This work was supported by a grant from the NIH (no. 1DP2GM126894-01), and by funds from the Pew Charitable Trusts and the Smith Family Foundation to R.S. The authors thank Dr. Shuo Jiang for his contribution toward development and optimization of the protocols.

<ol>
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E., von Dassow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+and+dynamic+microtubules+coordinately+shape+the+myosin+activation+zone+during+cytokinetic+furrow+formation.">Stable and dynamic microtubules coordinately shape the myosin activation zone during cytokinetic furrow formation.</a> The Journal of Cell Biology. 183 (3), 457-470 (2008).</li><li>Pous, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+specialization+of+stable+and+dynamic+microtubules+in+protein+traffic+in+WIF-B+cells.">Functional specialization of stable and dynamic microtubules in protein traffic in WIF-B cells.</a> The Journal of Cell Biology. 142 (1), 153-165 (1998).</li><li>Uehara, R., Goshima, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+central+spindle+assembly+requires+de+novo+microtubule+generation+in+the+interchromosomal+region+during+anaphase.">Functional central spindle assembly requires de novo microtubule generation in the interchromosomal region during anaphase.</a> The Journal of Cell Biology. 191 (2), 259-267 (2010).</li><li>Mitchison, T., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+instability+of+microtubule+growth.">Dynamic instability of microtubule growth.</a> Nature. 312 (5991), 237-242 (1984).</li><li>Bieling, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+a+microtubule+plus-end+tracking+system+in+vitro.">Reconstitution of a microtubule plus-end tracking system in vitro.</a> Nature. 450 (7172), 1100-1105 (2007).</li><li>Bieling, P., Telley, I. A., Surrey, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+minimal+midzone+protein+module+controls+formation+and+length+of+antiparallel+microtubule+overlaps.">A minimal midzone protein module controls formation and length of antiparallel microtubule overlaps.</a> Cell. 142 (3), 420-432 (2010).</li><li>Shimamoto, Y., Forth, S., Kapoor, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+pushing+and+braking+forces+generated+by+ensembles+of+Kinesin-5+crosslinking+two+microtubules.">Measuring pushing and braking forces generated by ensembles of Kinesin-5 crosslinking two microtubules.</a> Developmental Cell. 34 (6), 669-681 (2015).</li><li>Mani, N., Jiang, S., Neary, A. E., Wijeratne, S. S., Subramanian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+single+microtubules+and+bundles+by+a+three-protein+module.">Differential regulation of single microtubules and bundles by a three-protein module.</a> Nature Chemical Biology. 17 (9), 964-974 (2021).</li><li>Hyman, A. A., Salser, S., Drechsel, D., Unwin, N., Mitchison, T. 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A., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonblinking+and+long-lasting+single-molecule+fluorescence+imaging.">Nonblinking and long-lasting single-molecule fluorescence imaging.</a> Nature Methods. 3 (11), 891-893 (2006).</li><li>Wijeratne, S., Subramanian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometry+of+antiparallel+microtubule+bundles+regulates+relative+sliding+and+stalling+by+PRC1+and+Kif4A.">Geometry of antiparallel microtubule bundles regulates relative sliding and stalling by PRC1 and Kif4A.</a> eLife. 7, 32595 (2018).</li><li>Mani, N., Wijeratne, S. 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The authors declare no competing interests.

