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G.E. Bokinsky
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1
Into the Actin Cortex
A Step Towards Resolving Architecture, Dynamics & Mechanics of a Synthetic Cell's Reconstituted Actin Cortex
The actin cortex is a thin, dynamic, membrane-associated actin network present in most animal cells, where it plays a central role in cell shape, mechanical stability, and force transmission. However, the specific contributions of individual actin-binding proteins to cortex architecture, turnover, and mechanics are difficult to disentangle because many proteins act simultaneously within a mechanically active and biochemically complex environment. This study uses giant unilamellar vesicles (GUVs) as a minimal reconstituted system to investigate how two major actin nucleation pathways shape cortex organization, dynamics, and mechanical behavior. Two membrane-associated actin cortex systems were compared: an Arp2/3/VCA-nucleated branched cortex and a formin/profilin-nucleated linear cortex.
Confocal fluorescence microscopy showed that both systems could form cortex-like actin structures. Quantitative mesoscale organization analysis indicated that branched and linear cortices occupied distinct organizational states, differing in actin enrichment, cortical thickness, membrane localization, and spatial heterogeneity. Fluorescence recovery after photo-bleaching (FRAP) was then used to assess actin recovery in the lumen and cortex. Luminal FRAP revealed redistribution dynamics of fluorescent actin within the enclosed GUV lumen, while cortical FRAP shows limited recovery in both systems, suggesting low cortical turnover under the tested reconstitution conditions. During FRAP optimization, strong local photo-bleaching was found to mechanically perturb the cortex, causing shrinkage and contour deformation. This enabled an additional analysis of local and global mechanical responses, showing that local cortex damage could be accompanied by vesicle-scale deformation.
Together, these results show that branched and linear actin nucleation pathways generate distinguishable reconstituted cortex states with different mesoscale organization, apparent turnover behavior, and mechanical response. At the same time, the study highlights that FRAP-based turnover measurements must be interpreted carefully when photo-damage-induced mechanical perturbations are present. Exploratory plunge-freezing and dSTORM experiments further established first steps toward higher-resolution structural imaging, but also revealed current limitations in sample preparation and imaging conditions. Overall, this study provides a step toward understanding different actin nucleation pathways in light of their organization, dynamics, and mechanics in a minimal reconstituted environment, while laying groundwork for future nano-scale architectural visualization. ...
Confocal fluorescence microscopy showed that both systems could form cortex-like actin structures. Quantitative mesoscale organization analysis indicated that branched and linear cortices occupied distinct organizational states, differing in actin enrichment, cortical thickness, membrane localization, and spatial heterogeneity. Fluorescence recovery after photo-bleaching (FRAP) was then used to assess actin recovery in the lumen and cortex. Luminal FRAP revealed redistribution dynamics of fluorescent actin within the enclosed GUV lumen, while cortical FRAP shows limited recovery in both systems, suggesting low cortical turnover under the tested reconstitution conditions. During FRAP optimization, strong local photo-bleaching was found to mechanically perturb the cortex, causing shrinkage and contour deformation. This enabled an additional analysis of local and global mechanical responses, showing that local cortex damage could be accompanied by vesicle-scale deformation.
Together, these results show that branched and linear actin nucleation pathways generate distinguishable reconstituted cortex states with different mesoscale organization, apparent turnover behavior, and mechanical response. At the same time, the study highlights that FRAP-based turnover measurements must be interpreted carefully when photo-damage-induced mechanical perturbations are present. Exploratory plunge-freezing and dSTORM experiments further established first steps toward higher-resolution structural imaging, but also revealed current limitations in sample preparation and imaging conditions. Overall, this study provides a step toward understanding different actin nucleation pathways in light of their organization, dynamics, and mechanics in a minimal reconstituted environment, while laying groundwork for future nano-scale architectural visualization. ...
The actin cortex is a thin, dynamic, membrane-associated actin network present in most animal cells, where it plays a central role in cell shape, mechanical stability, and force transmission. However, the specific contributions of individual actin-binding proteins to cortex architecture, turnover, and mechanics are difficult to disentangle because many proteins act simultaneously within a mechanically active and biochemically complex environment. This study uses giant unilamellar vesicles (GUVs) as a minimal reconstituted system to investigate how two major actin nucleation pathways shape cortex organization, dynamics, and mechanical behavior. Two membrane-associated actin cortex systems were compared: an Arp2/3/VCA-nucleated branched cortex and a formin/profilin-nucleated linear cortex.
Confocal fluorescence microscopy showed that both systems could form cortex-like actin structures. Quantitative mesoscale organization analysis indicated that branched and linear cortices occupied distinct organizational states, differing in actin enrichment, cortical thickness, membrane localization, and spatial heterogeneity. Fluorescence recovery after photo-bleaching (FRAP) was then used to assess actin recovery in the lumen and cortex. Luminal FRAP revealed redistribution dynamics of fluorescent actin within the enclosed GUV lumen, while cortical FRAP shows limited recovery in both systems, suggesting low cortical turnover under the tested reconstitution conditions. During FRAP optimization, strong local photo-bleaching was found to mechanically perturb the cortex, causing shrinkage and contour deformation. This enabled an additional analysis of local and global mechanical responses, showing that local cortex damage could be accompanied by vesicle-scale deformation.
