Inhibition of mTORC1 by astrin and stress granules prevents apoptosis in cancer cells.

Mammalian target of rapamycin complex 1 (mTORC1) controls growth and survival in response to metabolic cues. Oxidative stress affects mTORC1 via inhibitory and stimulatory inputs. Whereas downregulation of TSC1-TSC2 activates mTORC1 upon oxidative stress, the molecular mechanism of mTORC1 inhibition remains unknown. Here, we identify astrin as an essential negative mTORC1 regulator in the cellular stress response. Upon stress, astrin inhibits mTORC1 association and recruits the mTORC1 component raptor to stress granules (SGs), thereby preventing mTORC1-hyperactivation-induced apoptosis. In turn, balanced mTORC1 activity enables expression of stress factors. By identifying astrin as a direct molecular link between mTORC1, SG assembly, and the stress response, we establish a unifying model of mTORC1 inhibition and activation upon stress. Importantly, we show that in cancer cells, apoptosis suppression during stress depends on astrin. Being frequently upregulated in tumors, astrin is a potential clinically relevant target to sensitize tumors to apoptosis.

Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However, for scientists wishing to publish obtained images and image-analysis results, there are currently no unified guidelines for best practices. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here, we present community-developed checklists for preparing light microscopy images and describing image analyses for publications. These checklists offer authors, readers and publishers key recommendations for image formatting and annotation, color selection, data availability and reporting image-analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby to heighten the quality and explanatory power of microscopy data.

Dissection of DNA damage and repair pathways in live cells by femtosecond laser microirradiation and free-electron modeling.

Understanding and predicting the outcome of the interaction of light with DNA has a significant impact on the study of DNA repair and radiotherapy. We report on a combination of femtosecond pulsed laser microirradiation at different wavelengths, quantitative imaging, and numerical modeling that yields a comprehensive picture of photon-mediated and free-electron-mediated DNA damage pathways in live cells. Laser irradiation was performed under highly standardized conditions at four wavelengths between 515 nm and 1,030 nm, enabling to study two-photon photochemical and free-electron-mediated DNA damage in situ. We quantitatively assessed cyclobutane pyrimidine dimer (CPD) and γH2AX-specific immunofluorescence signals to calibrate the damage threshold dose at these wavelengths and performed a comparative analysis of the recruitment of DNA repair factors xeroderma pigmentosum complementation group C (XPC) and Nijmegen breakage syndrome 1 (Nbs1). Our results show that two-photon-induced photochemical CPD generation dominates at 515 nm, while electron-mediated damage dominates at wavelengths ≥620 nm. The recruitment analysis revealed a cross talk between nucleotide excision and homologous recombination DNA repair pathways at 515 nm. Numerical simulations predicted electron densities and electron energy spectra, which govern the yield functions of a variety of direct electron-mediated DNA damage pathways and of indirect damage by •OH radicals resulting from laser and electron interactions with water. Combining these data with information on free electron-DNA interactions gained in artificial systems, we provide a conceptual framework for the interpretation of the wavelength dependence of laser-induced DNA damage that may guide the selection of irradiation parameters in studies and applications that require the selective induction of DNA lesions.

Micro-Meta App: an interactive tool for collecting microscopy metadata based on community specifications.

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.

A practical guide to bioimaging research data management in core facilities.

