Cellular & Molecular Imaging

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.

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

The files here are the source data for 4 main figures of Ma et al., Regional Ca2+ Wave, Hormone Responses upon Laser Ablation in Arabidopsis.

We here report an aberration-corrected 25-plane camera array Multifocus microscope (M25) for high-speed, high-resolution wide-field optical microscopy in three spatial dimensions (3D). We demonstrate live imaging of 25-plane 3D volumes of up to 180x180x50um at >100 volumes per second. 3D data is recorded simultaneously by an array of 25 small, sensitive, synchronized machine-vision cameras. M25 employs aberration-corrected Multifocus microscopy—an optical method where diffractive Fourier optics are used for multiplexing and refocusing light—with a simplified design for chromatic dispersion correction where a corrective diffractive gratings is placed on each camera in the array. This elegant architecture for chromatic correction will be applicable in a broad range of diffractive imaging applications. M25 is a powerful optical tool for high-speed 3D microscopy in that it allows both non-invasive, label-free bright-field and highly sensitive fluorescence microscopy. We showcase M25 capabilities in 3D particle tracking, bright-field, and fluorescence imaging in D. melanogaster, and locomotion and neural activity studies in C. elegans.

Sagittal section images showing cross-section of the stomodeal valve at point of greatest opening of 15 sand flies (Lutzomyia longipalpis) at various stages of infection with Leishmania mexicana. The images show the effects of an infection and of a second blood meal on the morphology of the foregut and midgut (especially thoracic midgut) compared to uninfected, control flies. Three flies were imaged by micro-CT scan in each of five groups: T7CON flies were 7 days after a non-infected blood meal. T9INF and T12INF flies were 9 and 12 days, respectively, after an infected blood meal. T6INF+ and T9INF+ flies were 6 and 9 days, respectively after an infected bloodmeal and 1 and 4 days, respectively, after a second, non-infected blood meal. The data are discussed in a paper published on 27th August 2021 in PLoS NTDs, which is accessible through the following link: https://doi.org/10.1371/journal.pntd.0009682

Our recent research, published in PNAS in 2024, highlights the significant role of the endoplasmic reticulum (ER) in modulating activity-driven postsynaptic signaling events. This signaling is associated with long-term potentiation of dendritic spine size, known as structural long-term potentiation (sLTP), which underpins the functional strengthening of glutamatergic synaptic transmission. Our findings demonstrate that high-frequency optical glutamate uncaging (HFGU) induces long-lasting sLTP in most ER-containing (ER+) spines, resulting in a sustained increase in spine ER content. This process is driven by a signaling cascade initiated by N-methyl-D-aspartate receptors (NMDARs), L-type Ca2+ channels (LTCCs), and Orai1 channels, the latter of which are activated by stromal interaction molecule 1 (STIM1) in response to Ca2+ release from the ER. In contrast, HFGU stimulation of ER-lacking (ER−) spines results in only transient sLTP and diminished Ca2+ signals, notably lacking contributions from Orai1 and the ER. Supporting the notion that spine ER governs structural metaplasticity, delivering a well-timed second HFGU stimulus to ER− spines recruited ER and produced persistent sLTP. However, repeated stimulation of previously potentiated ER+ spines failed to further enhance Ca2+ signaling, spine size, or spine ER content, potentially serving as a protective mechanism against runaway potentiation. Through our exploration of the distinct inhibitory effects of Orai1 inhibitors, AnCoA4 and Synta66, we found that the physical interaction between STIM1 and Orai1 at ER-plasma membrane junctions, rather than the subsequent Ca2+ entry, is essential for the persistence of sLTP.

The heterodimeric kinesin-2 motor (KIF3A/KIF3B with accessory protein KAP3) drives intraflagellar transport, essential for ciliogenesis and ciliary function. Three point mutations in the KIF3B subunit have recently been linked to disease in humans (E250Q and L523P) and Bengal cats (A334T) (Cogné et al., 2020). Patients display retinal atrophy and, in some cases, other ciliopathy phenotypes. However, the molecular mechanism leading to disease is currently unknown. Here, we used Kif3a-/-;Kif3b-/- (knockout) 3T3 cells, which cannot make cilia, to characterize these mutations. While reexpression of KIF3B(E250Q) and KIF3B(L523P) did not rescue ciliogenesis, reexpression of wildtype or KIF3B(A334T), restored ciliogenesis to wildtype levels. Fluorescent tagging revealed that the E250Q mutant decorated microtubules and thus is a rigor mutation. The L523P mutation, in the alpha-helical stalk domain, surprisingly did not affect formation of the KIF3A/KIF3B/KAP3 complex but instead impaired motility along microtubules. Lastly, expression of the A334T motor was reduced in comparison to all other conditions and displayed an impaired ability to disperse the Golgi complex when artificially linked to this high-load cargo. In summary, this work uses cell-based assays to elucidates the molecular effects of disease-causing mutations in the KIF3B subunit on the kinesin-2 holoenzyme.

This dataset contains comprehensive image-based morphological profiling data from a systematic drug repurposing screen for host-directed antivirals. The repository includes three major study components. The primary screen employed high-content Cell Painting analysis combined with viral immunostaining of 5,275 repurposable compounds screened against SARS-CoV-2 infection in Vero E6 monkey kidney cells. This initial morphological profiling identified compounds that reversed the infected cell phenotype towards non-infected controls. Hit validation was performed through dose-response morphological profiling experiments by Cell Painting and antibody staining in human A549 lung epithelial cells engineered to express the ACE2 receptor, confirming antiviral activity in a human-relevant cell model. A counter screen assessed compound antiviral activity that could be attributed to drug-induced phospholipidosis (DIPL) instead of specific engagement of viral or host targets. This component includes high-content data from human A549-ACE2 cells stained with HCS LipidTOX Green Phospholipidosis Detection Reagent, CellTracker Deep Red Dye, and Hoechst 33342. The dataset includes raw images and extracted morphological features. This resource enables identification of host-targeting antivirals with potential broad-spectrum activity and supports future pandemic preparedness efforts.

The Darwin Tree of Life project has the goal to sequence the genomes of 70,000 species of eukaryotic organisms in Britain and Ireland. This is a collection of photographs of the samples included in the study, provided by the National History Museum (NHM).