Motor processivity and speed determine structure and dynamics of microtubule-motor assemblies
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Curated by eLife
Evaluation Summary:
This study investigates on how weight loss by bariatric surgery or weight-matched dietary intervention impairs breast cancer growth as well as immunotherapy. This study can potentially provide some therapeutic intervention strategies on combining vertical sleeve gastrectomy and immunotherapy in treating breast cancer.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Active matter systems can generate highly ordered structures, avoiding equilibrium through the consumption of energy by individual constituents. How the microscopic parameters that characterize the active agents are translated to the observed mesoscopic properties of the assembly has remained an open question. These active systems are prevalent in living matter; for example, in cells, the cytoskeleton is organized into structures such as the mitotic spindle through the coordinated activity of many motor proteins walking along microtubules. Here, we investigate how the microscopic motor-microtubule interactions affect the coherent structures formed in a reconstituted motor-microtubule system. This question is of deeper evolutionary significance as we suspect motor and microtubule type contribute to the shape and size of resulting structures. We explore key parameters experimentally and theoretically, using a variety of motors with different speeds, processivities, and directionalities. We demonstrate that aster size depends on the motor used to create the aster, and develop a model for the distribution of motors and microtubules in steady-state asters that depends on parameters related to motor speed and processivity. Further, we show that network contraction rates scale linearly with the single-motor speed in quasi-one-dimensional contraction experiments. In all, this theoretical and experimental work helps elucidate how microscopic motor properties are translated to the much larger scale of collective motor-microtubule assemblies.
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Author Response
Reviewer #3 (Public Review):
The size of the excitation region and the size of the aster are linearly correlated but are drastically different in size. This provokes several questions.
• Why does only one aster form if the region of excitation is over 10x the size? Why are there not multiple asters formed within this activation region?
• A much larger excitation diameter than the size of the resultant structure suggests the amount of dimeric motor is not limiting. Why then does the size of the aster increase with excitation diameter?
• A linear relationship between excitation region and aster size may suggest a constant density of material within the aster. While the intensity profile of a single aster is given in Fig 1C, the magnitude of intensity versus the estimated size of the aster would determine …
Author Response
Reviewer #3 (Public Review):
The size of the excitation region and the size of the aster are linearly correlated but are drastically different in size. This provokes several questions.
• Why does only one aster form if the region of excitation is over 10x the size? Why are there not multiple asters formed within this activation region?
• A much larger excitation diameter than the size of the resultant structure suggests the amount of dimeric motor is not limiting. Why then does the size of the aster increase with excitation diameter?
• A linear relationship between excitation region and aster size may suggest a constant density of material within the aster. While the intensity profile of a single aster is given in Fig 1C, the magnitude of intensity versus the estimated size of the aster would determine whether the system is reduceable purely to changes in size/radial distribution.
We thank the reviewer for the careful consideration of our work. In the experiments performed for this study, we were careful to be in a regime in which a single aster formed within the excitation region. However, by varying the concentration of components in the system, it is possible for multiple asters to form. See Figure R2 for example images of cases in which multiple asters formed.
The increase in aster size with excitation region was also described previously in Ross, et al. 2019. In this, we found that the aster size scales with the volume of the excitation region, suggesting that the number of microtubules is limiting to aster size. This supports the hypothesis that there may be a density limit to the microtubules, likely due to steric interactions between the microtubules. We clarified this and added reference to the Ross, et al. findings in lines 115-118, as follows:
“In Ross, et al., it was determined that the aster size roughly scaled with the volume of the excitation area, suggesting that the number of microtubules limits the size of the aster. This hints that there may be a density limit to the microtubules in an aster.”
Is dimerization reversible after activation? If the motors cannot unbind from each other, and act as crosslinkers (for as long as they remain bound) are they likely to accumulate within the aster over time? This may challenge the steady state assumption.
We thank the reviewer for the thoughtful analysis. Dimerization is reversible after activation
- the lifetime of the optogenetic bond is about 20 seconds (Guntas et al., 2015). In order to form an aster, we repeatedly activate the sample at 20 second intervals, so there is a balance between motors unbinding from each other and ones becoming dimerized. This balance can create a non-equilibrium steady state. We have clarified this in lines 78-80, as follows:
“The optogenetic bond lasts for about 20 seconds before reverting to the undimerized state, thus in our experiments, we repeatedly illuminate the sample every 20 seconds (Guntas, et al. 2015).”
