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Time-Resolved FT-IR Microspectroscopy of Protein Aggregation Induced by Heat-Shock in Live Cells Elisa Mitri,†,‡ Saša Kenig,† Giovanna Coceano,‡ Diana E. Bedolla,† Massimo Tormen,‡ Gianluca Grenci,‡,§ and Lisa Vaccari*,† †

Elettra−Sincrotrone Trieste, Strada Statale 14 km 163.5, 34149 Basovizza, Trieste Italy IOM-CNR, TASC Laboratory, Strada Statale 14 km 163.5, 34149 Basovizza, Trieste Italy § MBI, National University of Singapore T-Lab, 5A Engineering Drive 1, Singapore ‡

S Supporting Information *

ABSTRACT: Maintaining the correct folding of cellular proteins is essential for preserving cellular homeostasis. Protein dishomeostasis, aberrant protein folding, and protein aggregation are indeed involved in several diseases including cancer, aging-associated, and neurodegenerative disorders. Accumulation of protein aggregates can also be induced from a variety of stressful conditions, such as temperature increase or oxidative stress. In this work, we monitored by Fourier transform-infrared (FT-IR) microspectroscopy the response of live breast cancer MCF-7 and mammary breast adenocarcinoma MDA-MB 231 cell lines to severe heat-shock (HS), caused by the rise of the cellular medium temperature from 37 ± 0.5 °C to 42 ± 0.5 °C. Through the study of the time-evolution of the second derivatives of the spectra and by the 2D correlation analysis of FT-IR absorbance data, we were able to identify a common sudden heat-shock response (HSR) among the two cell lines. The hyperfluidization of mammalian cell membranes, the transient increment of the signal lipids, as well as the alteration of proteome profile were all monitored within the first 40 min of stress application, while the persistent intracellular accumulation of extended β-folded protein aggregates was detected after 40 min up to 2 h. As a whole, this paper offers a further prove of the diagnostic capabilities of FT-IR microspectroscopy for monitoring in real-time the biochemical rearrangements undergone by live cells upon external stimulation.

O

regulation of heat shock proteins (HSPs)5 mediated by the activation of heat-shock factors (HSFs) genes. HSPs are expressed under normal conditions in a cell-cycle dependent manner and exert fundamental functions. The most notable and well-known is their role as molecular chaperones that, assisting in the synthesis and correct folding of constitutive proteins, help in preserving cellular homeostasis. Under cellular stress conditions, HSPs exhibit sophisticated protection of antiapoptotic mechanisms that prevent the formation of nonspecific protein aggregates and assist proteins in restoring and maintaining their native structures.6 Despite their designation, an increased expression level of HSPs may be prompted by a variety of stimuli, and this is an ubiquitous feature in those cells that cope not only with thermal stress but also with metabolic insults that induce denaturation of constitutive proteins. Among the others, HSR pathways are activated by oxidative and osmotic stress,

rganisms may suffer a large variety of stressful conditions from the surrounding environment, including sudden temperature variations. Heat-shock is triggered by a temperature increase of just a few degrees and it may induce a severe damage of important cellular structures, therefore interfering with essential cellular functions.1,2 The reason for the extreme cellular susceptibility even to small temperature changes resides on the kinetic lability of cellular proteins, thermodynamically stable at physiological temperature, that need to be conformationally flexible in order to perform their biological functions, such as enzymatic activity.3 Therefore, even a small increase in temperature results in the unfolding of proteins and exposure of hydrophobic amino acid residues, normally buried within the protein interior domains. The ultimate result of the conformational transition consists in unspecific intra- and intermolecular hydrophobic interactions that induce either protein misfolding or unspecific protein−protein aggregation of small oligomers up to extended aggregates,2 with consequent breakdown of the cellular machinery. In order to counteract the thermal stress, cells activate an ancient signaling pathway, called heat-shock response (HSR), that is highly conserved in all living beings, from prokaryotes to humans.4 HSR leads to the transient up© 2015 American Chemical Society

Received: October 15, 2014 Accepted: March 18, 2015 Published: March 18, 2015 3670

