Why did Rubens add a parrot to Titian's The Fall of Man? A pictorial manipulation of joint attention.

Almost 400 years ago, Rubens copied Titian's The Fall of Man, albeit with important changes. Rubens altered Titian's original composition in numerous ways, including by changing the gaze directions of the depicted characters and adding a striking red parrot to the painting. Here, we quantify the impact of Rubens's choices on the viewer's gaze behavior. We displayed digital copies of Rubens's and Titian's artworks-as well as a version of Rubens's painting with the parrot digitally removed-on a computer screen while recording the eye movements produced by observers during free visual exploration of each image. To assess the effects of Rubens's changes to Titian's composition, we directly compared multiple gaze parameters across the different images. We found that participants gazed at Eve's face more frequently in Rubens's painting than in Titian's. In addition, gaze positions were more tightly focused for the former than for the latter, consistent with different allocations of viewer interest. We also investigated how gaze fixation on Eve's face affected the perceptual visibility of the parrot in Rubens's composition and how the parrot's presence versus its absence impacted gaze dynamics. Taken together, our results demonstrate that Rubens's critical deviations from Titian's painting have powerful effects on viewers' oculomotor behavior.

Biogenesis, inheritance, and 3D ultrastructure of the microsporidian mitosome.

During the reductive evolution of obligate intracellular parasites called microsporidia, a tiny remnant mitochondrion (mitosome) lost its typical cristae, organellar genome, and most canonical functions. Here, we combine electron tomography, stereology, immunofluorescence microscopy, and bioinformatics to characterise mechanisms of growth, division, and inheritance of this minimal mitochondrion in two microsporidia species (grown within a mammalian RK13 culture-cell host). Mitosomes of Encephalitozoon cuniculi (2-12/cell) and Trachipleistophora hominis (14-18/nucleus) displayed incremental/non-phasic growth and division and were closely associated with an organelle identified as equivalent to the fungal microtubule-organising centre (microsporidian spindle pole body; mSPB). The mitosome-mSPB association was resistant to treatment with microtubule-depolymerising drugs nocodazole and albendazole. Dynamin inhibitors (dynasore and Mdivi-1) arrested mitosome division but not growth, whereas bioinformatics revealed putative dynamins Drp-1 and Vps-1, of which, Vps-1 rescued mitochondrial constriction in dynamin-deficient yeast (Schizosaccharomyces pombe). Thus, microsporidian mitosomes undergo incremental growth and dynamin-mediated division and are maintained through ordered inheritance, likely mediated via binding to the microsporidian centrosome (mSPB).

LRRK2 kinase activity is necessary for development and regeneration in Nematostella vectensis.

Background: The starlet sea anemone, Nematostella vectensis, is an emerging model organism with a high regenerative capacity, which was recently found to possess an orthologue to the human LRRK2 gene (nvLRRK2). The leucine rich repeat kinase 2 (LRRK2) gene, when mutated, is the most common cause of inherited Parkinson's Disease (PD). Its protein product (LRRK2) has implications in a variety of cellular processes, however, the full function of LRRK2 is not well established. Current research is focusing on understanding the function of LRRK2, including both its physiological role as well as its pathobiological underpinnings. Methods: We used bioinformatics to determine the cross-species conservation of LRRK2, then applied drugs targeting the kinase activity of LRRK2 to examine its function in development, homeostasis and regeneration in Nematostella vectensis. Results: An in-silico characterization and phylogenetic analysis of nvLRRK2 comparing it to human LRRK2 highlighted key conserved motifs and residues. In vivo analyses inhibiting the kinase function of this enzyme demonstrated a role of nvLRRK2 in development and regeneration of N. vectensis. These findings implicate a developmental role of LRRK2 in Nematostella, adding to the expanding knowledge of its physiological function. Conclusions: Our work introduces a new model organism with which to study LRRK biology. We show a necessity for LRRK2 in development and regeneration. Given the short generation time, genetic trackability and in vivo imaging capabilities, this work introduces Nematostella vectensis as a new model in which to study genes linked to neurodegenerative diseases such as Parkinson's.

Comparison of Blastema Formation after Injury in Two Cephalopod Species.

