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Ostreococcus tauri is an ancient phototrophic microalgae that possesses favorable genetic and cellular characteristics for reductionist studies probing biosystem design and dynamics. Here multimodal bioimaging and multi-omics techniques were combined to interrogate O. tauri cellular changes in response to variations in bioavailable nitrogen and carbon ratios. Confocal microscopy, stimulated Raman scattering, and cryo-soft x-ray tomography revealed whole cell ultrastructural dynamics and composition while proteomic and lipidomic profiling captured changes at the molecular and macromolecular scale.Despite several energy dense long-chain triacylglycerol lipids showing more than 40-fold higher abundance under N deprivation, only a few proteins directly associated with lipid biogenesis showed significant expression changes. However, the entire pathway for starch granule biosynthesis was highly upregulated suggesting much of the cellular energy is preferentially directed towards starch over lipid accumulation. Additionally, three of the five most downregulated and five of the ten most upregulated proteins during severe nitrogen depletion were unnamed protein products that warrant additional biochemical analysis and functional annotation to control carbon transformation dynamics in this smallest eukaryote.

Lipid droplet biogenesis, accumulation and secretion is an important field of research spanning biofuel feedstock production in algae and yeast to plant-microbe symbiosis or human metabolic disorders and other diseases. Here we evaluate the critical elements that influence lipid accumulation in the highly simplified and smallest known eukaryote Ostreococcus tauri and identify several conditions that satisfy its classification as an oleaginous green alga. In addition, these experiments revealed the release of excess lipids in pea-pod like structures where many dense lipid droplets are clustered in a linear fashion surrounded by an enveloping membrane which contrasts with known mechanisms from other eukaryotes. These results highlight the potential for Ostreococcus tauri to probe the evolution of lipid droplet dynamics as an emerging model organism with a compacted eukaryotic genome and also to impact lipid feedstock bioproduction applications either directly or using synthetic biology.One Sentence SummaryThe smallest known eukaryote Ostreococcus tauri is oleaginous and sheds lipid droplets as pea-pod like membrane enclosed clusters.

© 2016. Published by The Company of Biologists Ltd. Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.

Due to their interesting properties, research on colloidal nanocrystals has moved in the last few years from fundamental research to first applications in materials science and life sciences. In this review some recent biological applications of colloidal nanocrystals are discussed, without going into biological or chemical details. First, the properties of colloidal nanocrystals and how they can be synthesized are described. Second, the conjugation of nanocrystals with biological molecules is discussed. And third, three different biological applications are introduced: (i) the arrangement of nanocrystal–oligonucleotide conjugates using molecular scaffolds such as single-stranded DNA, (ii) the use of nanocrystal–protein conjugates as fluorescent probes for cellular imaging, and (iii) a motility assay based on the uptake of nanocrystals by living cells.

We have shown previously that epimorphin (EPM), a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which EPM mediates luminal morphogenesis. Treatment of cells with EPM to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein (C/EBP)β and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, since constitutive expression of LIP was sufficient to produce lumen formation, whereas constitutive expression of LAP blocked EPM-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which EPM expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPβ, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for EPM in luminal morphogenesis through control of C/EBPβ expression.

Soft X-ray microscopy is ideally suited to visualizing and quantifying biological cells. Specimens, including eukaryotic cells, are imaged intact, unstained and fully hydrated, and therefore visualized in a near-native state. The contrast in soft X-ray microscopy is generated by the differential attenuation of X-rays by the molecules in the specimen-water is relatively transmissive to this type of illumination compared to carbon and nitrogen. The attenuation of X-rays by the specimen follows the Beer-Lambert law, and therefore both linear and a quantitative measure of thickness and chemical species present at each point in the cell. In this chapter, we will describe the procedures and computational methods that lead to 50 nm (or better) tomographic reconstructions of cells using soft X-ray microscope data, and the subsequent segmentation and analysis of these volumetric reconstructions. In addition to being a high-fidelity imaging modality, soft X-ray tomography is relatively high-throughput; a complete tomographic data set can be collected in a matter of minutes. This new modality is being applied to imaging cells that range from small prokaryotes to stem cells obtained from mammalian tissues.

