Taking a LEAF out of the green lab book

With climate and environmental change a global concern, people and governments have looked to scientists to find ways to reduce carbon emissions and to live a more sustainable existence. In this context, scientific research does not only need to be reproducible and reliable, it must also itself be done sustainably. Both industrial research and academic research have huge carbon footprints: they need large capital equipment with high energy inputs, and day-to-day work in laboratories generates vast amounts of waste. For those working at the lab bench, the task of reducing the environmental impact of their work can appear daunting. But by setting data-driven objectives to reduce energy use and waste, and providing practical and realistic goals, we can carry out lab-based research more sustainably. Recently, the Biochemical Society hosted a webinar, titled ‘Environmental Sustainability in Biomedical Laboratories’ (Figure 1), outlining some of these sustainable labs initiatives, with an introduction to LEAF from Martin Farley. In this article, we hear from those working to implement lab sustainability programmes at the University of Oxford

The Needle Component of the Type III Secreton of Shigella Regulates the Activity of the Secretion Apparatus

Gram-negative bacteria commonly interact with eukaryotic host cells by using type III secretion systems (TTSSs or secretons). TTSSs serve to transfer bacterial proteins into host cells. Two translocators, IpaB and IpaC, are first inserted with the aid of IpaD by Shigella into the host cell membrane. Then at least two supplementary effectors of cell invasion, IpaA and IpgD, are transferred into the host cytoplasm. How TTSSs are induced to secrete is unknown, but their activation appears to require direct contact of the external distal tip of the apparatus with the host cell. The extracellular domain of the TTSS is a hollow needle protruding 60 nm beyond the bacterial surface. The monomeric unit of the Shigella flexneri needle, MxiH, forms a superhelical assembly. To probe the role of the needle in the activation of the TTSS for secretion, we examined the structure-function relationship of MxiH by mutagenesis. Most point mutations led to normal needle assembly, but some led to polymerization or possible length control defects. In other mutants, secretion was constitutively turned “on.” In a further set, it was “constitutively on” but experimentally “uninducible.” Finally, upon induction of secretion, some mutants released only the translocators and not the effectors. Most types of mutants were defective in interactions with host cells. Together, these data indicate that the needle directly controls the activity of the TTSS and suggest that it may be used to “sense” host cells

Evidence that a positive feedback loop drives centrosome maturation in fly embryos.

Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themselves. The PCM expands dramatically as cells prepare to enter mitosis (a process termed centrosome maturation), but it is unclear how this expansion is achieved. In flies, Spd-2 and Cnn are thought to form a scaffold around the mother centriole that recruits other components of the mitotic PCM, and the Polo-dependent phosphorylation of Cnn at the centrosome is crucial for scaffold assembly. Here, we show that, like Cnn, Spd-2 is specifically phosphorylated at centrosomes. This phosphorylation appears to create multiple phosphorylated S-S/T(p) motifs that allow Spd-2 to recruit Polo to the expanding scaffold. If the ability of Spd-2 to recruit Polo is impaired, the scaffold is initially assembled around the mother centriole, but it cannot expand outwards, and centrosome maturation fails. Our findings suggest that interactions between Spd-2, Polo and Cnn form a positive feedback loop that drives the dramatic expansion of the mitotic PCM in fly embryos

The function of Ana1 in centrosome assembly, and the regulation of centriole growth in Drosophila

Centrioles are microtubule-based structures that play a crucial role in the formation of centrosomes, cilia and flagella. Centriole defects are linked to several human diseases such as microcephaly, primordial dwarfism, cancer and ciliopathies. In Drosophila, Plk4, Sas-6, Ana2, Sas-4 and Asl form a conserved pathway that is essential for centriole assembly. Ana1 was identified as a potential centriole assembly protein in two genome-wide RNAi screens in Drosophila S2 cells, and ana1 mutant flies are uncoordinated, and their sperm are immotile. Thus, Ana1 appeared to have an important role in centriole assembly, but it was unclear how it functioned in the context of the known assembly pathway. The primary aim of my thesis was to characterise the function of fly Ana1 in vivo. I found that Ana1 is not essential for centriole assembly, but it helps recruit Asl to new-born centrioles to allow them to mature properly so that they can duplicate and organize mitotic PCM. Unexpectedly, I discovered that Ana1 also promotes centriole elongation in a dose-dependent manner. Several centriolar proteins can influence centriole length, but how they do so is unclear. In collaboration with several colleagues in the laboratory, we developed techniques that allowed us, for the first time, to monitor the kinetics of daughter centriole growth in Drosophila embryos. We found that Plk4 sets the size of the centriole cartwheel by enforcing an inverse relationship between the cartwheel growth rate and period. I found that Ana1 does not influence cartwheel growth, but I identified several other proteins that do so, most notably CP110 and Cep97, that normally bind to the distal end of the centrioles. These studies have provided several important new insights into how centriole length is regulated.</p

<i>Plp</i> mutant centrioles/basal bodies can recruit TZ proteins.

