DAAM mediates the assembly of long-lived, treadmilling stress fibers in collectively migrating epithelial cells in Drosophila
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Curated by eLife
Evaluation Summary:
This paper is of broad interest to readers who are interested in understanding cell migration and the cytoskeleton. It characterizes new behaviors of actin-based stress fibers in vivo during collective cell migration, and provides important observations that contribute to our fundamental understanding of these structures. The use of high-resolution live imaging in combination with Drosophila genetics and pharmacological inhibitors provides compelling data that supports the major claims of the paper.
(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. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
Stress fibers (SFs) are actomyosin bundles commonly found in individually migrating cells in culture. However, whether and how cells use SFs to migrate in vivo or collectively is largely unknown. Studying the collective migration of the follicular epithelial cells in Drosophila , we found that the SFs in these cells show a novel treadmilling behavior that allows them to persist as the cells migrate over multiple cell lengths. Treadmilling SFs grow at their fronts by adding new integrin-based adhesions and actomyosin segments over time. This causes the SFs to have many internal adhesions along their lengths, instead of adhesions only at the ends. The front-forming adhesions remain stationary relative to the substrate and typically disassemble as the cell rear approaches. By contrast, a different type of adhesion forms at the SF’s terminus that slides with the cell’s trailing edge as the actomyosin ahead of it shortens. We further show that SF treadmilling depends on cell movement and identify a developmental switch in the formins that mediate SF assembly, with Dishevelled-associated activator of morphogenesis acting during migratory stages and Diaphanous acting during postmigratory stages. We propose that treadmilling SFs keep each cell on a linear trajectory, thereby promoting the collective motility required for epithelial migration.
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Author Response:
Reviewer #1 (Public Review):
Sherrard and colleagues investigated the dynamics of stress fibers of the follicular epithelia during the migratory stages of egg chamber development. During the migratory stages of egg chamber development the follicular epithelium forms actin-based protrusions at the leading edge followed by a parallel array of stress fibers. As the follicular epithelia migrates along the basement membrane the encapsulates the egg chamber, the egg chamber rotates. At later stages of development the follicle cells stop migrating and the stress fibers are then used to form a contractile network which needed to help make the elongated shape of the egg. Using near total internal reflection microscopy, Sherrard and colleagues show that during this migratory period these stress fibers showed tread-milling …
Author Response:
Reviewer #1 (Public Review):
Sherrard and colleagues investigated the dynamics of stress fibers of the follicular epithelia during the migratory stages of egg chamber development. During the migratory stages of egg chamber development the follicular epithelium forms actin-based protrusions at the leading edge followed by a parallel array of stress fibers. As the follicular epithelia migrates along the basement membrane the encapsulates the egg chamber, the egg chamber rotates. At later stages of development the follicle cells stop migrating and the stress fibers are then used to form a contractile network which needed to help make the elongated shape of the egg. Using near total internal reflection microscopy, Sherrard and colleagues show that during this migratory period these stress fibers showed tread-milling behavior (growing in the front and shrinking in the back) and persisted for longer than the time for cell to travel at least one cell length (62%). Concomitant with these treadmilling stress fibers was the appearance of adhesions at the front of these stress fibers and their disappearance at the rear. This means that rather than just observing adhesions at the termini of the stress fibers they can be found all along their lengths. These dynamics were captured by the use of fluorescently tagged adhesion proteins (Paxillin,Talin) but could also been visualized by staining for endogenous proteins (betaPS integrin). The authors go on to correlate the treadmilling stress fibers with adhesion dynamics and observed that new adhesions are pulled reward, consistent with their maturing under tension. Blocking cell migration (with CK-666 or genetically with depletion of Abi) resulted in a conversion of these migratory (modular), tread-milling stress fibers to the conical stress fibers with adhesions on either termini rather than along the length. The authors go on to demonstrate the modular migratory stress fibers are dependent on the formin, DAAM, as depletion of this formin specifically led to 30% reduction in F-actin levels without affecting other actin-based structures. Furthermore, as the migratory phase of these follicle cells subsides, DAAM expression decreases with the more canonical stress fibers being dependent on the formin Dia.