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 dynamics, and those that are newly formed when two growing ends encounter each other and get crosslinked (Figure 3). Moreover, in addition to conventional experimental variables, such as protein concentrations and buffer conditions, these assays enable the evaluation of the effects of geometrical features of microtubule arrays, such as the lengths and angles between adjacent filaments in a bundle, which are emerging as important determinants of microtubule dynamics and MAP activity16.
In order to extend this experimental method for the in vitro reconstitution of multiple microtubule-based structures, the following key issues need to be addressed: (i) PRC1 preferentially crosslinks microtubules that are oriented anti-parallel to each other. While such bundles are found at the cell center during mitosis, bundles of parallel microtubules are a common feature in other microtubule-based structures within neuronal axons and the mitotic spindle. The protocol described above can be readily adapted to generate crosslinked parallel microtubules using recombinant crosslinkers such as Kinesin-1 and TRIM4610,17. (ii) In these reconstitution assays, differences in fluorescence intensity can be used to distinguish between single microtubules and pairs of microtubules11,15. Under the experimental conditions used here, intensity analyses indicate that most bundles contain two crosslinked microtubules, and line scan analyses provide information on their relative positioning. However, when there are more than two or three filaments in a bundle, the spatial resolution of standard TIRF-based imaging systems hinders identification of the ends and polarity of individual microtubules (~25 nm diameter)18. Moreover, while it is possible to identify the plus-ends of crosslinked anti-parallel microtubules from the direction of their growth, distinguishing the ends of cross-linked parallel microtubules growing in the same direction is hindered by the spatial resolution of optical microscopy. An extension of the experiment described here is to use polarity-marked microtubules or microtubule tip-binding proteins to position individual microtubules. For bundles with tens of microtubules, complementing the high temporal resolution of TIRF-based assays with techniques that have high spatial resolution, such as Atomic Force Microscopy19, promises to yield new insights into the dynamics of individual microtubules within a bundle.