<p>(<b>A,B</b>) Micrographs show images from fixed WT (A) or <i>Plp</i> mutant (B) spermatocytes stained with anti-Asl antibodies to reveal the centrioles (<i>red</i>), and anti-GFP antibodies (<i>green</i>) to reveal the distribution of GFP-fusions to the TZ proteins MKS1, Cby, Cep290 and CC2D2A (as indicated). Although EM studies show that the vast majority of <i>Plp</i> mutant centrioles are not connected to the PM, all of the centrioles appear to organise TZ proteins in a manner that appears to be very similar to the WT centrioles that are forming a cilium. Note that in some instances, the clustering of the centrioles can make this difficult to visualise, as multiple, prematurely separated, centrioles can be clustered one on top of the other, as appears to be the case in the panel showing the localisation of Cby-GFP in the mutant cells (B). Scale bar = 1μm.</p

Centrioles are not positioned properly relative to the cortex in <i>Plp</i> mutant nota.

<p>(<b>A,B</b>) Images from videos of living WT (A) or <i>Plp</i> mutant (B) SOPs expressing Jupiter-mCherry to reveal the MTs (<i>magenta</i>) and Asl to reveal the centrioles (<i>green</i>). Time in minutes relative to nuclear envelope breakdown (NEB) (t = 0) is indicated. These images are taken from the same videos shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007198#pgen.1007198.g001" target="_blank">Fig 1A and 1B</a>, but shown from a side-on view to the spindle. <i>White</i> arrow indicates a centriole pair separating prematurely. (<b>C,D</b>) Graphs compare the behaviour of WT (<i>blue</i>) and <i>Plp</i> mutant (<i>red</i>) cells, as indicated. (<b>E,F</b>) Images from electron tomograms (ETs) of WT (E) or <i>Plp</i> mutant (F) pupal notum cells, highlighting the position of the centrioles (arrows) relative to the cell cortex (dotted <i>green</i> line). (<b>G</b>) Graph quantifies the centriole-to-cortex distance in pupal notum cells. Scale bar = 2μm (A, B) or 100 nm (E, F) * p < 0.05.</p

Basal bodies appear to be specified properly in <i>Plp</i> mutant sensory neurons, but they are mis-positioned.

<p><b>(A-C)</b> Images from videos of living WT or <i>Plp</i> mutant sensory organs, as indicated, expressing Jupiter-mCherry to reveal the MTs (<i>magenta</i>) and Cep-104-GFP to reveal the centrioles and basal bodies (<i>green</i>). Time in hours:minutes after puparium formation (APF) (t = 0) is indicated. <b>(D)</b> Graph charts the position of the brightest Cep-104-GFP containing centriole (that will become the basal body) relative to the cortex in a single WT (<i>blue</i>) or <i>Plp</i> mutant (<i>red</i>) sensory organ, illustrating how the centriole appears to gradually drift away from the cortex. <b>(E)</b> Graph quantifies the distance of the brightest Cep-104-GFP containing centriole from the cortex at 30:00h AFP (when ciliogenesis is normally complete) in WT (<i>blue</i>), <i>Plp</i> mutant (<i>red</i>) or <i>Plp</i> mutant rescued by PLP-GFP (<i>green</i>) nota. (F,G) Images show 3D-reconstructions from SBF-SEM data of the cells in a WT (F) or <i>Plp</i> mutant (G) pupal notum sensory organ. The Sensory Neuron (<i>blue</i>) sends an extension (that would normally contain the axoneme close to its tip) through the cell body of the Bristle Cell (<i>green</i>); the Support Cell (<i>magenta</i>) is illustrated in the images, on the left, but not on the right, which are also rotated by 90^o. The overall organisation of the <i>Plp</i> mutant organ is not detectably perturbed. Scale bar = 2μm (A, B) or 5μm (C) or 500 nm (F,G).</p