microtubule tip for only ~0.8 sec, rapidly exchanging with the soluble pool26. In contrast, processive tip-trackers, like Dam1, travel with microtubule ends for many microns, and their association with microtubule tips can last for many seconds. The tip association time, as well as the resulting rate of tracking, depends strongly on the number of molecules that form the tip-tracking complex27. Larger protein ensembles are usually much better tip-trackers. For example, such complex assemblies as the isolated yeast kinetochores can remain coupled to microtubule ends for hours28. Some microtubule-binding proteins, e.g. Ndc80 kinetochore protein complex, have been found to be unable to track with microtubule ends at a single molecule level, yet Ndc80 is very efficient in coupling the motion of bead cargo19,29-31. Thus, to understand the mechanism of tip-tracking by different protein complexes, as well as their biological roles, it is important to examine tip-tracking as a function of the number of molecules in the tip-tracking complex, as well as to determine the ability of these complexes to exhibit collective motility on the surface of bead cargo.
Below we provide detailed protocols to prepare and conduct experiments with segmented microtubules (Figure 1A). First, the commercially available glass slides are modified to attach short polyethylene tubing (Protocol 1). The reusable microscopy flow chamber is then assembled from such a slide and the chemically or plasma-cleaned and silanized coverslip (protocol 2)32-34. The resulting chamber volume is only 20-25 &#956;l&#160;(or as small as 15 &#956;l, see Note 3 in Protocol 1), including the volume of the inlet tubing. Commercially available flow chambers can also be used, but their volume is usually larger, leading to the unnecessary waste of proteins. If a larger chamber is employed, the volume of all solutions in the protocols below should be scaled proportionally. Microtubule seeds are then prepared, for example using slowly hydrolysable GTP analog, GMPCPP (Guanosine-5&#8217;-[(&#945;,&#946;)-methyleno]triphosphate) (protocol 3, see also Hyman&#160;et al.35). The seeds are immobilized on a cleaned coverslip and the surface is subsequently blocked to prevent nonspecific absorption of other proteins32 (protocol 4 describes seeds immobilization using digoxigenin). The segmented microtubules can then be prepared using Protocol 5. The main rationale for this approach is that dynamic microtubule polymers, which form in the presence of GTP, can be stabilized temporarily by adding the short &#8220;caps&#8221; of stable tubulin segments, which contain GMPCPP. These caps also contain Rhodamine-labeled tubulin, so they can be removed simply by illuminating the field of view with a 530-550 nm laser or mercury arc lamp (Protocol 6)36. Fluorescence intensity of the tip-tracking signal can then be used to estimate the number of molecules that travel with the disassembling microtubule ends, taking into account the unevenness of the microscope field illumination (Protocol 7). A similar approach can be used to study interactions between depolymerizing microtubules and protein-coated beads, prepared as described in27 (Protocol 8). Some proteins will readily bind to the walls of segmented microtubules, but laser tweezers can also be used to hold the bead near the microtubule wall, thereby promoting its binding.

