A 3D organoid platform that supports liver-stage P.falciparum infection can be used to identify intrahepatic antimalarial drugs

Malaria, a major public health burden, is caused by Plasmodium spp parasites that first replicate in the human liver to establish infection before spreading to erythrocytes. Liver-stage malaria research has remained challenging due to the lack of a clinically relevant and scalable in vitro model of the human liver. Here, we demonstrate that organoids derived from intrahepatic ductal cells differentiated into a hepatocyte-like fate can support the infection and intrahepatic maturation of Plasmodium falciparum. The P.falciparum exoerythrocytic forms observed expressed both early and late-stage parasitic proteins and decreased in frequency in response to treatment with both known and putative antimalarial drugs that target intrahepatic P.falciparum. The P.falciparum-infected human liver organoids thus provide a platform not only for fundamental studies that characterise intrahepatic parasite-host interaction but can also serve as a powerful translational tool in pre-erythrocytic vaccine development and to identify new antimalarial drugs that target the liver stage infection.</p

Three-color dSTORM Imaging and Analysis of Recombination Foci in Mouse Spread Meiotic Nuclei

During the first meiotic prophase in mouse, repair of SPO11-induced DNA double-strand breaks (DSBs), facilitating homologous chromosome synapsis, is essential to successfully complete the first meiotic cell division. Recombinases RAD51 and DMC1 play an important role in homology search, but their mechanistic contribution to this process is not fully understood. Super-resolution, single-molecule imaging of RAD51 and DMC1 provides detailed information on recombinase accumulation on DSBs during meiotic prophase. Here, we present a detailed protocol of recombination foci analysis of three-color direct stochastic optical reconstruction microscopy (dSTORM) imaging of SYCP3, RAD51, and DMC1, fluorescently labeled by antibody staining in mouse spermatocytes. This protocol consists of sample preparation, data acquisition, pre-processing, and data analysis. The sample preparation procedure includes an updated version of the nuclear spreading of mouse testicular cells, followed by immunocytochemistry and the preparation steps for dSTORM imaging. Data acquisition consists of three-color dSTORM imaging, which is extensively described. The pre-processing that converts fluorescent signals to localization data also includes channel alignment and image reconstruction, after which regions of interest (ROIs) are identified based on RAD51 and/or DMC1 localization patterns. The data analysis steps then require processing of the fluorescent signal localization within these ROIs into discrete nanofoci, which can be further analyzed. This multistep approach enables the systematic investigation of spatial distributions of proteins associated with individual DSB sites and can be easily adapted for analyses of other foci-forming proteins. All computational scripts and software are freely accessible, making them available to a broad audience.</p

Dynamics and distribution of paxillin, vinculin, zyxin and VASP depend on focal adhesion location and orientation

Focal adhesions (FAs) are multiprotein structures that link the intracellular cytoskeleton to the extracellular matrix. They mediate cell adhesion and migration, crucial to many (patho-) physiological processes. We examined in two cell types

Multi-color dSTORM microscopy in Hormad1^-/spermatocytes reveals alterations in meiotic recombination intermediates and synaptonemal complex structure

Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy, and a mouse model with severe defects in meiotic DSB formation and synapsis (Hormad1-/-) to obtain more insight in the recombinase accumulation patterns in relation to repair progression. First, we used the known reduction in meiotic DSB frequency in Hormad1-/- spermatocytes to be able to conclude that the RAD51/DMC1 nanofoci that preferentially localize at distances of ~300 nm form within a single DSB site, whereas a second preferred distance of ~900 nm, observed only in wild type, represents inter-DSB distance. Next, we asked whether the proposed role of HORMAD1 in repair inhibition affects the RAD51/DMC1 accumulation patterns. We observed that the two most frequent recombinase configurations (1 DMC1 and 1 RAD51 nanofocus (D1R1), and D2R1) display coupled frequency dynamics over time in wild type, but were constant in the Hormad1-/- model, indicating that the lifetime of these intermediates was altered. Recombinase nanofoci were also smaller in Hormad1-/- spermatocytes, consistent with changes in ssDNA length or protein accumulation. Furthermore, we established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. Finally, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting coil formation in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. Using this large super-resolution dataset, we propose models with the D1R1 configuration representing one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or both ends loaded by the two recombinases, but in below-resolution proximity. This may then often evolve into D2R1, then D1R2, and finally back to D1R1, when DNA synthesis has commenced.</p

Live cell analyses of synaptonemal complex dynamics and chromosome movements in cultured mouse testis tubules and embryonic ovaries

During mammalian meiotic prophase, homologous chromosomes connect through the formation of the synaptonemal complex (SC). SYCP3 is a component of the lateral elements of the SC. We have generated transgenic mice expressing N- or C-terminal fluorescent-tagged SYCP3 (mCherry-SYCP3 (CSYCP) and SYCP3-mCherry (SYCPC)) to study SC dynamics and chromosome movements in vivo. Neither transgene rescued meiotic aberrations in Sycp3 knockouts, but CSYCP could form short axial element-like structures in the absence of endogenous SYCP3. On the wild-type background, both fusion proteins localized to the axes of the SC together with endogenous SYCP3, albeit with delayed initiation (from pachytene) in spermatocytes. Around 40% of CSYCP and SYCPC that accumulated on the SC was rapidly exchanging with other tagged proteins, as analyzed by fluorescent recovery after photobleaching (FRAP) assay. We used the CSYCP transgenic mice for further live cell analyses and observed synchronized bouquet configurations in living cysts of two or three zygotene oocyte nuclei expressing CSYCP, which presented cycles of telomere clustering and dissolution. Rapid chromosome movements were observed in both zygotene oocytes and pachytene spermatocytes, but rotational movements of the nucleus were more clear in oocytes. In diplotene spermatocytes, desynapsis was found to proceed in a discontinuous manner, whereby even brief chromosome re-association events were observed. Thus, this live imaging approach can be used to follow changes in the dynamic behavior of the nucleus and chromatin, in normal mice and different infertile mouse models

Group 1 metabotropic glutamate receptors 1 and 5 form a protein complex in mouse hippocampus and cortex

The group 1 metabotropic glutamate receptors 1 and 5 (mGluR1/5) have been implicated in mechanisms of synaptic plasticity and may serve as potential therapeutic targets in autism spectrum disorders. The interactome of group 1 mGluRs has remained largely unresolved. Using a knockout-controlled interaction proteomics strategy we examined the mGluR5 protein complex in two brain regions, hippocampus and cortex, and identified mGluR1 as its major interactor in addition to the well described Homer proteins. We confirmed the presence of mGluR1/5 complex by (i) reverse immunoprecipitation using an mGluR1 antibody to pulldown mGluR5 from hippocampal tissue, (ii) coexpression in HEK293 cells followed by coimmunoprecipitation to reveal the direct interaction of mGluR1 and 5, and (iii) superresolution microscopy imaging of hippocampal primary neurons to show colocalization of the mGluR1/5 in the synapse

Fast-spiking Parvalbumin Interneurons are Frequently Myelinated in the Cerebral Cortex of Mice and Humans

Myelination, the insulating ensheathment of axons by oligodendrocytes, is thought to both optimize signal propagation and provide metabolic support. Despite the well-established physiological importance of myelination to neuronal function, relatively little is known about the myelination of GABAergic interneurons in the cerebral cortex. Here, we report that a large fraction of myelin in mouse cerebral cortex ensheaths GABAergic interneurons, reaching up to 80% in hippocampal subregions. Moreover, we find that a very high proportion of neocortical and hippocampal parvalbumin (PV) interneurons exhibit axonal myelination. Using a combination of intracellular recordings and biocytin labeling of ex vivo human neocortex, we also confirm that axons of fast-spiking PV interneurons are extensively myelinated in the human brain. PV interneuron myelination in both mice and humans exhibits a stereotyped topography with a bias towards proximal axonal segments and relatively short internodes (∼27 μm) interspersed with branch points. Interestingly, myelin-deficient Shiverer mice exhibit an increased density and more proximal location of en passant boutons, suggesting that myelination might function in part to regu

Growth factor dependent changes in nanoscale architecture of focal adhesions

Focal adhesions (FAs) are flat elongated structures that mediate cell migration and link the cytoskeleton to the extracellular matrix. Along the vertical axis FAs were shown to be composed of three layers. We used structured illumination microscopy to examine the longitudinal distribution of four hallmark FA proteins, which we also used as markers for these layers. At the FA ends pointing towards the adherent membrane edge (heads), bottom layer protein paxillin protruded, while at the opposite ends (tails) intermediate layer protein vinculin and top layer proteins zyxin and VASP extended further. At the tail tips, only intermediate layer protein vinculin protruded. Importantly, head and tail compositions were altered during HGF-induced scattering with paxillin heads being shorter and zyxin tails longer. Additionally, FAs at protruding or retracting membrane edges had longer paxillin heads than FAs at static edges. These data suggest that redistribution of FA-proteins with respect to each other along FAs is involved in cell movement

Incomplete meiotic sex chromosome inactivation in the domestic dog

Background: In mammalian meiotic prophase, homologous chromosome recognition is aided by formation and repair of programmed DNA double-strand breaks (DSBs). Subsequently, stable associations form through homologous chromosome synapsis. In male mouse meiosis, the largely heterologous X and Y chromosomes synapse only in their short pseudoautosomal regions (PARs), and DSBs persist along the unsynapsed non-homologous arms of these sex chromosomes. Asynapsis of these arms and the persistent DSBs then trigger transcriptional silencing through meiotic sex chromosome inactivation (MSCI), resulting in formation of the XY body. This inactive state is partially maintained in post-meiotic haploid spermatids (postmeiotic sex chromatin repression, PSCR). For the human, establishment of MSCI and PSCR have also been reported, but X-linked gene silencing appears to be more variable compared to mouse. To gain more insight into the regulation and significance of MSCI and PSCR among different eutherian species, we have performed a global analysis of XY pairing dynamics, DSB repair, MSCI and PSCR in the domestic dog (Canis lupus familiaris), for which the complete genome sequence has recently become available, allowing a thorough comparative analyses. Results: In addition to PAR synapsis between X and Y, we observed extensive self-synapsis of part of the dog X chromosome, and rapid loss of known markers of DSB repair from that part of the X. Sequencing of RNA from purified spermatocytes and spermatids revealed establishment of MSCI. However, the self-synapsing region of the X displayed higher X-linked gene expression compared to the unsynapsed area in spermatocytes, and was post-meiotically reactivated in spermatids. In contrast, genes in the PAR, which are expected to escape MSCI, were expressed at very low levels in both spermatocytes and spermatids. Our comparative analysis was then used to identify two X-linked genes that may escape MSCI in spermatocytes, and 21 that are specifically re-activated in spermatids of human, mouse and dog. Conclusions: Our data indicate that MSCI is incomplete in the dog. This may be partially explained by extensive, but transient, self-synapsis of the X chromosome, in association with rapid completion of meiotic DSB repair. In addition, our comparative analysis identifies novel candidate male fertility genes