Strengths:
The claims of the manuscript are well supported by the data presented. The analyses rely on the dynamics of stress fibers being observed in the context of organism in vivo. This is in contrast to other studies where stress fiber dynamics have been limited to tissue culture cells in 2D on a single extracellular matrix protein. The finding that stress fibers a) treadmill in this context and b) form adhesions along their length and not just their termini will likely have a big impact on our understanding of cell migration and the role of stress fibers in general. This study also elegantly capitalizes on Drosophila genetics in order to identify which formins involved in stress fiber formation and dynamics and clearly shows that there is developmental shift from DAAM to Dia as the follicular epithelium stops migrating. They also show that modular stress fibers are also dependent on behavior, and when cell migration is inhibited there is shift to canonical stress fibers, thus modular stress fiber formation is both DAAM-dependent and dependent on migration.
Weaknesses:
The 30% reduction in F-actin in the DAAM amorphic allele does suggest that there is something else contributing to the formation of modular stress fibers which is not surprising but could also be more thoroughly addressed in the manuscript. While the authors went through great lengths to quantify their observations. Many of their claims are based on qualitative observations in particular the descriptions of adhesion dynamics when correlated to modular stress fiber dynamics (Figure 4) and the shift from modular stress fibers to canonical stress fibers (Figure 5). The near total internal reflection microscopy offers them an opportunity to derive rate constants to focal adhesion dynamics, and quantify the change in stress fiber type would have strengthen the manuscript.
We measured the lifetimes of both stationary and sliding adhesions and reported these data in Figure 4E. We also related these lifetimes to the distance required for the cell to travel one cell length within the text to provide the reader with additional context for these values. Although it would be possible to derive rate constants for focal adhesion assembly and disassembly, the relative lifetimes of the two adhesion types seemed to be a more pertinent piece of information for this first description of treadmilling stress fiber assembly.
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Evaluation Summary:
This paper is of broad interest to readers who are interested in understanding cell migration and the cytoskeleton. It characterizes new behaviors of actin-based stress fibers in vivo during collective cell migration, and provides important observations that contribute to our fundamental understanding of these structures. The use of high-resolution live imaging in combination with Drosophila genetics and pharmacological inhibitors provides compelling data that supports the major claims of the paper.
(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. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
Sherrard and colleagues investigated the dynamics of stress fibers of the follicular epithelia during the migratory stages of egg chamber development. During the migratory stages of egg chamber development the follicular epithelium forms actin-based protrusions at the leading edge followed by a parallel array of stress fibers. As the follicular epithelia migrates along the basement membrane the encapsulates the egg chamber, the egg chamber rotates. At later stages of development the follicle cells stop migrating and the stress fibers are then used to form a contractile network which needed to help make the elongated shape of the egg. Using near total internal reflection microscopy, Sherrard and colleagues show that during this migratory period these stress fibers showed tread-milling behavior (growing in the …
Reviewer #1 (Public Review):
Sherrard and colleagues investigated the dynamics of stress fibers of the follicular epithelia during the migratory stages of egg chamber development. During the migratory stages of egg chamber development the follicular epithelium forms actin-based protrusions at the leading edge followed by a parallel array of stress fibers. As the follicular epithelia migrates along the basement membrane the encapsulates the egg chamber, the egg chamber rotates. At later stages of development the follicle cells stop migrating and the stress fibers are then used to form a contractile network which needed to help make the elongated shape of the egg. Using near total internal reflection microscopy, Sherrard and colleagues show that during this migratory period these stress fibers showed tread-milling behavior (growing in the front and shrinking in the back) and persisted for longer than the time for cell to travel at least one cell length (62%). Concomitant with these treadmilling stress fibers was the appearance of adhesions at the front of these stress fibers and their disappearance at the rear. This means that rather than just observing adhesions at the termini of the stress fibers they can be found all along their lengths. These dynamics were captured by the use of fluorescently tagged adhesion proteins (Paxillin,Talin) but could also been visualized by staining for endogenous proteins (betaPS integrin). The authors go on to correlate the treadmilling stress fibers with adhesion dynamics and observed that new adhesions are pulled reward, consistent with their maturing under tension. Blocking cell migration (with CK-666 or genetically with depletion of Abi) resulted in a conversion of these migratory (modular), tread-milling stress fibers to the conical stress fibers with adhesions on either termini rather than along the length. The authors go on to demonstrate the modular migratory stress fibers are dependent on the formin, DAAM, as depletion of this formin specifically led to 30% reduction in F-actin levels without affecting other actin-based structures. Furthermore, as the migratory phase of these follicle cells subsides, DAAM expression decreases with the more canonical stress fibers being dependent on the formin Dia.