Regeneration is the ability to functionally replace significant amounts of lost tissue or whole appendages like arms, limbs or tentacles. The amount of tissue that can be regenerated varies among species, but regeneration is found in both invertebrate and vertebrate animals. Cephalopods have been broadly reported in the literature to regenerate their arms. There are over 800 species of Cephalopod; however, regeneration has only been documented in the literature in a few species (1). Here we compare arm regeneration in two species of cephalopod, the Octopus bimaculoides and the hummingbird bobtail squid Euprymna berryi.

The invasive cell coat at the microsporidian Trachipleistophora hominis-host cell interface contains secreted hexokinases.

Microsporidia are obligate intracellular parasites causing significant disease in humans and economically important animals. In parallel to their extreme genetic reduction, Microsporidia have evolved novel mechanisms for exploiting host metabolism. A number of microsporidians confer secretion of otherwise cytosolic proteins by coding for signal peptides that direct entry into the endoplasmic reticulum. The human pathogen Trachipleistophora hominis encodes for four hexokinases, three of which have signal peptides at the N-terminus. Here, we localized hexokinase 2 and hexokinase 3 through developmental stages of T. hominis using light and electron microscopy. Both proteins were concentrated in an extracellular coat previously termed the plaque matrix (PQM). The PQM (containing hexokinases) was morphologically dynamic, infiltrating the host cytoplasm predominantly during replicative stages. Throughout development the PQM interacted closely with endoplasmic reticulum that was demonstrated to be active in membrane protein biosynthesis and export. The impact of hexokinase on the host metabolism was probed using the fluorescent analog of glucose, 2-NBDG, which displayed spatially restricted increases in signal intensity at the parasite/vacuole surface, coincident with hexokinase/PQM distribution. Gross metabolic aberrations, measured using metabolic profiling with the Seahorse XF Analyzer, were not detectable in mixed stage cocultures. Overall, these results highlight a role for the extended cell coat of T. hominis in host-parasite interactions, within which secreted hexokinases may work as part of a metabolic machine to increase glycolytic capacity or ATP generation close to the parasite surface.

Quantifying Golgi structure using EM: combining volume-SEM and stereology for higher throughput.

Investigating organelles such as the Golgi complex depends increasingly on high-throughput quantitative morphological analyses from multiple experimental or genetic conditions. Light microscopy (LM) has been an effective tool for screening but fails to reveal fine details of Golgi structures such as vesicles, tubules and cisternae. Electron microscopy (EM) has sufficient resolution but traditional transmission EM (TEM) methods are slow and inefficient. Newer volume scanning EM (volume-SEM) methods now have the potential to speed up 3D analysis by automated sectioning and imaging. However, they produce large arrays of sections and/or images, which require labour-intensive 3D reconstruction for quantitation on limited cell numbers. Here, we show that the information storage, digital waste and workload involved in using volume-SEM can be reduced substantially using sampling-based stereology. Using the Golgi as an example, we describe how Golgi populations can be sensed quantitatively using single random slices and how accurate quantitative structural data on Golgi organelles of individual cells can be obtained using only 5-10 sections/images taken from a volume-SEM series (thereby sensing population parameters and cell-cell variability). The approach will be useful in techniques such as correlative LM and EM (CLEM) where small samples of cells are treated and where there may be variable responses. For Golgi study, we outline a series of stereological estimators that are suited to these analyses and suggest workflows, which have the potential to enhance the speed and relevance of data acquisition in volume-SEM.

Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals.

Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures.

Nanoparticle suspensions enclosed in methylcellulose: a new approach for quantifying nanoparticles in transmission electron microscopy.

Nanoparticles are of increasing importance in biomedicine but quantification is problematic because current methods depend on indirect measurements at low resolution. Here we describe a new high-resolution method for measuring and quantifying nanoparticles in suspension. It involves premixing nanoparticles in a hydrophilic support medium (methylcellulose) before introducing heavy metal stains for visualization in small air-dried droplets by transmission electron microscopy (TEM). The use of methylcellulose avoids artifacts of conventional negative stain-TEM by (1) restricting interactions between the nanoparticles, (2) inhibiting binding to the specimen support films and (3) reducing compression after drying. Methylcellulose embedment provides effective electron imaging of liposomes, nanodiscs and viruses as well as comprehensive visualization of nanoparticle populations in droplets of known size. These qualities facilitate unbiased sampling, rapid size measurement and estimation of nanoparticle numbers by means of ratio counting using a colloidal gold calibrant. Specimen preparation and quantification take minutes and require a few microliters of sample using only basic laboratory equipment and a standard TEM.