We like to thank the members of the Dogterom lab for valuable discussions. We acknowledge Stefan Diez and Marcel Janson for reagents, and Samara Reck-Peterson for help with the purification of dynein. This work was supported by funding from the European Research Council (ERC Synergy grant: MODEL-CELL).

<ol>
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The authors declare that they have no competing financial interests.

The methods described here present recent efforts to reconstitute basic mitotic spindles in water-in-oil emulsion droplets. Studying spindle assembly within geometrical boundaries provides numerous advantages. Important feedback mechanisms exist between microtubules of the mitotic spindle and the geometrical constraints in which they are assembled. Microtubules can generate pushing forces when they grow into geometrical boundaries, which can result in mitotic spindle displacement. In addition, growing into a physical barrier can induce microtubule catastrophes. Also, spindle assembly within a confined geometry can lead to a gradual depletion of individual components, such as tubulin, which in turn directly affects the microtubule growth rate36. These are all essential determinants of spindle assembly, which can be studied using the assays described here.
The described assays are very sensitive to small concentration differences of the droplet components. This is the case for tubulin, where minor concentration differences can result in either too slow or too rapid microtubule growth. In this case, microtubule growth rates can often still be corrected by adjusting the temperature during the first ~ 30 min of the experiment. Mitotic spindle assembly and positioning is also very sensitive to variations in the concentration of (cortical) force generators. This is likely to depend on the concentration, purity and activity of the purified components and needs to be titrated carefully for every newly purified protein.
In addition, certain trade-offs have to be made in imaging settings, depending on the nature of the assay. Initially, high levels of soluble fluorescent tubulin dimers make it difficult to image individual microtubules. As microtubules grow longer and soluble tubulin is slowly depleted, the signal-to-noise ratio gradually becomes higher and allows the imaging of individual microtubules. In contrast to setups with open systems, the geometrical confinement prevents the exchange of fluorescent molecules and oxygen scavenger system components within a bulk reservoir, resulting in significant bleaching. Especially for monitoring spindle assembly and positioning over time, it is required to image with low light intensities, even in the presence of a potent oxygen scavenger system.
These methods enable the bottom-up reconstitution of simplified spindle-like structures in vitro. In addition to the components introduced here, many other factors have been described to influence bipolar spindle formation, positioning, orientation, shaping, etc.3. This system can be further expanded to study the individual and combined effects of additional force generators. Important contributors to spindle formation that are currently absent from this system are the mitotic chromosomes. Mitotic chromatin has been shown to direct spindle formation by (1) chromokinesins that can generate so-called &#39;polar-ejection forces&#39;3, (2) the formation of a RanGTP gradient by the chromatin-bound RanGEF RCC138, (3) kinetochores that can interact with (depolymerizing) microtubules39 and (4) the chromatin itself, which can function as a mechanical spring in-between opposing microtubules40.
Finally, the described system currently provides limited temporal and spatial control over the activity and localization of introduced force-generators. In cells, many spindle assembly factors localize to specific compartments or are active within a restricted time-window. A striking example is the activity of dynein, which is specifically enriched at the posterior side of the C. elegans one-cell stage embryo to promote asymmetric spindle positioning and cell division. In this system, we are currently exploring the possibility of mimicking such temporal and asymmetric activity using methods borrowed from opto-genetic techniques. In addition, as is the case for the C. elegans embryo and cells with a rigid cell wall, many cells do not round up in mitosis. The shape of the geometrical confinement will have a fundamental impact on the forces acting on the spindle41. It will therefore be important to reconstitute spindle assembly and positioning in non-spherical droplets as well, potentially using more complex microfluidic systems that allow shaping and storing of emulsion droplets42,43. Finally, in tissues, cell-cell and cell-substrate signaling and attachment provide external cues that can be translated into directed spindle orientation, as is often observed in epithelial cells and stem cells. All of these specialized aspects have not been taken into account in this current study and might provide future challenges to build towards more in vivo-like reconstitutions of mitotic spindle assembly and positioning.