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 single microtubules in close spatial proximity. These microtubule subpopulations can display starkly different polymerization dynamics from each other, as needed for their proper function2,3,4,5. For instance, within the mitotic spindle, stable crosslinked bundles and dynamic single microtubules are present within a micron-scale region at the cell center6. Studying how the dynamic properties of coexisting microtubule populations are specified is, therefore, central to understanding the assembly and function of microtubule-based structures.
Microtubules are dynamic polymers that cycle between phases of polymerization and depolymerization, switching between the two phases in events known as catastrophe and rescue7. The dynamics of cellular microtubules are regulated by myriad Microtubule Associated Proteins (MAPs) that modulate the rates of microtubule polymerization and depolymerization and the frequencies of catastrophe and rescue events. It is challenging to investigate the activity of MAPs on spatially proximal arrays in cells, owing to the limitations of spatial resolution in light microscopy, especially in regions of high microtubule density. Moreover, the presence of multiple MAPs in the same cellular region hinders interpretations of cell biological studies. In vitro reconstitution assays, performed in conjunction with Total Internal Reflection Fluorescence (TIRF) microscopy, circumvent the challenges of examining mechanisms by which specific subsets of MAPs regulate the dynamics of proximal cellular microtubule arrays. Here, the dynamics of microtubules assembled in vitro are examined in the presence of one or more recombinant MAPs under controlled conditions8,9,10. However, conventional reconstitution assays are typically performed on single microtubules or on one type of array, precluding the visualization of coexisting populations.
Here, we present&#160;in vitro reconstitution assays that enable the simultaneous visualization of two microtubule populations under the same solution conditions11. We describe a&#160;method to simultaneously view the collective activity of multiple MAPs on single microtubules&#160;and on microtubule bundles crosslinked by the mitotic spindle-associated protein PRC1.&#160;The protein PRC1 preferentially binds at the overlap between anti-parallel microtubules, crosslinking them9. Briefly, this protocol consists of the following steps: (i) preparation of stock solutions and reagents, (ii) cleaning and surface treatment of coverslips used to create the imaging chamber for microscopy experiments, (iii) preparation of stable microtubule &#34;seeds&#34; from which polymerization is initiated during the experiment, (iv) specification of TIRF microscope settings to visualize microtubule dynamics, (v) immobilization of microtubule seeds and generation of crosslinked microtubule bundles in the imaging chamber, and (vi) visualization of microtubule dynamics in the imaging chamber through TIRF microscopy, upon addition of soluble tubulin, MAPs, and nucleotides. These assays enable the qualitative evaluation and quantitative examination of MAP localization and their effect on the dynamics of two microtubule populations. Additionally, they facilitate the evaluation of synergistic effects of multiple MAPs on these microtubule populations, across a wide range of experimental conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >(&#177;)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox)</td>
			<td >Sigma Aldrich</td>
			<td >238813</td>
			<td ></td>
		</tr>
		<tr >
			<td >1,4-piperazinediethanesulfonic acid (PIPES)</td>
			<td>Sigma Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr >
			<td >18x18 mm #1.5 coverslips&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>63787</td>
			<td></td>
		</tr>
		<tr >
			<td >2-Mercaptoethanol (BME)</td>
			<td>Sigma Aldrich</td>
			<td>M-6250</td>
			<td></td>
		</tr>
		<tr >
			<td >24x60 mm #1.5 coverslips</td>
			<td>Electron Microscopy Sciences</td>
			<td>63793</td>
			<td></td>
		</tr>
		<tr >
			<td >405/488/560/647 nm Laser Quad Band&#160;</td>
			<td>Chroma</td>
			<td>TRF89901-NK</td>
			<td></td>
		</tr>
		<tr >
			<td >Acetone</td>
			<td>Sigma Aldrich</td>
			<td>320110</td>
			<td></td>
		</tr>
		<tr >
			<td >Adenosine 5&#39;-triphosphate disodium salt hydrate (ATP)</td>
			<td>Sigma Aldrich</td>
			<td>A7699-5G</td>
			<td></td>
		</tr>
		<tr >
			<td >Avidin, NeutrAvidin&#174; Biotin-binding Protein (Molecular Probes&#174;)</td>
			<td>Thermo Fischer Scientific</td>
			<td>A2666</td>
			<td></td>
		</tr>
		<tr >
			<td >Bath sonicator: Branson 2800 Cleaner</td>
			<td>Branson</td>
			<td>CPX2800H</td>
			<td></td>
		</tr>
		<tr >
			<td >Beckman Coulter Polycarbonate Thickwall Tubes, 11 x 34 mm</td>
			<td>Beckman-Coulter&#160;</td>
			<td>343778</td>
			<td></td>
		</tr>
		<tr >
			<td >Beckman Coulter Polycarbonate Thickwall Tubes, 8 x 34 mm</td>
			<td>Beckman-Coulter&#160;</td>
			<td>343776</td>
			<td></td>
		</tr>
		<tr >
			<td >Biotin-PEG-SVA, MW 5,000</td>
			<td>Laysan Bio</td>
