Live cell imaging reveals 3 '-UTR dependent mRNA sorting to synapses

mRNA transport restricts translation to specific subcellular locations, which is the basis for many cellular functions. However, the precise process of mRNA sorting to synapses in neurons remains elusive. Here we use Rgs4 mRNA to investigate 3'-UTR-dependent transport by MS2 live-cell imaging. The majority of observed RNA granules display 3'-UTR independent bidirectional transport in dendrites. Importantly, the Rgs4 3'-UTR causes an anterograde transport bias, which requires the Staufen2 protein. Moreover, the 3'-UTR mediates dynamic, sustained mRNA recruitment to synapses. Visualization at high temporal resolution enables us to show mRNA patrolling dendrites, allowing transient interaction with multiple synapses, in agreement with the sushi-belt model. Modulation of neuronal activity by either chemical silencing or local glutamate uncaging regulates both the 3'-UTR-dependent transport bias and synaptic recruitment. This dynamic and reversible mRNA recruitment to active synapses would allow translation and synaptic remodeling in a spatially and temporally adaptive manner

Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion

The opportunistic pathogen Toxoplasma gondii is an obligate intracellular parasite part of the phylum Apicomplexa, able to infect all warm-blooded animals including humans. Invasion by apicomplexan parasites such as Plasmodium falciparum and Toxoplasma gondii to host cells requires the establishment and crossing through of a small ring-like junctional structure serving as an interface and stabiliser between the parasite and host cell plasma membrane. During the invasion process, the host cell possibly resist invasion to some degree, exerting force on the parasite’s entry point as de novo actin polymerisation has been characterised in this location (Gonzalez et al., 2009). Additionally, the parasite is required to generate force via an actomyosin motor to achieve host cell membrane penetration successfully, leading to mechanical deformation when the parasite is squeezing through the junctional ring. This actomyosin motor depends on a protein complex termed the glideosome, that pulls actin to achieve forward motility. Actin plays a key role in the parasite’s biology with important functions not only during invasion but also during replication, apicoplast maintenance and egress. Until recently, the lack of reliable F-actin sensors hampered the characterisation of actin dynamics during these processes. With the use of nanobodies with the potential to recognise actin (Periz et al., 2017), a complex actin behaviour was uncovered allowing the assessment of in vivo dynamics through the parasite’s lytic cycle. The uncovered flow of F-actin presented new opportunities to address debate over stablished hypothesis on parasite’s actin and to extend the initial roles attributed to actin including the establishment of cytoplasmic actin pool through the parasite’s life. Additionally, these F-actin dynamics were shown to be affected by traditional actin modulating drugs, as well as interference with actin binding factors resulting in abrogation of these dynamics and phenotypes associated with motility. Additionally in this thesis, it is suggested that F-actin’s role in invasion goes beyond powering the glideosome via force traction, but to facilitate nucleus passage and deformation. Real time and super resolution microscopy highlighted that during invasion events, the junction ring can oppose nucleus passage as parasites deficient of core components of the acto-myosin system have been shown to be incapable of withstand pressure exerted at the junction ring, leading to blebbing and collapse of the invading parasite (Bichet et al., 2016). Although some of these parasites are able to complete invasion, the dynamics are visibly affected suggesting more systems are at play during invasion. The literature shows that other eukaryotic systems deploy nucleus protection and displacement mechanisms to facilitate migration through tight spaces by the concerted action of actomyosin complexes and cytoskeletal structures (Petrie et al., 2012; Petrie and Yamada, 2015; McGregor, Hsia and Lammerding, 2016). This thesis proposes that the F-actin machinery facilitates nucleus passage through the junctional ring, offering a model fort the dual contribution of F-actin forces by constricting and pushing/pulling the nucleus during host cell invasion by these apicomplexan parasites, sharing similar mechanism with those of larger eukaryotes

Bidirectional intraflagellar transport is restricted to two sets of microtubule doublets in the trypanosome flagellum

Intraflagellar transport (IFT) is the rapid bidirectional movement of large protein complexes driven by kinesin and dynein motors along microtubule doublets of cilia and flagella. In this study, we used a combination of high-resolution electron and light microscopy to investigate how and where these IFT trains move within the flagellum of the protist Trypanosoma brucei. Focused ion beam scanning electron microscopy (FIB-SEM) analysis of trypanosomes showed that trains are found almost exclusively along two sets of doublets (3–4 and 7–8) and distribute in two categories according to their length. High-resolution live imaging of cells expressing mNeonGreen::IFT81 or GFP::IFT52 revealed for the first time IFT trafficking on two parallel lines within the flagellum. Anterograde and retrograde IFT occurs on each of these lines. At the distal end, a large individual anterograde IFT train is converted in several smaller retrograde trains in the space of 3–4 s while remaining on the same side of the axoneme

