Distinct mechanisms eliminate mother and daughter centrioles in meiosis of starfish oocytes

Centriole elimination is an essential process that occurs in female meiosis of metazoa to reset centriole number in the zygote at fertilization. How centrioles are eliminated remains poorly understood. Here we visualize the entire elimination process live in starfish oocytes. Using specific fluorescent markers, we demonstrate that the two older, mother centrioles are selectively removed from the oocyte by extrusion into polar bodies. We show that this requires specific positioning of the second meiotic spindle, achieved by dynein-driven transport, and anchorage of the mother centriole to the plasma membrane via mother-specific appendages. In contrast, the single daughter centriole remaining in the egg is eliminated before the first embryonic cleavage. We demonstrate that these distinct elimination mechanisms are necessary because if mother centrioles are artificially retained, they cannot be inactivated, resulting in multipolar zygotic spindles. Thus, our findings reveal a dual mechanism to eliminate centrioles: mothers are physically removed, whereas daughters are eliminated in the cytoplasm, preparing the egg for fertilization.European Molecular Biology Laboratory (EMBL)- EMBL International PhD Program; Laura and Arthur Colwin Endowed Summer Research Fellowship; Deutsche Forschungsgemeinschaft grant: (MU1423/4-1)

Structural rearrangements in the phage head-to-tail interface during assembly and infection

Many icosahedral viruses use a specialized portal vertex to control genome encapsidation and release from the viral capsid. In tailed bacteriophages, the portal system is connected to a tail structure that provides the pipeline for genome delivery to the host cell. We report the first, to our knowledge, subnanometer structures of the complete portal–phage tail interface that mimic the states before and after DNA release during phage infection. They uncover structural rearrangements associated with intimate protein–DNA interactions. The portal protein gp6 of bacteriophage SPP1 undergoes a concerted reorganization of the structural elements of its central channel during interaction with DNA. A network of protein–protein interactions primes consecutive binding of proteins gp15 and gp16 to extend and close the channel. This critical step that prevents genome leakage from the capsid is achieved by a previously unidentified allosteric mechanism: gp16 binding to two different regions of gp15 drives correct positioning and folding of an inner gp16 loop to interact with equivalent loops of the other gp16 subunits. Together, these loops build a plug that closes the channel. Gp16 then fastens the tail to yield the infectious virion. The gatekeeper system opens for viral genome exit at the beginning of infection but recloses afterward, suggesting a molecular diaphragm-like mechanism to control DNA efflux. The mechanisms described here, controlling the essential steps of phage genome movements during virus assembly and infection, are likely to be conserved among long-tailed phages, the largest group of viruses in the Biosphere

Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

<div><p>Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior <i>in vivo</i> with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis.</p></div

2PEM Imaging of D2.0R Cells Xenotransplanted in Mouse Ear Skin.

<p>A. Two weeks after injection with LifeAct-Ypet expressing D2.0R cells, a tumor is formed in the mouse ear skin (top left panel, outlined in green). The green fluorescent signal of the D2.0R cells can be observed in the tumor region (top right panel). A histological cross-section through the tumor region was obtained from the second ear injected at the same time with the same cell type. The Trichrome Masson staining colors the collagen in blue (bottom panel). B. Maximum projection of a z-stack obtained from an area in the periphery of the invasive tumor (top left panel A, white square). The D2.0R cells are shown in green, and the tumor is outlined in yellow. A background signal from the Evans Blue (red) can be observed in the vicinity of the blood vessels, potentially caused by the ‘leaky’ nature of the tumor vessels, its uptake by residing immune cells and the repeated injections. Scale bar: 50 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s006" target="_blank">Movie S4</a>. C. Higher magnification z-projection of the cell of interest boxed in B. Scale bar: 10 µm. D. Time-lapse imaging of a D2.0R tumor cell in vivo. The different panels depict, in black against a white background, the fluorescent signal of the actin in the cell at the indicated time-points. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s006" target="_blank">Movie S4</a>. E. Color-coded map of the structural changes of the cell of interest (boxed in B and C) over time. Scale bar: 10 µm.</p

Outline of the Correlative Workflow.

<p>A. The flowchart lists the general steps involved in the correlative workflow. B. Left panel: installation of the anesthetized mouse on the LM stage. The ear with the transplanted fluorescent tumor cells (right panel, cartoon) is mounted in a custom-built holder. C. Z-projection of a typical 2PEM dataset obtained from the mouse ear that was injected with GFP-expressing tumor cells (green). SHG signal of the collagen fibers is shown in blue. Evans Blue stains blood vessels and is depicted in red. Scale bar: 100 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s003" target="_blank">Movie S1</a>. D. Cartoon of the NIRB process representing the imaged volume containing a vessel (red), collagen fibers (blue), and tumor cells (green). Our NIRB-procedure entails drawing a frame at the surface of the skin with a high-powered laser, above and away from the ROI. The imaged volumes presented in this work were in between 60 and 200 µm in depth and ranging from 270 to 440 µm in xy. E. Following NIRB, the same volume is imaged again with 2PEM. The NIRB marks visible in this z-projection are traced in white. Scale bar: 100 µm. Asterisks point to hair follicles. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s003" target="_blank">Movie S1</a>. F. After chemical fixation of the mouse ear sample, NIRB markings remain temporarily autofluorescent and their location can be mapped. Asterisks pointing to hair follicles (as in E). G. Following EM processing, the embedded sample can be trimmed and sectioned by ultramicrotomy. Serial thick sections (500 nm) are placed on glass slides to be imaged by light microscopy (‘sections on LM slide’). Serial thin sections (60 to 240 nm) are mounted on slot grids (‘sections on TEM grid’) allowing for TEM observation and/or electron tomography.</p

Correlating 2PEM to 3D EM of D2.0R Cells using Collagen Fibers as a Reference.