DOI: 10.1021/ac5040659 Anal. Chem. 2015, 87, 3670−3677

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Analytical Chemistry

dissolved in dimethyl sulfoxide, and absorbance was measured at 570 nm with a Tecan infiniteF200PRO reader. Results are presented as the average of two experiments ± standard deviation. To follow cell survival at conditions more similar to FT-IR measurements, an aliquot of the same cells suspended in NaCl 0.9% for FT-IR microspectroscopy was stained with trypan blue before the vibrational analysis. The percentage of dead cells was estimated by counting in a Bürker chamber the number of stained versus the number of total cells. Once filled the device, remaining cells were left in a vial outside the incubator, kept at 37 ± 0.5 °C (for controls), and at 42 ± 0.5 °C (for heatstressed cells) for 2 h by immersion in a thermostated bath. Trypan blue vitality assay was then repeated after the measurement. Furthermore, at the end of FT-IR measurements, trypan blue solution was let to diffuse inside the fluidic device too, and the percentage of dead cells was estimated as the average of number of stained versus the total number of cells within three different regions (100 μm × 100 μm) of the device. Microfluidic Device and Experimental Setup. X-ARP 3100/10 based microfluidic devices with a thickness of 7.5 μm were produced on 0.5 mm thick CaF2 substrate according to the procedure detailed elsewhere15 at LILIT (Laboratory for Interdisciplinary LIThography) of IOM-CNR Trieste, Italy.21 The thickness of the windows was chosen in order to minimize the chromatic aberration induced by CaF2 windows.22 Once confined within the chip, cells were let to settle down onto the device surface and thermalized to 37 ± 0.5 °C for 30 min. FT-IR Microspectroscopy Data Collection. The experiments were carried out at the infrared beamline SISSI (Synchrotron Infrared Source for Spectroscopic and Imaging) at Elettra−Sincrotrone Trieste, Italy.23 FT-IR transmission spectra were acquired using a Hyperion 3000 Vis-IR microscope mounting an HgCdTe detector with a 100 μm infrared sensitive element, coupled with a Vertex 70 interferometer (Bruker Optics GmbH, Ettlingen, Germany). Repeated spectra on small groups of three cells were collected in transmission mode, setting microscope knife-edge apertures at 40 μm × 40 μm, using a 15X Schwarzschild condenser and objective (NA = 0.4), averaging 256 scans with a spectral resolution of 4 cm−1 and zero-filling factor of 2 in the range of frequencies between 800 and 5000 cm−1, in double side, forward/backward acquisition mode, with a scanner velocity of 40 kHz. The Fourier transform was carried out with Mertz phase correction, Blackman-Harris-3 terms apodization function. Air background and spectra of the buffer medium were collected using the same parameters. The first spectral point was acquired under physiological unperturbed conditions right after the thermalization at 37 ± 0.5 °C, and it is considered the t = 0 for each experiment. For each measurement run, four cell groups and one buffer point have been selected and monitored, with a time shift between the acquisition of one group and the following of 1 min. At t = 5 min, the thermal shock was applied by increasing the device temperature to 42 ± 0.5 °C, and the spectral profile of each cell group was recollected every 5 min for 2 h. The experiment was repeated 5 times for both cell lines. Control groups of MCF-7 and MDA-MB 231 cells were measured following the same scheme while keeping the device temperature constant at 37 ± 0.5 °C. Data from 12 heat-stressed groups and 4 control groups have been considered for data analysis.

starvation, pH shifts, exposure to metals or toxic substances, and several other types of physical, mechanical, and chemical offenses. Furthermore, recent studies pointed out that the aging process goes along with a decline in the cellular capability to maintain proteostasis, which involves also HSF genes and HSPs.7−9 FT-IR spectroscopy has been extensively employed for the assessment of secondary structure of proteins and protein aggregates in solution, especially exploiting the sensitivity of the Amide I band to protein conformation.10 However, these measurements are technically demanding, due to the need of absolute control of the optical path of the liquid chambers for accurately compensating the dominant water contribution to the Amide I band.11 Highlighting protein conformation on individual hydrated live cells is a task even more challenging.12,13 The exploitation of microfabrication techniques to produce visible-infrared (Vis-IR) transparent microfluidic devices has provided only in recent times the technological breakthrough for overcoming the constraints encountered in monitoring live cells by FT-IR microspectroscopy.14−17 Exploiting the newest advances in this field, Lisa Miller and co-workers recently demonstrated the feasibility of time-lapsed FT-IR imaging of protein aggregation in a cell culture model of amyotrophic lateral sclerosis.18 In this paper, we propose the use of FT-IR microspectroscopy as an analytical methodology for the investigation of the dynamics of HSR in live cells, with a temporal resolution of minutes, in order to complement most of the studies that are routinely performed on fixed or labeled cells.3,19,20 We investigated the response of MCF-7 breast cancer and MDAMB 231 breast adenocarcinoma cells upon a temperature increase to 42 ± 0.5 °C for 2 h. Spectral markers of cellular protein unfolding, misfolding, and aggregation have been elicited by FT-IR microspectroscopy that allowed to establish that the two cell lines activate HSR in the same time scale and following common pathways. Moreover, a higher sensitivity of MCF-7 cells could be deduced, since that cell line was the only one presenting preapoptotic spectral markers already during the 2 h experiment.



EXPERIMENTAL SECTION Cell Preparation. MDA-MB 231 (ATCC mammary gland adenocarcinoma cell line) and MCF-7 cells (ATCC breast cancer cell line) were chosen as models for the study. Both cell lines were grown in Dulbecco’s Modified Eagle’s Medium (PAA Laboratories), supplemented with fetal bovine serum 10% (Euroclone), in a incubator at 37.0 °C and 5% CO2 and routinely passaged every 3−4 days. Before measurements, cells were harvested from the flask using trypsin, washed twice with phosphate-buffered solution (PBS), then once with sterile physiological solution (NaCl 0.9%), and finally resuspended in the latter at a concentration of ∼1 × 106 cell/mL. The cell suspension was dropped into microfluidic devices thermalized at 37 ± 0.5 °C without further treatments. Cell Viability Assays. In order to ascertain the cell viability upon HS stress with respect to normally cultured cells, the MTT assay has been done. MCF-7 and MDA-MB 231 cells were plated in a 50 μL drop on CaF2 slides, (15 000 cells per window) left to attach for 30 min and topped up with DMEM media. After 24 h, they were exposed to 42 ± 0.1 °C for 2 h while the control cells were left at 37 ± 0.1 °C. MTT reagent (Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 0.5 mg/mL for 3 h. Formazan crystals were 3671

