Cellular & Molecular Imaging

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).

Transcription factors (TFs) efficiently locate their target DNA sequences by combining three-dimensional diffusion and one-dimensional sliding on nonspecific DNA. To balance rapid sliding with strong specific binding, TFs were proposed to switch between search and recognition conformations. For E. coli lac repressor (LacI), the folding of the hinge helices has been implicated in the conformational switch. Here, we tested how mutations in the hinge region impact the search speed and binding stability. Based on molecular dynamics simulations, we selected two LacI mutants favoring either search or recognition conformation. We measured the binding kinetics of the mutants both in vitro on DNA microarrays with 2,479 different Lac operators and in vivo via single-molecule experiments. We identified a mutation that enhances the specificity but reduces binding strength globally, and another mutation that makes the operator binding stronger but also reduces the specificity. However, the altered specificity impacts the search time less than expected. Instead, the major effect was impaired dissociation in response to IPTG induction for the strongly binding mutant. Together with earlier reports of affinity–inducibility trade-offs in LacI, our data support the model in which the trade-off is between binding stability and inducibility rather than between speed and binding stability. This post provides raw fluorescence imaging datasets from single-molecule live-cell experiments and Protein Binding Microarray (PBM) experiments, along with the info of all the codes necessary to replicate the analyses of our study. Code for analysis and figure generation is found in: 10.17044/scilifelab.29040599 Data: - Data Folders with prefix: 'Microscopy_Data_' are for Protein Binding Microarray experiments and Single Molecule experiments (raw fluorescence images) - Data Folders with prefix: 'MD_Data_' are for MD simulations, download it from the Scilifelab repo, see in the link section.

Manuscript abstract: Malaria transmission relies on sporozoite formation in the mosquito midgut and subsequent salivary gland invasion. Despite their importance, the cell biology of these processes remains poorly understood. We apply Mosquito Tissue Ultrastructure Expansion Microscopy (MoTissU-ExM), which physically expands infected mosquito tissues while preserving host and parasite ultrastructure. MoTissU-ExM reveals parasite structures and organelles, including features previously seen only by electron microscopy and novel structures not observed before. We use MoTissU-ExM to investigate sporozoite formation and salivary gland invasion, focusing on rhoptries - secretory organelles critical for host cell invasion. We establish a timeline for rhoptry biogenesis, show that two rhoptries are consumed during salivary gland invasion, and provide the first evidence that rhoptry pairs are specialized for different invasion events. We further characterize RON11 as the first protein involved in sporozoite rhoptry biogenesis; its disruption produces sporozoites that specifically fail to invade salivary gland epithelial cells, blocking parasite transmission. Dataset description: This dataset contains all microscopy data associated with the linked publication "Unlocking new understanding of Plasmodium sporozoite biology with expansion microscopy". All samples were prepared by ultrastructure-expansion microscopy (U-ExM). All samples were imaged on either a Zeiss LSM900 or LSM980 microscope, using either Airyscan-SR or Airyscan-MPLX modes. File names will include the magnification of the objective lens used as follows: 5x = EC Plan-Neofluar 5x/0.16NA Air 10x = Ziess Plan-Apochromat 10x/0.45NA air 20x = Ziess Plan-Apochromat 20x/0.8NA air 40x = Zeiss C-Apochromat 40x/1.2NA water-immersion autocorr M27 63x = Zeiss Plan-Apochromat 63x/1.4NA oil-immersion M27 Images are of mosquito tissues, or isolated parasites, from three Plasmodium species - berghei (Pb), falciparum (Pf), and yoelii (Py). Images are sorted and named as follows (folder name, file name) Plasmodium species > Tissue type/site of isolation > Parasite strain > Species abbreviation, MG/SG, Harvest day(dpi), Dye/Fluorophores (405nm -> 647nm), Objective, Image number (1->X), as (airyscan) For example, the third image taken of a P. berghei oocyst with the RON11iKD parasite line, that was harvested on Day 14 post infection, stained with NHS Ester AF405, BODIPY-FL, anti-Tubulin AF555, and Sytox Red, and imaged on the 40x-objective would be listed as follows: Plasmodium berghei > Infected midguts > RON11iKD > RON11KD MG 14dpi NHSBFlTub-SytR 40x 1 as The majority of images in this dataset are z-stacked images, but for many oocysts a single-slice image of the whole oocyst was taken. When this is the case, the single-slice image will be indicated with "SNAP". A list of the acronyms and abbreviations used in file names are as follows MG = Midgut SG = Salivary gland Spz = Sporozoite HC = Haemocoel dpi = Days post infection NHS = NHS Ester Alexa Fluor 405 BFl = Bodipy-FL-Ceramide BTRc = Bodipy-TR-Ceramide SytR = Sytox Deep Red Tub = anti-tubulin antibody CSP = anti-circumsporozoite protein antibody RAP1 = anti-rhoptry associated protein 1 antibody iKD = Inducible knockdown Ctrl = Control KD = Knockdown RON4 = anti-rhoptry neck protein 4 antibody GFP = anti-green fluorescent protein antibody WGA = Wheat germ aglutinnin BIP = anti-BiP antibody ERD2 = anti-ERD2 antibody

The images and complementary information presented here are related to the article: Simiyu, B. M. & R. Kurmayer, 2022. Response of planktonic diatoms to eutrophication in Nyanza Gulf of Lake Victoria, Kenya. Limnologica 93:125958 https://doi.org/10.1016/j.limno.2022.125958. The associated data article contains data and information on the identification of common diatoms using screening electron microscopy ( Phillips XL20 SEM). Diatoms presented belong in to six genera (11 species) and include: three chain forming centrics, four single cell centrics, two araphid diatoms originally described from L. Victoria, and one small highly variable preferentially epiphytic Nitzschia species. The method used for sample cleaning and analysis is presented.

