Meiotic cellular rejuvenation is coupled to nuclear remodeling in budding yeast.

Production of healthy gametes in meiosis relies on the quality control and proper distribution of both nuclear and cytoplasmic contents. Meiotic differentiation naturally eliminates age-induced cellular damage by an unknown mechanism. Using time-lapse fluorescence microscopy in budding yeast, we found that nuclear senescence factors - including protein aggregates, extrachromosomal ribosomal DNA circles, and abnormal nucleolar material - are sequestered away from chromosomes during meiosis II and subsequently eliminated. A similar sequestration and elimination process occurs for the core subunits of the nuclear pore complex in both young and aged cells. Nuclear envelope remodeling drives the formation of a membranous compartment containing the sequestered material. Importantly, de novo generation of plasma membrane is required for the sequestration event, preventing the inheritance of long-lived nucleoporins and senescence factors into the newly formed gametes. Our study uncovers a new mechanism of nuclear quality control and provides insight into its function in meiotic cellular rejuvenation

Viruses infecting a warm water picoeukaryote shed light on spatial co-occurrence dynamics of marine viruses and their hosts

The marine picoeukaryote Bathycoccus prasinos has been considered a cosmopolitan alga, although recent studies indicate two ecotypes exist, Clade BI (B. prasinos) and Clade BII. Viruses that infect Bathycoccus Clade BI are known (BpVs), but not that infect BII. We isolated three dsDNA prasinoviruses from the Sargasso Sea against Clade BII isolate RCC716. The BII-Vs do not infect BI, and two (BII-V2 and BII-V3) have larger genomes (~210 kb) than BI-Viruses and BII-V1. BII-Vs share ~90% of their proteins, and between 65% to 83% of their proteins with sequenced BpVs. Phylogenomic reconstructions and PolB analyses establish close-relatedness of BII-V2 and BII-V3, yet BII-V2 has 10-fold higher infectivity and induces greater mortality on host isolate RCC716. BII-V1 is more distant, has a shorter latent period, and infects both available BII isolates, RCC716 and RCC715, while BII-V2 and BII-V3 do not exhibit productive infection of the latter in our experiments. Global metagenome analyses show Clade BI and BII algal relative abundances correlate positively with their respective viruses. The distributions delineate BI/BpVs as occupying lower temperature mesotrophic and coastal systems, whereas BII/BII-Vs occupy warmer temperature, higher salinity ecosystems. Accordingly, with molecular diagnostic support, we name Clade BII Bathycoccus calidus sp. nov. and propose that molecular diversity within this new species likely connects to the differentiated host-virus dynamics observed in our time course experiments. Overall, the tightly linked biogeography of Bathycoccus host and virus clades observed herein supports species-level host specificity, with strain-level variations in infection parameters

Ultrastructural Characterization of Cellular Interactions Within Resting, Developing and Tumorigenic Breast Epithelium

Epithelial to mesenchymal transition (EMT) and collective cell migration events are of interest for understanding cancer progression and metastasis. The use of in vitro cell lines to model these processes are widely used and help to shed light upon the mechanisms that lead to tumorigenesis. Here we use two such models: the human mammary epithelial cell line HMT-3522 and ex vivo mouse mammary primary culture epithelial organoids. The HMT-3522 cell line provides an encapsulated system to study cancer progression, however detailed ultrastructural analyses of this cell line are lacking. To study collective cell migration we used the murine ex vivo epithelial "organoid" system, which upon growth factor stimulation undergoes branching morphogenesis. Growth of these systems in 3D cell culture is an important component in understanding the highly dynamic and complex processes of EMT and collective cell migration. High resolution imaging via transmission electron microscopy and three dimensional scanning electron microscopy provide a way to discern ultrastructural features of both collective cell migration and EMT. Electron microscopy imaging of the HMT-3522 S1 cell line, grown in 3D to form acini, demonstrated an abundance of membrane protrusions at all interior lateral and apical surfaces. Furthermore, these acinar structures were determined to have a semi-polarized state as defined by the appearance of a basement membrane, but lack all hallmarks of apical polarization. Three dimensional imaging of the membrane protrusions revealed they have a variety of lengths and an interdigitated nature. Determining this semi-polarized state to be more representative of a transitive tissue state, we sought to image the transient terminal end bud (TEB) of organoids undergoing branching morphogenesis. Ultrastructural analysis of in vivo murine mammary gland and ex vivo un-stimulated organoids revealed nearly identical tissue organization, with typical apico-basal polarity and almost no intercellular membrane protrusions. However, data of the stimulated organoids and specifically the TEB revealed a semi-polarized state of the cells very similar to the HMT-3522 S1 acini, with prolific membrane protrusions. Hypothesizing these membrane protrusions were aiding in normal collective cell migration, we investigated whether disruption of actin polymerization via drug treatments within the TEB would recapitulate the ultrastructure of the tumorigenic cell line HMT-3522 T4-2. RhoA kinase inhibition lead to a remarkably similar tissue and cellular morphology between the two cell types

