Dynamic full-field optical coherence tomography: 3D live-imaging of retinal organoids

Optical coherence tomography offers astounding opportunities to image the complex structure of living tissue, but lacks functional information. We present dynamic full-field optical coherence tomography to image living human induced pluripotent stem cell-derived retinal organoids non-invasively. Colored images with an endogenous contrast linked to organelle motility are generated, with sub-micrometer spatial resolution and millisecond temporal resolution, opening an avenue to identify specific cell types in living tissue via their function.Comment: 14 pages, 5 figures, 1 table, 6 video

Tools to study cytoadhesion of P. falciparum-infected erythrocytes under flow

Plasmodium falciparum infected erythrocytes are able to bind to a multitude of extracellular receptors through adhesins expressed on their membrane. The molecular interactions involved in parasite binding to these receptors are only partly understood, although the trait itself is well-known and used as an early indicator of severe malaria diagnosis and progress of infection. Binding to the microvasculature of blood vessels or the intervillous spaces of the placenta, ensures the survival of the parasite as it avoids splenic clearance in the later stages of maturation. In this thesis, the importance of sheer flow and cell morphology in binding dynamics of parasitized erythrocytes is further highlighted. While static adhesion assays would showcase similar amounts of cells adhering in different maturation stages, when observed in flow, this picture changes drastically. Trophozoite stage parasites seem to bind less frequently but more efficiently onto the simulated endothelium. In the last stage of maturation, schizont stage parasites alter the cell morphology to such an extent that adhesion is more likely but with less contact area and density of involved receptors. Changes in the membrane morphology between AA and AS erythrocytes also underline the significance of receptor presentation and accessibility influencing binding efficiency. The effects of a P. falciparum infection during primigravida are threatening to both the mother and the foetus, as infected erythrocytes sequester to the maternal side of the syncytiotrophoblast that lines the placenta. Despite the tremendous efforts in the field, the adhesive tropism of infected erythrocytes that leads to placental sequestration remains unsolved. In this thesis, I determined that measurements are not possible without the full length protein and interactions between the receptor and its erythrocyte-expressed ligand are not specific enough to distinguish from negative control experiments. Another form of cell adhesion investigated in this thesis is the formation of so-called rosettes, that form when an infected erythrocyte binds to non-infected erythrocytes. Rosette formation is considered either an indication of severe malaria or a symptom of progressed infection and is believed to propagate the severity of infection by obstructing smaller vasculature and normal blood flow. in this thesis, I developed a platform to study the position of rosettes within a channel in flow, in order to determine their margination tendance. In those experiments, I verified that regardless of haematocrit value and size of rosette, rosettes remain in flow and do not marginate towards the walls of the flow chamber

Development and Deployment of a Point-Source Digital Inline Holographic Microscope for the Study of Plankton and Particles to a Depth of 6000 m

Bochdansky, A. B., Jericho, M. H., & Herndl, G. J. (2013). Development and deployment of a point-source digital inline holographic microscope for the study of plankton and particles to a depth of 6000 m. Limnology and Oceanography: Methods, 11, 28-40. doi: 10.4319/lom.2013.11.2

Collective motion of cells: from experiments to models

Swarming or collective motion of living entities is one of the most common and spectacular manifestations of living systems having been extensively studied in recent years. A number of general principles have been established. The interactions at the level of cells are quite different from those among individual animals therefore the study of collective motion of cells is likely to reveal some specific important features which are overviewed in this paper. In addition to presenting the most appealing results from the quickly growing related literature we also deliver a critical discussion of the emerging picture and summarize our present understanding of collective motion at the cellular level. Collective motion of cells plays an essential role in a number of experimental and real-life situations. In most cases the coordinated motion is a helpful aspect of the given phenomenon and results in making a related process more efficient (e.g., embryogenesis or wound healing), while in the case of tumor cell invasion it appears to speed up the progression of the disease. In these mechanisms cells both have to be motile and adhere to one another, the adherence feature being the most specific to this sort of collective behavior. One of the central aims of this review is both presenting the related experimental observations and treating them in the light of a few basic computational models so as to make an interpretation of the phenomena at a quantitative level as well.Comment: 24 pages, 25 figures, 13 reference video link

Complexity in Developmental Systems: Toward an Integrated Understanding of Organ Formation