The images and complementary information presented here are related to the article: Simiyu, B. M. & R. Kurmayer, 2022. Response of planktonic diatoms to eutrophication in Nyanza Gulf of Lake Victoria, Kenya. Limnologica 93:125958 https://doi.org/10.1016/j.limno.2022.125958. The associated data article contains data and information on the identification of common diatoms using screening electron microscopy ( Phillips XL20 SEM). Diatoms presented belong in to six genera (11 species) and include: three chain forming centrics, four single cell centrics, two araphid diatoms originally described from L. Victoria, and one small highly variable preferentially epiphytic Nitzschia species. The method used for sample cleaning and analysis is presented.

The use of CRISPR-associated enzymes in iPSC-derived neurons for precise gene targeting and high-throughput gene perturbation screens offers great potential but presents unique challenges compared to dividing cell lines. CRISPRi screens in iPSC-derived neurons and glia have already been successful in relating gene function to neurological phenotypes; however, loss of dCas9-KRAB expression after differentiation has been observed by many labs and has been largely ascribed to transgene silencing after differentiation. Here, we investigated the expression levels of different CRISPR enzymes in iPSC and Ngn2-derived neurons using piggybac delivery. We found that the commonly used dCas9-KRAB (using the KOX1 domain) displayed dramatic reduction in protein expression levels following neuronal differentiation, yet surprisingly, nCas9 constructs retained comparable protein expression between iPSCs and neurons. We further found that CRISPR constructs, primarily relying on the SV40 Nuclear Localization Signal (NLS), fail to efficiently localize to the nuclei of neurons, despite having robust nuclear levels in iPSCs, leading to KRAB(KOX1)-specific cytoplasmic degradation. By testing other NLSs, we were able to correct neuronal nuclear localization and protein expression, confirming the contribution of mislocalization to the instability of dCas9-KRAB(KOX1) in neurons. As the lack of nuclear localization can have a profound impact on editing and gene perturbation efficiency, we suggest further investigation across both cultured and in-vivo post-mitotic cell models.

Tara Expeditions are global scientific voyages that probe morphological and molecular diversity, evolution and ecology of marine plankton to explore how they are impacted by changes in the Earth's climate. The first expeditions collected samples of marine plankton containing viruses, bacteria, archaea, protists and planktonic metazoans living in the photic layer of the world's oceans. These expeditions, the first taking place between 2009 and 2013, include Tara Oceans: a global view, and Tara Oceans Polar Circle, both of which followed the same sampling protocol. This dataset includes 1074 pictures of 284 planktonic taxa (mainly microalgae and other Ciliate and Radiolarian protists) collected from the vicinity of the Marquesas Islands in the Southern Pacific Ocean during the Tara Oceans expedition. Multiple samples particularly of the size fractions 5-20 and 20 180 um from four sites and two depths were processed with different methods and studied in detail using scanning electron microscopy.

In plants, a variety of stimuli trigger long-range calcium signals that travel rapidly along the vasculature to distal tissues via poorly understood mechanisms. Here, we use quantitative imaging and analysis to demonstrate that travelling calcium waves are mediated by diffusion and bulk flow of amino acid chemical messengers. Wounding triggers release of amino acids that diffuse locally through the apoplast, activating the calcium permeable channel GLUTAMATE-LIKE RECEPTOR3.3 as they pass. Over long distances through the vasculature, the wound-triggered dynamics of a fluorescent tracer show that calcium waves are likely driven by bulk flow of a channel activating chemical. We observed that multiple stimuli trigger calcium waves with similar dynamics, but calcium waves alone cannot initiate all systemic defence responses, suggesting that mobile chemical messengers are a core component of complex systemic signalling in plants. We present time-lapse images of whole leaves or plants, collected from plants (wild-type and a range of mutants) expressing calcium and glutamate reporters. We used these images to define the dynamics of calcium and glutamate waves that emanate from the sites at which different stimuli were triggered. These images are available as .czi and the scripts we used to analyse them are available via zenodo as described in the links.

Fibrosis is a key feature of a broad spectrum of cystic kidney diseases, especially autosomal recessive kidney disorders such as nephronophthisis (NPHP). However, its contribution to kidney function decline and the underlying molecular mechanism(s) remains unclear. Here, we show that kidney-specific deletion of Fbxw7, the recognition receptor of the SCFFBW7 E3 ubiquitin ligase, results in a juvenile-adult NPHP-like pathology characterized by slow-progressing corticomedullary cysts, tubular degeneration, severe fibrosis, and gradual loss of kidney function. Expression levels of SOX9, a known substrate of FBW7, and WNT4, a potent pro-fibrotic factor and downstream effector of SOX9, were elevated upon loss of FBW7. Heterozygous deletion of Sox9 in compound mutant mice led to the normalization of WNT4 levels, reduced fibrosis, and preservation of kidney function without significant effects on cystic dilatation and tubular degeneration. These data suggest that FBW7-SOX9-WNT4-induced fibrosis drives kidney function decline in NPHP and, possibly, other forms of autosomal recessive kidney disorders.

Light microscopy and scanning electron microscopy images of snow algae field samples containing Rosetta gen. nov., including type material.