During mitosis, the chromosomes of the replicated genome are organized at the cell equator to ensure equal distribution of sister chromatids over the newly formed daughter cells. Chromosome positioning and segregation is mediated by their attachment to spindle microtubules originating from two opposing centrosomes. In addition to ensuring faithful chromosome distribution, the orientation of the mitotic spindle also dictates the cell division plane1. Coordinated orientation of cell division is essential during many stages of development and for tissue homeostasis2. Specifically, regulated mitotic spindle positioning can result in the asymmetric distribution of cellular contents or even produce daughter cells of different sizes2. Mitotic spindle assembly and orientation starts in prophase and is orchestrated by a complex interplay between cooperating and antagonizing force-generators3,4. The generated forces consist of both pulling and pushing forces. These forces originate both from spindle contacts with the cell cortex as well as from within the spindle, and can be generated by microtubule dynamics as well as by molecular motors and cross-linkers (Figure 1).
Pushing forces can be generated when microtubules grow against rigid structures such as the cell cortex. Depending on the total resisting force generated within the system, this can result in repositioning of the mitotic spindle (low forces) or trigger microtubule buckling or catastrophe (high forces)5-8. The amount of force that can be generated by pushing depends on microtubule length, since length is a strong determinant of microtubule-buckling9. In addition, microtubules that originate from one centrosome can push against the other centrosome, a mechanism that has been suggested to drive centrosome separation in early prophase3,10.
In addition to pushing forces generated by growing microtubules, microtubule ends contacting the cell cortex can also mediate pulling forces11. Studies from multiple laboratories have shown that the controlled activity of cortical force generators is required for the asymmetric positioning of mitotic spindles in vivo1. Essential to these pulling forces is cortex-localized cytoplasmic dynein (hereafter referred to as &#39;dynein&#39;), a minus-end directed microtubule motor protein8,12,13. For example, in budding yeast, lateral interactions of cortical dynein with the microtubule lattice result in motor-dependent microtubule sliding along the cortex14. However, cortical pulling forces can also be generated by the ability of dynein to form end-on attachments to depolymerizing microtubules8. The energy generated by microtubule shrinkage may lead to forces that are generally an order of magnitude higher (~50 pN (reference15)) than the forces generated by individual motor proteins (~7-8 pN (ref. 16)). End-on attachment of cortical dynein to depolymerizing microtubules promotes spindle positioning in budding yeast17 and C. elegans13. Whether both motor-dependent sliding and microtubule depolymerization driven cortical pulling forces can cooperate or are mutually exclusive in vivo is at present unknown.
In addition to microtubule pushing forces and dynein-mediated cortical pulling forces, centrosome positioning is controlled from within the mitotic spindle by numerous other proteins. Kinesin-5 motors, for instance, exist as tetramers that can cross-link antiparallel interpolar microtubules, resulting in the generation of outward sliding forces18-20. Members of the kinesin-5 motor family are required for centrosome separation and bipolar spindle assembly in all eukaryotes studied, with the exception of C. elegans (reviewed in reference21).
Furthermore, diffusible cross-linkers of the Ase1/PRC1 family are also known to localize to overlapping microtubule regions of interpolar microtubules in vivo and in vitro22-27. The forces generated by Ase1-driven expansion of the overlapping microtubule-region are large enough to counterbalance kinesin-14 mediated sliding forces in vitro28,29. In cells, Ase1/PRC1 is required for stable formation of overlapping microtubule regions in the spindle midzone25-27,30, suggesting that the forces generated by diffusible cross-linkers can significantly contribute to spindle organization. Finally, forces from the mitotic chromatin and kinetochores influence mitotic spindle assembly and positioning through various pathways3. Since these components are not part of the reconstitution assays described here, they will not be discussed in detail.
Different theories exist on how the different molecular components and forces described above cooperate in spindle assembly and positioning, but we are still far from a quantitative understanding. In addition to experiments in living cells, in vitro experiments with purified components provide a powerful route to help reach this goal. Here we present a visual guide to an adapted and extended version of the recently published methods (Roth et al.31), in which basic spindles are reconstituted in water-in-oil emulsion droplets, starting with a minimal number of components. Using microfluidic techniques, spherical droplets are generated with sizes that are comparable to mitotic cells. Within these droplets, purified centrosomes and tubulin can be combined in order to study microtubule aster dynamics, force generation and aster-aster interactions. By introducing cortical (such as dynein) and inter-polar (such as kinesin-5/Ase1) force-generators, we reconstitute increasingly complex spindle-like structures that start to resemble the in vivo situation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1. Preparation of microfluidic chips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photomask (Photomask on film substrate)</td>