Strengths:
The claims of the manuscript are well supported by the data presented. The analyses rely on the dynamics of stress fibers being observed in the context of organism in vivo. This is in contrast to other studies where stress fiber dynamics have been limited to tissue culture cells in 2D on a single extracellular matrix protein. The finding that stress fibers a) treadmill in this context and b) form adhesions along their length and not just their termini will likely have a big impact on our understanding of cell migration and the role of stress fibers in general. This study also elegantly capitalizes on Drosophila genetics in order to identify which formins involved in stress fiber formation and dynamics and clearly shows that there is developmental shift from DAAM to Dia as the follicular epithelium stops migrating. They also show that modular stress fibers are also dependent on behavior, and when cell migration is inhibited there is shift to canonical stress fibers, thus modular stress fiber formation is both DAAM-dependent and dependent on migration.
Weaknesses:
The 30% reduction in F-actin in the DAAM amorphic allele does suggest that there is something else contributing to the formation of modular stress fibers which is not surprising but could also be more thoroughly addressed in the manuscript. While the authors went through great lengths to quantify their observations. Many of their claims are based on qualitative observations in particular the descriptions of adhesion dynamics when correlated to modular stress fiber dynamics (Figure 4) and the shift from modular stress fibers to canonical stress fibers (Figure 5). The near total internal reflection microscopy offers them an opportunity to derive rate constants to focal adhesion dynamics, and quantify the change in stress fiber type would have strengthen the manuscript.
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Reviewer #2 (Public Review):
In the present study, Sherrard et al. analyze the collective cell migration in Drosophila ovary follicle cells. The major new findings are that stress fibers show a novel form treadmilling behaviour during the migration and that the formin DAAM is required for the assembly of these stress fibers.
The authors describe the mechanism of stress fiber treadmilling and the remodeling of adhesions, in vivo, in great detail and find that is exhibits some unique features compared to cell culture models. The present study is one of the first studies addressing stress fibers in vivo in collective cell migration. Overall, this is a very well written manuscript featuring a well thought out, complete and exciting story.
There are three main novel contributions made in this beautiful study: First it is one of very few …
Reviewer #2 (Public Review):
In the present study, Sherrard et al. analyze the collective cell migration in Drosophila ovary follicle cells. The major new findings are that stress fibers show a novel form treadmilling behaviour during the migration and that the formin DAAM is required for the assembly of these stress fibers.
The authors describe the mechanism of stress fiber treadmilling and the remodeling of adhesions, in vivo, in great detail and find that is exhibits some unique features compared to cell culture models. The present study is one of the first studies addressing stress fibers in vivo in collective cell migration. Overall, this is a very well written manuscript featuring a well thought out, complete and exciting story.
There are three main novel contributions made in this beautiful study: First it is one of very few examples of studies of stress fibers in an in vivo context. Applying the wealth of knowledge about stress fibers from cell culture models requires us to gain better understanding of how they behave in the context of the whole organism. The present study does exactly that. Second, there are not many examples of studies that specifically seek to understand stress fiber behaviour during collective cell migration. Third, it combines live imaging of stress fiber with the genetic toolkit available in Drosophila, in the process identifying the formin DAAM as an important regulator of stress fiber assembly, and identifying a novel form of treadmilling. The sort of approach used in this manuscript, combining organ culture, quantitative live imaging, and genetic manipulation pushes the field forward in new directions.
This study is largely descriptive, and in that is self-contained and does not require many additional experiments. Although "descriptive" is an adjective that is typically used to diminish the importance of a paper this is absolutely not the case here. The novelty and contribution of this manuscript lies in the powerful insights derived from a high quality, genetics based, quantitative description of the behaviour of stress fibers. Although stress fibers have been widely studied in cell culture models the relation between these studies and what is actually seen in intact whole animals remains to be established. The present manuscript goes a long way towards this goal.
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