Cellular & Molecular Imaging

Asymmetric localization of oskar RNP granules to the oocyte posterior is crucial for abdominal patterning and germline formation in the Drosophila embryo. We show that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, we confirm in vitro that oskar granules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of client proteins. Genetic modification of scaffold granule proteins, or tethering the intrinsically disordered region of human Fused in Sarcoma (FUS) to oskar mRNA, allowed modulation of granule material properties in vivo. The resulting liquid-like properties impaired oskar localization and translation with severe consequences on embryonic development. Our study reflects how physiological phase transitions shape RNA-protein condensates to regulate localization and expression of a maternal RNA that instructs germline formation.

Two photon microscopy time series and lightsheet microscopy z-stack and time series of early Drosophila embryogenesis during posterior midgut invagination.

Bacteria occupy heterogeneous environments, attaching and growing within pores in materials, living hosts, and matrices like soil. Systems that permit high-resolution visualization of dynamic bacterial processes within the physical confines of a realistic and tractable porous media environment are rare. Here we use microfluidics to replicate the grain shape and packing density of natural sands in a 2D platform to study the flow-induced spatial evolution of bacterial biofilms underground. We introduce a wildtype strain (Pantoea sp. YR343, n=3) or an EPS-defective strain (Pantoea sp. YR343 ΔUDP, n=3) to the porous media platform and then simulate a rainfall event using gravity-driven flow of bacterial growth media.

Fluorescence imaging across the near-infrared (NIR, 700–1000 nm) and shortwave infrared (SWIR, 1000–2000 nm) regions offers significant advantages for biomedical applications, yet photophysical enhancements achieved with NIR and SWIR chromophores observed in solution often fail to translate to complex biological environments. Fluorous-soluble fluorophores, fluorofluorophores, face additional challenges, exhibiting poor brightness and photostability when dissolved in perfluorocarbons (PFCs) due to unfavorable interactions with the fluorous phase. Here, we report counterion exchange as a strategy to enhance the photophysical properties of two heptamethine cyanine fluorofluorophore for NIR and SWIR imaging. Exchanging the small chloride counterion with a large, fluorinated aryl borate counterions significantly improved the brightness (10-fold) and photostability (57-fold) in PFCs. These enhancements were successfully translated across multiple biological systems from macrophage cells to NIR imaging zebrafish retinal tissue and finally to SWIR imaging in mice. These results demonstrate that strategic counterion modification provides a straightforward approach to optimize fluorofluorophores, with solution-phase improvements that translate to in vivo NIR and SWIR imaging.

The Drosophila melanogaster olfactory system is one of the most intensively studied parts of the nervous system in any animal. Composed of ~50 independent olfactory neuron classes, with several associated hygrosensory and thermosensory pathways, it has been subject to diverse types of experimental analyses. However, synthesizing the available information is limited by the incomplete data and inconsistent nomenclature found in the literature. In this work, we first “complete” the peripheral sensory map through the identification of a previously uncharacterized antennal sensory neuron population expressing Or46aB, and the definition of an exceptional “hybrid” olfactory neuron class comprising functional Or and Ir receptors. Second, we survey developmental, anatomical, connectomic, functional and evolutionary studies to generate an integrated dataset and associated visualisations of these sensory neuron pathways, creating an unprecedented resource. Third, we illustrate the utility of the dataset to reveal relationships between different organizational properties of this sensory system, and the new questions these stimulate. Such examples emphasize the power of this resource to promote further understanding of the construction, function and evolution of these neural circuits.

"This dataset comprises processed images and class labels of UV microscopy images of human red blood cells infected with malaria at various degrees of parasitemia. The microscope used to caputure the images is a custom-built UV microscope employing a quartz Zeiss Ultrafluar 100x/0.85 finite conjugate objective. The cells are imaged at either one wavelength in deep UV (285 nm) or three simultaneous wavelengths (285 nm, 365 nm, 565 nm). Each condition contains many fields of view, extensive z-stacks, and a single time point. The directory structure is organized first by category: 'Training and validation', or 'Titration 2020-06-20'. Training and Validation is a collection of time-stamped data collection sessions acquired during development of the method. 'Titration 2020-06-20' is an experiment whereby a high parasitemia malaria culture was diluted serially into healthy red blood cells. Each dilution point was imaged and resides in a time-stamped directory."

OME-NGFF converted study from idr0090. Drug resistance threatens the effective prevention and treatment of an ever-increasing range of human infections. This highlights an urgent need for new and improved drugs with novel mechanisms of action to avoid cross-resistance. Current cell-based drug screens are, however, restricted to binary live/dead readouts with no provision for mechanism of action prediction. Machine learning methods are increasingly being used to improve information extraction from imaging data. Such methods, however, work poorly with heterogeneous cellular phenotypes and generally require time-consuming human-led training. We have developed a semi-supervised machine learning approach, combining human- and machine-labelled training data from mixed human malaria parasite cultures. Designed for high-throughput and high-resolution screening, our semi-supervised approach is robust to natural parasite morphological heterogeneity and correctly orders parasite developmental stages. Our approach also reproducibly detects and clusters drug-induced morphological outliers by mechanism of action, demonstrating the potential power of machine learning for accelerating cell-based drug discovery.

To establish infection, phytopathogens deploy effectors to compromise host defences and facilitate invasive growth. As part of this, the battle for control of symplastic connectivity via plasmodesmata is a key determinant of infection outcomes, yet little is known about how fungal effectors directly exploit these channels, and in turn, how hosts defend them. In this work, we identified ChEC108 as a plasmodesmal-targeting, cell-to-cell mobile effector from the anthracnose fungus, Colletotrichum higginsianum. ChEC108 binds the plasmodesmal protein HEAVY METAL-ASSOCIATED (HMA) ISOPRENYLATED PLANT PROTEIN 6 (HIPP6) from Arabidopsis via a tetrahedral metal ion coordination site with either of its HMA domains. Constitutive in planta expression of ChEC108 induces plasmodesmal closure and the upregulation of defence-associated genes in a manner dependent on its capacity to bind HIPP6. Further, HIPP6 binding impairs cell-to-cell mobility of ChEC108. Alongside the finding that loss of ChEC108 favoured C. higginsianum infection, this suggests ChEC108-HIPP6 interaction at plasmodesmata positively regulates defence.

The microtubule cytoskeleton consists of dynamic intracellular filaments and is involved in numerous processes, ranging from nuclear division to intracellular transport. Some of these microtubule-mediated processes are conserved in all eukaryotic lineages while others are specific for certain groups of organisms. Here, we focus on the microtubule cytoskeleton of oomycetes in the genus Phytophthora, a group of harmful plant pathogens. In Phytophthora palmivora lines expressing GFP-tagged α-tubulin we observed a conserved microtubule localization in the mitotic spindle and dynamic cytoplasmic microtubules between adjacent nuclei. These microtubules originated from the mitotic spindle and rapidly increased in length after mitosis. Since microtubule minus-ends are likely associated with MTOCs on the nuclear surface, the plus-ends of microtubules originating from adjacent nuclei likely maintain an antiparallel connection which may play a role in nuclear spacing. This idea was strengthened by erratic nuclear motility and positioning after microtubule depolymerization. Besides aberrant nuclear positioning we also observed less sustained tip growth in the absence of microtubules. This suggests that microtubules radiating into the hyphal tip from the apical nucleus function in sustaining tip growth. Altogether, this study provides novel insights in the localization, dynamics and functions of the microtubule cytoskeleton in the coenocytic Phytophthora hyphae.

Calcium imaging and electrophysiological datasets produced for and analyzed in https://doi.org/10.1101/2022.08.19.504510