			<td>#Biotin-PEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr >
			<td >Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>2905</td>
			<td></td>
		</tr>
		<tr >
			<td >Catalase</td>
			<td>Sigma Aldrich</td>
			<td>C40</td>
			<td></td>
		</tr>
		<tr >
			<td >Corning LSE Mini Microcentrifuge, AC100-240V</td>
			<td>Corning</td>
			<td>6670</td>
			<td></td>
		</tr>
		<tr >
			<td >Delicate Task Wipes</td>
			<td>Kimtech</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr >
			<td >Dithiothreitol (DTT)</td>
			<td>GoldBio</td>
			<td>DTT10</td>
			<td></td>
		</tr>
		<tr >
			<td >Emission filter</td>
			<td>Chroma</td>
			<td>ET610/75m</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethanol (200-proof)</td>
			<td>Decon Labs</td>
			<td>2705</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethylene glycol tetraacetic acid (EGTA)</td>
			<td>Sigma Aldrich</td>
			<td>3777</td>
			<td></td>
		</tr>
		<tr >
			<td >Glucose Oxidase</td>
			<td>Sigma Aldrich</td>
			<td>G2133</td>
			<td></td>
		</tr>
		<tr >
			<td >GMPCPP</td>
			<td>Jena Bioscience&#160;</td>
			<td>NU-405</td>
			<td></td>
		</tr>
		<tr >
			<td >Guanosine 5&#39;-triphosphate sodium salt hydrate (GTP)</td>
			<td>Sigma Aldrich</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr >
			<td >Hellmanex III detergent&#160;</td>
			<td>Sigma Aldrich</td>
			<td>Z805939</td>
			<td></td>
		</tr>
		<tr >
			<td >Immersion oil, Type A</td>
			<td>Fisher Scientific</td>
			<td>77010</td>
			<td></td>
		</tr>
		<tr >
			<td >Kappa-casein</td>
			<td>Sigma Aldrich</td>
			<td>C0406</td>
			<td></td>
		</tr>
		<tr >
			<td >Lanolin</td>
			<td>Fisher Scientific</td>
			<td>S25376</td>
			<td></td>
		</tr>
		<tr >
			<td >Lens Cleaning Tissue</td>
			<td>ThorLabs</td>
			<td>MC-5</td>
			<td></td>
		</tr>
		<tr >
			<td >Magnesium Chloride (MgCl2)</td>
			<td>Sigma Aldrich</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr >
			<td >Methylcellulose</td>
			<td>Sigma Aldrich</td>
			<td>M0512</td>
			<td></td>
		</tr>
		<tr >
			<td >Microfuge 16 Benchtop Centrifuge</td>
			<td>Beckman-Coulter&#160;</td>
			<td>A46474</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope Slides, Diamond White Glass, 25 x 75mm, 90&#176; Ground Edges, WHITE Frosted</td>
			<td>Globe Scientific</td>
			<td>1380-50W</td>
			<td></td>
		</tr>
		<tr >
			<td >mPEG-Succinimidyl Valerate, MW 5,000&#160;</td>
			<td>Laysan Bio</td>
			<td>#NH2-PEG-VA-5K</td>
			<td></td>
		</tr>
		<tr >
			<td >Optima&#8482; Max-XP Tabletop Ultracentrifuge</td>
			<td>Beckman-Coulter&#160;</td>
			<td>393315</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraffin</td>
			<td>Fisher Scientific</td>
			<td>P31-500</td>
			<td></td>
		</tr>
		<tr >
			<td >PELCO Reverse (self-closing), Fine Tweezers</td>
			<td>Ted Pella</td>
			<td>5377-NM</td>
			<td></td>
		</tr>
		<tr >
			<td >Petrolatum, White</td>
			<td>Fisher Scientific</td>
			<td>18-605-050</td>
			<td></td>
		</tr>
		<tr >
			<td >Plasma Cleaner, 115V</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td></td>
		</tr>
		<tr >
			<td >Potassium Hydroxide (KOH)</td>
			<td>Sigma Aldrich</td>
			<td>221473</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr >
			<td >Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermal-Lok 1-Position Dry Heat Bath</td>
			<td>USA Scientific</td>
			<td>2510-1101</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermal-Lok Block for 1.5 and 2.0 mL Tubes</td>
			<td>USA Scientific</td>
			<td>2520-0000</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermo Scientific&#8482; Pierce&#8482; Bond-Breaker&#8482; TCEP Solution, Neutral pH; 500mM</td>
			<td>Thermo Fischer Scientific</td>
			<td>PI-77720</td>
			<td></td>
		</tr>
		<tr >
			<td >TIRF 100X NA 1.49 Oil Objective</td>
			<td>Nikon</td>
			<td colspan="2">CFI Apochromat TIRF 100XC Oil</td>
		</tr>
		<tr >
			<td >TIRF microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr >
			<td >TLA 120.1 rotor</td>
			<td>Beckman-Coulter&#160;</td>
			<td>362224</td>
			<td></td>
		</tr>
		<tr >
			<td >TLA 120.2 rotor</td>
			<td>Beckman-Coulter&#160;</td>
			<td>357656</td>
			<td></td>
		</tr>
		<tr >
			<td >Tubulin protein (&#62;99% pure): porcine brain</td>
			<td>Cytoskeleton</td>
			<td>T240</td>
			<td></td>
		</tr>
		<tr >
			<td >Tubulin Protein (Biotin): Porcine Brain</td>
			<td>Cytoskeleton</td>
			<td>T333P</td>
			<td></td>
		</tr>
		<tr >
			<td >Tubulin protein (fluorescent HiLyte 647): porcine brain</td>
			<td>Cytoskeleton</td>
			<td>TL670M</td>
			<td></td>
		</tr>
		<tr >
			<td >Tubulin protein (X-rhodamine): bovine brain</td>
			<td>Cytoskeleton</td>
			<td>TL620M</td>
			<td></td>
		</tr>
		<tr >
			<td >VECTABOND&#174; Reagent, Tissue Section Adhesion</td>
			<td>Vector Biolabs</td>
			<td>SP-1800-7</td>
			<td></td>
		</tr>
		<tr >
			<td >VWR&#174; Personal-Sized Incubator, 120V, 50/60Hz, 0.6A</td>
			<td>VWR</td>
			<td>97025-630</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous visualization of the dynamics of crosslinked and single microtubules in vitro by tirf microscopy