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∼1516 cm−1 (intramolecular β-sheet31,32), and at ∼1574 cm−1 (unordered random coils and turns33). Symmetric (sym) and asymmetric (asym) stretching (ν) bands of methyl and methylene moieties are peaked at 2962 cm−1 (νas −CH3), 2923 cm−1 (νas −CH2), 2875 cm−1 (νs −CH3), 2852 cm−1 (νs CH2),34 while the CH stretching of vinyl moieties is not clearly discernible over the spectral noise (see Figure S1a in the Supporting Information). In the spectral region of bending modes of aliphatic chains, two contributions can be seen in Figure S1b in the Supporting Information, centered at 1468 and 1415 cm−1, related, respectively, to −CH2 scissoring and rocking vibration of aliphatic chains of the lipid bilayers in the liquid crystalline state.35 The second derivative of the asymmetric stretching band of phosphate moieties, PhI hereafter, of MDA-MB 231 cells displays two major components centered at 1241 and 1218 cm−1, conventionally assigned to the A and B helices of double stranded nucleic acids (Figure S1c in the Supporting Information).36,40 The symmetric stretching band of phosphates, PhII from here on, is centered at 1085 cm−1 and two shoulders related to stretching modes of ribose and polysaccharides are centered at 1120 and 1050 cm−1, respectively.37 MDA-MB 231 cells measured at a constant temperature of 37 ± 0.5 °C for 2 h do not show significant spectral changes in both position and relative intensities of the second derivative peaks, as can be appreciated from Figure S2a in the Supporting Information. As proven by trypan blue assays, the percentage of dead cells at the beginning of the experiment (10 ± 2%) is comparable within standard deviation with that measured at the end, both for cells kept at physiological temperature in the water bath (9 ± 2%) and cells into the microfluidic chamber (10 ± 3%). These data exclude possible adverse effects induced by the vertical confinement of the cells within the device at 7.5 μm, imposed by the optical path constraints.11 MDA-MB 231 cell viability is not affected also when the cells are kept at 42 ± 0.5 °C for 2 h, as highlighted by trypan blue tests. The percentage of dead cells at t = 0 min (14 ± 4%) is comparable to the one at t = 2 h (13 ± 6% in bath and 14% ± 5% in fluidic device). This evidence is further confirmed by the results of MTT test (see Figure S3a in the Supporting Information). However, FT-IR microspectroscopy data reveal a significant perturbation of the system, especially affecting the cellular proteome, as can be clearly appreciated from 1D time evolution of second derivatives for a representative group of MDA-MB 231 in the 1750−1480 cm−1 spectral region, shown in Figure 1a (see Figures S6a and S7a in the Supporting Information for 1D time evolution of second derivatives of two other sampled groups). Once applied the HS (brown curve in Figure 1a), the definition of the shoulder at 1633 cm−1 is lost, and a general broadening of the Amide I band toward 1640 cm−1 can be seen with respect to the unperturbed state. At t = 10 min (orange curve in Figure 1a), the same Amide I band components identified at t = 0 can be seen while at t = 15 min (yellow curve in Figure 1a) the Amide I component centered at 1655 cm−1 is less prominent, while a signal centered at ∼1635 cm−1 clearly shows up. After 20 min, a new shoulder is present, centered at ∼1627 cm−1 that downshifts to 1623 cm−1 at the next acquisition time (light green and olive green curves, respectively, in Figure 1a), related to misfolded structures. Between 25 and 40 min, the spectral profiles fluctuates through states characterized by a different degree of broadening of the 1655 cm−1 component, and the presence of one or more shoulders in the spectral region between 1640 and 1625 cm−1.

Data Preprocessing, Postprocessing, and Analysis. Raw cell and buffer spectra were corrected for carbon dioxide and water vapor using the OPUS 6.5 routines (Bruker Optics GmbH), then offset-corrected. In order to disclose the cellular spectral details hidden by the prominent water features, the spectral contribution of the water medium was subtracted from each cell spectrum by using the in-house optimized Matlab routine described by Vaccari et al.24 Band area integrals and second derivatives (Savitzky−Golay filter, 13 smoothing points) were computed with OPUS 6.5. The area integral of the following bands were considered: spectral region 3000− 2800 cm−1, representative of cellular lipids (Lipids); spectral region 1705−1480 cm−1, representative of cellular proteins (Proteins); spectral region 1590−1480 cm−1, Amide II (AmII); spectral region 1270−1185 cm−1, asymmetric stretching of phosphate moieties (PhI); spectral region 1150−1005 cm−1, symmetric stretching of phosphate moieties (PhII); the sum of the area integral of Lipids and the region 1760−950 cm−1 has been considered as representative of the overall cellular bimolecular content (Cell Mass).25 2D correlation analysis of absorbance spectra was performed using IR absorption data from t = 0 to t = 40 min with Δt = 5 min, by the application of the generalized 2D correlation algorithm of Noda in the entire range of frequencies of subtracted spectra (3000−1000 cm−1). Before 2D calculation, each spectrum was baseline corrected (Rubberband method, 16 baseline points) and normalized in the entire spectral range. Software used for 2D analysis is the freeware Matlab routine MIDAS2010.26 Rules proposed by Noda were exploited for the interpretation of the 2D IR plots.27



