Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint.

Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1Δ or mad2Δ strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation.

Determining growth rates from bright-field images of budding cells through identifying overlaps.

Much of biochemical regulation ultimately controls growth rate, particularly in microbes. Although time-lapse microscopy visualises cells, determining their growth rates is challenging, particularly for those that divide asymmetrically, like Saccharomyces cerevisiae, because cells often overlap in images. Here, we present the Birth Annotator for Budding Yeast (BABY), an algorithm to determine single-cell growth rates from label-free images. Using a convolutional neural network, BABY resolves overlaps through separating cells by size and assigns buds to mothers by identifying bud necks. BABY uses machine learning to track cells and determine lineages and estimates growth rates as the rates of change of volumes. Using BABY and a microfluidic device, we show that bud growth is likely first sizer- then timer-controlled, that the nuclear concentration of Sfp1, a regulator of ribosome biogenesis, varies before the growth rate does, and that growth rate can be used for real-time control. By estimating single-cell growth rates and so fitness, BABY should generate much biological insight.

PyOmeroUpload: A Python toolkit for uploading images and metadata to OMERO.

Tools and software that automate repetitive tasks, such as metadata extraction and deposition to data repositories, are essential for researchers to share Open Data, routinely. For research that generates microscopy image data, OMERO is an ideal platform for storage, annotation and publication according to open research principles. We present PyOmeroUpload, a Python toolkit for automatically extracting metadata from experiment logs and text files, processing images and uploading these payloads to OMERO servers to create fully annotated, multidimensional datasets. The toolkit comes packaged in portable, platform-independent Docker images that enable users to deploy and run the utilities easily, regardless of Operating System constraints. A selection of use cases is provided, illustrating the primary capabilities and flexibility offered with the toolkit, along with a discussion of limitations and potential future extensions. PyOmeroUpload is available from: https://github.com/SynthSys/pyOmeroUpload.

General calibration of microbial growth in microplate readers.

Optical density (OD) measurements of microbial growth are one of the most common techniques used in microbiology, with applications ranging from studies of antibiotic efficacy to investigations of growth under different nutritional or stress environments, to characterization of different mutant strains, including those harbouring synthetic circuits. OD measurements are performed under the assumption that the OD value obtained is proportional to the cell number, i.e. the concentration of the sample. However, the assumption holds true in a limited range of conditions, and calibration techniques that determine that range are currently missing. Here we present a set of calibration procedures and considerations that are necessary to successfully estimate the cell concentration from OD measurements.

Inferring time derivatives including cell growth rates using Gaussian processes.

Often the time derivative of a measured variable is of as much interest as the variable itself. For a growing population of biological cells, for example, the population's growth rate is typically more important than its size. Here we introduce a non-parametric method to infer first and second time derivatives as a function of time from time-series data. Our approach is based on Gaussian processes and applies to a wide range of data. In tests, the method is at least as accurate as others, but has several advantages: it estimates errors both in the inference and in any summary statistics, such as lag times, and allows interpolation with the corresponding error estimation. As illustrations, we infer growth rates of microbial cells, the rate of assembly of an amyloid fibril and both the speed and acceleration of two separating spindle pole bodies. Our algorithm should thus be broadly applicable.

A microfluidic system for studying ageing and dynamic single-cell responses in budding yeast.

Recognition of the importance of cell-to-cell variability in cellular decision-making and a growing interest in stochastic modeling of cellular processes has led to an increased demand for high density, reproducible, single-cell measurements in time-varying surroundings. We present ALCATRAS (A Long-term Culturing And TRApping System), a microfluidic device that can quantitatively monitor up to 1000 cells of budding yeast in a well-defined and controlled environment. Daughter cells are removed by fluid flow to avoid crowding allowing experiments to run for over 60 hours, and the extracellular media may be changed repeatedly and in seconds. We illustrate use of the device by measuring ageing through replicative life span curves, following the dynamics of the cell cycle, and examining history-dependent behaviour in the general stress response.

Unmixing of fluorescence spectra to resolve quantitative time-series measurements of gene expression in plate readers.