Bioimage data are generated in diverse research fields throughout the life and biomedical sciences. Its potential for advancing scientific progress via modern, data-driven discovery approaches reaches beyond disciplinary borders. To fully exploit this potential, it is necessary to make bioimaging data, in general, multidimensional microscopy images and image series, FAIR, that is, findable, accessible, interoperable and reusable. These FAIR principles for research data management are now widely accepted in the scientific community and have been adopted by funding agencies, policymakers and publishers. To remain competitive and at the forefront of research, implementing the FAIR principles into daily routines is an essential but challenging task for researchers and research infrastructures. Imaging core facilities, well-established providers of access to imaging equipment and expertise, are in an excellent position to lead this transformation in bioimaging research data management. They are positioned at the intersection of research groups, IT infrastructure providers, the institution´s administration, and microscope vendors. In the frame of German BioImaging - Society for Microscopy and Image Analysis (GerBI-GMB), cross-institutional working groups and third-party funded projects were initiated in recent years to advance the bioimaging community's capability and capacity for FAIR bioimage data management. Here, we provide an imaging-core-facility-centric perspective outlining the experience and current strategies in Germany to facilitate the practical adoption of the FAIR principles closely aligned with the international bioimaging community. We highlight which tools and services are ready to be implemented and what the future directions for FAIR bioimage data have to offer.

Cilia-localized LKB1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney.

Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy-related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell-autonomous manner and results in peritubular accumulation of CCR2+ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.

Ift88, but not Kif3a, is required for establishment of the periciliary membrane compartment.

The primary cilium is a sensory organelle at the cell surface with integral functions in cell signaling. It contains a microtubular axoneme that is rooted in the basal body (BB) and serves as a scaffold for the movement of intraflagellar transport (IFT) particles by Kinesin-2 along the cilium. Ift88, a member of the anterograde moving IFT-B1 complex, as well as the Kinesin-2 subunit Kif3a are required for cilia formation. To facilitate signaling, the cilium restricts the access of molecules to its membrane ("ciliary gate"). This is thought to be mediated by cytoskeletal barriers ("subciliary domains") originating from the BB subdistal/distal appendages, the periciliary membrane compartment (PCMC) as well as the transition fibers and zone (TF/TZ). The PCMC is a poorly characterized membrane domain surrounding the ciliary base with exclusion of certain apical membrane proteins. Here we describe that Ift88, but not Kinesin-2, is required for the establishment of the PCMC in MDCK cells. Likewise, in C. elegans mutants of the Ift88 ortholog osm-5 fail to establish the PCMC, while Kinesin-2 deficient osm-3 mutants form PCMCs normally. Furthermore, disruption of IFT-B1 into two subcomplexes, while disrupting ciliogenesis, does not interfere with PCMC formation. Our findings suggest that cilia are not a prerequisite for the formation of the PCMC, and that separate machineries with partially overlapping functions are required for the establishment of each.

Divergent function of polycystin 1 and polycystin 2 in cell size regulation.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2, the genes encoding polycystin 1 (PC1) and polycystin 2 (PC2), respectively. PC1 and PC2 localize to the primary cilium and form a protein complex, which is thought to regulate signaling events. PKD1 mutations are associated with a stronger phenotype than PKD2, suggesting the existence of PC1 specific functions in renal tubular cells. However, the evidence for diverging molecular functions is scant. The bending of cilia by fluid flow induces a reduction in cell size through a mechanism that involves the kinase LKB1 but not PC2. Here, using different in vitro approaches, we show that contrary to PC2, PC1 regulates cell size under flow and thus phenocopies the loss of cilia. PC1 is required to couple mechanical deflection of cilia to mTOR in tubular cells. This study pinpoints divergent functions of the polycystins in renal tubular cells that may be relevant to disease severity in ADPKD.

A Joint Action in Times of Pandemic: The German BioImaging Recommendations for Operating Imaging Core Facilities During the SARS-Cov-2 Emergency.

Operating shared resource laboratories (SRLs) in times of pandemic is a challenge for research institutions. In a multiuser, high-turnover working space, the transmission of infectious agents is difficult to control. To address this challenge, imaging core facility managers being members of German BioImaging discussed how shared microscopes could be operated with minimal risk of spreading SARS-CoV-2 between users and staff. Here, we describe the resulting guidelines and explain their rationale, with a focus on separating users in space and time, protective face masks, and keeping surfaces virus-free. These recommendations may prove useful for other types of SRLs. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Progesterone modulates microtubule dynamics and epiboly progression during zebrafish gastrulation.