RESULTS Live cells belonging to the cell lines MCF-7 and MDA-MB 231 were subjected to a sudden temperature increase of 5 °C, from 37 ± 0.5 °C to 42 ± 0.5 °C. Small groups of three cells were repeatedly measured for 2 h every 5 min. To limit the variability of the sampled populations, induced by the specific HS tolerance and the different biochemical content at different stages of cellular progression,28 only cells grown to confluence, and therefore mostly in the resting state, were measured. We focused our attention on the analysis of time-dependent spectral trends: time-evolution of second derivative spectra and 2D correlation analysis of absorbance spectra have been exploited as complementary methods to increase the molecular specificity of FT-IR microspectroscopy. In addition, relative variations of several band ratios have also been considered as a diagnostic of the overall behavior of both cell lines in-time. Three time intervals were defined in order to better describe the phenomenon: immediate (within 10 min upon thermal stress, from t = 5 min to t = 15 min), early (within 40 min upon thermal stress, from t = 15 min to t = 45 min), and late (within almost 2 h of HS, from t = 45 min to t = 135 min) HSR. HSR of MDA-MB 231 Cells. The peculiar spectral features of unperturbed MDA-MB 231 cells have been retrieved from the second derivatives of the water-subtracted spectra, averaged from all the heat-stressed sampled groups at t = 0 min (unperturbed conditions). Data are provided in the Supporting Information for brevity and summarized in the following. The Amide I band of MDA-MB 231 cell line (see Figure S1b in the Supporting Information) is peaked at ∼1655 cm−1 (α-helix29); a shoulder is centered at 1680 cm−1 (unordered random coils and turns 29 ) and a second one at around 1633 cm −1 (intramolecular β-sheet30). Accordingly, the Amide II band has three components centered at ∼1550 (α-helix29) and 3672

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The mutually dependent intensity variations between Amide I and Amide II band components could be better highlighted by 2D correlation analysis on FT-IR absorption data. The synchronous 2D IR correlation plot, Syn plot hereafter, of the same representative group of MDA-MB 231 in the spectral region of Amide I and Amide II bands is shown in Figure 1b and it correlates the variations of spectral intensities upon thermal stress application occurring in-phase within the first 40 min of HS. It pops up that the autopeak related to the Amide I component centered at ∼1620 cm −1 , associated with intermolecular β-aggregates, is more prominent and therefore it is responsible for the deepest fluctuation in the IR profiles of these cells during early HSR. Other autopeaks are observed for protein signals at ∼1655 cm−1 (AmI, α-helix), at ∼1555 cm−1 (AmII, α-helix), and at ∼1525 cm−1 (AmII, tentatively assigned to both intra- and intermolecular β-structures, see later). Two negative cross-peaks, ∼1620 and ∼1655 cm−1 and ∼1620 and ∼1555 cm−1, as well as two positive ones, ∼1620 and ∼1525 cm−1 and ∼1620 and ∼1690/AmI, β-aggregates18/cm−1, can be seen, revealing that the intensity variations of these spectral components have, respectively, opposite and common timetrends. Most of the published reports assign the low-frequency component of Amide II to tyrosine side chain contributions38 and to intramolecular β-structures, and only few authors have tentatively assigned it also and to β-folded aggregates.31 Our results suggests that the ∼1525 cm−1 component of Amide II band may be assigned not only to intramolecular β-sheets but also to the same type of protein folding described by the 1620 cm−1 frequency, and therefore to intermolecular β-aggregates. Moreover the negative Syn cross peak observed between the components of the Amide II band centered at ∼1555 and ∼1525 cm−1 further strengthens this hypothesis, allowing to draw a clear parallelism between the 1655/1620 cm −1 components of the Amide I band and the 1555/1525 cm−1 components of Amide II. The latter is known to be less affected by unavoidable uncertainties in water-subtraction with respect to Amide I; therefore, the possibility to establish a clear parallelism supports our hypothesis. Figure S5 in the Supporting Information reports the asynchronous 2D correlation plot for the same group of MDA-MB 231 cells, Asyn plot hereafter, and a detailed analysis is also provided in the Supporting Information. From a general point of view, the Asyn plot detects variations of spectral intensities that occur out-of-phase (delayed or accelerated), as a function of time. It is noteworthy to highlight that, even if the Syn plots of all the sampled groups exhibit a high similarity (see the Supporting Information, Figures S6b and S7b), the Asyn ones slightly differ between samples (see Figures S5, S6c, and S7c in the Supporting Information), while the common message behind all of them is that the event associated with the peak at ∼1655 cm−1 occurs before the one at ∼1620 cm−1. The applicability of 2D correlation analysis for the investigation of a complex system, such as a live cell, has been reported by other authors.39,40 It is however undoubtable that establishing the fine dynamic of protein folding by this technique is challenging even for pure proteins, and it is further complicated by the complexity of the cellular machinery. The task is even more challenging in our case, since, as it will be better clarified in the Discussion, the nature of the events related to HSR is regulated by a dynamic equilibrium that can be shifted to reagents (native proteins) or products (β-aggregates) depending on a multitude of factors, first of all the individual cellular susceptibility to the stress. Therefore, because of the transient

