Letter pubs.acs.org/NanoLett
The Photosynthetic Eukaryote Nannochloris eukaryotum as an Intracellular Machine To Control and Expand Functionality of Human Cells Cara K. Black, Doina M. Mihai, and Ilyas Washington* Ophthalmology, Columbia University Medical Center, New York, New York 10032, United States S Supporting Information *
ABSTRACT: To construct an intracellular machine, we sought a symbiotic relationship between a photosynthetic green alga and human cells. Human cells selectively take up the minimal eukaryote Nannochloris eukaryotum and the resulting symbionts are able to survive and proliferate. Host cells can utilize N. eukaryotum’s photosynthetic apparatus for survival, and expression of cellular vascular endothelial growth factor can be controlled with input of photonic energy. This seemingly rare spontaneous association provides an opportunity to fabricate light-controlled, intracellular machines.
KEYWORDS: Symbiosis, nanomachine, endosymbiosis, molecular machine, nanomedicine
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within mammalian cells. This seeming incompatibility limits the number of biological platforms that can be engineered to carry out a particular task inside the cellular milieu to fine-tune cell function. We thus sought to identify unicellular guests that can form a stable union with human cells. Here, we report a spontaneous endosymbiotic association between a photosynthetic green alga and human-derived cells. Cultured human retinal pigment epithelial (RPE) cells, fibroblasts, keratinocytes and HeLa cells all take up the algae, Nannochloris eukaryotum, but rat- or mouse-derived cells do not. Conversely, 10 other species of algae do not form intracellular associations with any of the above cells. Results suggest that N. eukaryotum does not significantly harm human host cells, and human cells and algae maintain their ability to replicate. Nutrient-deprived human cells containing N. eukaryotum survive longer than cells without N. eukaryotum, and the guest could be used to manipulate its host, as exemplified by controlling the synthesis of vascular endothelial growth factor. Endosymbiosis between photosynthetic and nonphotosynthetic organisms is known to occur in protists, metazoan invertebrates and in one vertebrate amphibian host, the larvae of the spotted salamander, Ambystoma maculatum.2−4 The invasion of a eukaryotic alga into human cells represents a unique association with implications for the construction of nanoscale machines to increase human cell function.
he construction of machines capable of functioning on a subcellular level and their application to expand function of complex biological systems are persistent goals for future technology. Current efforts to construct molecular machines are largely centered on top-down and bottom-up approaches. Top-down approaches can be described as the minimization of current technologies, while bottom-up approaches can be described as the creation of molecular-sized parts to assemble into a larger working apparatus. However, our current inability to control motion at a molecular level and the lack of technologies to power that motion make the construction of well-controlled microscale machines a distant goal. To achieve the goal of fabrication of microsized structures that can accomplish programed tasks on a subcellular level in the next decade, additional approaches are needed. Nature expands biological function by the creation of stable polygenetic systems. Endosymbiosis, as exemplified by modern day mitochondria and chloroplasts, is a prime example and has been a major driving force of evolutionary change.1 In such systems, symbiotic guests can be considered to be molecular machines that impart an advantage to their host. By extension, the identification of distinct genetic systems that can cohabit within mammalian cells and be programmed to accomplish a task at will would expedite the development of microscale machines that can act intracellularly to dictate cellular fate. In theory, such machines can be readily programmed through current genetic engineering practices to synthesize molecular instructions (DNA, RNA, small molecules, carbohydrates, proteins, and so forth) to prevent or cure almost any disease or extend the life span or the capabilities of a cell or tissue type. However, there are few organisms that are known to cohabit © 2014 American Chemical Society
Received: February 19, 2014 Revised: April 15, 2014 Published: April 28, 2014 2720
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Figure 1. Uptake of photosynthetic green algae by mammalian cells. (A) Light microscopy image of RPE-N. eukaryotum symbionts cocultured for 3 weeks. About 0.5 algae per cell (15 cells and 8 algae are in the image); algae are seen as lighter dots. (B) Light microscopy image of RPE-N. eukaryotum symbionts. Algae are seen multiplying by budding (arrows). (C) Fluorescence image of symbionts. Chloroplasts are seen as U-shapes. (D,E) Electron micrographs of symbionts at 24 h after coincubation. Algae are seen as electron-dense spheres. Lipid inclusions were abundant inside algal cells and in the cytoplasm of host cells (F). Algae were observed in perialgal vacuoles (E) and in direct contact with the cytoplasm (F). Algal cell walls also appeared either smooth (F) or frayed (E), see arrows. Ch, chloroplast; L, lipid inclusions; N, host cell’s nucleus; PV, perialgal vacuole.
