The positioning mechanics of microtubule asters in Drosophila embryo explants

Reviewer #1 (Public Review):

Summary:
This paper addresses the mechanisms positioning microtubule asters in Drosophila explants. Taking advantage of a genetic mutant, blocking the cell cycle in early embryos, the authors generate embryos with centrosomes detached from nuclei and then study the positioning mechanisms of such asters in explants. They conclude that asters interact via pushing forces. While this is an artificial system, understanding the mechanics of asters positioning, in particular, whether forces are pushing or pulling is an important one.

Strengths:
The major strength of this paper is the series of laser cutting experiments supporting that asters position via pushing forces acting both on the boundary (see below for a relevant comment) and between asters. The combination of imaging, data analysis and mathematical modeling is also powerful.

Weaknesses:
This paper has weaknesses, mainly in the presentation but also in the quality of the data which do not always support the conclusions satisfactorily (this might in part be a presentation issue).

In Figure 2, it is difficult for me to understand what is being tracked. I believe that the authors track the yolk granules (visible as large green blobs) and not lipid droplets. There is some confusion between the text, legends and methods so I could not tell. If the authors are tracking yolk granules as a proxy for hydrodynamics flows it seems appropriate to cite previous papers that have used and verified these methods. More notably, this figure is somewhat disconnected with the rest of the paper. I find the analysis interesting in principle but would urge the authors to propose some interpretation of the experiments in the context of their big-picture message. At this point, I cannot understand what the Figure adds.

In Figure 3, it is not surprising that the aster-aster interactions are different from interactions with the boundary which is likely more rigid. It is also hard to understand why the force and thus velocity should scale as microtubule length. This Figure should be better conceptualized. I think that it becomes clear at the end of the paper that the authors are trying to derive an effective potential to use in a mathematical model in Figure 5 to test their hypotheses. I think that should be told from the start, so a reader understands why these experiments are being shown.

The experiments in Figure 4 are very nice in supporting a pushing model. However, it would help if the authors could speculate what the single aster is pushing against in this experiment. The experiments reported in Figure 1 seemed to suggest that the aster mainly pushed against the boundary. In the experiments in Figure 4 do the individual asters touch the boundary on both sides? I think that readers need more information on what the extract looks like for those experiments.

Figure 4F could use some statistics. I doubt that the acceleration in the pink curves would be significant. I believe that the deceleration is and that is probably the most crucial result. Since the authors present only 3 asters pairs it is important to be sure that these conclusions are solid.

Reviewer #2 (Public Review):

Summary:
The aster, consisting of microtubules, plays important roles in spindle positioning and the determination of the cleavage site in animals. The mechanics of aster movement and positioning have been extensively studied in several cell types. However, there is no unified biophysical model, as different mechanisms appear to predominate in different model systems. In the present manuscript, the authors studied aster positioning mechanics in the Drosophila syncytial embryo, in which short-ranged aster repulsion generates a separation force. Taking advantage of the ex vivo system developed by the group and the fly gnu mutant, in which the nuclear number can be minimized, the authors performed time-lapse observations of single asters and multiple asters in the explant. The observed aster dynamics were interpreted by building a mathematical model dealing with forces. They found that aster dissociation from the boundary depends on the microtubule pushing force. Additionally, laser ablation targeting two separating asters showed that aster-aster separation is also mediated by the microtubule pushing force. Furthermore, they built a simulation model based on the experimental results, which reproduced aster movement in the explant under various conditions. Notably, the actual aster dynamics were best reproduced in the model by including a short-ranged inhibitory term when asters are close to the boundary or each other.

Strengths:
This study reveals a unique aster positioning mechanics in the syncytial embryo explant, which leads to an understanding of the mechanism underlying the positioning of multiple asters associated with nuclei in the embryo. The use of explants enabled accurate measurement of aster motility and, therefore, the construction of a quantitative model. This is a notable achievement.

Weaknesses:
The main conclusion that aster repulsion predominates in this system has already been drawn by the same authors in their recent study (de-Carvalho et al., Development, 2022). As the present work provides additional support to the previous study using different experimental system, the authors should emphasize that the present manuscripts adds to it (but the conceptual novelty is limited). The molecular mechanisms underlying aster repulsion remain unexplored since the authors were unable to identify specific factor(s) responsible for aster repulsion in the explant.

Specific suggestions:
Microtubules should be visualized more clearly (either in live or fixed samples). This is particularly important in Figure 4E and Video 4 (laser ablation experiment to create asymmetric asters).