Reconstitution of actomyosin networks in cell-sized liposomes reveals distinct mechanical roles of cytoskeletal organization in membrane shape remodeling
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Abstract
The actin cortex, a thin layer of actomyosin network beneath the plasma membrane, regulates various cell functions by generating active forces and inducing membrane deformations, including blebs. Although upstream signaling is involved in regulating cell shape, the extent to which downstream actomyosin molecules can control the shape remains elusive. Here, using a minimal reconstituted system with a combination of agent-based computational model, we show that while actin-membrane coupling strength determines the magnitude of membrane deformation, its balance with actin network connectivity governs the bleb initiation mechanism, either by detachment of the cortex from the membrane or by rupture of the cortex. This balance also regulates whether single or multiple blebs form. Furthermore, both experiments and simulations suggest that not only the dense cortical network but also the sparse volume-spanning network actively contributes to regulating bleb number. These findings provide mechanical insights into how cells tune actin network organization to control their shape.
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spatial heterogeneity of bulk cytoskeletal networks affect the number and position of blebs
This is a very interesting idea. You nicely demonstrated that blebs can form from basic elements of the cytoskeleton. It seems that an idea that follows is that more 'control' or determination on whether or where to form the bleb comes from higher-level signaling and localization.
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“Multiple blebs”
Why do you think that there wasn't the clear distribution along the periphery in Fig 5g like you saw for your 2uM His-actinin control? Does this distribution have any impact on multiple bleb formation?
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coupled to the membrane
What relevance does this have to a physiological process? Do you think it is just that there are multiple mechanisms for blebs to form, or do you think they are initiated in certain ways dependent on context?
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contraction
Why do the blebs fade or resolve at t31 or t26 in figs 3e and 3f, respectively? The actin network seems to become even more concentrated with this event.
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distribution
But the distribution was significantly different at 2uM of each. Do you have concerns about the distribution at these lower concentrations?
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CH and CN
This seems mostly due to the methylcellulose, correct? I'm wondering if there is a way to determine the actual number of anchor points in the liposome? Perhaps some staining against the His tag? It might be interesting to see where deformations lie in relation to clusters of anchor points.
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liposomes
This is a really clever approach! I love how you separated out variables like this.
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F-actin is 1.4 μM
Do you also have the Kd of untagged actinin for F-actin? It could be nice to know if the tag has any impact on binding. I'm also curious if the membrane tethered actinin has a different affinity for actin filaments compared to free-floating actinin.
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ake place sequentially
What do you think is in the bottom right corner of the liposome in figure 1b? It looks like that's where there is a cluster of actin filaments - is that a premature bleb or something? It shows up right at the beginning, before myosin contractility.
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the membrane
This is a super beautiful figure! Very clear distinction.
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membrane and the corte
How much do the anchor points actually move within the membrane? Is it enough to generate just local changes in force, or larger scale changes in the overall relationship between the cortex and the membrane?
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