<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 1: Microscopy and other equipment.</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss 
			Nikon</td>
			<td>Axio Imager 2 
			Eclipse Ti</td>
			<td>other microscope models capable of DIC and epi-fluorescence-imaging can be used</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Zeiss 
			Nikon</td>
			<td>420490-9900-000 
			CFI Apo 100x Oil 1.49</td>
			<td>100X, DIC, 1.3-1.49 NA</td>
		</tr>
		<tr>
			<td>Objective heater</td>
			<td>Bioptechs</td>
			<td>150803, 150819-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent filter cube</td>
			<td>Chroma</td>
			<td>49004 or 49008 
			41017 or 49020</td>
			<td>optimized for Rhodamine fluorescence&#160; 
			optimized for GFP fluorescence</td>
		</tr>
		<tr>
			<td>Acquisition software</td>
			<td>freeware MicroManager 
			Molecular Devices</td>
			<td>not applicable 
			MetaMorph 7.5</td>
			<td>http://valelab.ucsf.edu/~MM/MMwiki/ 
			other software can be used to acquire images and for a particle tracking</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon3, DU-897E-cs0-#BV</td>
			<td>Highly sensitive EMCCD camera</td>
		</tr>
		<tr>
			<td>Trapping laser</td>
			<td>IPG Photonics</td>
			<td>YLR-10-1064-LP</td>
			<td>1064 nm laser, 10 W</td>
		</tr>
		<tr>
			<td>Fluorescence excitation lasers</td>
			<td>Coherent, Inc. 
			Coherent, Inc.</td>
			<td>Sapphire 488 LP 
			Sapphire 552 LP</td>
			<td>excitation of green fluorophores 
			excitation of red fluorophores</td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial flow chambers</td>
			<td>Warner Instruments</td>
			<td>RC-20 or RC-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump</td>
			<td>Cole Palmer 
			Harvard Apparatus</td>
			<td>Masterflex 77120-52 
			Pico Plus</td>
			<td>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.&#160;</td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 2: Microscopy chamber preparation.</td>
		</tr>
		<tr>
			<td>Modified microscope slides for reusable chambers</td>
			<td>Precision Glassblowing of Colorado</td>
			<td>Custom order www.precisionglassblowing.com</td>
			<td>Sonic slots in slides using schematics in Figure 1</td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427410</td>
			<td>I.D. 0.58 mm, O.D. 0.965 mm; use these tubes to connect assembled chamber to the pump and waste container</td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427400</td>
			<td>I.D. 0.28 mm, O.D. 0.61 mm; use these tubes to make the reusable chamber</td>
		</tr>
		<tr>
			<td>Regular microscope slides</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>Other similar slides can be used</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>VWR</td>
			<td>48393-150, 48366-067</td>
			<td>Other similar coverslips can be used</td>
		</tr>
		<tr>
			<td>Silicon sealant</td>
			<td>World Precision Instruments</td>
			<td>KIT, SILICON SEALANT 5 MIN CURE</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy glue</td>
			<td>Loctite</td>
			<td>83082</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate adhesive</td>
			<td>Scotch 3M</td>
			<td>AD114</td>
			<td>Or cyanoacrylate adhesive from other manufacturers</td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 3: Coverslips cleaning and coating.</td>
		</tr>
		<tr>
			<td>Molecular Sieves, Grade 564</td>
			<td>Macron</td>
			<td>4490-04</td>
			<td width="200px"></td>
		</tr>
		<tr>
			<td>Coverglass Staining Jar</td>
			<td>Ted Pella, Inc.</td>
			<td>21036</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip Ceramic Holder</td>
			<td>Thomas Scientific</td>
			<td>8542e40</td>
			<td></td>
		</tr>
		<tr>
			<td>PlusOne Repel Silane</td>
			<td>GE Healthcare Biosciences</td>
			<td>17-1332-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-digoxigenin AB</td>
			<td>Roche applied science</td>
			<td>11093274910</td>
			<td></td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 4: Preparation of seeds and segmented microtubules.</td>
		</tr>
		<tr>
			<td>Tubulin</td>
			<td>purified from cow brains 
			Cytoskeleton, Inc</td>
			<td>&#160; 
			T238P</td>
			<td>For purification protocols see 49&#8211;51 
			Unlabeled porcine tubulin</td>
		</tr>
		<tr>
			<td>Labeled tubulin</td>
			<td>Cytoskeleton, Inc 
			Invitrogen 
			Invitrogen</td>
			<td>TL590M 
			C1171 (Rhodamine) 
			A-2952 (Digoxigenin)</td>
			<td>Rhodamine-labeled porcine tubulin 
			Tubulin can be labeled with any amine-reactive dye as in reference52.</td>
		</tr>
		<tr>
			<td>GMPCPP</td>
			<td>Jena Biosciences</td>
			<td>NU-405</td>
			<td>Aliquot and store at -70 &#176;C</td>
		</tr>
		<tr>
			<td>VALAP</td>
			<td>vaseline, lanolin, and paraffin at 1:1:2 by mass</td>
			<td></td>
			<td>see ref. 9</td>
		</tr>
	</tbody>
</table>

preparation of segmented microtubules to study motions driven by the disassembling microtubule ends

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.

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 cells18,38, 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...

Watch this Scientific Journal Video about Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends at JoVE.com

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

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.

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are ...

preparation-segmented-microtubules-to-study-motions-driven

Research

JoVE Journal

Biology

14.3K 조회수.  Russian Academy of Sciences.  미세 소관은 본질적으로 불안정 폴리머, 그리고 성장과 단축 사이에 자신의 전환 확률 통제하기가 어렵습니다. 여기에서 우리는 photoablatable 안정 캡으로 분할 미세 소관을 사용하여 프로토콜을 설명합니다. 분할 된 미세 소관 해중합함으로써 분해 미세 소관의 끝 동작의 분석을 지원, 높은 공간적 해상도로 트리거 될 수 있습니다. 