Actin gel dynamics:Matthias Bussonnier

Materials and Methods

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Materials and Methods

Buffers

G-Buffer

G-Buffer is used to conserve actin in the monomeric form. Actin is diluted in G-Buffer and kept on ice for at least 12 hours before further use. G-buffer is aliquoted and stored at -20°C. For weekly use it is thawed and conserved on ice for up to a week. G-buffer is never refrozen. pH is adjusted to 7.4.

Composition of G-Buffer:

  • 0.2 mM \(CaCl_2\)
  • 0.5 mM DTT (Dithiothreitol, or (2S,3S)-1,4-bis(sulfanyl)butane-2,3-diol)
  • 2.0 mM Tris (tris(hydroxymethyl)aminomethane or 2-Amino-2-hydroxymethyl-propan)
  • 0.2 µM ATP (Adenosine triphosphate)

Polymerisation Buffer

Polymerisation buffer or X-Buffer is used for the polymerisation of actin gels on beads as well as bead dilution and buffer cleaning. It is aliquoted and conserved at -20°C. During experiments, it is stored on ice for up to a week. X-Buffer is never refrozen.

Composition of X-Buffer :

  • 10 mM Hepes (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid)
  • 0.1 M \(KCl\)
  • 1 mM \(MgCl_2\)
  • 1 mM ATP (Adenosine triphosphate)
  • 0.1 mM \(CaCl_2\)

X-Buffer with BSA

Same as X-Buffer, with the addition of 1% BSA (10 mg/ml). BSA is used to prevent non specific adsorption. X-BSA buffer is used in place of X-Buffer for probe-beads conservation.

ATP-Mix Buffer

ATP-Mix buffer or simply Mix contains the required ATP for actin polymerisation. It is aliquoted and stored at -20°C. Kept on ice for weekly use. pH is adjusted to 7.4.

  • 12.0 mM ATP,
  • 20,0 mM DDT
  • 0.88 mM Dabco
  • 24.0 mM \(MgCl_2\)

Protein preparation

pWA (also called pVCA)

pWA is used as a nucleation promoting factor. It is expressed from Human pVCA (verprolin homology central and acidic domain) into Rosetta 2(DE3) pLysS (Novagen) Cell. Purified pWA is aliquoted and conserved at -80°C, never refrozen, and conserved on ice for daily use.

Actin

Actin and biotinylated actin are purchased from Cytoskeleton (Denver, CO, USA), and stored at -80°C. Fluorescent Alexa-488 actin is obtained from Molecular Probes, stored at -80°C, and prepared according to manufacturer recommendation.

Actin is stored in 5µL aliquots at a concentration of ~238 µM, and fluorescent actin in 3µL aliquots at a concentration of ~106 µM.

G-actin with 20% fluorescently labeled actin monomers is prepared the day before the experiment, by mixing 1 aliquot of actin with 1 aliquot of fluorescently labeled actin, and by diluting the mix with G-Buffer until the desired concentration is reached.

Profilin

Human profilin is expressed by competent cells and purified in our laboratory as described in [Carvalho et al. 13a]. Profilin is conserved at 4°C for a few months and kept on ice for daily use.

Arp2/3

Bovine Arp2/3, complex, is purchased from Cytoskeleton, prepared as recommended by the manufacturer, aliquoted at 1µM and conserved at -80°C. Aliquots are never refrozen and stored on ice for weekly use.

Capping protein

Mouse capping protein (CP; a1/b2) is purified as previously described in [Soeno et al. 98]. CP was a gift from Laurent Blanchoin.

Myosin II

Myosin II is purified from rabbit skeletal muscle and fluorescent myosin II is prepared as previously described in [SoareseSilva et al. 11]. The Myosin II functionality is confirmed by motility assays. Gliding speed shows an average of 4.5 + 1.5 µm/s (N = 27).