Figure 2. Both algae and cells maintained their ability to proliferate. (A) Nonconfluent RPE cells with algae guest. The field of view shows 7.5 RPE cells. (B) Same dish and area 11 days later, showing 64 RPE cells in the field of view.
algal strains, Picocystis sp., Micromonas pusilla, Ostreococcus sp., Pycnococcus provasolii, Choricystis sp., Meyerella planktonica, Picochlorum oklahomense, Nannochloropsis oculata, Chlorella protothecoides, and Chlorella vulgaris were not engulfed by any of the human cells tested after up to 7 days of coincubation. For further studies, we used RPE cells because of our ongoing interest in understanding and preventing diseases of the eye. Uptake of N. eukaryotum by the RPE cells was further confirmed with fluorescence (Figure 1C) and transmission electron microscopy (Figures 1D,E). Images revealed that the algae resided in the cell’s cytoplasm and in vacuoles similar in appearance to perialgal vacuoles seen in paramecium-algae
To determine whether photosynthetic green algae could form a symbiotic relationship with human cells, we screened 11 algal strains for their ability to enter into human cells in culture. N. eukaryotum was engulfed by RPE, fibroblasts, keratinocytes, and HeLa cells within 30 min after coincubation (Figure 1A, and Supporting Information Figure 1). Cells readily took up the algae. For example, all algae added to the plate were found inside cells until the cell’s cytoplasmic space was completely filled up to approximately 30 algae per cell (Supporting Information Figure 2). In contrast, rat-derived liver and spleen cells and mouse-derived embryonic fibroblast, RPE, and conjunctival cells did not take up N. eukaryotum. Ten other 2721
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Figure 3. Cells with N. eukaryotum are more viable under cyclic light. Cell viability as measured by resazurin reduction. Curves for control RPE cells and RPE cells with an average of 5 algae per cell kept under cyclic light (A) or constant dark (B) for 3 days. Means with 95% confidence intervals are shown. When kept in cyclic light, cells with algae were more viable. However, in darkness, cells with algae were less viable. P values compare total amounts of resazurin reduced.
Figure 4. N. eukaryotum guests are seemingly not toxic to host cells. (A) Cellular ATP concentrations and (B) mitochondrial membrane potentials for RPE cells alone or RPE cells with algal guests, cultured under cyclic light. Means with 95% confidence intervals are shown. (C) Relative proliferation as measured by the rate of resazurin reduction after 4 days in RPE cells with and without algae guests cultured under cyclic light. Proliferation was calculated as the difference in reduction rates at days 2 and 4 from the reduction rate at day zero. Means with 95% confidence intervals are shown. *P value was less than 0.05 when compared to control cells at the same day. (D) Reversible increase and decrease in chlorophyll fluorescence upon light and dark adaptation (photoinhibition) in RPE cells with and without algal guest. Means with 95% confidence intervals are shown.
