Imaging spotlight – How to divide without a ring: a mechanical ratchet drives unilateral cytokinesis

In this paper highlight, Jan Brugués and Alison Kickuth discuss the microscopy and microrheology techniques that they used in their research dissecting the mechanics of cytokinesis.

What are the key results from your paper?

Many early embryonic cells divide without forming a closed actomyosin ring, raising a fundamental mechanical puzzle: how can an open-ended, contractile actin band remain stable and generate force to drive cytokinesis?

Using zebrafish embryos as a model system, we show that the solution to this puzzle is cell-cycle–dependent changes in cytoplasm rheology. During interphase, large microtubule asters stiffen the cytoplasm, mechanically anchoring the contractile band along its length and allowing it to grow despite having loose ends. In the subsequent M-phase, microtubule disassembly fluidizes the cytoplasm, enabling rapid furrow ingression. Repeated cycles of stabilization and fluidization act as a temporal mechanical ratchet, driving unilateral cytokinesis over several rapid cell cycles without ever forming a closed ring.

Which imaging and image analysis techniques have you used for this research?

To capture physical mechanisms of cytokinesis, we combined confocal spinning disk microscopy with mechanical perturbations and microrheology in vivo:

Spinning disk confocal microscopy opened the door to study actin and microtubule dynamics in large (~700 µm) zebrafish embryos across multiple cell cycles. In addition, we combined this imaging technique with several mechanical measurements and perturbations.

Femtosecond laser ablation with a far-red pulsed laser and a pulse picker allowed us to make multiple consecutive laser cuts while imaging microtubules and actin with a time resolution of 300 ms on the spinning disk confocal microscope. The laser ablation revealed that the band is anchored along its length, and that microtubules splay when the contractile band recoils, suggesting their mechanical role in supporting the band. In addition, it gave us insights into band tension across the cell cycle.

For rheological measurements, we built a magnetic tweezers setup to quantitatively infer cytoplasm material properties in the presence or absence of large microtubule structures spanning the cytoplasm (in interphase and M-phase, respectively), while simultaneously imaging microtubules.

Further, we equipped our spinning disk confocal microscope with an Impetux optical tweezers setup (yes, this is all on the same microscope). This method allowed us to validate and extend the rheology measurements from magnetic tweezers, providing frequency-resolved measurements of the elastic (G′) and viscous (G″) moduli of the cytoplasm.

Finally, we probed the cytoplasm rheology at a larger scale by using ferrofluid droplets. Because they are fluid, custom sizes of droplets can be injected into the cytoplasm using standard injection techniques. By applying a homogeneous magnetic field to the sample, we were able to see relative changes in droplet extension across the cell cycle at length scales relevant to furrow ingression.

Our image analysis approaches included bead tracking for magnetic tweezers, microtubule orientation analysis, as well as particle image velocimetry and kymograph measurements (for recoil analysis), carried out using Fiji, Matlab, and Python.

In summary, combining high-resolution imaging with a variety of mechanical measurements, we uncovered how cytoplasm mechanics power open-ended cytokinesis across rapid embryonic cell cycles.

Are there any technical tricks that you have learnt during doing this research?

One puzzling result arose from measuring rheology using both magnetic tweezers and optical tweezers. The creep response of the magnetic tweezers is well fitted with a rheological model that is fluid on long time scales, while optical tweezer measurements of  the elastic and viscous moduli (G’, G’’) are consistent with models that are solid on the long time scales. To directly check the consistency between these two types of measurements, we converted the creep response of the magnetic tweezer experiments into frequency dependent elastic and viscous moduli (G’, G’’). Remarkably, these two measurements were consistent in magnitude and trend, and both showed a ~3-fold increase of both the viscous and elastic moduli that was consistent for all measured frequencies. In light of this finding, we decided to use the measured G’ and G’’ as direct descriptors of the material properties, instead of fitting a rheological model. For the magnetic tweezers creep response, several models could fit the data (Jeffreys or Kelvin-Voigt fractional for example), with different mechanical interpretations that could be extrapolated at temporal scales beyond the measured time. We believe that the conversion to G’ and G’’ together with the optical tweezers gives a more accurate picture of the rheology of the system. We were excited to provide these complementary measurements that are typically performed separately within one system, and we hope that providing evidence of their consistency will also be a valuable contribution to the field. 

Are you open to researchers contacting you for collaboration on the methodology?

Of course, we are always open to collaborate with other groups, and we are also happy to provide our custom-built magnetic tweezer. 

Are there any advances in imaging that would help your research?

After completing the work for this paper, we updated our laser ablation system. Now we can make cuts by simply using a mirror to move the laser spot, whilst previously we used a custom software to move the sample during cuts. We are very excited about this upgrade and hope to do exciting science with it in the future!

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