Final Report Summary - PALMSEP (Septin organisation by multiparameter photoactivated localisation microscopy)
The septins are a family of polymerising molecules that are essential for successful cell division. They have first been discovered in yeast, but are involved in cell division in humans as well, where they assemble a structure around the abscission site. How exactly this polymeric structure is assembled from the septin building blocks is unclear, but if assembly fails, cells fail to divide properly. In humans, various septin genes play important roles in several forms of cancer and neurodegenerative diseases. Understanding the basis for the polymerisation of septins into polymers is thus of fundamental importance in the cell biology of these diseases. The main objective of this study is thus to understand how individual septin molecules assemble to form filaments and how these filaments are organised in the striking ring-shaped structures which are visible both in mammalian cells and in yeast cells.
The building blocks of septin filaments are dimeric rods of 40-nm length which are assembled of several different septin proteins. The ring-shaped structures have a diameter of approximately 500 nm and are barely resolvable with fluorescence microscopy. Single-molecule localisation based superresolution microscopy is based on the activation and subsequent localisation of single fluorophores and reaches a resolution that is 10-20 fold higher than that of diffraction limited microscopes. In principle, the resolution should be sufficient to resolve the ends of the septin rods, which would allow us to directly see the orientation of the rods within the filaments.
To investigate the organisation of septin complexes we established single-molecule localisation based superresolution microscopy. The custom built microscope was designed for flexibility and stability and featured four laser lines for imaging and activation of photoswitchable fluorophores, active focus stabilisation, full computer control, three-dimensional resolution and two spectral channels for simultaneous dual-colour imaging. We developed a fast and comprehensive software package for analysis and visualisation of superresolution data.
The resolution of localisation microscopy depends mainly on the brightness of the fluorescent labels. To be able to use bright dyes instead of photoswitchable proteins while keeping the specificity of genetic tagging, we developed a new labelling strategy, the so-called nanobody labeling. Nanobodies are tiny antibodies which bind to green fluorescent protein (GFP)-labelled proteins with a very high affinity and which we labeled with a bright organic dye. With these nanobodies, any already existing GFP construct can directly be used for superresolution microscopy. For instance, we were able to measure superresolution localisation maps of more than 25 different proteins in budding yeast.
In yeast, septins assemble into amazing hourglass and ring-shaped structures. We investigated Cdc11, one of the septin proteins in yeast, in greater detail. Superresolution images with nanobodies showed filamentous structures, similar to what has been observed in electron microscopy. With dual-colour superresolution microscopy we investigated the spatial relationship between septin rings and the yeast cell wall. The brightness of the labels allowed us to perform three-dimensional superresolution imaging, which we used to investigate how the septin rings form at the mother bud neck in yeast. We found that these structures directly form as rings and not develop out of patches and could measure the dependence of the ring size on the development stage of the cell.
Our approach to three-dimensional (3D) superresolution imaging was not sufficient to resolve filaments at the side of the structures. To obtain data with maximal three-dimensional resolution we performed measurements using interference PALM in collaboration with Harald Hess (Howard Hughes Institute, VA, USA). Septin filaments were clearly visible in the 3D reconstructions. As a next step, we will perform a statistical analysis on these structures using spatial correlation analysis to determine the orientations of the individual septin rods. These results will clarify key aspects of the septin complexes in yeast cell division and will help us to establish a comprehensive model on the organisation and function of septin in yeast.
The building blocks of septin filaments are dimeric rods of 40-nm length which are assembled of several different septin proteins. The ring-shaped structures have a diameter of approximately 500 nm and are barely resolvable with fluorescence microscopy. Single-molecule localisation based superresolution microscopy is based on the activation and subsequent localisation of single fluorophores and reaches a resolution that is 10-20 fold higher than that of diffraction limited microscopes. In principle, the resolution should be sufficient to resolve the ends of the septin rods, which would allow us to directly see the orientation of the rods within the filaments.
To investigate the organisation of septin complexes we established single-molecule localisation based superresolution microscopy. The custom built microscope was designed for flexibility and stability and featured four laser lines for imaging and activation of photoswitchable fluorophores, active focus stabilisation, full computer control, three-dimensional resolution and two spectral channels for simultaneous dual-colour imaging. We developed a fast and comprehensive software package for analysis and visualisation of superresolution data.
The resolution of localisation microscopy depends mainly on the brightness of the fluorescent labels. To be able to use bright dyes instead of photoswitchable proteins while keeping the specificity of genetic tagging, we developed a new labelling strategy, the so-called nanobody labeling. Nanobodies are tiny antibodies which bind to green fluorescent protein (GFP)-labelled proteins with a very high affinity and which we labeled with a bright organic dye. With these nanobodies, any already existing GFP construct can directly be used for superresolution microscopy. For instance, we were able to measure superresolution localisation maps of more than 25 different proteins in budding yeast.
In yeast, septins assemble into amazing hourglass and ring-shaped structures. We investigated Cdc11, one of the septin proteins in yeast, in greater detail. Superresolution images with nanobodies showed filamentous structures, similar to what has been observed in electron microscopy. With dual-colour superresolution microscopy we investigated the spatial relationship between septin rings and the yeast cell wall. The brightness of the labels allowed us to perform three-dimensional superresolution imaging, which we used to investigate how the septin rings form at the mother bud neck in yeast. We found that these structures directly form as rings and not develop out of patches and could measure the dependence of the ring size on the development stage of the cell.
Our approach to three-dimensional (3D) superresolution imaging was not sufficient to resolve filaments at the side of the structures. To obtain data with maximal three-dimensional resolution we performed measurements using interference PALM in collaboration with Harald Hess (Howard Hughes Institute, VA, USA). Septin filaments were clearly visible in the 3D reconstructions. As a next step, we will perform a statistical analysis on these structures using spatial correlation analysis to determine the orientations of the individual septin rods. These results will clarify key aspects of the septin complexes in yeast cell division and will help us to establish a comprehensive model on the organisation and function of septin in yeast.