The working buffer for Myosin contains

  • 25 mM imidazole
  • 50 mM \(KCl\)
  • 70 mM sucrose
  • 1mM Tris
  • 2 mM \(MgCl_2\)
  • 1 mM ATP
  • 0.1 mM DTT
  • 0.02 mg/ml β-casein,

Then, pH is adjusted to 7.4. In the working buffer, myosin II forms monofilaments about 0.7 µm long, which roughly correspond to about 100 motors.

Lipids, reagent and proteins

Chemicals are purchased from Sigma Aldricht (St-Louis, Mo, USA, unless stated otherwise. EPC (l-\(\alpha\)-phosphatidylcholine) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl polyethylene glycol 2000] (biotinylated lipids), 1,2-dioleoyl-sn-glycero-3-phosphocholine are purchased from Avanti Polar Lipids (Alabaster, USA). Monomeric actin containing 10% or 20% of labeled Alexa-488 actin and 0.25 % of biotinylated actin is diluted in G-Buffer

Doublet preparation

Cell-sized liposomes are formed by electro formation [Angelova et al. 86]. A 20 µL mix of EPC lipids and PEG-biotin lipids (present at 0.1 %, mol ), at a 2.5 mg/ml in chloroform/methanol 5:3 concentration, is deposited on glass plates coated with ITO. Glass is then dried with nitrogen and placed under vacuum for 2 hours.

A chamber is formed, using the ITO plates with their conductive sides facing inside, then filled with sucrose buffer (200mM sucrose, 2mM Tris adjusted to a pH of 7.4). The. Chamber is finally sealed with hematocrit paste (Vitrex Medical, Denmark).

An alternate current voltage of 1V at 10 Hz is applied between the ITO-coated surfaces for 75 minutes, to form liposomes.

The same preparation is done a second time and, by adding 0.9µm sulphorhodamin to the sucrose buffer, the liposomes inside buffer are marked fluorescently.

The two solutions are mixed in order to have the inside buffer of half the liposomes marked in red, and being to be able to distinguish the interface in some of the formed doublets.

The formed liposomes are incubated 15 minutes with 160 nM streptavidin in order to get them coated with it. Streptavidin-coated liposomes tend to aggregate. The solution containing doublets is then diluted 30 times. Waiting 15 minutes increases the ratio doublets/single liposomes, by still avoiding the aggregates of more liposomes.

A bulk solution of 40 µM actin monomers — 10% fluorescent and 0.25% biotinylated — is diluted 40 times in a working buffer (25 mM imidazole, 50 mM KCl, 70 mM sucrose, 1mM Tris, 2 mM \(MgCl_2\), 1 mM ATP, 0.1 mM DTT, 0.02 mg/ml β-casein, adjusted at to a pH of 7.4) and polymerized for one hour. The adjunction of 1 µm of phalloidin after 1 hour prevents further depolymerisation.

Actin filaments are diluted to 0.1 µM (10x), mixed with streptavidin-coated doublets of liposomes, and incubated for 15 min. The mix is diluted 5 times to reduce the fluorescent background due to actin monomers in solution.

Bead Preparation

Carboxylated polystyrene beads (Polysciences, Philadelphia, PA) of 4.34 ± 0.239 μm (Standard deviation) diameter were used as actin-beads and probe-beads.

Beads are stored at 4°C.

Before being coated by BSA (probe-bead) or pWA (actin-bead), the bead solution is cleaned by centrifugation at 5000 rpm, 2min. After removing the supernatant, the pellet is resuspended in X-Buffer. This procedure is repeated twice.

Actin-Bead Preparation

Cleaned polystyrene beads are incubated for 20 min at 20°C under agitation with 2 μM pVCA. Centrifuged at 5000rpm 2min, the supernatant is removed and the pellet diluted 4 times in X-buffer. The beads are stored on ice for one day.

Probe Bead Preparation

Cleaned polystyrene beads are incubated under agitation with 10 mg/ml BSA at room temperature for 30 minutes. Passivated beads are then centrifuged, separated from supernatant, the pellet is resuspended in X-BSA buffer and stored at 4°C for weekly use.