Figure 1. For the representative group of MDA-MB 231 cells are shown: (a) second derivatives at different sampled times within 2 h of HS application in the spectral region 1750−1480 cm−1; (b) synchronous 2D correlation plots of absorbance spectra within the first 40 min of HS application in the same spectral region of part a; (c) trend of Proteins/Cell Mass (squares), Lipids/Cell Mass (circles), PhI/AmII (up triangle), and PhII/AmII (down triangle) ratios (red markers) compared with a control group of MDA-MB 231 (blue markers) over the 2 h experimental time course, normalized to the corresponding value at t = 0 min. Each trend is offset by 0.5 au.

After 40 min of severe HS, a shoulder within the Amide I band centered at 1621 cm−1 arises, characteristic of intermolecular protein β-aggregates30 (navy curve in Figure 1a). The steady presence of β-aggregates characterizes the late HSR of MDAMB 231 cells (blue curve in Figure 1a). At the end of the experiment (black curve in Figure 1a), the presence of βaggregates is even more pronounced and a further broadening of the α-helix component is detected. In addition, no detectable shifts in the position of the symmetric and asymmetric stretching bands of both methyl and methylene moieties has been revealed. Instead, the profile of bending modes of methylene moieties changes immediately after the application of the thermal stress, and the two components identified in the unperturbed state merge to a single component centered at ∼1455 cm−1 (for more details, see Figure S8 in the Supporting Information). 3673

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not show immediate changes; however, an uptrend of PhI/ AmII characterizes the late HSR of MDA-MB 231 cells while an almost monotone downtrend for the PhII/AmII band ratio can be appreciated and confirmed from data reported in Table 1. HSR of MCF-7 Cells. The peculiar spectral features of unperturbed MCF-7 cells have been obtained from second derivatives of the water-subtracted spectra, averaged on all the heat-stressed sampled groups under unperturbed conditions at t = 0 min (Data are provided in the Supporting Information, Figure S1). From a spectroscopic point of view, the two unperturbed cell lines share many common spectral features: in the spectral ranges 3050−2800 (see Figure S1a in the Supporting Information) and 1750−1400 cm−1 (see Figure S1b in the Supporting Information) significant spectral differences could not be seen, with the exception of the detectability of the CH stretching of vinyl moieties centered at 3008 cm−1. More differences, although small, among the two cell lines can be appreciated in the low wavenumber region, from 1300 to 1000 cm−1 (see Figure S1c in the Supporting Information): the spectral component of the PhI band (Bhelices of DNA) is here centered at 1224 cm−1, while the two shoulders of the PhII band are upshifted of few wavenumbers with respect to MDA-MB 231 cells. The same approach used for the analysis of HSR to MDAMB 231 cells was applied also to MCF-7 cell line. The inspection of the second derivatives for the representative control groups highlights that there are no significant changes in the spectral shape of these cells upon 2 h of measurements inside the fluidic chamber (see Figure S2b in the Supporting Information). Moreover, the trypan blue dye-exclusion tests performed at the end of the FT-IR measurement, directly into the microfluidic chamber (9 ± 4%) as well as on cells kept at 42 ± 0.5 °C in a thermalized bath (10 ± 2%), reveal that the percentage of dead cells is comparable to the one obtained before the measurements (10 ± 3%) (see Figure S4 in the Supporting Information). MCF-7 cell viability is not affected even when the cells are kept at 42 ± 0.5 °C for 2 h, as highlighted by trypan blue tests and confirmed by the results of the MTT test (see Figure S4 in the Supporting Information). However, also for this cell line, FT-IR data reveal significant changes in the cellular vibrational profile as a consequence of the temperature increase. Figure 2 reports the 1D time evolution of second derivatives of a representative group of MCF-7 cells in the spectral region 1750−1400 cm−1 during the entire experimental time, and the 2D correlation plots in the region of Amide I and Amide II bands within the first 40 min of HS application. The detailed data analysis for the representative cell group and two others is reported in the Supporting Information (see Figures S9−S11). The obtained results can be summarized as follows: all the sampled MCF-7 groups elicit spectral variations in the proteome profile comparable to the ones highlighted for MDA-MB 231 cells. They immediately respond to HS by the partial unfolding of native α-helix and β-sheet domains of cellular proteins and the immediate HSR is characterized by the trend of the system to re-establish proteostasis. The early response is still characterized by the oscillation of spectral features of misfolded protein structure in the 1640−1625 cm−1 spectral region, by the stable appearance of the spectral components related to intermolecular β-sheets (centered at ∼1620 and 1525 cm−1) for HS longer than 40 min, and by its enhancement during late HSR. Considerations similar to the ones drawn out for MDA-MB 231 cells can be also done with