<p>A. Cartoon of the sectioning approach. Following 2PEM, NIRB and processing as described before, a few thick sections were produced from the sample to verify the orientation of the block and the presence of the NIRB markings (‘Approach’). The sectioning direction is indicated with an arrow. As soon as the first NIRB marks were spotted in the thick sections, we proceeded with obtaining thin 240 nm sections for electron tomography (‘Correlation+EM imaging ROI’). These sections were not only employed for EM imaging of the ROI, but also provided the structural features that were used for correlation between 2PEM and EM. B. Graphic representation of the landmark-based correlation procedure. The prisms with the blue and green ribs represent similar 3D volumes that differ in size, shape and orientation, representing the samples pre- and post-EM-processing. In order to predict the position of the ROI (ball, visible in volume 1) in volume 2, similar points (landmarks) in both datasets are identified (‘Correlation’). These landmarks are overlaid in 3D (‘Registration’) enabling warping of volume 1 into volume 2 and thus also projecting the position of the ROI in volume 2 (‘Docking’). C. Collagen fibers can be observed in both the 2PEM z-stack (top row panels, 2PEM) and the EM images of the serial sections (middle row, TEM). In the TEM images, the collagen is visible as electron dense fibers running in between the cellular material. Each column shows a 2PEM and an EM image obtained at a similar position in the sample, pre- and post EM processing. The numbers in the top left corner of the panels indicate their position in z to the image shown in the first column. The 2PEM dataset is stretched in z relative to the TEM dataset, causing the larger z-steps between the 2PEM images in the different rows. The bottom row shows the overlay of the 2PEM image (blue) and the TEM image (grey levels). Scale bars: 100 µm. D-F. The landmark-registration procedure is performed in Amira, using 3D models of collagen fibers as a reference. The selected landmarks (colored ‘targets’) indicate corresponding points in both datasets. D. 3D model of the collagen fibers (blue) and the cell of interest (green), obtained from a high magnification 2PEM z-stack of the cell of interest. E: 3D model of the collagen fibers (grey), obtained from a z-stack of EM images. F. The 3D visualization software Amira was used to overlay the landmarks and perform non-linear transformations to the 2PEM model to warp it into the EM model. G-I: Landmark-registration between the 2PEM and EM datasets enables retrieval and electron tomography of the cell of interest (green). G. Following the registration of the 2PEM in the EM dataset, the cell of interest could be docked within the z-stack of EM images. Scale bar: 10 µm. H. The docked cell of interest is outlined in green on a TEM section of the ROI. The nucleus of the cell is visible. Scale bar: 3 µm. K. Virtual slice through the tomogram obtained in the area that is boxed in I. The collagen fibers in the top left corner are clearly recognizable owing to their striated structure and high electron density. Scale bar: 500 nm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s006" target="_blank">Movie S4</a>.</p

Nintedanib and a bi-specific anti-VEGF/Ang2 nanobody selectively prevent brain metastases of lung adenocarcinoma cells

Brain metastases (BM) are an ever-increasing challenge in oncology, threatening quality of life and survival of many cancer patients. The majority of BM originate from lung adenocarcinoma, and stage III patients have a risk of 40-50% to develop BM in the first years of disease onset. As therapeutic options are limited, prevention of their occurrence is an attractive concept. Here we investigated whether Nintedanib (BIBF 1120), a tyrosine kinase inhibitor (TKI) targeting the VEGF pathway approved for lung adenocarcinoma, and the dual anti-VEGF-A/Ang2 nanobody BI836880 have the potential to prevent BM formation. A mouse model of brain metastasis from lung adenocarcinoma was used in which tumor cells were injected intracardially. Metastases formation occurred inside and outside of the brain and was followed by MRI, IVIS, and immunohistochemistry. BM were reduced in volume and number by both Nintedanib and the dual anti-VEGF-A/Ang2 nanobody, which translated into improved survival. Both compounds were able to normalize cerebral blood vessels at the site of brain metastatic lesions. Extracranial metastases, however, were not reduced, and meningeal metastases only partially. Interestingly, unspecific control IgG also lead to brain vessel normalization and reduction of brain and meningeal metastases. This data indicates a brain-specific group effect of antiangiogenic compounds with respect to metastasis prevention, most likely by preventing an early angiogenic switch. Thus, Nintedanib and BI836880 are promising candidates for future BM preventive study concepts in lung adenocarcinoma patients

Hemodynamic forces tune the arrest, adhesion and extravasation of circulating tumor cells

International audienc

Hemodynamic forces tune the arrest, adhesion and extravasation of circulating tumor cells

International audienc