A stable microtubule bundle formed through an orchestrated multistep process controls quiescence exit

Reviewer #1 (Public Review):

In their manuscript, Laporte et al. analyze the process of formation of the quiescent-cell nuclear microtubule (Q-nMT) bundle, a set of parallel MTs that emanate from the nuclear side of the spindle pole bodies (SPBs) upon the entry of Saccharomyces cerevisiae cells in quiescence. Based on their results, the authors propose that Q-nMT bundle formation is a multistep process that comprises three distinct sequential phases. The authors further evaluate the role of different factors during the growth of the Q-nMT bundle upon quiescence entry, as well as during the disassembly of this structure once cells resume their proliferation.

The Q-nMT is an interesting cellular structure whose physiological function is still widely unknown. Hence, providing new insights into the dynamics of Q-nMT bundle formation and identifying new factors involved in this process is an interesting topic of relevance in the field. The authors made a substantial effort in order to evaluate Q-nMT bundle formation and provide a considerable amount of data, mainly obtained from microscopy analyses. Overall, the experiments are well described and properly executed, and the data in the manuscript are clearly presented.

Despite the interest in the study, there are important issues that could affect the validity of the conclusions drawn in the manuscript. In this way, regarding the analysis of the dynamics of Q-nMT bundle formation, the experimental set up described in some of the experiments raises certain concerns, which mostly derive from the nocodazole treatments and the use of this microtubule-depolymerizing agent as the only approach to evaluate the stability of the Q-nMT bundle. On the other hand, regarding the factors involved in Q-nMT formation, the differences in microtubule length with the wild-type strain, despite being statistically significant, are really subtle for many of the mutants analyzed (e.g., bir1, slk19, etc.). Additionally, there are proteins that are proposed to participate in the process of Q-nMT formation and whose expression during quiescence needs to be demonstrated. Finally, although the cell viability defects observed for some of the mutants in these factors could be certainly associated with the lack of expression or mutation of the specific gene under evaluation, in none of the cases can they be directly attributed to a defect in aberrant Q-nMT bundle formation.

Based on the aforementioned reasons, and despite the considerable effort by the authors, it is my impression that many of the conclusions of the manuscript are not sufficiently justified by the data provided. Additional evidence, including the incorporation of key experimental controls that are currently missing, would be required in order to more strongly support the conclusions of the manuscript.

Reviewer #2 (Public Review):

Summary: The authors investigate the assembly of the Q-nMT, a stable microtubule structure that is assembled during quiescence. Notably, the authors show that the formation of the Q-nMT cannot be solely explained by changes in the physicochemical properties of quiescent cells. The authors report that Q-nMT assembly occurs in three regulated steps and identify kinesin motor proteins involved in the assembly and disassembly of the structure.

Strengths: The findings provide new insight into the assembly and possible function of the Q-nMT with respect to the response of haploid budding yeast to glucose starvation.

Weaknesses: The manuscript would benefit from more precise language and requires additional clarification regarding how claims are supported by the evidence. Clear definitions are also required, for example, "active process" is not defined. Some conclusions are not supported by the results, for example, the claim that the Q-nMT functions as a checkpoint effector that inhibits re-entry into the cell cycle.

Reviewer #3 (Public Review):

In this study, the authors analyzed a unique and very stable microtubule bundle that is formed in yeast cells entering quiescence. They show that the structure is required for yeast cells to maintain viability during quiescence and that it needs to be disassembled for cell cycle re-entry. They identify different stages during the assembly process and focus on the molecular players required for microtubule bundle formation and stabilization. They identify kinetochore as well as molecular motors such as auroraB, kinesin-14, and kinesin-5 that assemble, stabilize and crosslink the microtubules of the bundle. The paper also investigates the disassembly of the structure and shows that disassembly is required for cell cycle re-entry.

The study is very comprehensive, provides quantifications to support claims, and identifies various players involved in these processes, providing mechanistic insight. It also presents various control experiments to exclude alternative explanations and support the proposed model.

It is the first molecular-level insight into how this very stable microtubule structure can be assembled, maintained, and disassembled, and how this is coordinated with cell cycle exit and re-entry. This information may be very useful when analyzing similarly stable, microtubule-based structures in other organisms such as cilia in animals, which also display cell cycle-coordinated dynamics.

Overall, this is a nicely presented study that provides important insight into the field and beyond, but there are a few points that need to be addressed regarding methods used for quantifications and data presentation.