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Evaluation Summary:
This study investigates on how weight loss by bariatric surgery or weight-matched dietary intervention impairs breast cancer growth as well as immunotherapy. This study can potentially provide some therapeutic intervention strategies on combining vertical sleeve gastrectomy and immunotherapy in treating breast cancer.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This study provides relatively convincing in vivo phenotype data in mice related to vertical sleeve gastrectomy (VSG) and provides some potential mechanistic insight. This study can potentially provide some therapeutic intervention strategies on combining VSG and immunotherapy in treating breast cancer. On the other hand, this paper also has some weaknesses especially related to the detailed molecular mechanism and characterization as described below:
1. The major weakness lies on the detailed characterization on which inflammatory response factors that may mediate the phenotype of HFS VSG mice when compared to WM Sham mice. The data presented currently is mainly limited to RNA-Seq data, which lacks detailed characterization.
2. The other significant weakness also is related to the descriptive nature on …
Reviewer #1 (Public Review):
This study provides relatively convincing in vivo phenotype data in mice related to vertical sleeve gastrectomy (VSG) and provides some potential mechanistic insight. This study can potentially provide some therapeutic intervention strategies on combining VSG and immunotherapy in treating breast cancer. On the other hand, this paper also has some weaknesses especially related to the detailed molecular mechanism and characterization as described below:
1. The major weakness lies on the detailed characterization on which inflammatory response factors that may mediate the phenotype of HFS VSG mice when compared to WM Sham mice. The data presented currently is mainly limited to RNA-Seq data, which lacks detailed characterization.
2. The other significant weakness also is related to the descriptive nature on characterizing the effect of immune features in Fig.4 for these mice. What is the potential mechanism on regulating T cell signaling or Cytolysis in HFS VSG mice vs WM sham mice? This at least needs some preliminary exploration and characterization.
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Reviewer #2 (Public Review):
This is a study based on the clinical observation that bariatric surgery in patients appears to be beneficial to reduce breast cancer risk. In mice with diet-induced obesity, followed by vertical sleeve gastrectomy (VSG) or dietary weight loss, tumor graft growth and response to immune checkpoint blockade were investigated. Bariatric surgery was found to be not as effective as dietary interventions in suppressing tumor growth despite achieving a similar extent of weight and adiposity loss. Leptin-mediated signaling was ruled out as a potential mechanism that could account for that difference. Notably, tumors in mice that received VSG displayed elevated inflammation and expression of the immune checkpoint ligand, PD-L1. In addition, mice that received VSG had reduced tumor-infiltrating T lymphocytes and …
Reviewer #2 (Public Review):
This is a study based on the clinical observation that bariatric surgery in patients appears to be beneficial to reduce breast cancer risk. In mice with diet-induced obesity, followed by vertical sleeve gastrectomy (VSG) or dietary weight loss, tumor graft growth and response to immune checkpoint blockade were investigated. Bariatric surgery was found to be not as effective as dietary interventions in suppressing tumor growth despite achieving a similar extent of weight and adiposity loss. Leptin-mediated signaling was ruled out as a potential mechanism that could account for that difference. Notably, tumors in mice that received VSG displayed elevated inflammation and expression of the immune checkpoint ligand, PD-L1. In addition, mice that received VSG had reduced tumor-infiltrating T lymphocytes and cytolysis suggesting an ineffective anti-tumor microenvironment. Anti-PD-L1 immunotherapy suppressed tumor progression after VSG but not in control obese mice. Genomic analysis of adipose tissue after bariatric surgery from both patients and mouse models revealed a conserved gene expression signature.
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Reviewer #3 (Public Review):
In this manuscript, the authors have investigated how weight loss by bariatric surgery or weight-matched dietary intervention impairs breast cancer growth. They have shown that post-bariatric surgery, the tumors show augmented inflammation and an immune checkpoint; PD-L1 expression, which suppresses the anti-tumor immune responses. In addition, anti-PD-L1 therapy in these mice has shown to be more effective at slowing tumor growth. The authors report interesting observations, and the findings are well supported by the data, however, the use of only one syngeneic model tampers the reviewer's enthusiasm. Overall, the study is clinically important and helps in stratifying obese breast cancer patients that may respond to anti-PD-L1 immune checkpoint inhibition.
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