Bioimage Data Analysis Workflows ‒ Advanced Components and Methods

This open access textbook aims at providing detailed explanations on how to design and construct image analysis workflows to successfully conduct bioimage analysis. Addressing the main challenges in image data analysis, where acquisition by powerful imaging devices results in very large amounts of collected image data, the book discusses techniques relying on batch and GPU programming, as well as on powerful deep learning-based algorithms. In addition, downstream data processing techniques are introduced, such as Python libraries for data organization, plotting, and visualizations. Finally, by studying the way individual unique ideas are implemented in the workflows, readers are carefully guided through how the parameters driving biological systems are revealed by analyzing image data. These studies include segmentation of plant tissue epidermis, analysis of the spatial pattern of the eye development in fruit flies, and the analysis of collective cell migration dynamics. The presented content extends the Bioimage Data Analysis Workflows textbook (Miura, Sladoje, 2020), published in this same series, with new contributions and advanced material, while preserving the well-appreciated pedagogical approach adopted and promoted during the training schools for bioimage analysis organized within NEUBIAS – the Network of European Bioimage Analysts. This textbook is intended for advanced students in various fields of the life sciences and biomedicine, as well as staff scientists and faculty members who conduct regular quantitative analyses of microscopy images

Bioimage Data Analysis Workflows ‒ Advanced Components and Methods

This open access textbook aims at providing detailed explanations on how to design and construct image analysis workflows to successfully conduct bioimage analysis. Addressing the main challenges in image data analysis, where acquisition by powerful imaging devices results in very large amounts of collected image data, the book discusses techniques relying on batch and GPU programming, as well as on powerful deep learning-based algorithms. In addition, downstream data processing techniques are introduced, such as Python libraries for data organization, plotting, and visualizations. Finally, by studying the way individual unique ideas are implemented in the workflows, readers are carefully guided through how the parameters driving biological systems are revealed by analyzing image data. These studies include segmentation of plant tissue epidermis, analysis of the spatial pattern of the eye development in fruit flies, and the analysis of collective cell migration dynamics. The presented content extends the Bioimage Data Analysis Workflows textbook (Miura, Sladoje, 2020), published in this same series, with new contributions and advanced material, while preserving the well-appreciated pedagogical approach adopted and promoted during the training schools for bioimage analysis organized within NEUBIAS – the Network of European Bioimage Analysts. This textbook is intended for advanced students in various fields of the life sciences and biomedicine, as well as staff scientists and faculty members who conduct regular quantitative analyses of microscopy images

Mitochondrial function and dynamics in demyelinated axons

Demyelination is a pathological process which causes profound changes in the physiology of axons. Post mortem evidence suggests that mitochondrial content is increased in demyelinated axons which has led to the widely accepted theory that loss of myelin causes an increase in axonal energy demand which in turn can be satisfied by a larger number of mitochondria. However, demyelinated axons are known to undergo a series of changes from conduction block early on in demyelination to altered modes of conduction and finally remyelination and return of normal conduction. Little is known about how these different states influence axonal mitochondria. In this thesis, mitochondrial function and dynamics were investigated throughout the time course of lysolecithin-mediated de- and remyelination in the saphenous nerve in vivo. First, the time course of anatomical and electrophysiological changes after application of lysolecithin was mapped out using semi-thin resin sections and recordings of compound action potentials. Then, mitochondrial dynamics and membrane potential were measured at various time points during the onset and resolution of demyelination. Mitochondrial transport was significantly reduced during the first week of demyelination, preceding the accumulation of stationary mitochondria in the axon. At the same time, mitochondrial membrane potential was significantly increased, particularly at the earliest time points investigated (days 2 and 4). Interestingly, all changes including the abovementioned accumulation of mitochondria took place before the return of conduction and putative increase in energy demand. In order to understand better the processes underlying these changes, the role of two known modifiers of mitochondrial dynamics were investigated: action potential conduction and intra-axonal calcium. A 6h conduction block was induced using bupivacaine and confirmed using electrophysiological stimulation but did not lead to any of the changes seen in the early phases of demyelination. On the other hand, calcium imaging using the genetically encoded calcium sensor Tn-XXL revealed a slight but consistent increase in intra-axonal calcium in demyelinated axons, both at the time point with the highest increase in mitochondrial membrane potential (day 2) and at the time point with the highest mitochondrial density (day 8). Taken together, these findings point to impaired axonal calcium homeostasis, rather than changes in energy demand, as the main driving force behind mitochondrial changes in demyelination. The fact that mitochondrial transport remained impaired until later in the remyelination process may have implications for the long term survival of chronically demyelinated axons

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin