University of Groningen Fluorescent Nanodiamonds as Free Radical Sensors in Aging Yeast Cells van der Laan, Kiran

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Fluorescent Nanodiamonds as Free Radical Sensors in Aging Yeast Cells

van der Laan, Kiran

DOI:

10.33612/diss.112906297

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.

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Publication date: 2020

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Citation for published version (APA):

van der Laan, K. (2020). Fluorescent Nanodiamonds as Free Radical Sensors in Aging Yeast Cells: a baker’s yeast response to small diamonds with great potential!. Rijksuniversiteit Groningen.

https://doi.org/10.33612/diss.112906297

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Toward using Fluorescent Nanodiamonds to Study

Chronological Aging in Saccharomyces Cerevisiae

Kiran J. van der Laan1_{, Julie Naulleau}1_{, Viraj G. Damle}1_{, Alina Sigaeva}1_{, Nicolas Jamot}1_, Felipe P. Perona-Martinez1_{, Mayeul Chipaux}1_{, Romana Schirhagl}1_*

1_{University of Groningen, University Medical Center Groningen, Antonius Deusinglaan} 1, 9713 AW Groningen

ACS Analytical Chemistry 90: 13506-13513 (2018).

Abstract

One of the theories aiming to explain cellular aging is the free radical theory of aging, which describes the possible role of increased production and accumulation of free radicals. Fluorescent nanodiamonds (FNDs) are proposed to provide a tool to detect these radicals, as they function as magnetic sensors that change their optical properties depending on their magnetic surrounding. Therefore they could enable the study for aging at a molecular level and unravel the exact role of free radicals in this process. In this study, important steps toward this goal are made. FNDs are introduced in chronologically aging yeast cells. Furthermore, the behavior of FNDs in these aging cells is studied, to demonstrate the potency of using FNDs in the search for causes of cellular aging.

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Toward using Fluorescent Nanodiamonds to Study

Chronological Aging in Saccharomyces Cerevisiae

Kiran J. van der Laan1_{, Julie Naulleau}1_{, Viraj G. Damle}1_{, Alina Sigaeva}1_{, Nicolas Jamot}1_, Felipe P. Perona-Martinez1_{, Mayeul Chipaux}1_{, Romana Schirhagl}1_*

1_{University of Groningen, University Medical Center Groningen, Antonius Deusinglaan} 1, 9713 AW Groningen

ACS Analytical Chemistry 90: 13506-13513 (2018).

Abstract

One of the theories aiming to explain cellular aging is the free radical theory of aging, which describes the possible role of increased production and accumulation of free radicals. Fluorescent nanodiamonds (FNDs) are proposed to provide a tool to detect these radicals, as they function as magnetic sensors that change their optical properties depending on their magnetic surrounding. Therefore they could enable the study for aging at a molecular level and unravel the exact role of free radicals in this process. In this study, important steps toward this goal are made. FNDs are introduced in chronologically aging yeast cells. Furthermore, the behavior of FNDs in these aging cells is studied, to demonstrate the potency of using FNDs in the search for causes of cellular aging.

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Understanding the mechanisms of cellular aging is one of the emerging challenges in the current aging population. The causes of aging at a molecular level are still largely unknown, although some involved mechanisms have been appointed. Free radical formation and accumulation are believed to be one of the causes for cellular aging, but their exact role is strongly debated in the past decades.(1,2) We are aiming to use a new method called diamond magnetometry to monitor free radical activity in the future, by using fluorescent nanodiamonds (FNDs). FNDs can function as quantum sensors that change their optical properties depending on their magnetic surrounding, with single electron-spin sensitivity.(3) Since unpaired electrons in free radicals are expected to influence coherence times, these FNDs could potentially enable the study on the role of free radicals in cellular aging.

The FNDs used in diamond magnetometry harbor a defect in the diamond structure, in which two carbon atoms in the lattice are substituted by a nitrogen atom (N) and an adjacent vacancy (V). Magnetic information from the environment can be read out optically because of these NV centers, causing the fluorescence signal of the nanodiamonds to change in the presence of electron spins. The stability of the NV centers and their fluorescence (4,5), together with the excellent biocompatibility of FNDs, provide great promises to use its magneto-optical behavior as sensors in biomedical model systems.(5–9)

Until now FNDs have been used for several applications, such as labeling,(10) monitoring temperature alterations (7,11,12) and studying drug delivery.(9,13–15) To provide important prerequisites to study aging, in this work the FNDs are introduced into yeast cells. Yeast is one of the most successful model organisms to study aging for several reasons, including the conservation of aging process among different organisms and its suitability for genetic manipulation.(16) For proper functioning of the FNDs, the distance between the diamond probe and the target molecules needs to be as small as possible.(17) Therefore it is necessary to introduce the nanodiamonds into the cells using a previously shown effective method.(18) In this study, we have analyzed the internalization and behavior of nanodiamonds in aging yeast cells. Furthermore, we have determined where exactly diamonds end up after internalization. All together, these results demonstrate the potency of using FNDs in yeast cells to study aging at a molecular level.

Results

An effective method to obtain fluorescent nanodiamond (FND) internalization in young yeast cells was earlier shown to be both biocompatible and highly effective.(18) Here the cells were monitored after performing the uptake protocol in aged cells, in order to work toward the goal of using FNDs in the search for causes of cellular aging.

FND Uptake in Stationary Phase Yeast Cells.

In the earlier mentioned study, the effectiveness of FND uptake was shown in yeast cells in their active growth phase (mid log phase). In order to study chronological aging however, cells were grown until the stationary growth phase of yeast. Cells were restricted to chronological aging by transferring them to water after they had reached the stationary growth phase. In this research, the effectiveness of the uptake method in stationary phase cells was checked, as well as the viability of aged cells after uptake of FNDs.

First, the effectiveness of the uptake method in stationary phase cells was checked, as well as the viability of aged cells after uptake of FNDs. The number of internalized FNDs per cell was increased after performing the chemical transformation protocol, although the increase was not in all cases significant (Figure 1a,b). Cells were additionally categorized based on the number of internalized objects or particles and the distribution of the percentage of cells per category is shown in Figure 1c,d. An object is defined as connected FND positive pixels, which means it can also be an aggregate. Out of the total intensity of the objects, the number of single particles is calculated using the intensity of a single bright pixel. Showing both numbers gives an impression on the state of aggregation of particles. Compared to the control sample, there was a greater percentage of cells with a high amount of both objects and particles. The low number of particles and objects that are found in the control sample, could be attributed to large aggregates on the cell surface which give false positives. For the transformation protocol we find particles that are unambiguously inside the cells, stressing the need for the transformation protocol. Moreover, the viability of the cells was not changed as a result of performing the chemical transformation and adding the FNDs (Figure 1e). The cell samples subjected to FNDs and the uptake protocol did not show altered viability compared to the untreated control sample.

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Understanding the mechanisms of cellular aging is one of the emerging challenges in the current aging population. The causes of aging at a molecular level are still largely unknown, although some involved mechanisms have been appointed. Free radical formation and accumulation are believed to be one of the causes for cellular aging, but their exact role is strongly debated in the past decades.(1,2) We are aiming to use a new method called diamond magnetometry to monitor free radical activity in the future, by using fluorescent nanodiamonds (FNDs). FNDs can function as quantum sensors that change their optical properties depending on their magnetic surrounding, with single electron-spin sensitivity.(3) Since unpaired electrons in free radicals are expected to influence coherence times, these FNDs could potentially enable the study on the role of free radicals in cellular aging.

The FNDs used in diamond magnetometry harbor a defect in the diamond structure, in which two carbon atoms in the lattice are substituted by a nitrogen atom (N) and an adjacent vacancy (V). Magnetic information from the environment can be read out optically because of these NV centers, causing the fluorescence signal of the nanodiamonds to change in the presence of electron spins. The stability of the NV centers and their fluorescence (4,5), together with the excellent biocompatibility of FNDs, provide great promises to use its magneto-optical behavior as sensors in biomedical model systems.(5–9)

Until now FNDs have been used for several applications, such as labeling,(10) monitoring temperature alterations (7,11,12) and studying drug delivery.(9,13–15) To provide important prerequisites to study aging, in this work the FNDs are introduced into yeast cells. Yeast is one of the most successful model organisms to study aging for several reasons, including the conservation of aging process among different organisms and its suitability for genetic manipulation.(16) For proper functioning of the FNDs, the distance between the diamond probe and the target molecules needs to be as small as possible.(17) Therefore it is necessary to introduce the nanodiamonds into the cells using a previously shown effective method.(18) In this study, we have analyzed the internalization and behavior of nanodiamonds in aging yeast cells. Furthermore, we have determined where exactly diamonds end up after internalization. All together, these results demonstrate the potency of using FNDs in yeast cells to study aging at a molecular level.

Results

An effective method to obtain fluorescent nanodiamond (FND) internalization in young yeast cells was earlier shown to be both biocompatible and highly effective.(18) Here the cells were monitored after performing the uptake protocol in aged cells, in order to work toward the goal of using FNDs in the search for causes of cellular aging.

FND Uptake in Stationary Phase Yeast Cells.

In the earlier mentioned study, the effectiveness of FND uptake was shown in yeast cells in their active growth phase (mid log phase). In order to study chronological aging however, cells were grown until the stationary growth phase of yeast. Cells were restricted to chronological aging by transferring them to water after they had reached the stationary growth phase. In this research, the effectiveness of the uptake method in stationary phase cells was checked, as well as the viability of aged cells after uptake of FNDs.

First, the effectiveness of the uptake method in stationary phase cells was checked, as well as the viability of aged cells after uptake of FNDs. The number of internalized FNDs per cell was increased after performing the chemical transformation protocol, although the increase was not in all cases significant (Figure 1a,b). Cells were additionally categorized based on the number of internalized objects or particles and the distribution of the percentage of cells per category is shown in Figure 1c,d. An object is defined as connected FND positive pixels, which means it can also be an aggregate. Out of the total intensity of the objects, the number of single particles is calculated using the intensity of a single bright pixel. Showing both numbers gives an impression on the state of aggregation of particles. Compared to the control sample, there was a greater percentage of cells with a high amount of both objects and particles. The low number of particles and objects that are found in the control sample, could be attributed to large aggregates on the cell surface which give false positives. For the transformation protocol we find particles that are unambiguously inside the cells, stressing the need for the transformation protocol. Moreover, the viability of the cells was not changed as a result of performing the chemical transformation and adding the FNDs (Figure 1e). The cell samples subjected to FNDs and the uptake protocol did not show altered viability compared to the untreated control sample.

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Figure 1. Efficiency and viability of FND uptake in stationary phase yeast cells. Cells were

grown until stationary phase of cell growth, before performing the chemical transformation and adding the FNDs. The particle uptake was quantitatively analyzed directly after the transformation protocol. (a-d) Quantitative analysis of FND internalization. Objects are composed of adjacent FND signal positive pixels and particles reflect the actual number of FNDs, derived from the total intensity of the objects. For each sample, 124-125 cells were analyzed (black n=125, green n=125, blue n=124). a and b show the average number of FNDs per cell. Error bars represent standard errors of the mean. Statistical differences were tested by comparing to the control (untreated cells with FNDs) with an unpaired, two-tailed t-test with Welch’s correction. c and d display a histogram of cells containing a range of FNDs. (e) Survival of stationary phase cells. Colony forming units (CFUs) are shown as a measure for the viability of stationary phase yeast cells after the chemical transformation procedure. Bars represent averages out of three independent experiments (n=3), each performed in triplicate. The grey bar indicates the area that differs 20% with the control sample, in which the viability is considered as normal. Significance is tested compared to the control situation by performing an unpaired, two-tailed t-test with Welch’s correction; no statistical differences were found. Error bars represent standard errors of the mean.

FND Behavior during Chronological Aging.

In order to investigate the effect of FND internalization on the aging process, a chronological life span assay was performed. Cells were restricted to age chronologically by keeping them in a non-dividing state. After the addition of FNDs at day 1, the cells were kept in water and the survival of the cells was measured by assessing the proliferative ability every two days. This resulted in the chronological lifespan curve (Figure 2a). The decline in viability we see in all the samples is the chronological aging effect. There are several theories about the reasons and mechanisms involved in this decline, one of them is the above mentioned free radical theory1,2_{. In the first week, the functional decline of the} cells seemed to happen at a similar rate, despite small differences. At later aging time points though, the differences in the percentage of remaining viable cells became greater but not significant. Interestingly, from day 2 the viability decreased in parallel with the control sample (Figure 2b). Similar decline rates were detected, but starting from a different percentage of cells at day 2 and thus ending up at lower number of cells at day 6-day10.

Figure 2. Chronological life span. The number of viable cells was analyzed in the days

after chemical transformation was performed to achieve uptake. Cells were kept in water, to keep them in a non-dividing state. (a) Chronological survival of cells. After FND uptake, the survival was determined by testing the proliferative capacity every 2 days. Per condition, the value of a time point was compared to the number of viable cells with that condition at day 1, which was set as 100%. (b) Decline rate per measured time period. This part shows the absolute numbers of viable cells that are lost in between the time points that are measured. Given values of both (a) and (b) are averages out of three biologically independent experiments (n=3), each performed in three technical replicates. Error bars represent standard errors of the mean. Significance is tested compared to the control sample (untreated cells without FNDs), with an unpaired, two-tailed t-test with Welch’s correction (no significant differences).

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Figure 1. Efficiency and viability of FND uptake in stationary phase yeast cells. Cells were

grown until stationary phase of cell growth, before performing the chemical transformation and adding the FNDs. The particle uptake was quantitatively analyzed directly after the transformation protocol. (a-d) Quantitative analysis of FND internalization. Objects are composed of adjacent FND signal positive pixels and particles reflect the actual number of FNDs, derived from the total intensity of the objects. For each sample, 124-125 cells were analyzed (black n=125, green n=125, blue n=124). a and b show the average number of FNDs per cell. Error bars represent standard errors of the mean. Statistical differences were tested by comparing to the control (untreated cells with FNDs) with an unpaired, two-tailed t-test with Welch’s correction. c and d display a histogram of cells containing a range of FNDs. (e) Survival of stationary phase cells. Colony forming units (CFUs) are shown as a measure for the viability of stationary phase yeast cells after the chemical transformation procedure. Bars represent averages out of three independent experiments (n=3), each performed in triplicate. The grey bar indicates the area that differs 20% with the control sample, in which the viability is considered as normal. Significance is tested compared to the control situation by performing an unpaired, two-tailed t-test with Welch’s correction; no statistical differences were found. Error bars represent standard errors of the mean.

FND Behavior during Chronological Aging.

In order to investigate the effect of FND internalization on the aging process, a chronological life span assay was performed. Cells were restricted to age chronologically by keeping them in a non-dividing state. After the addition of FNDs at day 1, the cells were kept in water and the survival of the cells was measured by assessing the proliferative ability every two days. This resulted in the chronological lifespan curve (Figure 2a). The decline in viability we see in all the samples is the chronological aging effect. There are several theories about the reasons and mechanisms involved in this decline, one of them is the above mentioned free radical theory1,2_{. In the first week, the functional decline of the} cells seemed to happen at a similar rate, despite small differences. At later aging time points though, the differences in the percentage of remaining viable cells became greater but not significant. Interestingly, from day 2 the viability decreased in parallel with the control sample (Figure 2b). Similar decline rates were detected, but starting from a different percentage of cells at day 2 and thus ending up at lower number of cells at day 6-day10.