			<td>Selba S.A. Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3025</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer (4 inch silicon wafer p/Boron &#60;1-0-0&#62; 10-20 &#937;-cm, 500-550&#956;m, SSP, w/2 flats)</td>
			<td>WRS Materials</td>
			<td>4P0SSP-005</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater&#160;</td>
			<td>Polos</td>
			<td>SPIN150I</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS pre-polymer RVT615 A+B</td>
			<td>Lubribond</td>
			<td>9481</td>
			<td></td>
		</tr>
		<tr>
			<td>Corona treater</td>
			<td>Electro-technic products</td>
			<td>BD-20ACV</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Microfluidic setup</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DOPS (1,2-Dioleoyl-sn-glycero-3-phospho-L-serine)</td>
			<td>Avanti Polar Lipids</td>
			<td>840035</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinyl PE (1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl))</td>
			<td>Avanti Polar Lipids</td>
			<td>870285</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>650498</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pipets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Laboport</td>
			<td>KNF</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum chamber</td>
			<td>Kartell</td>
			<td>Polypropylene Vacuum Desiccator</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr>
			<td>Surfactant (Span80)</td>
			<td>Sigma-Aldrich</td>
			<td>85548</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Branson</td>
			<td>M2800H</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td></td>
			<td></td>
			<td>24x60mm, thickness 1.5</td>
		</tr>
		<tr>
			<td>Glass-slides</td>
			<td></td>
			<td></td>
			<td>26x76mm</td>
		</tr>
		<tr>
			<td>Puncher</td>
			<td>Harris Uni-Core</td>
			<td>135840/135841/135843</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory sealing film</td>
			<td>Parafilm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Valap (Vaseline, lanolin, paraffin wax melted at equal concentrations)</td>
			<td></td>
			<td></td>
			<td>Home made</td>
		</tr>
		<tr>
			<td>Brightfield Microscope</td>
			<td>Leica</td>
			<td>PMIRB&#160;</td>
			<td>Any inverted brightfield would suffice</td>
		</tr>
		<tr>
			<td>Pressure controler</td>
			<td>Fluigent</td>
			<td>MFCS-FLEX-4C-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>PEEK Tubing</td>
			<td>Cluzeau Info. Labo, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidics vials (Tubes Micrew 0.5 ml and 1 ml)</td>
			<td>VWR, The Netherlands</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3. Reconstituting spindle formation and positioning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dextran-647nm (Dextran, Alexa Fluor 647, 10,000 MW, Anionic, Fixable)</td>
			<td>Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA - Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (D-(+)-Glucose)</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose-oxidase (Glucose oxidase from Aspergillus niger)</td>
			<td>Sigma-Aldrich</td>
			<td>G6125</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT (DL-dithioltheitol)</td>
			<td>Sigma-Aldrich</td>
			<td>646563</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase (Catalase form bovine liver)</td>
			<td>Sigma-Aldrich</td>
			<td>C9322</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubulin (Tubulin from bovine brain)</td>
			<td>Cytoskeleton Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tubulin-488/561 (HiLyte-488/Rhodamine-labeled tubulin from porcine brain)</td>
			<td>Cytoskeleton Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GTP (Guanosine-&#39;5-triphosphate, sodium salt hydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>51120</td>
			<td></td>
		</tr>
		<tr>
			<td>&#954;-casein (&#954;-casein from bovine serum milk)</td>
			<td>Sigma-Aldrich</td>
			<td>C0406</td>
			<td></td>
		</tr>
		<tr>
			<td>Airfuge (Air-driven ultracentrifuge)</td>
			<td>Beckman-Coulter</td>
			<td>CLS</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Introducing spindle-assembly factors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Neutravidin</td>
			<td>Sigma-Aldrich</td>
			<td>A2666</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP (Adenosine-&#39;5-triphosphate, disodium salt hydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>PEP (Phospho(enol)pyruvic acid monosodium salt hydrate &#8805; 97% (enzymatic))</td>
			<td>Sigma-Aldrich</td>
			<td>P0564</td>
			<td></td>
		</tr>
		<tr>
			<td>PK/LDH -(Pyruvate kinase/lactic dehydrogenase enzymes from rabbit muscle)</td>
			<td>Sigma-Aldrich</td>
			<td>P0294</td>
			<td></td>
		</tr>
	</tbody>
</table>

reconstitution of basic mitotic spindles in spherical emulsion droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

The methods presented in the previous sections allow the reconstitution of spindle-like structures with increasing complexity using the geometrical confinement of water-in-oil emulsion droplets. This section describes representative results that qualitatively demonstrate the capability of these assays.
During mitosis, when the bipolar spindle is assembled, cells round up to form spheres with a diameter of roughly 15 &#181;m, as measured for human cells. This characteristic mitotic cell shape provides a geometrical boundary that both restricts and directs spindle size and orientation35,36. Microfluidic techniques, provide a level of geometrical confinement that accurately resembles the situation observed in living cells and therefore permits the bottom-up reconstruction of mitotic spindles in vitro.
Microtubule asters are by themselves already capable of complex behavior when microtubule growth is restricted by geometrical boundaries. As microtubules grow, pushing forces generated by the incorporation of new tubulin dimers drive the two centrosomes to opposing sides of the emulsion droplets. At first, centrosomes freely diffuse within the confined volume (Figure 4a, left panel). After about 20-30 min, the first microtubules become visible and centrosome diffusion becomes restricted as microtubules grow against the cortex in all directions. Asters with microtubules of intermediate length (roughly 50% of the droplet diameter) can (sterically) repel each another, with microtubules pushing against each other, the other centrosomes and the cortex. This results in a typical &#39;bipolar&#39; spindle-like arrangement with the two centrosomes opposing each other (Figure 4a, middle panel). When microtubules grow longer than ~50% of the droplet-diameter, the centrosomes get pushed further to opposing boundaries of the droplet, with microtubules growing along the droplet cortex (Figure 4a, right panel). It is important to note that microtubule growth rates in these assays are very sensitive to both temperature and tubulin-concentrations. These parameters will therefore strongly affect the time when nucleation is first observed and when steady-state aster positions are reached.
In cells, cortical pulling forces are generated through the formation of load-bearing attachments between microtubule plus-ends and cortex-associated dynein. In animal cells, this association depends on the G&#945;i/LGN/NuMA complex, which is targeted to the plasma membrane via N-terminal myristoylation of G&#945;i1. In these reconstitution assays, the requirement of the G&#945;i/LGN/NuMA complex is bypassed by directly coupling dynein to biotinylated lipids through the formation of biotin-streptavidin-biotin complexes. These bonds are relatively stable (KD ~10-14 M)37 and form rapidly (usually within 10 min after emulsion droplet formation, before microtubule nucleation becomes apparent) (Figure 4b). In the presence of cortical dynein, centrosomes typically retain a more central position, whereas in the absence of dynein, centrosomes are pushed to opposite sides of the droplet cortex (Figure 4c). We reason this is the result of two effects, which prevent and counteract microtubule pushing forces. (1) Dynein directly promotes microtubule catastrophes, thereby restricting microtubule length and preventing excessive microtubule buckling, and (2) cortical pulling forces lead to net centering forces on the individual asters8, which counteract the aster-aster repulsion forces.
The diffusible cross-linker Ase1 induces forces that tend to increase the overlapping region of anti-parallel microtubules29. Consistent with this, in the presence of Ase1 (and absence of dynein) centrosomes are found close together with Ase1 localizing to bundled interpolar microtubules (Figure 4d). Members of the kinesin-5 family drive centrosome separation by providing pushing forces from within the spindle (see introduction). In the presence of Cut7 (the S. pombe kinesin-5 ortholog), centrosomes are pushed to opposite sides of the emulsion droplets, even in the presence of Ase1 (Figure 4e). By combining cortical and inter-polar force generators, the level of complexity can be further increased in these experiments, eventually leading to a comprehensive understanding of bipolar mitotic spindle assembly. A detailed quantitative description of these results will be available in the future (Roth et al., manuscript in preparation).
