Introduction of a leaky stop codon as molecular tool in Chlamydomonas reinhardtii.

Expression of proteins in the chloroplast or mitochondria of the model green alga Chlamydomonas reinhardtii can be achieved by directly inserting transgenes into organellar genomes, or through nuclear expression and post-translational import. A number of tools have been developed in the literature for achieving high expression levels from the nuclear genome despite messy genomic integration and widespread silencing of transgenes. Here, recent advances in the field are combined and two systems of bicistronic expression, based on ribosome reinitiation or ribosomal skip induced by a viral 2A sequence, are compared side-by-side. Further, the small subunit of Rubisco (RBCS) was developed as a functional nuclear reporter for successful chloroplast import and restoration of photosynthesis: To be able to combine RBCS with a Venus fluorescent reporter without compromising photosynthetic activity, a leaky stop codon is introduced as a novel molecular tool that allows the simultaneous expression of functional and fluorescently tagged versions of the protein from a single construct

Supplementary Code from Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, short SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets, e.g. nuclear proteins such as cbp1 and mis16 and the centromere-specific histone protein cnp1. The SExY protocol optimizations increasing protein yield could be beneficial for many studies, i.e. when targeting low abundance proteins or studies that rely on genetic labelling for various reasons, e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality

SI figure 1 from Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, short SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets, e.g. nuclear proteins such as cbp1 and mis16 and the centromere-specific histone protein cnp1. The SExY protocol optimizations increasing protein yield could be beneficial for many studies, i.e. when targeting low abundance proteins or studies that rely on genetic labelling for various reasons, e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality

SI figure 2 from Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, short SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets, e.g. nuclear proteins such as cbp1 and mis16 and the centromere-specific histone protein cnp1. The SExY protocol optimizations increasing protein yield could be beneficial for many studies, i.e. when targeting low abundance proteins or studies that rely on genetic labelling for various reasons, e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality

SI figure 4 from Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, short SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets, e.g. nuclear proteins such as cbp1 and mis16 and the centromere-specific histone protein cnp1. The SExY protocol optimizations increasing protein yield could be beneficial for many studies, i.e. when targeting low abundance proteins or studies that rely on genetic labelling for various reasons, e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality

SI figure 3 from Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, short SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets, e.g. nuclear proteins such as cbp1 and mis16 and the centromere-specific histone protein cnp1. The SExY protocol optimizations increasing protein yield could be beneficial for many studies, i.e. when targeting low abundance proteins or studies that rely on genetic labelling for various reasons, e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality

Endosymbiont costs and benefits in a parasitoid infected with both Wolbachia and Cardinium

Theory suggests that maternally inherited endosymbionts can promote their spread and persistence in host populations by enhancing the production of daughters by infected hosts, either by improving overall host fitness, or through reproductive manipulation. In the doubly infected parasitoid wasp Encarsia inaron, Wolbachia manipulates host reproduction through cytoplasmic incompatibility (CI), but Cardinium does not. We investigated the fitness costs and/or benefits of infection by each bacterium in differentially cured E. inaron as a potential explanation for persistence of Cardinium in this population. We introgressed lines infected with Wolbachia, Cardinium or both with the cured line to create a similar genetic background, and evaluated several parasitoid fitness parameters. We found that symbiont infection resulted in both fitness costs and benefits for E. inaron. The cost was lower initial egg load for all infected wasps. The benefit was increased survivorship, which in turn increased male production for wasps infected with only Cardinium. Female production was unaffected by symbiont infection; we therefore have not yet identified a causal fitness effect that can explain the persistence of Cardinium in the population. Interestingly, the Cardinium survivorship benefit was not evident when Wolbachia was also present in the host, and the reproduction of doubly infected individuals did not differ significantly from uninfected wasps. Therefore, the results of our study show that even when multiple infections seem to have no effect on a host, there may be a complex interaction of costs and benefits among symbionts