Force indentation experiments

Preparation of sample

An equal amount of both actin and probe beads are placed in the polymerization mix consisting of :

  • 2µL BSA at 10%
  • 3µL of ATP-Mix Buffer
  • 1.5 µL Profilin (114µM)
  • 1 µL beads (50% actin-bead 50% probe bead)
  • 0.5 µL Arp2/3 (22,3 µM)
  • between 0 and 2 µL CP (0.5 µM)
  • Completed to 15 µL using X-Buffer.

5 µL of G-Actin (20% fluorescent) is then added to the previous mix. This moment marks the time t=0 for the experiment and recording. The experimental chamber is made of 2 coverslips, separated by VaLaP, which is a mix of vaseline (33%) Lanoline (33%) and Parafine(33%) in equal mass proportion. The chamber is prepared by gently depositing 20 µL of the final beads mix at the lower coverslip center and 4 drops of VaLaP where the corner of the upper (18x18mm) coverslip will rest. The VaLaP, acting as a spacer, prevents the sample from being squashed. The upper coverslip is then placed on top of the sample and the chamber is sealed with VaLaP.

QPD positioning and calibration of microscope

The prepared sample is placed on the microscope and a drop of water is deposited on top of the upper coverslip to assure the immersion of the light collecting objective. The collecting objective and the quadrant photodiode are placed on top of the sample (Optical tweezer).

The trapping laser is then aligned with the photodiode, checking in the meantime that no object is trapped during the process. The conjugation of the objective back focal plane with the AODs and the QPD, is optimized by adjusting the distance of both objectives with respect to the sample.

A trapping laser is positioned near the center of the microscope field of view, using the custom written LabView program (Fig 433). The QPD is adjusted in X and Y direction to \(\Delta X = \Delta Y = 0V\). This has to be done while no object is trapped in the laser focus.

Initial bead trapping

Two maximum strength traps (~50mW/trap) are created near the center of the microscope field of view, separated by 15 to 20 µm. The sample plane is then moved in the Z-direction, by displacing the 3D piezo controlled sample stage, to position the traps near the chamber middle plane. A temporary removal of the infrared filter from the microscope allows to see the trapping lasers reflection on both the upper and lower coverslips and to determine the localisation of the observation chamber middle plane .

../_images/frontend.png

Figure 433: Software interface responsible for controlling the optical tweezer. Sample image showing 2 polystyrene beads and a single trap (A, white cross) holding one bead. Cursors (B,C) are available to displace the optical trap(s). Cursors can control the position of the stage: X (D), Y (E, blue) and Z (E,red). The blue rectangle highlights the slider that allows the control of the traps power. The red rectangle highlights the area where the different parameters of the experiment can be set (approach speed and resting time at closest point). 3 indicators at the bottom of the screen indicate the voltage on the QPD.

The operator then captures one probe-bead and one actin-bead in each trap. Both types of beads can be recognized using fluorescent microscopy, as actin-beads, promptly covered with a fluorescent actin, can clearly be distinguished from the probe-beads, which remain dark. If two identical beads are trapped, one of the two traps can selectively be disabled or decreased in stiffness, letting the bead escape from it , and the procedure can be repeated.

The operator will then roughly move the two traps one micrometer in each direction, to check that the two beads are effectively trapped in the tweezer and that no external forces act on the beads.

For practical reasons, the traps are aligned along one of the principal axis of the AOD, before starting the indentation experiments.

Indentations

The operator sets the experiment parameters in the software:

  • Average bead radius,
  • Approach/Retraction Speed.
  • Resting Time
  • Laser Power

For each pair of actin/probe beads, the initial minimum approach distance of the traps is set to 5 to 8 µm, before doing a single indentation cycle. If the maximum measured force between the two beads is not higher than 8 to 10 pN, the minimum approach distance is reduced by 0.25 to 1 µm and the procedure repeated. Once the maximum force measured is in the 10-15pN range, the right distance is found and up to 10 automatic force-indentation experiments are performed (Fig 434) . Before each indentation, the software automatically does a “scan” of each bead, to ensure correct calibration. An indentation cycle has the following steps :

  • Probe trap is approaching the actin-bead at constant speed until the minimal approach distance has been reached.
  • At the minimal distance, the traps remain stationary for the predefined (typically 3 seconds) resting time.
  • Probe trap returns to its initial position at constant speed.
  • Cycle is repeated as many times as set.