During animal development, embryonic cells assemble into intricately structured organs by working together in organized groups capable of implementing tightly coordinated collective behaviors, including patterning, morphogenesis and migration. Although many of the molecular components and basic mechanisms underlying such collective phenomena are known, the complexity emerging from their interplay still represents a major challenge for developmental biology. Here, we first clarify the nature of this challenge and outline three key strategies for addressing it: precision perturbation, synthetic developmental biology, and data-driven inference. We then present the results of our effort to develop a set of tools rooted in two of these strategies and to apply them to uncover new mechanisms and principles underlying the coordination of collective cell behaviors during organogenesis, using the zebrafish posterior lateral line primordium as a model system. To enable precision perturbation of migration and morphogenesis, we sought to adapt optogenetic tools to control chemokine and actin signaling. This endeavor proved far from trivial and we were ultimately unable to derive functional optogenetic constructs. However, our work toward this goal led to a useful new way of perturbing cortical contractility, which in turn revealed a potential role for cell surface tension in lateral line organogenesis. Independently, we hypothesized that the lateral line primordium might employ plithotaxis to coordinate organ formation with collective migration. We tested this hypothesis using a novel optical tool that allows targeted arrest of cell migration, finding that contrary to previous assumptions plithotaxis does not substantially contribute to primordium guidance. Finally, we developed a computational framework for automated single-cell segmentation, latent feature extraction and quantitative analysis of cellular architecture. We identified the key factors defining shape heterogeneity across primordium cells and went on to use this shape space as a reference for mapping the results of multiple experiments into a quantitative atlas of primordium cell architecture. We also propose a number of data-driven approaches to help bridge the gap from big data to mechanistic models. Overall, this study presents several conceptual and methodological advances toward an integrated understanding of complex multi-cellular systems

Membrane proteins in the outer mebrane of plastids and mitochondria

Channels of the plastid and mitochondrial outer membranes facilitate the turnover of molecules and ions via these membranes. Although channels have been studied many questions pertaining to the whole diversity of plastid and mitochondrial channels in Arabidopsis thaliana and Pisum sativum remain unanswered. In this thesis I studied OEP16, OEP37 and VDAC families in two model plants, in Arabidopsis and pea. The Arabidopsis OEP16 family represents four channels of α-helical structure, similar to the pea OEP16 protein. These channels are suggested to transport amino acids and compounds with primary amino groups. Immunoblot analysis, GFP/RFP protein fusion expression, as well as proteomic analysis showed that AtOEP16.1, AtOEP16.2 and AtOEP16.4 are located in the outer envelope membrane of plastids, while AtOEP16.3 is in mitochondria. The gene expression and immunoblot analyses revealed that AtOEP16.1 and AtOEP16.3 proteins are highly abundant and ubiquitous; expression of AtOEP16.1 is regulated by light and cold. AtOEP16.2 is highly expressed in pollen, seeds and seedlings. AtOEP16.4 is a low expressed housekeeping protein. Single knockout mutants of AtOEP16.1, AtOEP16.2 and AtOEP16.4, and double mutants of AtOEP16 gene family did not show any remarkable phenotype. However, macroarray analysis of Atoep16.1-p T-DNA mutant revealed 10 down-regulated and 6 up-regulated genes. In contrast to the α-helical OEP16 proteins, the OEP37 and VDAC proteins are of β-barrel structure. The PsOEP37 and AtOEP37 channel proteins form a selective barrier in the outer envelope of chloroplasts. Electrophysiological studies in lipid bilayer membranes showed that the PsOEP37 channel is permeable for cations. Specific expression profiles showed that AtOEP37 and PsOEP37 are highly expressed in the entire plant. The isolated PsVDAC gene encodes a protein, which is located in mitochondria. In Arabidopsis gene database, five Arabidopsis genes, which code for VDAC-like proteins were announced. One gene was not detected, whereas four of these genes expressed in leaves, roots, flower buds and pollen

Aqueous foams and aerated oil-in-water emulsions partly stabilised by edible particles