The nucleus, although it has been studied for many decades, remains the body of many mysteries. In eukaryotic cells, DNA is found in two coexistent readable forms, an open, gene-rich region (euchromatin) that allows transcription of DNA into RNA, and a compacted region (heterochromatin) that contains silenced genes. In response to numerous extracellular stimuli, the cell must express specific proteins, which means that the DNA coding for these proteins must be in a transcriptionally active form. Consequently the balance between euchromatin and heterochromatin must be regulated during cell differentiation. Understanding this regulation requires fine-tuned biochemical and biophysical analyses and cutting-edge imaging techniques. Thanks to the soft X-ray microscope of the National Center for X-ray Tomography (Uchida et al., 2010; Larabell et al. 2010), high-resolution images of the 3D organization of the nucleus in the native state are now achievable. Cryo-immobilization of the cells (fast freezing) for x-ray imaging avoids chemical fixation artifacts associated with TEM. Imaging with X-rays in the water window energy range allows a natural contrast between water and biomolecules. X-ray imaging avoids perturbations caused by chemicals and osmotic changes associated with dehydration required for TEM, which cause deformation of nuclear structures (Finan and Guilack, 2010). As a result, x-ray tomography yields the first 3D views of nuclear organization in intact cells with a resolution of 50 nm. Our study indicates that heterochromatin forms a continuous network in 3D space, with no evidence of the chromatin patches described in TEM. Likewise, euchromatin regions are continuous. In addition distinct 3D chromatin patterns are linked to the differentiation/maturation state of the cell. Studying the 3D pattern of the nucleus using soft x-ray tomography is shedding new light on our understanding of cell differentiation.

The ability of cancer cells to migrate and metastasize is known to be directly related to tumor cell motility. Therefore, assaying the level of tumor cell motility is an excellent indicator of metastatic potential. We have developed an efficient and sensitive two-dimensional cell motility assay to image the phagokinetic uptake of colloidal CdSe/ZnS semiconductor nanocrystals (quantum dots [QDs]). As cells move across a thin, homogeneous layer of QDs, they engulf and uptake the nanocrystals and leave behind a fluorescent-free trail. By measuring the ratio of trail area to cell area we have discovered that it is possible to distinguish between noninvasive and invasive cancer cells lines. This technique has, therefore, the potential to be used as a rapid, robust, and quantitative in vitro measure of metastatic potential. Because the technique only relies on fluorescence detection, requires no significant data processing, and is used with live cells, it is both rapid and straightforward.

For years the existence of nuclear actin has been heavily debated, but recent data have clearly demonstrated that actin, as well as actin-binding proteins (ABPs), are located in the nucleus. We examined live EGFP-actin-expressing cells using confocal microscopy and saw the presence of structures strongly resembling actin filaments in the nuclei of MDA-MB-231 human mammary epithelial tumor cells. Many nuclei had more than one of these filamentous structures, some of which appeared to cross the entire nucleus. Extensive analysis, including fluorescence recovery after photobleaching (FRAP), showed that all EGFP-actin in the nucleus is monomeric (G-actin) rather than filamentous (F-actin) and that the apparent filaments seen in the nucleus are invaginations of cytoplasmic monomeric actin. Immunolocalization of nuclear pore complex proteins shows that similar invaginations are seen in cells that are not overexpressing EGFP-actin. To determine whether there is a correlation between increased levels of invagination in the cell nuclei and the state of de-differentiation of the cell, we examined a variety of cell types, including live Xenopus embryonic cells. Cells that were highly de-differentiated, or cancerous, had an increased incidence of invagination, while cells that were differentiated had few nuclear invaginations. The nuclei of embryonic cells that were not yet differentiated underwent multiple shape changes throughout interphase, and demonstrated numerous transient invaginations of varying sizes and shapes. Although the function of these actin-filled invaginations remains speculative, their presence correlates with cells that have increased levels of nuclear activity.

Colloidal nanocrystals are building blocks of the “nanoworld”. Their electronic properties enable the building of single-electron transistors, their optical properties can be used to generate fluorescence labels with many different colors. Based on the principles of molecular recognition and self assembly biological molecules can be used to arrange nanoscale building blocks. Two applications will be discussed. Colloidal gold nanocrystals were conjugated with a controlled number of DNA molecules per nanocrystal. By using complementary sequences of DNA molecules that were attached to different nanocrystals, small groupings of gold nanocrystals could be formed. Biomolecule conjugated colloidal semiconductor nano-crystals also have been used to fluorescence label structural compartments of cells. These nanocrystals were found to be actively incorporated by living cells. It will be described how cells “eat” nanocrystals and an assay for cell mobility based on this fact will be introduced.