BACKGROUND: To connect gene expression with cellular physiology, we need to follow levels of proteins over time. Experiments typically use variants of Green Fluorescent Protein (GFP), and time-series measurements require specialist expertise if single cells are to be followed. Fluorescence plate readers, however, a standard in many laboratories, can in principle provide similar data, albeit at a mean, population level. Nevertheless, extracting the average fluorescence per cell is challenging because autofluorescence can be substantial. RESULTS: Here we propose a general method for correcting plate reader measurements of fluorescent proteins that uses spectral unmixing and determines both the fluorescence per cell and the errors on that fluorescence. Combined with strain collections, such as the GFP fusion collection for budding yeast, our methodology allows quantitative measurements of protein levels of up to hundreds of genes and therefore provides complementary data to high throughput studies of transcription. We illustrate the method by following the induction of the GAL genes in Saccharomyces cerevisiae for over 20 hours in different sugars and argue that the order of appearance of the Leloir enzymes may be to reduce build-up of the toxic intermediate galactose-1-phosphate. Further, we quantify protein levels of over 40 genes, again over 20 hours, after cells experience a change in carbon source (from glycerol to glucose). CONCLUSIONS: Our methodology is sensitive, scalable, and should be applicable to other organisms. By allowing quantitative measurements on a per cell basis over tens of hours and over hundreds of genes, it should increase our understanding of the dynamic changes that drive cellular behaviour.

The Drosophila actin regulator ENABLED regulates cell shape and orientation during gonad morphogenesis.

Organs develop distinctive morphologies to fulfill their unique functions. We used Drosophila embryonic gonads as a model to study how two different cell lineages, primordial germ cells (PGCs) and somatic gonadal precursors (SGPs), combine to form one organ. We developed a membrane GFP marker to image SGP behaviors live. These studies show that a combination of SGP cell shape changes and inward movement of anterior and posterior SGPs leads to the compaction of the spherical gonad. This process is disrupted in mutants of the actin regulator, enabled (ena). We show that Ena coordinates these cell shape changes and the inward movement of the SGPs, and Ena affects the intracellular localization of DE-cadherin (DE-cad). Mathematical simulation based on these observations suggests that changes in DE-cad localization can generate the forces needed to compact an elongated structure into a sphere. We propose that Ena regulates force balance in the SGPs by sequestering DE-cad, leading to the morphogenetic movement required for gonad compaction.

Fibroblast growth factor signalling controls successive cell behaviours during mesoderm layer formation in Drosophila.

Fibroblast growth factor (FGF)-dependent epithelial-mesenchymal transitions and cell migration contribute to the establishment of germ layers in vertebrates and other animals, but a comprehensive demonstration of the cellular activities that FGF controls to mediate these events has not been provided for any system. The establishment of the Drosophila mesoderm layer from an epithelial primordium involves a transition to a mesenchymal state and the dispersal of cells away from the site of internalisation in a FGF-dependent fashion. We show here that FGF plays multiple roles at successive stages of mesoderm morphogenesis in Drosophila. It is first required for the mesoderm primordium to lose its epithelial polarity. An intimate, FGF-dependent contact is established and maintained between the germ layers through mesoderm cell protrusions. These protrusions extend deep into the underlying ectoderm epithelium and are associated with high levels of E-cadherin at the germ layer interface. Finally, FGF directs distinct hitherto unrecognised and partially redundant protrusive behaviours during later mesoderm spreading. Cells first move radially towards the ectoderm, and then switch to a dorsally directed movement across its surface. We show that both movements are important for layer formation and present evidence suggesting that they are controlled by genetically distinct mechanisms.

Differential and overlapping functions of two closely related Drosophila FGF8-like growth factors in mesoderm development.

Thisbe (Ths) and Pyramus (Pyr), two closely related Drosophila homologues of the vertebrate fibroblast growth factor (FGF) 8/17/18 subfamily, are ligands for the FGF receptor Heartless (Htl). Both ligands are required for mesoderm development, but their differential expression patterns suggest distinct functions during development. We generated single mutants and found that ths or pyr loss-of-function mutations are semi-lethal and mutants exhibit much weaker phenotypes as compared with loss of both ligands or htl. Thus, pyr and ths display partial redundancy in their requirement in embryogenesis and viability. Nevertheless, we find that pyr and ths single mutants display defects in gastrulation and mesoderm differentiation. We show that localised expression of pyr is required for normal cell protrusions and high levels of MAPK activation in migrating mesoderm cells. The results support the model that Pyr acts as an instructive cue for mesoderm migration during gastrulation. Consistent with this function, mutations in pyr affect the normal segmental number of cardioblasts. Furthermore, Pyr is essential for the specification of even-skipped-positive mesodermal precursors and Pyr and Ths are both required for the specification of a subset of somatic muscles. The results demonstrate both independent and overlapping functions of two FGF8 homologues in mesoderm morphogenesis and differentiation. We propose that the integration of Pyr and Ths function is required for robustness of Htl-dependent mesoderm spreading and differentiation, but that the functions of Pyr have become more specific, possibly representing an early stage of functional divergence after gene duplication of a common ancestor.