The objectives of this thesis are to enhance the understanding of edible solid particle behaviour at oil-water and at air-water interfaces, and how their adsorption at these interfaces affects the emulsion and foam stability. Materials stabilised solely by solid particles are of great interest due to long-term stability, lower emulsifier content and also in order to replace surfactants and oils, which are often used in food and pharmaceutical products. The project is funded by Rich Products, a leading manufacturer of non-dairy icings and toppings. The interest of Rich Products in this research is in application of calcium carbonate nanoparticles in whipped cream formulations. Air bubbles in whipped cream are coated and stabilised by adsorption of fat droplets during aeration. Particular interest of the company was to see whether calcium carbonate particles could fulfil all or part of the role played by fat droplets in stabilising air bubbles in whipped cream. In addition, this may decrease the fat content of such whipped cream products, desirable for certain consumers.Aeration properties of aqueous dispersions of particles and an anionic food-grade surfactant, sodium stearoyl lactylate, are studied. It is shown that the hydrophilic nature of the particles prevented their adsorption at air-water interfaces. Oppositely charged surfactant molecules were used to modify the surface properties of the particles. The in situ surface modification of the particles upon adsorption of the surfactant molecules is shown to promote the adsorption of particles at air-water interfaces. The particles were observed to form a rigid barrier around air bubbles and provide long-term stability to aqueous foams. The effect of surfactant concentration, particle addition and aeration method are all presented. The progress in preparation of model whipping cream emulsions and aerated emulsions was then continued by preparation of palm kernel oil-in-water emulsions. It is shown how aeration of palm kernel oil-in-water emulsions in the presence of surface modified calcium carbonate particles provides long-term stability to aerated emulsions. The results from characterisation of the model whipped cream foams are presented and discussed in relation to foams stability. It is shown that adsorption of calcium carbonate particles together with palm kernel oil droplets around air bubbles prevents foam collapse and enhances the foam life-time. The effect of particle concentration, oil volume fraction and whipping duration on several properties of the model whipped creams are presented. Together with preparation and characterisation of model samples, aeration properties of Vanilla Bettercreme®, a commercial non-dairy ready-to-whip cream manufactured by the sponsor of the project are also presented. This product has been used as a reference in different stages of this project.Palm kernel oil-in-water emulsions stabilised by calcium carbonate particles or by sodium stearoyl lactylate are then compared in terms of their stability towards coalescence. It is observed that in the emulsions stabilised by surfactants, oil droplets joined together and resulted in emulsion destabilisation. On the other hand, in particle-stabilised emulsions adsorption of the particles around oil droplets creates a rigid barrier around them and prevents the droplets joining together. The effect of emulsifier concentration and oil volume fraction are studied in detail.Finally, preparation of oil-in-water emulsions and aerated oil-in-water emulsions containing non-crystallisable oils are investigated. The aim was to understand the interactions of fluid oil droplets with an air-water interface. Both the emulsions and aerated emulsions were stabilised by sodium stearoyl lactylate. It is shown that the oil type, surfactant concentration and the oil volume fraction affect the stability of the emulsions and aerated emulsions prepared. The size of oil droplets in aerated oil-in-water emulsions is shown to be the determining factor for their stability

Characterization of a soybean BAG gene and its potential role in nematode resistance

Title from PDF of title page; abstract from short PDF (University of Missouri--Columbia, viewed on June 26, 2014).Plants resistant to the soybean cyst nematode (SCN) mount a hypersensitive cell death-like response upon nematode feeding, but the genes regulating this process are not known. Laser-assisted microdissection of nematode feeding cells coupled with microarray analysis identified a soybean gene upregulated 87-fold in plants resistant to SCN that shared sequence similarity with the Arabidopsis BAG6 (Bcl-2 associated athanogene 6) gene. BAG genes encode an evolutionarily conserved family of proteins in animals, yeast and plants. These proteins contain a conserved BAG domain which mediates interaction with the molecular chaperone HSP70/HSC70. Members of the BAG protein family in animals and yeast function in apoptosis to regulate a range of activities from inhibition to promotion of cell death. However, much less is known about the role of BAG proteins in plants. A family of seven BAG genes (AtBAG1-7) has been identified in Arabidopsis. AtBAG6 was shown to induce programmed cell death in both yeast and Arabidopsis. Expression of a truncated version of the protein, spanning a calmodulin-binding IQ domain and the BAG domain, enhanced the cell death phenotype. Here, we demonstrate that similar to AtBAG6, overexpression of the full length GmBAG6A protein or a truncated version spanning the IQ and BAG domains, induced cell death in yeast and plants, with the truncated form showing an enhanced cell death phenotype. Expression of the truncated form in Arabidopsis and soybean under the control of a nematode-inducible promoter significantly reduced nematode development demonstrating its potential use in engineering a novel form of nematode resistance in crop plants

Building a light-sheet microscope to study the early development of the polyclad flatworm Maritigrella crozieri (Hyman, 1939)