Control of microtubule dynamics is crucial for cell migration. We analyzed regulation of microtubule network dynamics in the zebrafish yolk cell during epiboly, the earliest coordinated gastrulation movement. We labeled microtubules with EMTB-3GFP and EB3-mCherry to visualize and measure microtubule dynamics by TIRF microscopy live imaging. Yolk cell microtubules dynamics is temporally modulated during epiboly progression. We used maternal zygotic Pou5f3 mutant (MZspg) embryos, which develop strong distortions of microtubule network organization and epiboly retardation, to investigate genetic control of microtubule dynamics. In MZspg embryos, microtubule plus-end growth tracks move slower and are less straight compared to wild-type. MZspg embryos have altered steroidogenic enzyme expression, resulting in increased pregnenolone and reduced progesterone levels. We show that progesterone positively affects microtubule plus-end growth and track straightness. Progesterone may thus act as a non-cell-autonomous regulator of microtubule dynamics across the large yolk cell, and may adjust differing demands on microtubule dynamics and stability during initiation and progression phases of epiboly.

Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens.

Protein arginylation is a posttranslational modification of both N-terminal amino acids of proteins and sidechain carboxylates and can be crucial for viability and physiology in higher eukaryotes. The lack of arginylation causes severe developmental defects in moss, affects the low oxygen response in Arabidopsis thaliana and is embryo lethal in Drosophila and in mice. Although several studies investigated impact and function of the responsible enzyme, the arginyl-tRNA protein transferase (ATE) in plants, identification of arginylated proteins by mass spectrometry was not hitherto achieved. In the present study, we report the identification of targets and interaction partners of ATE in the model plant Physcomitrella patens by mass spectrometry, employing two different immuno-affinity strategies and a recently established transgenic ATE:GUS reporter line (Schuessele et al., 2016 New Phytol. , DOI: 10.1111/nph.13656). Here we use a commercially available antibody against the fused reporter protein (ß-glucuronidase) to pull down ATE and its interacting proteins and validate its in vivo interaction with a class I small heatshock protein via Förster resonance energy transfer (FRET). Additionally, we apply and modify a method that already successfully identified arginylated proteins from mouse proteomes by using custom-made antibodies specific for N-terminal arginine. As a result, we identify four arginylated proteins from Physcomitrella patens with high confidence.Data are available via ProteomeXchange with identifier PXD003228 and PXD003232.

Calcium buffer proteins are specific markers of human retinal neurons.

Ca(2+)-buffer proteins (CaBPs) modulate the temporal and spatial characteristics of transient intracellular Ca(2+)-concentration changes in neurons in order to fine-tune the strength and duration of the output signal. CaBPs have been used as neurochemical markers to identify and trace neurons of several brain loci including the mammalian retina. The CaBP content of retinal neurons, however, varies between species and, thus, the results inferred from animal models cannot be utilised directly by clinical ophthalmologists. Moreover, the shortage of well-preserved human samples greatly impedes human retina studies at the cellular and network level. Our purpose has therefore been to examine the distribution of major CaBPs, including calretinin, calbindin-D28, parvalbumin and the recently discovered secretagogin in exceptionally well-preserved human retinal samples. Based on a combination of immunohistochemistry, Neurolucida tracing and Lucifer yellow injections, we have established a database in which the CaBP marker composition can be defined for morphologically identified cell types of the human retina. Hence, we describe the full CaBP make-up for a number of human retinal neurons, including HII horizontal cells, AII amacrine cells, type-1 tyrosine-hydroxylase-expressing amacrine cells and other lesser known neurons. We have also found a number of unidentified cells whose morphology remains to be characterised. We present several examples of the colocalisation of two or three CaBPs with slightly different subcellular distributions in the same cell strongly suggesting a compartment-specific division of labour of Ca(2+)-buffering by CaBPs. Our work thus provides a neurochemical framework for future ophthalmological studies and renders new information concerning the cellular and subcellular distribution of CaBPs for experimental neuroscience.

hMSC-Derived VEGF Release Triggers the Chemoattraction of Alveolar Osteoblasts.