nature of the variations monitored and the 1 min acquisition delay between one group and the following, the small monitored differences are not surprising and, more importantly, do not affect the overall picture of the phenomenon. Summarizing, the findings reported here demonstrate that the sampled groups of MDA-MB 231 cells immediately respond to HS through a relative increase of the unordered domains in cellular proteins. The immediate HSR is however characterized by the tendency of the system to re-establish the native protein state. During the early HSR, spectral features that reveal the broadening of the spectral components associated with α-helix folded proteins and the appearance spectral features related to misfolded ones in the 1640−1625 cm−1 spectral region are detected. The late HSR is dominated by the accumulation of large β-folded protein aggregates. The trend of several band ratios as a function of time has also been considered in order to get more insight on the system transformations, and they are plotted in Figure 1c for the representative group of MDA-MB 231 (red colored data series). For comparison purposes, the time-trend of the same integral ratios for a Control group is also plotted (blue colored data series). The percentage changes of the integral ratios with respect to t = 0 at representative experimental time points are reported in Table 1. Standard deviation of the data has been Table 1. Percentage Changes ± Standard Deviation (s.d.) of Integral Ratios for Control and Heat-Stressed MDA-MB 231 Cell Groups at Representative Time Points percentage change ± s.d. integral ratio

a

Proteins/CellMass (I) Proteins/CellMass (F) Lipids/CellMass (I) Lipids/CellMass (F) PhI/AmII (F) PhII/AmII (F)

control (n = 4) 1.01 0.98 0.99 0.99 1.01 0.96

± ± ± ± ± ±

0.03 0.03 0.07 0.02 0.03 0.03

stressed (n = 12)

Pa

± ± ± ± ± ±

** † * † ** **

1.12 1.01 1.08 0.99 1.14 0.77

0.06 0.01 0.07 0.10 0.06 0.11

a Time points: Immediate, I (within 10 min from t = 0 min), and final, F (at t = 135 min). Differences among the two groups, as obtained by unpaired two-sample Student’s t test, significant for P < 0.01 are labeled as **, for P < 0.05 as *, nonsignificant as †.

obtained averaging on all the sampled groups. The significance of the differences between control (37 ± 0.5 °C) and heatstressed (42 ± 0.5 °C) MDA-MB 231 populations was verified by the unpaired two-sample t test. As can be seen from Figure 1c, for the Control group the considered integral ratios follows sinusoidal-like fluctuations around the initial value during the entire experiment. This behavior well reflects the biochemical cycles of live cells as previously reported by the authors.15 The percentage variations are below the standard deviation for all the considered integral ratios, as reported in Table 1, describing the stationary time series. The only exception is represented by the PhII/AmII ratio that slightly decreases in time. Conversely, for stressed MDA-MB 231 cells, the Proteins/ Cell Mass ratio shows a significant sudden and transient increase of the relative cellular protein content that later undergoes sinusoidal-like fluctuations around values comparable to the ones of the unperturbed state. Moreover, a sudden increase of the Lipids/Cell Mass ratio is also detected upon the application of the thermal shock. For the early/late HSR, this ratio settles at values comparable to the ones of the unperturbed state. The PhI/AmII and PhII/AmII ratios do 3674

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Table 2. Percentage Changes ± Standard Deviation (s.d.) of Integral Ratios for Control and Heat-Stressed MCF-7 Cell Groups at Representative Time Points percentage change ± s.d. integral ratio

a

Proteins/CellMass (I) Proteins/CellMass (F) Lipids/CellMass (I) Lipids/CellMass (F) PhI/AmII (F) PhII/AmII (F)

control (n = 4) 1.01 1.00 0.98 0.99 0.98 0.98

± ± ± ± ± ±

0.05 0.01 0.02 0.02 0.03 0.01

stressed (n = 12)

Pa

± ± ± ± ± ±

* † * * † *

1.15 1.00 1.17 1.12 1.02 0.81

0.12 0.04 0.14 0.09 0.09 0.09

a Time points: Immediate, I (within 10 min from t = 0 min), and final, F (at t = 135 min). Differences among the two groups, as obtained by unpaired two-sample Student’s t test, significant for P < 0.01 are labeled as **, for P < 0.05 as *, nonsignificant as †.

231 cells, the only exception is represented by the PhII/AmII ratio that slightly decreases in time. The behavior of heat-stressed MCF-7 cells exhibit peculiar features with respect to MDA-MB 231 cells. Both Proteins/Cell Mass and Lipids/Cell Mass ratios undergo a significant sudden and transient increase upon the application of the thermal shock. However, while the relative protein content settles at values comparable to the one of the unperturbed state at the end of the experiment, the relative lipid content increases. The PhI/AmII and PhII/AmII ratios do not suffer immediate changes. However, while the latter integral ratio significantly diminishes at the end of the experiment, the former is almost stationary over the entire measurement time.