Together, these results show that branched and linear actin nucleation pathways generate distinguishable reconstituted cortex states with different mesoscale organization, apparent turnover behavior, and mechanical response. At the same time, the study highlights that FRAP-based turnover measurements must be interpreted carefully when photo-damage-induced mechanical perturbations are present. Exploratory plunge-freezing and dSTORM experiments further established first steps toward higher-resolution structural imaging, but also revealed current limitations in sample preparation and imaging conditions. Overall, this study provides a step toward understanding different actin nucleation pathways in light of their organization, dynamics, and mechanics in a minimal reconstituted environment, while laying groundwork for future nano-scale architectural visualization.
Confocal fluorescence microscopy showed that both systems could form cortex-like actin structures. Quantitative mesoscale organization analysis indicated that branched and linear cortices occupied distinct organizational states, differing in actin enrichment, cortical thickness, membrane localization, and spatial heterogeneity. Fluorescence recovery after photo-bleaching (FRAP) was then used to assess actin recovery in the lumen and cortex. Luminal FRAP revealed redistribution dynamics of fluorescent actin within the enclosed GUV lumen, while cortical FRAP shows limited recovery in both systems, suggesting low cortical turnover under the tested reconstitution conditions. During FRAP optimization, strong local photo-bleaching was found to mechanically perturb the cortex, causing shrinkage and contour deformation. This enabled an additional analysis of local and global mechanical responses, showing that local cortex damage could be accompanied by vesicle-scale deformation.
Together, these results show that branched and linear actin nucleation pathways generate distinguishable reconstituted cortex states with different mesoscale organization, apparent turnover behavior, and mechanical response. At the same time, the study highlights that FRAP-based turnover measurements must be interpreted carefully when photo-damage-induced mechanical perturbations are present. Exploratory plunge-freezing and dSTORM experiments further established first steps toward higher-resolution structural imaging, but also revealed current limitations in sample preparation and imaging conditions. Overall, this study provides a step toward understanding different actin nucleation pathways in light of their organization, dynamics, and mechanics in a minimal reconstituted environment, while laying groundwork for future nano-scale architectural visualization.
Master thesis
(2023)
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J.J. van Heuckelum, R.M. de Kruijff, P.L. Hagedoorn, I. Apachitei, G.E. Bokinsky
Implant-associated infections by antibiotic-resistant biofilm-forming pathogenic bacteria such as Staphylococcus aureus have become a growing concern as they are difficult to treat and lead to implant revision surgery. Implant coatings consisting of superparamagnetic iron oxide nanoparticles may prevent the attachment to and infection of implants by bacteria. However, these nanoparticles release iron, which is also a nutrient necessary for bacterial growth, and may inadvertently contribute to infection. In this thesis the uptake of iron from iron nanoparticles by S. aureus is investigated.
To achieve this goal, nanoparticles were characterized, separation methods were developed and growth curves were constructed to determine the influence of nanoparticles on the growth of S. aureus. The release and uptake of iron from nanoparticles was investigated through the irradiation of iron nanoparticles and the measurement of the remaining 59Fe after nanoparticle-supplemented growth.
It was found that only 3.7 ± 2.4% of nanoparticle iron was taken up by S.aureus after 24 hours, while 53 ± 12% of free iron was taken up. The percentage of iron that was taken up was found to be greater than the weight percentage of iron that was released from the nanoparticles.
While this demonstrates that iron remains with S. aureus after separation. It is not yet known whether this iron is truly internalized, or simply attached to the membrane. It is also not conclusively known whether nanoparticle iron contributes to determine bacterial growth. Therefore, future experiments should be conducted to determine the internalization and the subcellular distribution of radioactive nanoparticle iron and its effects on bacterial metabolism.
...
Implant-associated infections by antibiotic-resistant biofilm-forming pathogenic bacteria such as Staphylococcus aureus have become a growing concern as they are difficult to treat and lead to implant revision surgery. Implant coatings consisting of superparamagnetic iron oxide nanoparticles may prevent the attachment to and infection of implants by bacteria. However, these nanoparticles release iron, which is also a nutrient necessary for bacterial growth, and may inadvertently contribute to infection. In this thesis the uptake of iron from iron nanoparticles by S. aureus is investigated.
To achieve this goal, nanoparticles were characterized, separation methods were developed and growth curves were constructed to determine the influence of nanoparticles on the growth of S. aureus. The release and uptake of iron from nanoparticles was investigated through the irradiation of iron nanoparticles and the measurement of the remaining 59Fe after nanoparticle-supplemented growth.
It was found that only 3.7 ± 2.4% of nanoparticle iron was taken up by S.aureus after 24 hours, while 53 ± 12% of free iron was taken up. The percentage of iron that was taken up was found to be greater than the weight percentage of iron that was released from the nanoparticles.
While this demonstrates that iron remains with S. aureus after separation. It is not yet known whether this iron is truly internalized, or simply attached to the membrane. It is also not conclusively known whether nanoparticle iron contributes to determine bacterial growth. Therefore, future experiments should be conducted to determine the internalization and the subcellular distribution of radioactive nanoparticle iron and its effects on bacterial metabolism.