Figure 2. Chronological life span. The number of viable cells was analyzed in the days

after chemical transformation was performed to achieve uptake. Cells were kept in water, to keep them in a non-dividing state. (a) Chronological survival of cells. After FND uptake, the survival was determined by testing the proliferative capacity every 2 days. Per condition, the value of a time point was compared to the number of viable cells with that condition at day 1, which was set as 100%. (b) Decline rate per measured time period. This part shows the absolute numbers of viable cells that are lost in between the time points that are measured. Given values of both (a) and (b) are averages out of three biologically independent experiments (n=3), each performed in three technical replicates. Error bars represent standard errors of the mean. Significance is tested compared to the control sample (untreated cells without FNDs), with an unpaired, two-tailed t-test with Welch’s correction (no significant differences).

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To record quantum measurements with the purpose of measuring free radical activity and its role in aging, measurements will have to be taken at different aging time points. Therefore, the persistence of FND internalization in aged cells was examined. In Figure 3, the number of internalized FNDs in the cells is depicted as a function of time. Although there are still cells with internalized FNDs in the days after the initial uptake attempt at day 1, the number of FNDs inside cells got lower over time. This could be explained by either excretion of FNDs in some way, or by cell death of the cells with internalized FNDs. The latter explanation did not seem to be the case, since an additional experiment showed that the addition of diamonds did not cause decreased viability at the time-points were the number of internalized diamonds was diminished (Figure S2).

Figure 3. FND presence over time. The number of internalized FNDs was analyzed in the

days after chemical transformation was performed to achieve uptake. After one day, the biggest drop in number of internalized objects (a) and particles (b) was observed. This effect was most obvious in the sample were the cells were additionally treated with a Triton wash, directly after the chemical transformation. Given values are relative numbers that are normalized against the control by subtraction of the values found in the control sample, in which diamond uptake was not stimulated. For all samples, per condition and time point, approximately 100 cells were analyzed: (green n=125 at day 1, n=103 at day 2, n=111 at day 3, n=99 at day 4; blue n=124 at day 1, n=104 at day 2, n=107 at day 3, n=82). Error bars represent standard errors of the mean.

Visual inspection of scanning electron microscopy (SEM) images revealed the subcellular localization of the nanodiamonds. Cells were embedded directly after performing the uptake protocol and adding the nanodiamonds, therefore images represent the location of FNDs shortly after uptake. Qualitative analysis of the images revealed that the nanodiamonds were predominantly located around the nucleus (Figure 4a) and other membrane-enclosed, spherical structures (Figure S1). Interestingly, budding yeast cells with internalized FNDs

were captured (Figures 4a and S1), once more indicating that cells remained functional after uptake of FNDs. Using a focused ion beam SEM, a detailed 3D visualization of a cell with FNDs was obtained (Figure 5), with the suspected nanodiamonds again localized around these spherical structures and the nucleus.

Figure 4. Subcellular localization of FNDs. (a) SEM visualization of yeast cells with

internalized FNDs shows the location of FNDs within the cells. A representative image is shown wherein FNDs are located around in close proximity of the nucleus. Furthermore, FNDs were found in budding yeast cells. The scale bar represents 1.00 µm and applies to all three subfigures (a, b and c). (b) Confirmation of the localization in AFM stiffness mode. In stiffness mode, diamonds show a significantly higher stiffness (3: 37 GPa) than the surrounding cell material (2: 17 GPa). The outside of the cell (1) has an even lower stiffness of 13 GPa. The circled line shows a vesicle structure, visible in the height image that is constructed by taking the height at every point in AFM. (c) Confirmation of localization in AFM deformation mode. This shows the same AFM image, but in deformation mode. Here, the sharp edge of the diamond is clearly visible.

To confirm that the structures observed in SEM images are indeed nanodiamonds - and not holes through the sections as an artifact resulting from the sample preparation, or other cellular structures - atomic force microscopy (AFM) analysis was conducted. As expected, the structures that were identified as diamonds were very stiff compared to the rest of the cell, three times as stiff, and did not deform at al. The size of the suspected diamonds was also in reasonable agreement with what was expected. Additionally, the characteristic sharp edge structure of diamonds was found. By measuring the height of the diamond particles, they could be differentiated from through holes in the section which would also have very high stiffness.

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