Microtubules are polymers of &#945;&#946;-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&#160;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.

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 were identified by their fluorescence signal in the 647 nm channel. Microtubules with fluorescence signals in both channels were identified as pre-formed bundles (Figure 4). If experiments are performed with biotinylated and non-biotinylated microtubules with the same fluorescent label, detected fluorescence intensities for bundles will be around two-fold or higher than that of single microtubules. Based on the proportion and density of each population, the concentration of microtubule seeds in steps 5.6 and 5.10 can be optimized.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63377/63377fig04.jpg" /> 
Figure 4: Identification of single microtubules and crosslinked microtubules in the field of view. Representative field of view showing 647 nm (left), 560 nm (center), and merged (right) channels. Single microtubules (yellow arrowheads) and bundles (white arrowheads) are indicated in the merged channel. Scale bar represents 2 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63377/63377fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Dynamics of single microtubules and PRC1-crosslinked bundles. Representative video showing microtubule dynamics, with 647 nm fluorophore-labeled biotinylated microtubule seeds (blue), 560 nm fluorophore-labeled non-biotinylated microtubule seeds and 560 nm fluorophore-labeled soluble tubulin (red), and GFP-labeled PRC1 (green). Single and crosslinked microtubules are indicated by white and yellow arrows, respectively. The movie was recorded over 10 min (61 frames) and displayed at a rate of 12 frames/second. Assay conditions: 0.5 nM GFP-PRC1, 50 mM KCl and 37 &#176;C. Scale bar: 5 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63377/Video1_rev1.mov" target="_blank">Please click here to download this Video.</a>
After an imaging sequence has been acquired, analyze the video to ensure that microtubules are dynamic (Video 1). Adjust assay components (tubulin volume in soluble tubulin mix, nucleotide stocks, protein concentrations) according to observations. For example, if microtubules do not polymerize, increase concentration of soluble tubulin and/or GTP in soluble tubulin mix. Similarly, increase working concentrations of fluorescently labeled MAPs if they are not visible on microtubules, and decrease concentrations if their background fluorescence intensity in the field of view is comparable to their intensity on microtubules. When visualizing motile motor proteins, increase ATP concentrations if motors do not exhibit motility on microtubules. Adjust Laser intensity for the excitation channels corresponding to microtubule fluorescence to ensure that differences in fluorescence intensity between single microtubules and bundles can be captured within the dynamic range of the detector.
Video 2: Differences in dynamics of single microtubules and PRC1-crosslinked bundles in the presence of two MAPs:&#160;CLASP1&#160;and Kif4A. Representative video showing microtubule dynamics, with 647 nm fluorophore-labeled biotinylated microtubule seeds (blue), and 560 nm fluorophore-labeled non-biotinylated microtubule seeds and 560 nm fluorophore-labeled soluble tubulin (red). Single and crosslinked microtubules are indicated by white and yellow arrows, respectively. The movie was recorded over 20 min (121 frames) and displayed at a rate of 20 frames/second. Assay conditions: 200 nM CLASP1-GFP, 0.5 nM PRC1, and 10 nM Kif4A. Scale bar: 2 &#181;m. Video is reproduced from reference11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63377/Video2.mov" target="_blank">Please click here to download this Video.</a>
In the representative example shown in Video 2, a field of view containing single microtubules and crosslinked bundles is shown. It is found that under these assay conditions (assay mix containing 0.5 nM PRC1, 50 mM KCl, and MAPs of interest: 200 nM GFP-labeled CLASP1, 10 nM Kinesin Kif4A), single microtubules elongate over the course of the assay, whereas the growth of crosslinked microtubules is stalled.
For quantitative analysis of microtubule dynamics, open microscopy files in the FIJI software, and select single microtubules and bundles for analysis. Use the following criteria to exclude single microtubules and bundles from further analysis: exclude microtubules or bundles (i) found at the edges of the field of view, (ii) obscured by protein aggregates, or (iii) whose filaments move in the z-direction out of the TIRF range. Parameters of microtubule dynamics, such as length, growth rate, rescue frequency, and catastrophe frequency, can be obtained by constructing and analyzing kymographs for each single microtubule or microtubule pair11,15.