symbionts.5 Lipid inclusions could be seen in the algae and in the surrounding host’s cytoplasm by electron microscopy (Figures 1E,F), suggesting the transfer of lipids from the algae to their host. We could influence the stability of the union by manipulating light and CO2 levels. For example, when an RPE cell contained approximately 10 algal cells, the algae replicated and eventually took over the entire cell’s cytoplasm and killed the host cell within 4 weeks if exposed to periodic light. However, if kept it in the dark, the union was stable for up to 4 weeks, when we stopped the experiment. When algae were added to confluent RPE cells at less than an average of six algal cells per RPE cell, and when the resulting symbionts were kept under cyclic light, the union remained stable for up to 6 weeks at which time we stopped observations. In this latter situation, algae could be seen budding, a sign of replication (Figure 1B), but they did not overtake the host cell. The ratio of algae to cells did not significantly change over time. When we added N. eukaryotum to nonconfluent cells, the resulting symbionts proliferated until the entire surface of the plate was covered. Figure 2A shows nonconfluent RPE cells, some cells having up to 13 algae per cell. After 11 days in culture, both cells and algae proliferated so that the entire plate was covered with cells and nearly every cell contained multiple
algae guests (Figure 2B), suggesting that both cells and algae were able to proliferate. To evaluate viability of host cells, we employed four commonly used assays. First, we used the dye resazurin to measure metabolic activity. Metabolically active cells take up resazurin and reduce it to the fluorescent resorufin. The rate of resazurin reduction is proportional to cell fitness.6 N. eukaryotum alone did not reduce resazurin; thus, resazurin reduction was selective for the RPE host’s metabolic activity. We added known amounts of algae to the human cells (algae dose). All algae added were taken up, as determined by observation of the entire wells by light microscopy. Algae were incubated with RPE cells at an average dose of five algae per cell for 3 days under either cyclic light or constant darkness, prior to evaluation of viability. Control cells contained no algae. Light-exposed cells with algae were able to reduce 12% more resazurin compared to light-exposed cells without algae (P = 0.01, Figure 3A). However, this trend was reversed when the cells were kept in the dark, where treated cells now reduced 15% less resazurin compared to control cells (P = < 0.001, Figure 3B). As a second measure of host viability, we measured the concentrations of host adenosine triphosphate (ATP) in symbionts raised under cyclic light. To release ATP, we 2722
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Figure 5. N. eukaryotum guests increase host cell survival. Cell viability as measured by resazurin reduction curves for starved RPE cells and RPE cells with algae either kept under cyclic light (A) or kept in the dark (B). Means are shown; confidence intervals have been omitted for clarity. Numbers represent average number of algae per RPE cell. P values compare total amounts of resazurin reduced in comparison to control curves in red. Cell viability is positively correlated with algal concentration upon light exposure. Symbiont viability diminished in the dark.
able to reduce decreased by 1,263-fold after starvation compared to control cells incubated in nutrient-replete media (P = < 0.001).9 However, for cells with five algae per cell, the amount of reduced resazurin decreased by only 29-fold after starvation when kept under cyclic light (Figure 5A). Symbionts with five algae per cell were more viable than symbionts with fewer algae, which, in turn, were more viable than cells without algae. In darkness, when photosynthesis did not take place, survival of the symbionts was closer to that of cells without algae (Figure 5B). However, cells with algae were still more viable then cells without algae. RPE cells play a central role in retina homeostasis by regulating the retinal vascular network through the expression of vascular endothelial growth factor (VEGF) in response to decreases in oxygen and cellular metabolism.10,11 Aberrant angiogenesis of retinal blood vessels is responsible for multiple types of ocular pathology. Currently, antibodies to VEGF are injected in the eye to prevent this pathologic angiogenesis.12 However, this line of treatment has shown limited long-term value in preventing vision loss.12 We reasoned that the introduction of oxygen producing N. eukaryotum might be a viable strategy to increase cellular oxygenation and metabolism, thereby leading to a reduction of VEGF synthesis. Figure 6