DISCUSSION Heat-shock response, HSR, is an ubiquitous and highly conserved defense mechanism that is involved in the cellular response to several injuries including oxidative stress, exposure to heavy metals or toxic substances, inflammation, and thermalstress.41,42 The precise sequence of events that occurs within the cell in response to HS and the cellular fate primarily depends on the severity of the stress.43 For mammalian cells, mild, severe, and deleterious conditions are conventionally distinguished and are characterized by temperatures in the range(s) of 39.5 ± 1 °C, 42.5 ± 1 °C, and 45 ± 1 °C, respectively. In this study, the response to severe HS has been investigated in two different breast cancer cell lines: MCF-7, a breast cancer cell line which does not have metastatic activity, and MDA-MB 231, an aggressive metastatic-like adenocarcinoma cell line. FT-IR results reveal that MDA-MB 231 and MCF-7 cells activate HSR in the same time scale following comparable pathways that can be harmonized in a unified scheme as follows. Both cell lines immediately sense the temperature increase from 37 to 42 deg and exert a response through variations in the lipid spectral profile. Cellular membranes are not simple passive barriers, but they represent the interface between the intra and extracellular world. The sensing of environmental changes, both chemical and physical, are first monitored by plasma membranes and changes in membrane physical state and composition are involved in cellular responses and modulate intracellular signaling.43 We did observe no changes in the position of the symmetric stretching band of methylene groups, a peak considered diagnostic of the state of order of the membranes. However, since an upshift of two to four wavenumbers is generally detected for the gel to liquid crystalline phase transition,35 a

Figure 2. For the representative group of MCF-7 cells are shown: (a) second derivatives at different sampled times within the 2 h of HS application in the spectral region 1750 and 1480 cm−1; (b) synchronous 2D correlation plots of absorbance spectra within the first 40 min of HS application in the same spectral region of part a; (c) trend of Proteins/Cell Mass (squares), Lipids/Cell Mass (circles), PhI/AmII (up triangle), and PhII/AmII (down triangle) ratios (red markers) compared with a control group of MCF-7 (blue markers) over the 2 h experimental time course, normalized to the corresponding value at t = 0 min. Each trend is offset by 0.5 au.

relation to methyl and methylene stretching and bending mode (see Figure S12 in the Supporting Information). The trends of several band ratios as a function of time have been considered for MCF-7 cells too, and they are plotted in Figure 2c for the representative heat-stressed and control groups (red and blue colored data series, respectively). The percentage changes of the integral ratios with respect to t = 0 at representative experimental time points averaged on all the sampled MCF-7 groups are reported in Table 2. As can be seen from Figure 2c, for the controls, the considered integral ratios follows sinusoidal-like fluctuations around the initial value during the entire experiment and the percentage changes are below the standard deviation, as can be deduced from Table 2, describing a stationary time series. Comparably to MDA-MB 3675

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in misfolded monomers. Monomers are then transformed into the relative unfolded structure from another class of HSPs, called unfoldases, like GroEL, and then correctly refold.42 The spectroscopic evidence of this dynamic equilibrium resides in the alternation/coexistence of the spectral components in the 1640−1625 cm−1 spectral region in the first 40 min of thermal stress. These spectral components are related to misfolded protein domains, possibly rich in intramolecular β-structures,49 and/or protein aggregates intercepted by HSP chaperones. In other words, during the first 40 min of HS application, the HSR machinery reaches the higher efficiency, as documented in literature47 and supported by the FT-IR results that prove the continuous balance between the protein misfolding/aggregation and the refolding action of HSPs. It is possible to clearly assert that it is after 40 min of stress application that the protein folding becomes unbalanced toward the formation of extended protein aggregates for both cell lines, as revealed by the permanent presence of the spectral components centered at 1620/1525 cm−1. The late HSR of both MDA-MB 231 and MCF-7 cells is characterized by a sort of steady state where extended βaggregates persist and accumulate in the final state. In addition, a downtrend in the PhII over Amide II ratio is detected for both cell lines. It is known that severe thermal stress arrests constitutive protein synthesis and reduces cell metabolism. PhII band contains contributions from C−OH stretching of ribose and carbohydrate, and therefore it seems reasonable to link the downtrend of PhII/AmII ratio to the impairment of carbohydrate metabolism in these cell lines. A synergic effect in this respect could be elicited by the absence of nutrients in the physiological solution, which could also justify the slight decline of carbohydrate metabolism highlighted for the controls. Both cell lines are still alive upon 2 h of stress application, as proven by viability assays as well as supported by literature data.50,51 However, an uptrend of the PhI/AmII is observed only for MDA-MB 231 cells and possibly related to the maintenance of an altered transcriptome activity, associated with the de novo HSPs synthesis due to the persistence of the HS conditions. On the contrary, the invariance over time of the PhI/AmII band for MCF-7 cells suggests a lower transcription rate for these cells. This could be ascribed to a higher sensibility toward HS of MCF-7 cell line. Indeed, if the system is not able to further thwart the aggregation of proteins, all the transcription pathways are blocked and cells undergo through apoptotic or autophagy commitment.43 In this scenario, the increase in the Lipids/Cell Mass in the final state could be ascribed to the formation of lipid droplets, as elsewhere reported by the authors,25 and then to the apoptosis commitment of these cells promoted by the persistence of stressful conditions. Indeed, the same behavior is not observed for MDA-MB 231 cells, in which the Lipids/Cell Mass ratio remains almost constant and the transcriptome activity is maintained. Actually, the metastatic nature of this cell line should make it more resistant to stressful conditions and able to survive even after severe stress conditions up to 44 °C.50

