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Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2021)

Iron (Fe) is vital to growth and energy production of living organisms because it serves as a critical cofactor in many enzymatic reactions. Understanding Fe homeostasis in the plant lineage, including Fe storage and recycling mechanisms, provides a pathway to improve global primary productivity in agriculture and carbon capture capabilities. Chlamydomonas reinhardtii is a eukaryotic, unicellular green alga that has been used as a photosynthetic reference organism for the study of Fe metabolism, biofuel production, and other processes. In many organisms, Fe storage is often realized using vacuoles or the prototypical Fe storage protein, ferritin. In Chlamydomonas, ferritin appears to play a minor role in Fe storage, as its abundance is reduced with increasing amounts of extracellular Fe. Separately, the acidocalcisome, a lysosome-related vacuole characterized by acidic pH, high calcium (Ca) and polyphosphate (polyP) content, has been shown to house various trace metal ions in over-accumulating situations and is a candidate reservoir for Fe storage. Yet, the exact location for excess Fe in Chlamydomonas is uncertain. The pathway for and the regulation of sequestration, distribution, and mobilization of excess Fe taken up under Fe luxury conditions are not well understood either. Targeting these issues, my project was set to address three specific aims on Fe storage and transport in Chlamydomonas: 1) Systematic analysis of secondary experimental variables that determine cellular Fe accumulation; 2) Distinguishing the role of acidocalcisomes in Fe storage; and 3) Identifying the molecular mechanisms for excess Fe sequestration and mobilization. In Aim 1, six common experimental variables relating to various environmental aspects were surveyed to examine their impact on cellular growth and Fe content. The results revealed that cells over-accumulate Fe during stationary phase (3-fold increase) and in alkaline condition (10-fold increase). These Fe over-accumulating conditions were used to address questions in Aim 2 and 3. In Aim 2, various types of elemental images consistently showed Fe in cells grown at alkaline pH colocalizing strongly with Ca and P, markers of acidocalcisomes. In contrast, the Fe accumulated in cells during stationary phase is mostly sequestered into foci that do not contain Ca or P, suggesting an Fe storage site other than the acidocalcisome. Thus, cells may be selectively housing Fe in the acidocalcisome under specific conditions. Interestingly, investigation over the bioavailability of stored Fe suggested that the Fe accumulated under alkaline condition is not readily accessible, as indicated by the slow growth of cells from such cultures upon transfer to Fe-free medium. In Aim 3, a comparative transcriptomic analysis of cells grown in alkaline vs. neutral media exposed a significant increase (138-fold) in the abundance of FEA2 transcripts, encoding a periplasmic Fe-assimilating protein, hence strongly implicating FEA2 in playing a specific role in Fe accumulation under alkaline condition. In addition, a set of ten mutants involved in Fe homeostasis was tested for the capability to accumulate and re-distribute excess Fe. Surprisingly, the fer1 strain, lacking the predominant ferritin subunit, over-accumulated even more Fe than did the wild-type and other tested mutant strains, but failed to benefit from its greater Fe content. This outcome implies that ferritin has a role in Fe acquisition and mobilization. Altogether, my studies provide a methodical analysis of the experimental stimuli resulting in Fe accumulation and the underlying mechanisms of Fe storage and transport in Chlamydomonas at a fundamental level.

    Cover page: Functional Analysis of Iron Storage and Transport in the Green Alga Chlamydomonas reinhardtii
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2021)
    The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass spectrometry (MS)-based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared with MS-based imaging techniques, XFM offers the additional advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in Chlamydomonas reinhardtii to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal overaccumulation (Fe and Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the vtc1 mutant, defective in polyphosphate synthesis and failing to accumulate Ca, to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.
      Cover page: Single-cell visualization and quantification of trace metals in Chlamydomonas lysosome-related organelles
      • Article
      UCLA Previously Published Works (2021)
      The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass-spectrometry (MS)-based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared to MS-based imaging techniques, XFM offers the advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in Chlamydomonas reinhardtii , to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal over-accumulation (Fe, Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions, and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the vtc1 mutant, defective in polyphosphate synthesis and failing to accumulate Ca to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.

      Significance statement

      Transition metals are of crucial importance for primary productivity; their scarcity limits crop yield in agriculture and carbon sequestration at global scale. Copper (Cu), iron (Fe) and manganese (Mn) are among the most important trace elements that enable the redox chemistry in oxygenic photosynthesis. The single-celled, eukaryotic green alga Chlamydomonas reinhardtii is a choice experimental system for studying trace metal homeostasis in the context of phototrophy, offering all the advantages of a classical microbial system with a well-characterized photosystem and trace metal metabolism machinery of relevance to plants. This project identifies and differentiates different trace metal storage sites in Chlamydomonas and uncovers the dynamics of trace metal storage and mobilization in situations of fluctuating resources.
        Cover page: Single-cell Visualization and Quantification of Trace Metals in Chlamydomonas Lysosome-Related Organelles
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2022)
        Even subtle modifications in growth conditions elicit acclimation responses affecting the molecular and elemental makeup of organisms, both in the laboratory and in natural habitats. We systematically explored the effect of temperature, pH, nutrient availability, culture density, and access to CO2 and O2 in laboratory-grown algal cultures on growth rate, the ionome, and the ability to accumulate Fe. We found algal cells accumulate Fe in alkaline conditions, even more so when excess Fe is present, coinciding with a reduced growth rate. Using a combination of Fe-specific dyes, X-ray fluorescence microscopy, and NanoSIMS, we show that the alkaline-accumulated Fe was intracellularly sequestered into acidocalcisomes, which are localized towards the periphery of the cells. At high photon flux densities, Zn and Ca specifically over-accumulate, while Zn alone accumulates at low temperatures. The impact of aeration was probed by reducing shaking speeds and changing vessel fill levels; the former increased the Cu quota of cultures, the latter resulted in a reduction in P, Ca, and Mn at low fill levels. Trace element quotas were also affected in the stationary phase, where specifically Fe, Cu, and Zn accumulate. Cu accumulation here depends inversely on the Fe concentration of the medium. Individual laboratory strains accumulate Ca, P, and Cu to different levels. All together, we identified a set of specific changes to growth rate, elemental composition, and the capacity to store Fe in response to subtle differences in culturing conditions of Chlamydomonas, affecting experimental reproducibility. Accordingly, we recommend that these variables be recorded and reported as associated metadata.
          Cover page: Simple steps to enable reproducibility: culture conditions affecting Chlamydomonas growth and elemental composition
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