selectively lysed RPE cells and quantified ATP in the lysate using a luciferase ATP reporter assay. The presence of N. eukaryotum guests did not significantly affect intracellular ATP levels (P values >0.05; Figure 4A). Next, we evaluated mitochondrial membrane potential of the host cell using a mitochondria-selective dye, JC-1. Membrane potential decreased by as much as 14% upon addition of algae when cultures were kept under cyclic light. Increasing the amount of algae did not further decrease the hosts cells’ membrane potential (Figure 4B). To confirm our observation that host cells were able to proliferate (Figure 2), we quantified host cell proliferation over 4 days in the presence of N. eukaryotum. We used the rate of resazurin reduction to measure the relative increase in RPE cells after 2 and 4 days of growth, as this rate is also proportional to cell number. After 2 days, proliferation was slightly faster for cells with an average of 1.25 algae per cell (Figure 4C). At the highest concentration tested, 6.25 algae per cell, proliferation at day 4 was approximately 50% reduced compared to control cells. After establishing the viability of the host cells, we measured the photosynthetic activity of the RPE-N. eukaryotum symbionts. To do this, we analyzed changes in chlorophyll fluorescence in response to light: photoinhibition.7 For symbionts, chlorophyll fluorescence increased by 26 to 40% after exposure to bright light. Furthermore, chlorophyll fluorescence returned to baseline (P < 0.05) when the symbionts were kept in the dark for 30 min (Figure 4D). This observed reversible reduction of chlorophyll fluorescence by light is consistent with viable photosynthetic chloroplasts undergoing photosynthesis. The word “symbiosis” describes physical associations between different species of organisms.8 A given symbiotic interaction can vary from mutualism to one-sided mutualism to antagonism, depending on the environment. To show that N. eukaryotum could potentially impart a biological advantage to its host cell, we incubated RPE cells and RPE symbionts under cyclic light or in darkness in media devoid of nutrients (i.e., in phosphate buffered saline, PBS) for 3 days and then measured cell viability using the resazurin reduction assay. In addition, we incubated cells without algae in nutrient-replete cell media as controls. The amount of resazurin that cells without algae were
Figure 6. Manipulation of VEGF synthesis with light. VEGF production after 12 h in cells and symbionts kept in the dark or under cyclic light (6 h on/6 h off). Averages and standard deviations of eight replicates are shown. *P value was less than 0.05 in comparison to cells without algae. 2723
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plots VEGF expression after 12 h in RPE cells harboring various amounts of algae and kept under cyclic light (6 h light/ 6 h dark) or constant darkness. Cells kept in the dark produced the same amount of VEGF regardless of the amount of algae incorporated. However, in the presence of light symbionts produced up to 35% less VEGF compared to control cells in a dose-depended manner. Total excreted protein remained the same in all groups. Thus, in the presence of the algae VEGF production could be modulated with light, demonstrating that intracellular algae could be successfully employed to fine-tune cell function. Photosynthetic green algae excrete carbohydrates, fats, hormones and vitamins in a light-dependent manner, and have become an attractive platform for recombinant protein and peptide expression.13 Thus, we wondered whether microalgae could be used as light-controlled, intracellular machinery that can be programmed to manufacture cellular components to tailor and expand biological functionality. Algae represent a diverse group of organisms from different phylogenetic groups and taxonomic divisions. N. eukaryotum (also known as N. eukaryota, Nanochlorum eucaryotum, and Picochlorum eukaryotum) is a marginal eukaryote containing one mitochondrion, chloroplast, nucleus, and dictyosome in its cytoplasm.14−16 N. eucaryotum also shows a high degree of adaptability,16−18 such as to salt concentration (0.25−12% NaCl), low light, elevated CO2, and decreased O2 levels, characteristics that may support its intracellular persistence, as observed in this study. All 11 algae species tested were between 1 and 3 μm in diameter. The observation that one out of the 11 algae species tested was taken up by human cells and not by rat- or mousederived cells suggests that this symbiotic association is perhaps selective and rare. There are no known mammalian algae endosymbionts. Chick fibroblasts and macrophages have been reported to take up Chlorella; however, the Chlorella was subsequently digested.19 Here, the two Chlorella species tested were not taken up. A photosynthetic bacterium could invade cultured mammalian macrophages; however, this was only when the bacterium was genetically engineered to escape the autophagic endosome.20 During mammalian algae infections, algae seem to reside extracellularly.21 Furthermore, only a small number of bacteria are able to replicate in the mammalian cytoplasm, suggesting that general endosymbiosis with mammalian cells is rare.22 Further research is needed