Jorgens et al., JCS 2015 Movie 10

<b>Movie 10:</b> <b>Lamin B1 stain of S1 acinus illuminates multiple nuclear tunnels and the NR.</b> Confocal stack of staining for lamin B1 (green) and DNA (DAPI, blue) in growth arrested S1 acini.  NR type II is observed in multiple cells as well as nuclear tunnels transversing the nucleus.<div><br><div><p>J Cell Sci 2016 : doi: 10.1242/jcs.190967</p></div></div

Self-organizing actin networks drive sequential endocytic protein recruitment and vesicle release on synthetic lipid bilayers.

Forces generated by actin assembly assist membrane invagination during clathrin-mediated endocytosis (CME). The sequential recruitment of core endocytic proteins and regulatory proteins, and assembly of the actin network, are well documented in live cells and are highly conserved from yeasts to humans. However, understanding of CME protein self-organization, as well as the biochemical and mechanical principles that underlie actins role in CME, is lacking. Here, we show that supported lipid bilayers coated with purified yeast Wiskott Aldrich Syndrome Protein (WASP), an endocytic actin assembly regulator, and incubated in cytoplasmic yeast extracts, recruit downstream endocytic proteins and assemble actin networks. Time-lapse imaging of WASP-coated bilayers revealed sequential recruitment of proteins from different endocytic modules, faithfully replicating in vivo behavior. Reconstituted actin networks assemble in a WASP-dependent manner and deform lipid bilayers, as seen by electron microscopy. Time-lapse imaging revealed that vesicles are released from the lipid bilayers with a burst of actin assembly. Actin networks pushing on membranes have previously been reconstituted; here, we have reconstituted a biologically important variation of these actin networks that self-organize on bilayers and produce pulling forces sufficient to bud off membrane vesicles. We propose that actin-driven vesicle generation may represent an ancient evolutionary precursor to diverse vesicle forming processes adapted for a wide array of cellular environments and applications

Cytonemes with complex geometries and composition extend into invaginations of target cells

Cytonemes are specialized filopodia that mediate paracrine signaling in Drosophila and other animals. Studies using fluorescence confocal microscopy (CM) established their general paths, cell targets, and essential roles in signaling. To investigate details unresolvable by CM, we used high-pressure freezing and EM to visualize cytoneme structures, paths, contents, and contacts. We observed cytonemes previously seen by CM in the Drosophila wing imaginal disc system, including disc, tracheal air sac primordium (ASP), and myoblast cytonemes, and identified cytonemes extending into invaginations of target cells, and cytonemes connecting ASP cells and connecting myoblasts. Diameters of cytoneme shafts vary between repeating wide (206 ± 51.8 nm) and thin (55.9 ± 16.2 nm) segments. Actin, ribosomes, and membranous compartments are present throughout; rough ER and mitochondria are in wider proximal sections. These results reveal novel structural features of filopodia and provide a basis for understanding cytoneme cell biology and function