Cell division is the most important and common events in life. However, the entire mechanism of cell division is still mysterious for us. Recently, scientists (Carlos Garzon-Coral, Horatiu A. Fantana, Jonathon Howard) found A force-generating machinery that can maintain the spindle at the cell center during mitosis, and they published their findings on Science Magazine (Science 27 May 2016: Vol. 352, Issue 6289, pp. 1124-1127  DOI: 10.1126/science.aad9745). Here, let us to appreciate their wonderful article. (Don’t forget that Creative Biomart can provide you molecular tools in cell division research area.)

The position and orientation of the mitotic spindle determines the plane of cell division, which, in turn, determines how the cytoplasmic contents are partitioned to the daughter cells and how the daughter cells are localized within the tissue. After the spindle reaches the cell center before metaphase, its position and orientation must be precisely maintained until the cell enters anaphase. The molecular forces underlying the maintenance of spindle position and orientation are not known.

Although much is known about how force is generated by purified proteins and in cell extracts, little is understood about how molecular forces are integrated in vivo to serve complex cellular processes, such as spindle positioning. This is due to the difficulties of exerting and measuring forces in intact cells. Indeed, with the exception of the landmark paper by Nicklas in 1983 that measured forces associated with spindle elongation during anaphase, there has been no direct quantitative measurement of forces on mitotic spindles in cells.

Fig.1

To measure mitotic forces in vivo, we injected 1.0-μm–diameter superparamagnetic beads (9) and used magnetic tweezers (10) to exert calibrated forces of up to 200 pN to mitotic spindles in one- and two-cell Caenorhabditis elegans embryos, a model system for studying mitosis (Fig. 1A ). We applied forces of 20 to 60 pN to the centrosome at one of the spindle’s poles for up to 20 s during metaphase, when the spindle is in a relatively quiescent phase at the cell center (Fig. 1A). In response to force, the spindle rotated as the centrosome was displaced up to 3 μm from the anterior-posterior (A-P) axis (Fig. 1, B and C). Thus, it was possible to perturb the position and orientation of the spindle by using magnetic forces.

Fig. 2

The kinetics of spindle displacement indicated that the mitotic spindle is held at the cell center by viscoelastic forces. First, after the onset of the force, the centrosome moved with an approximately constant velocity during the first few seconds (Fig. 2A, average displacement), which suggested that the spindle is subject to viscous forces. Second, the displacement speed decreased after several seconds (Figs. 1C and 2A), which indicated that there is an elastic force (i.e., a spring) that opposes the external force. Third, after cessation of the force, the centrosome partially relaxed back toward its initial position (Figs. 1C and 2B), which suggested that the elastic element returns part of its stored mechanical energy. Finally, higher external forces were required to displace the centrosome through larger distances, as expected for an elastic element.

To estimate the stiffness of the elastic element, we fit the spindle’s rising phase (Fig. 2A) with a Voigt model (Fig. 2A, left inset), in which a spring and a viscous damper are in parallel. A curve fit of the data to the Voigt model gave a stiffness (κ) of 16.4 ± 2.1 pN/μm: 16 pN force, on average, was needed to displace the centrosome 1 μm from the A-P axis. We call the force the centering force and the stiffness the centering stiffness. The drag coefficient (γ) of the damper was 134 ± 27 pN·s/μm. The associated time constant (κ/γ) was 8.1 ± 1.5 s. The time constant of the relaxation phase was 14.5 ± 2.8 s (Fig. 2B, solid red line), longer than the rising phase. The dynamics of the spindle are very different from the dynamics of beads in the cytoplasm, which relax incompletely and much more quickly (0.65 ± 0.08 s) (Fig. 2B). Thus, a centering machinery opposes motion of the spindle away from the cell center and has viscoelastic properties distinct from those of the cytoplasm.

We propose that the centering machinery acts like a set of four damped springs that oppose movements transverse to the A-P axis (Fig. 2B, right inset, black). These springs orient the spindle so that when one centrosome is perturbed, the spindle pivots around the other centrosome (Fig. 1B). Correct orientation along the A-P axis ensures that the cleavage plane is perpendicular to the A-P axis during cytokinesis.

Fig. 3

As the cell cycle progresses from metaphase through anaphase, several morphological and mechanical changes take place. Concomitant with these changes, we found that the centering stiffness increased fivefold (Fig. 3A): During anaphase, forces on the order of 100 pN were required to displace the spindle 1 μm. These forces are similar in magnitude to the forces measured during chromosome segregation by Nicklas in grasshopper cells. An increase in the centering force may help to stabilize spindle position against high centrifugal forces that occur during the anaphase, such as those driving transverse oscillations.

 

In a remarkable adaptation of mechanical properties to cellular function, the magnitudes of the stiffness and damping of the centering machinery are ideally suited for cellular function. A centering spring with stiffness 16 pN/μm is rigid enough to stabilize the spindle against thermal forces: The displacement fluctuations of a spring due to Brownian motion have a standard deviation of √(k_BT/κ) where k_B is the Boltzmann constant and T is absolute temperature), which is about 16 nm for the single-cell embryo. Thus, the precision of centration is not limited by thermal fluctuations. Indeed, fluctuations from other sources, such as stochastic variation in the number of force generators (i.e., microtubules) with a predicted standard deviation of R/√M ≈ 1000 nm, are expected to exceed the thermal fluctuations.

On the other hand, the centering spring is compliant enough to allow adjustments of spindle position by a small number of motor proteins. During metaphase, the spindle moves through displacement d ≈ 3 μm along the A-P axis into the posterior half of the embryo to set up asymmetric cell division. If the centering stiffness is similar along the A-P axis as transverse to it (Fig. 2B, gray springs), which is reasonable given the symmetry of the microtubule asters, then such a posterior displacement requires a force imbalance of κd ≈ 50 pN. This could be exerted by as few as 10 to 20 cortical force generators. The drag coefficient is also well adapted. If it were much lower, then transient force imbalances due to motor stochasticity would not be smoothed out; if it were much higher, then it would prevent posterior displacement from being completed on the minute time scale.

In conclusion, a force-generating centration apparatus with spring-like properties maintains the spindle at the cell center. The centering stiffness is high enough to ensure the precise maintenance of spindle position against thermal and other fluctuations while spindle assembly is completed and the cell prepares for chromosome segregation. Yet it is low enough to allow force generators to fine-tune the position of the spindle to facilitate asymmetric cell division.

For more details, you can visit www.sciencemag.org.

 

Reference：

Carlos Garzon-Coral1, Horatiu A. Fantana1, Jonathon Howard, A force-generating machinery maintains the spindle at the cell center during mitosis, Science, Vol. 352, Issue 6289, pp. 1124-1127.
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