Background-deflection Brillouin microscopy reveals altered biomechanics of intracellular stress granules by ALS protein FUS

Altered cellular biomechanics have been implicated as key photogenic triggers in age-related diseases. An aberrant liquid-to-solid phase transition, observed in in vitro reconstituted droplets of FUS protein, has been recently proposed as a possible pathogenic mechanism for amyotrophic lateral sclerosis (ALS). Whether such transition occurs in cell environments is currently unknown as a consequence of the limited measuring capability of the existing techniques, which are invasive or lack of subcellular resolution. Here we developed a non-contact and label-free imaging method, named background-deflection Brillouin microscopy, to investigate the three-dimensional intracellular biomechanics at a sub-micron resolution. Our method exploits diffraction to achieve an unprecedented 10,000-fold enhancement in the spectral contrast of single-stage spectrometers, enabling, to the best of our knowledge, the first direct biomechanical analysis on intracellular stress granules containing ALS mutant FUS protein in fixed cells. Our findings provide fundamental insights on the critical aggregation step underlying the neurodegenerative ALS disease

Direct conversion of human pluripotent stem cells into cranial motor neurons using a piggyBac vector

Human pluripotent stem cells (PSCs) are widely used for in vitro disease modeling. One of the challenges in the field is represented by the ability of converting human PSCs into specific disease-relevant cell types. The nervous system is composed of a wide variety of neuronal types with selective vulnerability in neurodegenerative diseases. This is particularly relevant for motor neuron diseases, in which different motor neurons populations show a different susceptibility to degeneration. Here we developed a fast and efficient method to convert human induced Pluripotent Stem Cells into cranial motor neurons of the branchiomotor and visceral motor subtype. These populations represent the motor neuron subgroup that is primarily affected by a severe form of amyotrophic lateral sclerosis with bulbar onset and worst prognosis. This goal was achieved by stable integration of an inducible vector, based on the piggyBac transposon, allowing controlled activation of Ngn2, Isl1 and Phox2a (NIP). The NIP module effectively produced electrophysiologically active cranial motor neurons. Our method can be easily extended to PSCs carrying disease-associated mutations, thus providing a useful tool to shed light on the cellular and molecular bases of selective motor neuron vulnerability in pathological conditions

FUS mutant human motoneurons display altered transcriptome and microRNA pathways with implications for ALS pathogenesis

The FUS gene has been linked to amyotrophic lateral sclerosis (ALS). FUS is a ubiquitous RNA-binding protein, and the mechanisms leading to selective motoneuron loss downstream of ALS-linked mutations are largely unknown. We report the transcriptome analysis of human purified motoneurons, obtained from FUS wild-type or mutant isogenic induced pluripotent stem cells (iPSCs). Gene ontology analysis of differentially expressed genes identified significant enrichment of pathways previously associated to sporadic ALS and other neurological diseases. Several microRNAs (miRNAs) were also deregulated in FUS mutant motoneurons, including miR-375, involved in motoneuron survival. We report that relevant targets of miR-375, including the neural RNA-binding protein ELAVL4 and apoptotic factors, are aberrantly increased in FUS mutant motoneurons. Characterization of transcriptome changes in the cell type primarily affected by the disease contributes to the definition of the pathogenic mechanisms of FUS-linked ALS

Importin-beta and CRM1 control a RANBP2 spatiotemporal switch essential for mitotic kinetochore function

Protein conjugation with small ubiquitin-related modifier (SUMO) is a post-translational modification that modulates protein interactions and localisation. RANBP2 is a large nucleoporin endowed with SUMO E3 ligase and SUMO-stabilising activity, and is implicated in some cancer types. RANBP2 is part of a larger complex, consisting of SUMO-modified RANGAP1, the GTP-hydrolysis activating factor for the GTPase RAN. During mitosis, the RANBP2–SUMO-RANGAP1 complex localises to the mitotic spindle and to kinetochores after microtubule attachment. Here, we address the mechanisms that regulate this localisation and how they affect kinetochore functions. Using proximity ligation assays, we find that nuclear transport receptors importin-β and CRM1 play essential roles in localising the RANBP2–SUMO-RANGAP1 complex away from, or at kinetochores, respectively. Using newly generated inducible cell lines, we show that overexpression of nuclear transport receptors affects the timing of RANBP2 localisation in opposite ways. Concomitantly, kinetochore functions are also affected, including the accumulation of SUMO- conjugated topoisomerase-IIα and stability of kinetochore fibres. These results delineate a novel mechanism through which nuclear transport receptors govern the functional state of kinetochores by regulating the timely deposition of RANBP2

Protein clustering in chemically stressed HeLa cells studied by infrared nanospectroscopy

