Periodic Reporting for period 1 - ALTER e-GROW (Strategies of 3-D growth in brown algae)
Berichtszeitraum: 2023-02-01 bis 2025-07-31
Living organisms come in all shapes and sizes. Most grow in thickness (animals, plants), but some develop in only one dimension (1D), such as filaments with cells stacked on top of each other, or in two dimensions (2D), such as a disc made of a single layer of cells. In contrast, multicellular algae, mosses, and fungi are organisms that can limit their growth to one or two axes.
During embryogenesis, brown algae develop according to specific spatio-temporal patterns. In space, they grow first in one dimension, then in two, before some of them shift to three dimensions. In time, the stages during which the cells divide along the same axes last several days. This way of developing 3D embryos is unique among multicellular organisms. and brown algae show a high diversity in this mode of 3D body construction: the time at which the cell division orientations are switched, the duration of the 1D or 2D growth periods, and the order in which the body axes are set all differ between species.
The aim of the project is to identify the mechanisms that control 3D growth. In particular, the project will focus on the mechanisms allowing cells to maintain the same division axis and those that trigger cell division in another spatial dimension. It will also investigate whether these mechanisms are similar between four different algae, and to what extent they can be compared with those developed by plants, animals or fungi.
Three lines of research will be developed. In line 1, the growth of brown algae embryos needs to be observed over time and in 3D. This will require 4D microscopy technologies such as light sheet and multiphoton microscopy, together with the development of fluorescent vital probes to mark the outlines of cells specifically involved in establishing 3D growth. It will also require a better understanding of cytoskeletal dynamics and the mapping of anisotropic markers, such as chemical or mechanical markers of the cell wall. In line 2, cell growth and division will be modelled based on the cellular data obtained in line 1. We will use microtubule dynamics models to simulate the position of the cell division plane and viscoplastic models of the cell wall to simulate cell growth. In line 3, the models will be tested by changing the experimental conditions, e.g. by applying mechanical forces (turgidity, compression) or by modifying the dynamics of cell growth and division (e.g. by genetic mutation or drug-mediated inhibition).
During embryogenesis, brown algae develop according to specific spatio-temporal patterns. In space, they grow first in one dimension, then in two, before some of them shift to three dimensions. In time, the stages during which the cells divide along the same axes last several days. This way of developing 3D embryos is unique among multicellular organisms. and brown algae show a high diversity in this mode of 3D body construction: the time at which the cell division orientations are switched, the duration of the 1D or 2D growth periods, and the order in which the body axes are set all differ between species.
The aim of the project is to identify the mechanisms that control 3D growth. In particular, the project will focus on the mechanisms allowing cells to maintain the same division axis and those that trigger cell division in another spatial dimension. It will also investigate whether these mechanisms are similar between four different algae, and to what extent they can be compared with those developed by plants, animals or fungi.
Three lines of research will be developed. In line 1, the growth of brown algae embryos needs to be observed over time and in 3D. This will require 4D microscopy technologies such as light sheet and multiphoton microscopy, together with the development of fluorescent vital probes to mark the outlines of cells specifically involved in establishing 3D growth. It will also require a better understanding of cytoskeletal dynamics and the mapping of anisotropic markers, such as chemical or mechanical markers of the cell wall. In line 2, cell growth and division will be modelled based on the cellular data obtained in line 1. We will use microtubule dynamics models to simulate the position of the cell division plane and viscoplastic models of the cell wall to simulate cell growth. In line 3, the models will be tested by changing the experimental conditions, e.g. by applying mechanical forces (turgidity, compression) or by modifying the dynamics of cell growth and division (e.g. by genetic mutation or drug-mediated inhibition).
The first task was to successfully produce 3D images of the cells of the four brown algae with different 3D growth strategies, which was not possible at the start of the project. To achieve this, we worked with chemists to characterise two key fluorescent dyes specific to the cell wall and plasma membrane, allowing live brown algae cells to be observed in 3D over several days. This is a major advance, as the ability to image live cells in 3D during the development of brown seaweed embryos was a sine qua non for the success of the project.
The second challenge was to develop 4D imaging techniques that would allow live embryos to be observed over several days. To grow the algae embryos under appropriate growth conditions (cool temperature, light intensity and photoperiod), additional technological developments of the light sheet and confocal/bi-photon microscopes were necessary, and recently carried out by the supplier. Thanks to the installation of a light ring to illuminate the algae with adjustable light intensity and photoperiod, and a cooling chamber capable of maintaining temperatures as low as 13°C, we can image cell growth and division in brown algae embryos in real time and over several days. The ability to rotate the sample through 360° during imaging is also a major advantage of this equipment, allowing a complete reconstruction of the embryo body in 3D.
These two technological developments, the development of fluorescent probes and the development of suitable 4D microscopy technologies, were major technical challenges at the outset of the project. Now that they have been solved, the project can move on to its core, the mechanical modelling of cell growth and division.
The second challenge was to develop 4D imaging techniques that would allow live embryos to be observed over several days. To grow the algae embryos under appropriate growth conditions (cool temperature, light intensity and photoperiod), additional technological developments of the light sheet and confocal/bi-photon microscopes were necessary, and recently carried out by the supplier. Thanks to the installation of a light ring to illuminate the algae with adjustable light intensity and photoperiod, and a cooling chamber capable of maintaining temperatures as low as 13°C, we can image cell growth and division in brown algae embryos in real time and over several days. The ability to rotate the sample through 360° during imaging is also a major advantage of this equipment, allowing a complete reconstruction of the embryo body in 3D.
These two technological developments, the development of fluorescent probes and the development of suitable 4D microscopy technologies, were major technical challenges at the outset of the project. Now that they have been solved, the project can move on to its core, the mechanical modelling of cell growth and division.
Vital probes that allow visualisation of the contours of living and growing cells open up the world of 3D in developing brown algae tissues. The impact of these probes can be significant for any cell biology project that requires a view of the cell as a whole and over a period of time during which important events take place (such as the switching of division planes).
Work done before the development of these probes, and therefore only in 2D, has shown that cells often divide along the shortest division plane, perpendicular to the longest cell axis and in the centre of the cell (Errera's rule). By modelling how microtubule growth can influence the orientation of cell division, we found that the dynamics of microtubules in a homogeneous cytosol very approximately account for the position and orientation of the cell division plane in apical and subapical cylindrical cells in the filament of the brown alga Sphacelaria and in cuboidal cells of the Saccharina embryo. Interestingly, preliminary simulations were able to simulate the tilting of the cell division plane during shape changes resulting from cell divisions, but only partially. Therefore, the mechanism of microtubule-mediated “cortical pushing” alone does not seem to be sufficient to explain the cell division patterns observed in these algae. Other factors must therefore modify the dynamic forces of the microtubules. The spatio-temporal pattern of cortical actin accumulation in these cells is currently being investigated.
Furthermore, models taking into account the mechanical properties of the cell wall have shown that isotropic growth of the Saccharina embryo lamina could result from a self-organising mechanism of cell division and growth, as long as a mechanical anisotropy is established and maintained at the level of the whole embryo. This anisotropy could be inherited from the previous embryonic stage where only transverse cell walls are formed. In addition to this 2D model, we are currently developing 3D simulation models of cell growth in response to tensile stress, fed by the 3D cell shape and growth data obtained from the microscopy techniques described above.
In short, the project has overcome the main challenge of obtaining the 3D shapes of cells in growing brown algae embryos. With these data, the project can now move on to the core of the project, which is to model the processes of alternating cell division orientations based on both microtubule dynamics and cellular mechanical factors.
Work done before the development of these probes, and therefore only in 2D, has shown that cells often divide along the shortest division plane, perpendicular to the longest cell axis and in the centre of the cell (Errera's rule). By modelling how microtubule growth can influence the orientation of cell division, we found that the dynamics of microtubules in a homogeneous cytosol very approximately account for the position and orientation of the cell division plane in apical and subapical cylindrical cells in the filament of the brown alga Sphacelaria and in cuboidal cells of the Saccharina embryo. Interestingly, preliminary simulations were able to simulate the tilting of the cell division plane during shape changes resulting from cell divisions, but only partially. Therefore, the mechanism of microtubule-mediated “cortical pushing” alone does not seem to be sufficient to explain the cell division patterns observed in these algae. Other factors must therefore modify the dynamic forces of the microtubules. The spatio-temporal pattern of cortical actin accumulation in these cells is currently being investigated.
Furthermore, models taking into account the mechanical properties of the cell wall have shown that isotropic growth of the Saccharina embryo lamina could result from a self-organising mechanism of cell division and growth, as long as a mechanical anisotropy is established and maintained at the level of the whole embryo. This anisotropy could be inherited from the previous embryonic stage where only transverse cell walls are formed. In addition to this 2D model, we are currently developing 3D simulation models of cell growth in response to tensile stress, fed by the 3D cell shape and growth data obtained from the microscopy techniques described above.
In short, the project has overcome the main challenge of obtaining the 3D shapes of cells in growing brown algae embryos. With these data, the project can now move on to the core of the project, which is to model the processes of alternating cell division orientations based on both microtubule dynamics and cellular mechanical factors.