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Abstract
Active and dynamic migration and deformation of cells are universal research targets in cell biology. The leader cells that develop at the wound edge are required to actively reorganize their structure for migration and deformation. Such active reorganization of cellular morphology possibly affects the temperature inside the cells via biochemical reactions. On the other hand, it is also possible that changes of intracellular temperature may first trigger and induce active migration and cellular deformation. Recent development of temperature sensors for cells enables the display of temperature difference between two adjacent cell populations such as the leader and follower cells around a wound. We used two different temperature measurement methods to check the temperature of leader and follower cells in a wound healing assay system and found that leader cells were 10° ∼ 101°C warmer than follower cells. We also confirmed that, when the cells were artificially warmed with an infrared laser, they started moving, and when the laser was stopped, the cells also stopped moving. These results suggest that the warmth of the leader cells is not simply a result of their active movement, but also can play the role of a trigger for cell migration and deformation. Our results shed light on the possibility that various observed cases of intracellular temperature increase may trigger associated biological phenomena.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Single cell-scale temperature difference is one of the newest topic in these days of biology [1–7]. Though temperature is one of the most basic physical indices, analysing the temperature of a small area like a cell was not possible until today and has not yet fully discussed the meaning of the temperature difference in a small space [8–10].
Especially its meaning for organisms are supposed to be bidirectional; they produce heat and are affected by the produced heat. Organisms use part of the energy obtained from metabolism in biochemical reactions, and unused energy is released as heat.
For example, mitochondria store the energy from acetyl CoA in the form of a proton gradient across the inner mitochondrial membrane. This energy is used for ATP synthesis. However, the energy stored in the proton gradient is greater than the energy required for ATP synthesis, and most of the energy is discarded as heat. This heat is thought to be the source of heat production in mitochondria [11].
On the other hand, there are biochemical reactions that are greatly affected by temperature. For example, the polymerization of cytoskeleton such as microtubules and actin filaments, and the migration rate of myosin are known to be biochemical reactions that are greatly affected by temperature [12,13].
These bidirectional flow of heat logically structures a feedback regulation in the biochemical network of cells. In another word, the produced heat, which can be detected as a temperature difference, will trigger or induce the next biochemical reactions, and will give and effect on the morphology or behaviour of cells.
The rapid development of temperature sensors for cells reveals such temperature differences [14–11]. For example, intracellular temperature distribution dependent on each organelle were detected such as higher temperature of nuclei than cytoplasm [14]. This analysis was performed with a polyacrylamide polymer-conjugated temperature sensing small fluorescent molecule, fluorescent polymeric thermometer (FPT). The study used a characteristics of FPT of which fluorescence lifetime gets elongate when temperature gets higher. In another study, researchers monitored the time-course of temperature fluctuation with particular triggers such as mitochondrion depolarizing agent [20]. They developed GFP-based thermosensors (tsGFPs), which includes Salmonella thermosensing protein TlpA in its structure for temperature sensing by fluorescence intensity, and genetically can target specific organelle such as mitochondria. In order to target specific organella, another GFP-based thermosensor was developed (gTEMP), which uses the ratiometric measurement of two fluorescent proteins according to the difference of fluorescent sensitivity against temperature of each protein [19]. There is the other type of study which visualized the temperature of mitochondria by using a small molecule fluorescent thermometer targeting mitochondria (Mito thermo yellow) [16]. The same group also developed a fluorescent thermometer which targets endoplasmic reticulum [15].
These studies used various probes to analyse intracellular temperature, and each probe has different advantages and disadvantages [21,22]. By finding efficient combinations of different types of probes based on their properties is desired to obtain reliable results.
The diversity of the various characteristics of genetically or environmentally single-originated cell population is another topic of recent cell biology [23]. However, it is rare to see the investigation of diversity of temperature-relating characteristics within a cell population. One of the few examples indicated the heterogeneity of cellular responses toward temperature, which appeared on the difference of cancer cells malignancy dependent on the environmental temperature [24]. The study suggested that cells migrating faster than the other cells are affected differently by environmental temperature. The study indicated the importance of environmental temperature for changing cellular motility, but did not test and discuss intracellular temperature.
Here we show the temperature difference within a cell population, which is given a signature by scraping off the cells as the leader cells on the boundary of the wound and next to the leaders as followers in a single culturing environment. By using two different temperature probes independently, we succeeded to show the higher temperature of leaders than followers. One of the measurement using fluorescent lifetime of sensor probe succeeded to exhibit the different temperature cells in single image at one moment. Another measurement using the shift of fluorescence wavelength showed the time course fluctuation dependent on infrared laser on and off. We confirmed these two different methods indicated the same tendency of temperature difference between leaders and followers in our experimental system. We also exhibit the leader cells warmed with laser changed their shape and started to move toward laser spot.
These results suggest that warming up of leader cells happens physiologically and triggers further movements and deformation of those cells.
2. Results
2.1 Higher temperature of leader cells than follower cells exhibited by two independent temperature detection systems
First, we performed temperature measurement of the NIH-3T3 cells under wound healing assay with two different methods. Figure 1(A) is the image of a cell which was successfully incorporated FPT, and Fig. 1(B) is the fluorescence decay curve.
Fig. 1. Fluorescence lifetime of FPT incorporated in NIH-3T3 cell. (A) Fluorescence lifetime image of FPT in a NIH-3T3 cell taken by fluorescence lifetime imaging microscopy (FLIM). Bar = 5 μm. (B) Fluorescence decay curve of FPT to calculate its fluorescence lifetime.