Photo-Thermal Induced Resonance (PTIR) nanospectroscopy, tuned towards amide-I absorption, was used to study the distribution of proteic material in 34 different HeLa cells, of which 18 were chemically stressed by oxidative stress with Na3AsO3. The cell nucleus was found to provide a weaker amide-I signal than the surrounding cytoplasm, while the strongest PTIR signal comes from the perinuclear region. AFM topography shows that the cells exposed to oxidative stress undergo a volume reduction with respect to the control cells, through an accumulation of the proteic material around and above the nucleus. This is confirmed by the PTIR maps of the cytoplasm, where the pixels providing a high amide-I signal were identified with a space resolution of ∼300 × 300 nm. By analyzing their distribution with two different statistical procedures we found that the probability to find protein clusters smaller than 0.6 μm in the cytoplasm of stressed HeLa cells is higher by 35% than in the control cells. These results indicate that it is possible to study proteic clustering within single cells by label-free optical nanospectroscopy

ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cells- derived motoneurons

Patient-derived induced Pluripotent Stem Cells (iPSCs) provide an opportunity to study human diseases mainly in those cases where no suitable model systems are available. Here we have taken advantage of in vitro iPSCs derived from patients affected by Amyotrophic Lateral Sclerosis and carrying mutations in the RNA-binding proteins FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUS(R514S) and FUS(R521C) patients' fibroblasts, while in the case of the severe FUS(P525L) mutation, where fibroblasts were not available, a heterozygous and a homozygous iPSC lines were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, nicely correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis

Guiana Dolphin (Sotalia guianensis) in the Maracaibo Lake System, Venezuela: conservation, threats, and population overview

The Guiana dolphin (Sotalia guianensis) home range is located among Central and South American countries, in coastal habitats in the Caribbean and Atlantic Ocean. Its distribution is scattered, with multiple population centres which are under threats that vary based on local realities. We compiled and assessed biological data from multiple sources (published and unpublished data) to improve our understanding regarding the Maracaibo Lake Management Unit, which is an isolated and unique population core of this species. We identified at least two distinguishable population centres throughout the Maracaibo Lake System, one in the northern portion – in the Gulf of Venezuela, and another in the southern portion of the Maracaibo Lake itself. Both centres have differences in some biological aspects (e.g. group size and habitat use), but similarities in the human-induced pressures (e.g. intentional take, habitat degradation, and traditional use). We detailed the uses of Guiana dolphin (consumptive and non-consumptive) by community members, including the use as talismans for indigenous fishers and consumption of its meat as a religious belief (Easter period), and dolphin watching tours carried out by local companies. In one artisanal port, at least 15 animas are intentionally taken annually to be used for local consumption, shark-bait, or trade; however, we acknowledge that this annual take is likely an underestimate. Further research is needed to clarify how and at what magnitude mentioned and other key-threats are impacting over Guiana dolphin MU in the Maracaibo Lake System

3D bioprinted human cortical neural constructs derived from induced pluripotent stem cells

Bioprinting techniques use bioinks made of biocompatible non-living materials and cells to build 3D constructs in a controlled manner and with micrometric resolution. 3D bioprinted structures representative of several human tissues have been recently produced using cells derived by differentiation of induced pluripotent stem cells (iPSCs). Human iPSCs can be differentiated in a wide range of neurons and glia, providing an ideal tool for modeling the human nervous system. Here we report a neural construct generated by 3D bioprinting of cortical neurons and glial precursors derived from human iPSCs. We show that the extrusion-based printing process does not impair cell viability in the short and long term. Bioprinted cells can be further differentiated within the construct and properly express neuronal and astrocytic markers. Functional analysis of 3D bioprinted cells highlights an early stage of maturation and the establishment of early network activity behaviors. This work lays the basis for generating more complex and faithful 3D models of the human nervous systems by bioprinting neural cells derived from iPSCs

Visualization of the three-dimensional structure of the human centromere in mitotic chromosomes by super-resolution microscopy

The human centromere comprises large arrays of repetitive alpha-satellite DNA at the primary constriction of mitotic chromosomes. In addition, centromeres are epigenetically specified by the centromere-specific histone H3 variant CENP-A that supports kinetochore assembly to enable chromosome segregation. Since CENP-A is bound to only a fraction of the alpha-satellite elements within the megabase-sized centromere DNA, correlating the three-dimensional (3D) organization of alpha-satellite DNA and CENP-A remains elusive. To visualize centromere organization within a single chromatid, we used a combination of the Centromere Chromosome Orientation Fluorescent In Situ Hybridization (Cen-CO-FISH) technique together with Structured Illumination Microscopy (SIM). Cen-CO-FISH allows the differential labeling of the sister chromatids without the denaturation step used in conventional FISH that may affect DNA structure. Our data indicate that alpha-satellite DNA is arranged in a ring-like organization within prometaphase chromosomes, in presence or absence of spindle's microtubules. Using expansion microscopy (ExM), we found that CENP-A organization within mitotic chromosomes follows a rounded pattern similar to that of alpha-satellite DNA, often visible as a ring thicker at the outer surface oriented towards the kinetochore-microtubules interface. Collectively, our data provide a 3D reconstruction of alpha-satellite DNA along with CENP-A clusters that outline the overall architecture of the mitotic centromere. [Media: see text] [Media: see text] [Media: see text] [Media: see text