Molecular dissection of Penelope transposable element regulatory machinery.

Penelope-like elements (PLEs) represent a new class of retroelements identified in more than 80 species belonging to at least 10 animal phyla. Penelope isolated from Drosophila virilis is the only known transpositionally active representative of this class. Although the size and structure of the Penelope major transcript has been previously described in both D. virilis and D. melanogaster transgenic strains, the architecture of the Penelope regulatory region remains unknown. In order to determine the localization of presumptive Penelope promoter and enhancer-like elements, segments of the putative Penelope regulatory region were linked to a CAT reporter gene and introduced into D. melanogaster by P-element-mediated transformation. The results obtained using ELISA to measure CAT expression levels and RNA studies, including RT-PCR, suggest that the active Penelope transposon contains an internal promoter similar to the TATA-less promoters of LINEs. The results also suggest that some of the Penelope regulatory sequences control the preferential expression in the ovaries of the adult flies by enhancing expression in the ovary and reducing expression in the carcass. The possible significance of the intron within Penelope for the function and evolution of PLEs, and the effect of Penelope insertions on adjacent genes, are discussed.

Live imaging of Drosophila gonad formation reveals roles for Six4 in regulating germline and somatic cell migration.

BACKGROUND: Movement of cells, either as amoeboid individuals or in organised groups, is a key feature of organ formation. Both modes of migration occur during Drosophila embryonic gonad development, which therefore provides a paradigm for understanding the contribution of these processes to organ morphogenesis. Gonads of Drosophila are formed from three distinct cell types: primordial germ cells (PGCs), somatic gonadal precursors (SGPs), and in males, male-specific somatic gonadal precursors (msSGPs). These originate in distinct locations and migrate to associate in two intermingled clusters which then compact to form the spherical primitive gonads. PGC movements are well studied, but much less is known of the migratory events and other interactions undergone by their somatic partners. These appear to move in organised groups like, for example, lateral line cells in zebra fish or Drosophila ovarian border cells. RESULTS: We have used time-lapse fluorescence imaging to characterise gonadal cell behaviour in wild type and mutant embryos. We show that the homeodomain transcription factor Six4 is required for the migration of the PGCs and the msSGPs towards the SGPs. We have identified a likely cause of this in the case of PGCs as we have found that Six4 is required for expression of Hmgcr which codes for HMGCoA reductase and is necessary for attraction of PGCs by SGPs. Six4 affects msSGP migration by a different pathway as these move normally in Hmgcr mutant embryos. Additionally, embryos lacking fully functional Six4 show a novel phenotype in which the SGPs, which originate in distinct clusters, fail to coalesce to form unified gonads. CONCLUSION: Our work establishes the Drosophila gonad as a model system for the analysis of coordinated cell migrations and morphogenesis using live imaging and demonstrates that Six4 is a key regulator of somatic cell function during gonadogenesis. Our data suggest that the initial association of SGP clusters is under distinct control from the movements that drive gonad compaction.

D-six4 plays a key role in patterning cell identities deriving from the Drosophila mesoderm.

Patterning of the Drosophila embryonic mesoderm requires the regulation of cell type-specific factors in response to dorsoventral and anteroposterior axis information. For the dorsoventral axis, the homeodomain gene, tinman, is a key patterning mediator for dorsal mesodermal fates like the heart. However, equivalent mediators for more ventral fates are unknown. We show that D-six4, which encodes a Six family transcription factor, is required for the appropriate development of most cell types deriving from the non-dorsal mesoderm - the fat body, somatic cells of the gonad, and a specific subset of somatic muscles. Misexpression analysis suggests that D-Six4 and its likely cofactor, Eyes absent, are sufficient to impose these fates on other mesodermal cells. At stage 10, the mesodermal expression patterns of D-six4 and tin are complementary, being restricted to the dorsal and non-dorsal regions respectively. Our data suggest that D-six4 is a key mesodermal patterning mediator at this stage that regulates a variety of cell-type-specific factors and hence plays an equivalent role to tin. At stage 9, however, D-six4 and tin are both expressed pan-mesodermally. At this stage, tin function is required for full D-six4 expression. This may explain the known requirement for tin in some non-dorsal cell types.