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><tbody><tr><td>&lt;strong&gt;1. Preparation of microfluidic chips&lt;/strong&gt;</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 &amp;lt;1-0-0&gt;10-20 &amp;Omega;-cm, 500-550 &amp;mu;m, SSP, with 2 flats)</td><td>WRS Materials</td><td>4P0SSP-005</td><td></td></tr><tr><td>Spin coater&amp;nbsp;</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>&lt;strong&gt;2. Microfluidic setup&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</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>24 mm x 60 mm, thickness 1.5</td></tr><tr><td>Glass-slides</td><td></td><td></td><td>26 mm x 76 mm</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&amp;nbsp;</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>&lt;strong&gt;3. Reconstituting spindle formation and positioning&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Dextran-647 nm (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-&#039;5-triphosphate, sodium salt hydrate)</td><td>Sigma-Aldrich</td><td>51120</td><td></td></tr><tr><td>&amp;kappa;-casein (&amp;kappa;-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>&lt;strong&gt;4. Introducing spindle-assembly factors&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Neutravidin</td><td>Sigma-Aldrich</td><td>A2666</td><td></td></tr><tr><td>ATP (Adenosine-&#039;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 &amp;ge;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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Biochemistry

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

Chemistry

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Self-Assembly of Microtubule Tactoids

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is especially the case during mitosis or meiosis, where the microtubules form the spindle during cell division. The spindle is the machinery used to segregate genetic material during cell division. Toward creating self-organized spindles in vitro, we recently developed a technique to reconstitute microtubules into spindle-like assemblies with a minimal set of microtubule-associated proteins and crowding agents. Specifically, MAP65 was used, which is an antiparallel microtubule crosslinker from plants, a homolog of Ase1 from yeast and PRC1 from mammalian organisms. This crosslinker self-organizes microtubules into long, thin, spindle-like microtubule self-organized assemblies. These assemblies are also similar to liquid crystal tactoids, and microtubules could be used as mesoscale mesogens. Here, protocols are presented for creating these microtubule tactoids, as well as for characterizing the shape of the assemblies using fluorescence microscopy and the mobility of the constituents using fluorescence recovery after photobleaching.

This article presents a protocol for the formation of microtubule assemblies in the shape of tactoids using MAP65, a plant-based microtubule crosslinker, and PEG as a crowding agent.

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell ...

Bioengineering

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the requirement of microfluidics to form controlled droplets imposes a barrier to non-experts, limiting their use. Here, we describe particle templated emulsification, an approach to generate monodisperse droplets without microfluidics. Using templating hydrogel spheres, we encapsulate samples in monodispersed droplets by simple vortexing. We demonstrate the approach by using it to perform microfluidic-free digital PCR.

Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays.

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the ...

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...

Impact of Cytoskeleton on Nuclear Shape and Condensate Dynamics in Mouse Oocytes

Capturing Cytoskeleton-Based Agitation of the Mouse Oocyte Nucleus Across Spatial Scales

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named oocytes. Their development is marked by cell growth and subsequent divisions, two critical phases that prepare the oocyte for fusion with sperm to initiate embryogenesis. During growth, oocytes reorganize their cytoplasm to position the nucleus at the cell center, an event predictive of successful oocyte development in mice and humans and, thus, their embryogenic potential. In mouse oocytes, this cytoplasmic reorganization was shown to be driven by the cytoskeleton, the activity of which generates mechanical forces that agitate, reposition, and penetrate the nucleus. Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear RNA-processing organelles known as biomolecular condensates. This protocol provides an experimental framework to document, with high temporal resolution, the impact of the cytoskeleton on the nucleus across spatial scales in mouse oocytes. It details the imaging and image analysis steps and tools necessary to evaluate i) cytoskeletal activity in the oocyte cytoplasm, ii) cytoskeleton-based agitation of the oocyte nucleus, and iii) its effects on biomolecular condensate dynamics in the oocyte nucleoplasm. Beyond oocyte biology, the methods elaborated here can be adapted for use in somatic cells to similarly address cytoskeleton-based tuning of nuclear dynamics across scales.

Author Spotlight: Elucidating the Dynamics of Mechano-Transduction and Nuclear Agitation in Mouse Oocytes

This protocol provides an experimental framework to document the physical impact of the cytoskeleton on nuclear shape and the internal membrane-less organelles in the mouse oocyte system. The framework can be adapted for use in other cell types and contexts.

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named ...

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

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Chapters in this video

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

Self-Assembly of Microtubule Tactoids

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

Capturing Cytoskeleton-Based Agitation of the Mouse Oocyte Nucleus Across Spatial Scales