more modest upshift is expected to be related to the thermalinduced hyperfluidization of the cell membranes, characterized by the formation of nonbilayer structures at specific location but not involving the remodeling of the entire cellular lipidome. Conversely, the scissoring band of the methylene groups is more sensitive to cellular packing; it has a forked shape under unperturbed condition and it loses definition and broadens upon temperature rise for both cell lines (see Figures S8 and S12 in the Supporting Information), revealing an increased hydrocarbon chain molecular disorder and higher mobility.20,35 Spectroscopic data also show that both cell lines tend to reestablish and preserve the membrane integrity during thermal fluctuations, as proven by the methylene bending modes, inclined to reassume the spectral shape under unperturbed conditions during early and late HSR. A crucial role in this respect is played by the so-called small HPSs that, interacting with plasma membranes, stabilize the bilayer liquid-crystalline phase relative to the inverted hexagonal phase in lipids, counteracting the HS-induced hyperfluidization.44 It has been reported that changes in membrane fluidity induced by HS determine the increase of cytosolic concentration of Ca2+, which in turn contributes to HSR induction. Dishomeostasis of calcium ions takes place in both Ca2+containing and Ca2+-free media, like the one used for this experiment, due to the mobilization of specific pools of cytosolic Ca2+.45,46 Interestingly, hyperfluidization of plasma membranes happens at the same time as the transient increase of the relative lipid concentration, measured by FT-IR microscopy during the first minutes of thermal shock application. This is probably due to de novo synthesis of lipids that act as secondary mediators of HSR.47 It is known that the accumulation of small signal lipids, and ceramides in particular, happens within the first 5 min from the stimulation and it is necessary for the translation initiation and synthesis of HSPs.43 In accordance with literature data, a transient increase on the relative protein cellular content is detected for both cell lines, within the same time scale the lipid response is evoked.43 The transient nature of the phenomenon is well justified by the contemporary arrest of constitutive protein synthesis that is documented to occur in severe HS. Transcription of HSP is mediated by the activation of heat-shock factor genes (HSFs) and reaches its maximum rate within the first 30 min of HS application.48 HSPs act in stoichiometric manner, trying to reestablish protein homeostasis, but overall their synthesis is counterbalanced in-time by the arrest of the synthesis of newpeptides and the digestion of older ones.42 The broadening of the Amide I band toward lower wavenumbers within the first 5 min of HS application reveals the partial unfolding of native α-helix and β-sheet structures to random/unfolded protein fragments, and therefore an almost immediate unfolding of constitutive proteins can be deduced. Under physiological conditions, unfolded proteins spontaneously misfold into β-sheet rich monomers and aggregate in small misfolded oligomers, a process driven by hydrophobic forces.6 HS chaperones counteract the aggregation by correctly refolding misfolded proteins. Indeed, in the 10−15 min of HS application, the broadening of α-helix component slightly diminishes, and the spectral shape is more similar to the one of the unperturbed conditions. However, since the stimulation persists, the efficiency of the HS machinery is not sufficient to completely reverse the process, and unfolded/misfolded proteins tend to aggregate. HSP disaggregases (like HSp70 or HSp 110) bind disordered protein oligomers and divide them



CONCLUSIONS The results here presented do not pretend to provide a comprehensive overview of the specificity of HSR among different cell lines and they are not conclusive. However, to the best of our knowledge, they represent one of the first18,39,40 examples of time-resolved FT-IR microspectroscopy of protein 3676

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aggregation in live cells and further confirm the capability of this vibrational microscopy technique to follow in-depth variations of the cellular biochemical profile in real-time and specifically of the patterns of proteome rearrangement. A large variety of insults, different for thermal stress, induces the denaturation of cellular constitutive proteins and activates HSR pathways. Therefore, the possibility to identify and monitor intermediates of protein aggregation in live cells can offer great advantages for biomedical studies, suggesting the possibility to use FT-IR microspectroscopy as a tool for the early diagnosis of cellular stress conditions.



ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Phone: +39 0403758465. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS The authors want to thank Dr. Giovanni Birarda for the proofreading. REFERENCES

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Time-resolved FT-IR microspectroscopy of protein aggregation induced by heat-shock in live cells.

Maintaining the correct folding of cellular proteins is essential for preserving cellular homeostasis. Protein dishomeostasis, aberrant protein foldin...
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