Quantitative elemental imaging in eukaryotic algae Stefan Schmollinger12 Si Chen3 and Sabeeha S. Merchant12 1California Institute for Quantitative Biosciences QB3 University of California Berkeley CA

2025-05-02 3 0 1.39MB 33 页 10玖币

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Quantitative elemental imaging in eukaryotic algae

Stefan Schmollinger1,2,*, Si Chen3 and Sabeeha S. Merchant1,2

1California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA

94720

2Departments of Molecular and Cell Biology and Plant and Microbial Biology, Berkeley, CA 94720

33X-ray Science Division, Argonne National Laboratory, Lemont, IL 60439

* Present address: MSU-DOE Plant Research Laboratory, Michigan State University, East

Lansing, MI 48824

Corresponding author: Stefan Schmollinger

612 Wilson Road, MSU-DOE Plant Research Laboratory, Michigan State University, East

Lansing, MI 48824, USA, +1 (310) 779-0687, schmolli@msu.edu

Keywords: trace metal sequestration, heavy metal detoxification, iron, copper, manganese, XRF,

SXRF, Chlamydomonas

Stefan Schmollinger (https://orcid.org/0000-0002-7487-8014)

Si Chen (https://orcid.org/0000-0001-6619-2699)

Sabeeha S. Merchant (https://orcid.org/0000-0002-2594-509X)

Abstract

All organisms, fundamentally, are made from the same raw material, namely the elements of the

periodic table. Biochemical diversity is achieved with how these elements are utilized, for what

purpose and in which physical location. Determining elemental distributions, especially those of

trace elements that facilitate metabolism as cofactors in the active centers of essential enzymes,

can determine the state of metabolism, the nutritional status or the developmental stage of an

organism. Photosynthetic eukaryotes, especially algae, are excellent subjects for quantitative

analysis of elemental distribution. These microbes utilize unique metabolic pathways that require

various trace nutrients at their core to enable its operation. Photosynthetic microbes also have

important environmental roles as primary producers in habitats with limited nutrient supply or toxin

contaminations. Accordingly, photosynthetic eukaryotes are of great interest for biotechnological

exploitation, carbon sequestration and bioremediation, with many of the applications involving

various trace elements and consequently affecting their quota and intracellular distribution. A

number of diverse applications were developed for elemental imaging allowing subcellular

resolution, with X-ray fluorescence microscopy (XFM) being at the forefront, enabling quantitative

descriptions of intact cells in a non-destructive method. This Tutorial Review summarizes the

workflow of a quantitative, single-cell elemental distribution analysis of a eukaryotic alga using

XFM.

The aim of this review is to (1) highlight the contributions of different elements to photosynthetic

life and the concepts of how organisms control their elemental composition, (2) introduce the

methodologies involved in studying elemental distributions in cells, especially X-ray fluorescence

microscopy (XFM, XRF), (3) review the current state of XFM studies in eukaryotic algae, and (4)

to extract a methodology frame-work for conducting XFM studies from these works. It is our goal

to facilitate the entry into the field of elemental research for algae scholars encountering questions

of metal homeostasis and elemental heterogeneity for the first time, and to encourage the use of

quantitative elemental imaging approaches for the purpose of determining biological function.

Elemental composition of cells

The elements of the periodic table are the indivisible foundation of all matter, including all

biological life of our planet (Figure 1). Every component of a cell is assembled from a selection of

elements, most prominently C, H, N, O, P, S, which are essential and constitute the backbone of

proteins, carbohydrates, nucleic acids and lipids (1-3). Essentiality of an element is defined by its

irreplaceability and its requirement in metabolism to complete the vegetative or reproductive life

cycle (4), while beneficial elements only improve the organisms fitness. The set of essential

elements therefore can vary between different organisms, depending on the organism’s

environmental niche and its required enzyme portfolio. It is estimated that ~ 40 % of all enzymes

utilize a uniquely suited element outside the group of macronutrients (CHNOPS) within their

catalytic centers to enabling catalysis (5, 6). Most organisms employ several cations (K, Mg, Ca

and to a lesser degree Na) and the Cl anion, all of which are abundant constituents of biological

matter, to regulate osmotic pressure and pH, build gradients across membranes that facilitate

energy production, transport processes or signal transduction, or serve as cofactors or allosteric

regulators in metabolites or proteins (7). And lastly, organisms require various additional sets of

elements in trace amounts (including many metal ions), to enable chemical functionalities that are

not provided from metabolites or amino acids (3, 8). These micronutrients/trace elements are

required co-factors for metabolically-essential enzymes, or for enzymes that enable new

biochemical capabilities, and consequently their acquisition, intracellular distribution and

utilization are a crucial aspect of cellular metabolism (3). Trace elements typically present the

most interesting targets for elemental imaging, because of the impact of their chemistry on cell

health and metabolism, the range of their abundances in an organism and the dynamic regulation

involved in their utilization (3, 9). A set of trace elements is essential for most organisms, including