Human mesenchymal stem cells (hMSCs) are promising candidates for regenerative periodontal strategies, due to the broad spectrum of supportive effects on cells and tissues at the site of application. Although positive effects are visible, the understanding of their underlying mechanisms still requires further elucidation. Recently, we have shown that hMSCs are capable to prompt osteogenic differentiation of alveolar osteoblasts, thereby presumably contributing to alveolar bone regeneration. Another issue that is critical in this context is the attraction of hard tissue-forming cells to regeneration sites, but it is an open question whether hMSCs can afford this. In the present manuscript, we show by life cell imaging that in interactive cocultures, hMSCs successfully trigger osteoblast chemotaxis. Gene expression analysis for hMSC-innate chemoattractive biomolecules, orchestrating this process, revealed vascular endothelial growth factor (VEGF), PgE synthase, osteoprotegerin (OPG), monocyte colony-stimulating factor, and transforming growth factor ß1, which was confirmed for VEGF and OPG on the protein level. Noteworthy, we showed that only corresponding levels of VEGF but not OPG attracted alveolar osteoblasts similar to hMSC coculture, while VEGF inhibitor abolished both the VEGF and the hMSC-triggered chemoattraction. In summary, we have identified secreted OPG and VEGF proteins as potential chemoattractants, of which further characterization yielded VEGF as a causative for hMSC-directed osteoblast chemotaxis. With respect to the better understanding of potential hMSC-based periodontal regeneration strategies, we propose hMSC-derived VEGF release as a mechanism in the recruitment of hard tissue-forming cells to alveolar bone sites in need of regeneration.

Measuring Local Viscosities near Plasma Membranes of Living Cells with Photonic Force Microscopy.

The molecular processes of particle binding and endocytosis are influenced by the locally changing mobility of the particle nearby the plasma membrane of a living cell. However, it is unclear how the particle's hydrodynamic drag and momentum vary locally and how they are mechanically transferred to the cell. We have measured the thermal fluctuations of a 1 µm-sized polystyrene sphere, which was placed in defined distances to plasma membranes of various cell types by using an optical trap and fast three-dimensional (3D) interferometric particle tracking. From the particle position fluctuations on a 30 µs timescale, we determined the distance-dependent change of the viscous drag in directions perpendicular and parallel to the cell membrane. Measurements on macrophages, adenocarcinoma cells, and epithelial cells revealed a significantly longer hydrodynamic coupling length of the particle to the membrane than those measured at giant unilamellar vesicles (GUVs) or a plane glass interface. In contrast to GUVs, there is also a strong increase in friction and in mean first passage time normal to the cell membrane. This hydrodynamic coupling transfers a different amount of momentum to the interior of living cells and might serve as an ultra-soft stimulus triggering further reactions.

ViBE-Z: a framework for 3D virtual colocalization analysis in zebrafish larval brains.

Precise three-dimensional (3D) mapping of a large number of gene expression patterns, neuronal types and connections to an anatomical reference helps us to understand the vertebrate brain and its development. We developed the Virtual Brain Explorer (ViBE-Z), a software that automatically maps gene expression data with cellular resolution to a 3D standard larval zebrafish (Danio rerio) brain. ViBE-Z enhances the data quality through fusion and attenuation correction of multiple confocal microscope stacks per specimen and uses a fluorescent stain of cell nuclei for image registration. It automatically detects 14 predefined anatomical landmarks for aligning new data with the reference brain. ViBE-Z performs colocalization analysis in expression databases for anatomical domains or subdomains defined by any specific pattern; here we demonstrate its utility for mapping neurons of the dopaminergic system. The ViBE-Z database, atlas and software are provided via a web interface.