Watch this Scientific Journal Video about Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy at JoVE.com

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

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.

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

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

simultaneous-visualization-dynamics-crosslinked-single-microtubules

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.5K Views.  Massachusetts General Hospital. 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.

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

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient association of pore-forming and auxiliary subunits, lateral diffusion, and clustering with other proteins. However, the relationship between lateral diffusion and function is poorly understood. To approach this problem, we describe how lateral mobility and activity of individual channels in supported lipid membranes can be monitored and correlated using total internal reflection fluorescence (TIRF) microscopy. Membranes are fabricated on an ultrathin hydrogel substrate using the droplet interface bilayer (DIB) technique. Compared to other types of model membranes, these membranes have the advantage of being mechanically robust and suitable for highly sensitive analytical techniques. This protocol measures Ca2+ ion flux through single channels by observing the fluorescence emission of a Ca2+-sensitive dye in close proximity to the membrane. In contrast to classical single-molecule tracking approaches, no fluorescent fusion proteins or labels, which can interfere with lateral movement and function in the membrane, are required. Possible changes in ion flux associated with conformational changes of the protein are only due to protein lateral motion in the membrane. Representative results are shown using the mitochondrial protein translocation channel TOM-CC and the bacterial channel OmpF. In contrast to OmpF, the gating of TOM-CC is very sensitive to molecular confinement and the nature of lateral diffusion. Hence, supported droplet-interface bilayers are a powerful tool to characterize the link between lateral diffusion and the function of ion channels.

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence TIRF Microscopy

This protocol describes how to use TIRF microscopy to track individual ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient ...

Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the posttranslational modifications, microtubules can form complexes with various interacting proteins. These microtubule-protein complexes are often implicated in human diseases. Understanding the structure of such complexes is useful for elucidating their mechanisms of action and can be studied by cryo-electron microscopy (cryo-EM). To obtain such complexes for structural studies, it is important to extract microtubules containing or lacking specific posttranslational modifications. Here, we describe a simplified protocol to extract endogenous tubulin from genetically modified mammalian cells, involving microtubule polymerization, followed by sedimentation using ultracentrifugation. The extracted tubulin can then be used to prepare cryo-electron microscope grids with microtubules that are bound to a purified microtubule-binding protein of interest. As an example, we demonstrate the extraction of fully tyrosinated microtubules from cell lines engineered to lack the three known tubulin-detyrosinating enzymes. These microtubules are then used to make a protein complex with enzymatically inactive microtubule-associated tubulin detyrosinase on cryo-EM grids.

Here, we describe a protocol to extract endogenous tubulin from mammalian cells, which can lack or contain specific microtubule-modifying enzymes, to obtain microtubules enriched for a specific modification. We then describe how the extracted microtubules can be decorated with purified microtubule-binding proteins to prepare grids for cryo-electron microscopy.

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the ...