The Lophotrochozoa is an evolutionary interesting clade comprising many and mostly marine phyla. Despite their diverse morphology, these animals often have a biphasic life-cycle in form of a free-swimming, ciliated larval stage, the trochophore, and seemingly retained a highly conserved developmental pattern called spiral cleavage. This opens the door for comparative studies to better understand the shared mechanisms of the spiralian developmental program, its deviations and the evolution of lophotrochozoan body plans. Here I studied the early development and larva formation in the polyclad flatworm Maritigrella crozieri and compared it with other members of this evolutionarily diverse group of animals. In order to conduct this research, I first built a light-sheet microscope (OpenSPIM) that would allow me to follow the development of the polyclad embryo from the zygote into the larval stage and to acquire sophisticated 3d-reconstructions of fixed embryonic stages. I then used this and other techniques to characterise in detail the early development of M. crozieri and its conserved spiral cleavage pattern. Precise volume measurements of 3d-reconstructed early blastomeres and the investigations of associated cleavage patterns indicate that this polyclad worm may not follow a strictly equal spiral cleavage type, as was previously thought. I investigated the cleavage pattern and fate of micromere 4d, which in M. crozieri gives rise to mesoderm and generates bilaterally symmetric embryos at a cellular level. A first cell lineage analysis of this organism involved long-term live imaging recordings and point to a conserved fate of blastomeres that, like in other spiral cleavers, give rise to ectodermally derived structures, specifically the larva’s locomotion system (ciliary band) and a sensory organ at the apical point (apical organ). These findings strengthen the idea that these structures may be homologous to those found in other trochophore larvae. This work increased the current knowledge of the early development of the polyclad flatworm M. crozieri, which facilitates evolutionary comparisons of the development of different flatworms and lophotrochozoans more broadly and can contribute to addressing the homology of marine larvae

Characterizing the Function of CSLD Proteins During Plant Cell Wall Deposition in Arabidopsis

As one of the most significant features of plant cells, the cell wall not only defines plant cell shape but also provides strength and rigidity to the plant. During plant development, changes in cell shape are primarily driven by cell expansion, which is controlled by cell wall deposition and modification. The two major mechanisms that control these changes are called diffuse growth and tip growth. During diffuse expansion, cell wall materials are synthesized and integrated in a polarized fashion along the entire expanding face of the cells. In contrast, during tip growth new cell wall deposition is restricted to a limited plasma membrane domain, leading to the highly polarized cell expansion associated with this directed cell wall construction. As the major load-bearing component in plant cell walls, cellulose is also the most abundant biopolymer on earth. Unlike other cell wall polysaccharides, cellulose is synthesized in the plasma membranes by large integral membrane protein complexes called cellulose synthase complexes (CSCs). The catalytic subunits of the CSCs are encoded by members of the Cellulose Synthase (CESA) family. Previous research showed that CESA1, CESA3, and CESA6 are required for the formation of active CSCs involved in the synthesis of cellulose in the primary cell wall of cells undergoing diffuse growth in Arabidopsis. Interestingly, our laboratory previously demonstrated that CSCs containing CESA3 and CESA6 did not appear to be required for new cellulose synthesis at the apical plasma membranes of root hair cells undergoing tip growth. Instead, members of a related family of Cellulose Synthase-Like D (CSLD) proteins showed tip-specific localization in these membranes and provided cell wall synthase activity required for maintenance of structural integrity of the cell wall in these tip-growing root hairs. However, while these CSLD cell wall synthases are essential, the nature of the polysaccharides generated by CSLD proteins has remained elusive. Here, I use genetic and biochemical approaches to characterize the catalytic activity of one member of the CSLD family, CSLD3. Genetic complementation of a cesa6 mutant with a chimeric CESA6 protein containing a CSLD3 catalytic domain demonstrated that the CSLD catalytic domains can successfully generate β-1,4-glucan polymers for cellulose synthesis. Time-lapse fluorescence microscopy demonstrated that these CESA6-CSLD3 chimeric proteins assembled into CSC complexes with similar mobility as CESA6-labeled complexes in hypocotyl cells. Proteoliposomes containing purified, detergent-solubilized CSLD3 and CESA6 proteins could specifically utilize UDP-glucose as an enzymatic substrate and synthesize products that are only sensitive to endo-β-1,4-glucanase. Taken together, these data strongly support the conclusion that CSLD3 represents a UDP-glucose-dependent β-1,4-glucan synthase. However, whether CSLD proteins require the formation of higher-order complexes to perform β-1,4 glucan synthase activities remained unclear. Here, I used genetic methods to demonstrate that CSLD2 and CSLD3 proteins are functionally interchangeable with each other during root hair elongation and cell plate formation. CSLD5 could partially rescue the root hair elongation defects in csld3 mutants. However, it plays a unique and essential function during cell plate formation. Proteoliposomes containing CSLD2 and CSLD5 displayed conserved β-1,4 glucan synthases activities similar to those described for CSLD3. Taken together, these results indicate that while all three vegetatively expressed CSLD proteins possess conserved β-1,4 glucan synthases activities, CSLD5 has a more complicated and specialized role during cell plate formation. To sum up, my dissertation research further supports that CSLD proteins represent a distinct family of cellulose synthase in Arabidopsis.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/162885/1/jiyuany_1.pd