In addition to observing the position of the centrosomes at fixed moments in time, and relating their behavior to the observed lengths of the microtubules, valuable information can be obtained by following the positioning process over time. By reducing exposure times and laser intensities, it is now possible to follow aster positioning at 2 min intervals for at least 1 hr with only ~30% bleaching. This allows monitoring of aster assembly and positioning from freely diffusing centrosomes (0 min.) via centralized (12 min.) to decentralized asters (36 min and further) (Figure 5c). This facilitates the study of both steady-state behavior and spindle assembly kinetics. By combining droplets with different contents in the same sample, it is, for example, possible to directly compare centrosome positioning and spindle assembly kinetics in droplets with and without cortical force generators (Figure 5b).
<img alt="Figure 1" src="/files/ftp_upload/54278/54278fig1.jpg" /> 
Figure 1: Forces Acting on the Mitotic Spindle. Several force-generating molecules act on microtubules of the mitotic spindle to promote spindle formation and positioning. Microtubules that grow into the cell cortex generate a pushing force on the centrosome. Cortical dynein (purple) captures depolymerizing microtubules and generates a pulling force on the centrosomes. Within the spindle, Kinesin-5/Cut7 motor proteins provide an outward sliding force on anti-parallel microtubules, whereas cross-linkers of the PRC1/Ase1 family generate an opposing outward sliding force. <a href="https://www.jove.com/files/ftp_upload/54278/54278fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54278/54278fig2.jpg" /> 
Figure 2: Microfluidic Chip Design. (A) Design of 4 inch photomask containing 4 microfluidic chips. (B) Detailed representation of a single chip. Chips contain one outlet channel and three inlet channels followed by a dust-filter. Inlet channel 1 contains the water-phase, channel 2 the lipid/oil-phase and channel 3 can be used to dilute the formed droplets with additional lipid/oil-phase. (C) Detailed representation of the junction where the lipid/oil-phase (coming from the top and bottom) meets with the water-phase (coming from the left). At the junction, droplets will form and flow towards the outlet channel on the right. (D) Detailed representation of a dust-filter with 2 &#181;m channels. <a href="https://www.jove.com/files/ftp_upload/54278/54278fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54278/54278fig3.jpg" /> 
Figure 3: Methodology for Water-in-Oil Emulsion Droplet Formation. (A) Microfluidic chip and microfluidics tubing on a bright-field microscope. Both the water- and lipid/oil-phase tubes are connected to the chip inlets 1 and 2 respectively. (B) Restriction on microfluidics chip where the water- and lipid/oil-phases meet. By changing the pressures, droplets size can be controlled. (C) Design of PDMS-coated flow-cells. After loading the droplets into the flow-cell, the open ends are closed with additional Valap. (D) Schematic of droplet formation. Droplets are used to encapsulate centrosomes, tubulin and additional components required for mitotic spindle formation. (E) Schematic of cortical-dynein targeting. Biotinylated (Bio) dynein is targeted to biotinylated lipids through streptavidin (Strp). <a href="https://www.jove.com/files/ftp_upload/54278/54278fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54278/54278fig4.jpg" /> 
Figure 4: Representative Results of Spindle-Reconstitutions with Increasing Complexity. (A) Maximum intensity projections of droplets containing two centrosomes showing examples of short, intermediate, and long microtubules. Centrosomes and microtubules are visualized by the addition of 10% HiLyte-488 labeled Tubulin. Scale bars = 10 &#181;m. (B) Localization of GFP-biotin-dynein-TMR in the absence (-, left) and presence (+, right) of streptavidin (Strp). (C) Microtubule aster positioning in the absence (-, left) or presence (+, right) of cortical GFP-biotin-dynein-TMR. (D) Microtubule aster (left panel, green) positioning in the presence of Ase1 (middle panel, red). (E) Microtubule aster (left panel, green) positioning in the presence of Ase1 and Cut7 (kinesin-5) (middle panel, red). <a href="https://www.jove.com/files/ftp_upload/54278/54278fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54278/54278fig5.jpg" /> 
Figure 5: Imaging Aster Positioning Dynamics in vitro. (A) Design of a PDMS cup that allows droplet imaging for up to several hr. (B) Generation, storage and imaging of droplets (transmitted) containing different contents, illustrated by absence (left) inclusion (right) of fluorescent dextran (Alexa 647). (C) Single plane images of a 60 min time-lapse (2 min intervals) taken from a z-stack (2 &#181;m distances) of a droplet containing a single centrosome. <a href="https://www.jove.com/files/ftp_upload/54278/54278fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets at JoVE.com

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...

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9.7K Views.  Delft University of Technology. The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

Video: Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Title Cell Encapsulation by Droplets

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.