to establish the selectivity of the N. eucaryotum−human cell interaction. The surface elements on N. eucaryotum that orchestrate its rapid uptake may be elucidated and exploited to control the delivery of nanosized constructs into cells. This line of research also has implications for understanding cell-tocell recognition, the potential congruence of host and guest and the establishment of models to understand how coordination is maintained in polygenetic multicellular systems. The increase in resazurin reduction rates in light-exposed symbionts compared to dark-raised symbionts may reflect changes in intracellular carbohydrate composition. Algae, such as N. eucaryotum, are known to secrete carbohydrates, such as saccharose, glucose, fructose, galactose, mannose, arabinose, xylose, ribose, fucose, and rhamnose.23 Changes in the cell’s carbohydrate composition would alter the balance of glycolytic and oxidative metabolism and the ability of cells to reduce resazurin. Specifically, a higher concentration of carbohydrates appears to increase the cell’s ability to reduce resazurin.24
Enhanced survival of symbionts during nutrient deprivation further suggests that N. eucaryotum can exchange products synthesized through carbon fixation with its host cell and thus act as machines to increase cell survival. In addition to carbohydrates, algae are known to secrete an abundance of lipids, which can also be used as an energy source by the host cell. The observation of secreted lipids by electron microcopy is of particular interest because lipid remodeling is a key mechanism by which cells adapt to their environment. As such, lipid exchange suggests that algae can be employed as a tool to alter the cell’s lipid composition without engineering the host’s lipid synthesis apparatus. N. eucaryotum-to-host nutrient exchange is also consistent with the observed decrease in host viability in symbionts kept in the dark, when algae do not carry out photosynthesis. The slight advantage observed in the darkraised symbionts during starvation could be due to nonphotosynthetic fixation or exchange of carbohydrate or lipid reserves. Although intracellular algae were not able to provide enough biogenic substances to completely stop cell death, this work suggests that the introduction of algae synthesizing and excreting recombinant proteins is a viable strategy to increase the cellular proteome to expand and/or correct biological function. The regulation of VEGF expression, which is tightly linked to cellular metabolism, is particularly interesting given that aberrant metabolism is thought to drive a majority of intractable diseases. As algae can augment metabolic pathways by several mechanisms, their introduction into tissues could potentially serve as a tool to impede pathological progression resulting from decreases in metabolism. We note that light of wavelengths that drive photosynthesis readily penetrates into the human body.25 Further work needs to be done to evaluate the long-term stability of this symbiosis. For example, quantitatively determining whether the ratio of algae to human cells significantly changes over time. In biomedical research, biological function is often expanded in attempts to correct disease and cellular deficiencies. In engineering, biological function is often expanded to adapt an organism to a particular task or environment. At present, our ability to expand the function of complex biological systems is largely limited to the paradigms of direct manipulation of the target organism’s genomes or the introduction of transient mRNA or subsequent protein products. Evolution has demonstrated that the creation of stable polygenetic systems can be a powerful means to increase biological complexity and, by extension, offers an opportunity to expand our repertoire to manipulate and control biological systems. To this end, we propose that N. eucaryotum can be exploited as programmable intracellular machines that can be controlled by light to increase function of human cells.
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ASSOCIATED CONTENT
S Supporting Information *
Methods and figures of (1) uptake of photosynthetic green algae by mammalian cells and (2) representative light microscopy image of human RPE cells engulfed with N. eukaryotum. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. 2724
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Author Contributions
K.B. designed the study, carried out all experimental procedures, performed the data analysis, and drafted the manuscript. D.M. participated in the design and maintained cell cultures. I.W. conceived and designed the study and wrote the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Supported by the Office of Naval Research (N00014-08-10150) and Nanoscale Science and Engineering Initiative of the National Science Foundation (NSF CHE-0117752 and CHE0641523).
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REFERENCES
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