Fe, Cu, Co, Ni, Mn, Zn, Se, V, I and Mo. Redox biochemistry is a central aspect of trace metal

utilization. Mn, Cu, and Fe, are therefore among the most abundant and important trace elements

for all organisms; at lower abundance Ni, Co and Mo are also required as cofactors by many

organisms (5, 10-17). Fe, Cu and Mn, among many important contributions, are critical for

photosynthetic electron transfer (18). Ni is used in a wide range of organisms, for example in

ureases and hydrogenases (19). Mo, outside of bacterial Mo-nitrogenase, is usually found in the

Molybdenum cofactor Moco, which is used in many enzymes, most prominently nitrate reductase

(17). Zn is similarly abundant and widespread as a trace element as Fe, Cu and Mn, but is used

as a Lewis acid and a structural component for proteins in most organisms (20-22). Se, most

prominently, is utilized as selenocysteine in specific enzymes requiring the element in their

catalytic centers, for example glutathione peroxidases (23-26). Co is used as a cofactor in a few

enzymes directly, but most famously is at the center of cobalamin (also known as vitamin B12),

which is critical to nitrogen-fixing bacteria (27, 28). V is used in vanadium-dependent

nitrogenases and haloperoxidases, and has an important role as an electron acceptor in

bacterial respiration (29). I is used in thyroid hormones in vertebrates and haloperoxidases in

algae (30, 31).

Othertrace elements (B, Si, As, Br, Sr, Cd, Ba, W, Hg, Pb, La, Ce and Nd) are either employed

in very specialized roles in select organisms in a specific environmental niche, or in individual

enzymes with a specific beneficial, but non-essential function (32-35).

Many of the trace elements, among other bioactive elements, can also accumulate in organisms

involuntarily, using uptake routes for intended cofactors, and interfere with biological processes

either in an advantageous, or more commonly a detrimental way.

Therefore, utilization of, especially the redox-active trace elements, comes with inherent risks.

The chemical reactivities that make these elements useful in the first place must be controlled

intracellularly to avoid unintended reactions (36-38). The concentration of many of these elements

in the environment of cells is a critical parameter, determining if the organism is starving for the

element as a nutrient, if either abundance or bioavailability is low, or if cell health is threatened by

overexposure (39). The toxicity can either be directly attributed to the detrimental reactivities of

the elements when uncontrolled, to the production of secondary toxic products, for example

reactive oxygen species, or via enzyme mis-metalation. Mis-metalation is largely attributed to the

inherent flexibility in proteins and the similar physical properties (ionic radii, charge and

coordination preferences) of the biologically common trace metal (40, 41). Most enzymes are

tuned to function with a specific metal cofactor. Binding of a different, similar metal at the active

site can result in loss-of-function, or worse, the production of unintended products or promotion

of side-reactions (38, 42, 43). All organisms therefore carefully control their elemental composition

at the point of uptake, resulting in specific cellular quotas, especially in the case of redox active

trace elements. Cells also employ elaborate strategies to avoid mis-metalation intracellularly,

including the compartmentalization of specific elements to ensure that the correct metal binds to

newly-synthesized proteins, or the use of metallochaperones to ensure correct delivery through

protein-protein interactions (44-46). Some metals are associated with organic groups or build into

large clusters (for example Fe in heme and Fe-S clusters) for similar reasons.

The pathways for trace element metabolism are among the most ancient in biology (47), and the

general concepts involved in trace metal utilization are well conserved across organisms.

Photosynthetic organisms specifically have unique requirements with respect to the elemental

composition, because of the metabolic demand of the photosynthetic apparatus and specific

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QuantitativeelementalimagingineukaryoticalgaeStefanSchmollinger1,2,*,SiChen3andSabeehaS.Merchant1,21CaliforniaInstituteforQuantitativeBiosciences(QB3),UniversityofCalifornia,Berkeley,CA947202DepartmentsofMolecularandCellBiologyandPlantandMicrobialBiology,Berkeley,CA9472033X-rayScienceDivision,Argonn...

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Quantitative elemental imaging in eukaryotic algae Stefan Schmollinger12 Si Chen3 and Sabeeha S. Merchant12 1California Institute for Quantitative Biosciences QB3 University of California Berkeley CA

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