During this cycle, the deflection of the laser induced by both probe-bead and actin-bead are recorded by the QPD.

After an indentation cycle is finished, the experimenter can try to perform the indentation on the actin-bead from another direction, or release the actin-bead, proceeding to a new one.

In case the indented actin network shows signs of inhomogeneity or symmetry breaking, the experiments are stopped and not taken into account for further analysis.

The date and time of each indentation cycle is recorded, to extract the time of polymerisation for each sample.

indent experiment

Figure 434: Schematic of indentation experiment. On the left is the actin-bead, covered with actin, in the static trap, on the right the probe-bead in the mobile trap. At the beginning of the experiment (A) the probe-bead is situated far from the actin-bead. During the approach phase (I) the moving trap approaches the static trap at 10µm/sec until it reaches the minimal approach distance (B). The moving trap stays at the minimal approach distance for 3sec (II), which constitutes the relaxation phase.C) The actin gels are relaxed, the distance between bead is smaller than on B. III), the moving trap retracts at 10 µm/sec back to its initial position.

Time Shared Optical Traps

The optical trap is built on an inverted microscope (Olympus, IX71) equipped with a fluorescence (200W mercury lamp, Osram, Munich, Germany). The sample is observed through an Olympus 60X water immersion objective (Olympus) with numerical aperture NA=1.2, that also serves at the entry point of the optical tweezer laser. The light source is an infrared fiber laser (\(\lambda=1064nm\), YLP-1-1064, IPG, Germany). The X, Y positionings and the trapping force stiffness are controlled by 2 Acousto Optic Deflectors (AODs, AA-Optoelectronics, France) that are placed in the conjugated plane of the objective back focal plane . Multiple traps can be achieved by switching the laser between multiple positions within a switching time, in the order of 5 µs, and resting on each position 20µs or more.

Light refracted by the trapped sample is collected by a 40X (N.A:0.9, Olympus) water immersion objective, and imaged on a quadrant photodiode (QPD) conjugated with the back focal plane of the light collection objective. Signals from the QPD (\(\Delta X, \Delta Y\) and \(\Sigma\)) are sampled at 500kHz, by a Digital To Analogic Aquisition card (NI PCIe-6363, National Instruments, Austin, Texas) and controlled by a custom written Labview software (National Instruments) coupled with Matlab (Mathworks, Natick, MA). Raw signals are preprocessed by binning all voltages measured during the laser resting time (typically 20µs, at one position). Finally, the mean and standard deviation for each trap visit is stored for further processing.

The trap stiffness is inferred from bead radius, laser power, number of present traps and controlled experiment data. In controlled experiments, the trap stiffness was calibrated using the power spectral density method, and was determined to be as high as 80 pN/µm at full laser power (119mW) for a single trap. In the case of multiplexing, both traps as used in this work, were calibrated before the experiment. The sample coarse positioning was achieved through a pair of micrometer precision screws, capable of translating the microscope stage in X and Y, and finer positionings in X,Y and Z directions with the help of a 3D piezo stage, with an accessible range of 80 µm in each direction and a sub-micrometer accuracy.

Oocyte

Oocyte obtention

Oocyte culture, collection, and micro injection?, were done at the College de France by Maria Almonacid.

Oocytes were collected from 11 to 15 week old mice (WT), fmn2-/- as previously described in [Holubcova et al. 13] and maintained in Prophase I in M2+BSA supplemented with 1µM Milrinone. Oocyte were then injected with cRNA, using a micro-injector Eppendorf FemtoJet. Imaging was carried out at \(37^\circ{}C\).

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