We calibrated the linear function between temperature and the fluorescence lifetime (FLIM) of FPT in NIH-3T3 cells at 330°C in a stagetop incubator (supplementary Fig. S1). The regression line showed good linearity between 26°C to 40°C (R2 = 0.929; Temperature [°C] = 2.31 × Lifetime [ns] + 7.292). We used this regression line for further analyses to calculate temperature from the lifetime of FPT. We performed wound healing assay with FPT-treated NIH-3T3 cells, and took the FLIM images (Fig. 2(A); supplementary Fig. S2). We defined the cells on the first line from wound is the leader cells, and the others are follower cells (Fig. 2(A), the cells marked with white arrowhead are the leader cells). As the images show the leaders are warmer than followers.
Fig. 2. Temperature imaging of the leader and follower cells of wound healing assay analysed with FPT and FLIM. A. FLIM images of NIH-3T3 under wound healing assay. Each image includes wound area (at lower-left corner), the line of leader cells(indicated with white arrow head), and the followers area (upper-right area). bar = 10 μm. B. The histogram of the temperature distribution of cells (red bar: leaders; blue bar: followers). The temperature of cell medium was kept at 33°C to enhance the sensitivity of FPT-based thermometry.
By repeating the same experiments, we confirmed that leader cells significantly warmer than follower cells. The mean temperature of leader cells was 33.74°C (95% C.I.= 0.582°C (n = 120)), the mean temperature of follower cells was 32.73°C (95% C.I.= 0.695°C (n = 46)) ($\Delta T$ = 1.01, Welch t-test p = 0.02885) (Fig. 2(B) and supplementary Fig. S2).
We performed wound healing assay with the same cell line and analysed the temperature of leader and follower cells with another temperature measuring method, which uses quantum dot [18]. For this analyses, we calibrated the linear function between temperature [°C] and the fluorescence intensity ratio of two particular fluorescence wavelength ranges (Fig. 3) at 37°C stagetop incubator. In order to improve the precision of the approximate line for the fluorescence intensity ratio of quantum dot against temperature, we used Gaussian blur for noise reduction of original image (Supplement 1), and numerically searched efficient ranges for calculating the ratio by Bayesian optimization (Materials and Methods). We chose first the range of $\lambda _{fmin}$ and $\lambda _{fmax}$ for one-dimensional Gaussian fitting of fluorescence spectral, after that three wavelength values to define the fluorescence intensity ratio ($\lambda _{rmin}$, $\lambda _{rmid}$ and $\lambda _{rmax}$) were chosen. By using the new approximation line, the temperature of leader cells were estimated 7.3°C higher (mean = 43.9°C, 95% C.I.= 4.043°C) than follower cells (mean = 36.6°C, 95% C.I.= 4.94°C) [Fig. 4, p = 0.018 (Welch’s t-test)].
Fig. 3. Quantum dots spectrum imaging for temperature detection. (A) The spectral intensity of quantum dot at different temperature. The left panel shows the spectrum at 26°C, and the middle panel shows the spectrum of quantum dot at 42°C. The right panel shows the distribution of peak wavelength of 3 quantum dots at each temperature. (B) Regression lines of Temperature and the peak wavelength(left panel) or Intensity Ratio(right panel). The range of $\lambda$ for each numerator and denominator to calculate ratio was identified by the Bayesian optimization. Error bars in the left panel are mean ± 95% C.I. of peak wavelengths at each temperature condition; at 30°C environment, 651.59±0.596 nm, at 37°C environment, 652.83±0.097, at 44°C environment, 652.74±0.287. Error bars in the right panel indicate mean ± 95% C.I. of fluorescence intensity ratios at each temperature condition; at 30°C environment, 0.379±0.0259, at 37°C environment, 0.400±0.0256, at 44°C environment, 0.426±0.0209.
Fig. 4. Temperature of the leader and follower cells in wound healing assay analysed with quantum dot. (A) NIH-3T3 cells images incorporated quantum dots in leader cells. The scale bars indicate 10 μm. Left panel shows green fluorescence of actin-GFP in NIH-3T3; middle panel shows the fluorescence of quantum dots; the right panel was produced by merging the image of cells and quantum dots, and the square area indicated with yellow line shows as an expanded image of an example cell. (B) NIH-3T3 cells images incorporated quantum dots in follower cells, and arranged as the same with upper panels. (C) The detected temperature of leader cells and follower cells. The detected mean temperature of leader cells in 37°C environment was 43.9°C (95% C.I.= 4.94°C; follower cells was 36.6°C (95% C.I.= 4.04°C). Blue circles are the results of each experiment, black lines indicate the mean temperature of Leaders or Followers, respectively. The p-value of Welch’s t-test between Leaders and Followers was 0.018.
These results showed that warmer cells than the average of the population more frequently appear in the leader cells than the follower cells, and the averaged temperature difference of leader cells and follower cells is in the range of 100 ∼ 101°C.
Our study revealed that leader cells are frequently warmer a few degree than follower cells. This phenomenon was confirmed with Fluorescent Life time elongation of FPT and wavelength red-shift of quantum dots.
Next, we examined that when we warmed cells with exogenous methods such as using infrared laser (IR laser), if cells started to elongate or to move like leader cells, in order to know if the temperature difference is just a result of leader cells activity, or if the temperature difference can work as a trigger to behaving like a leader cells.
2.2 Cells warmed with infrared laser changed their shape and started migration
First, we confirmed if our IR system can warm up cells by analysing intracellular temperature with quantum dot (Fig. 5). The system we constructed using W-VIEW GEMINI-2C (Hamamatsu Photonics) is indicated in Fig. 5(A). By using this system, we can detect distributed light intensity with single imaging dependent on the wavelength which was distinguished by the mirror combination. We analysed if each quantum dot in water shows red-shift by IR laser [Fig. 5(B)]. All quantum dots showed gaining the ratio which is the evidence of red-shift. We also checked the area distribution of heating by IR laser by using quantum dot. IR laser is known that it can be assumed cylindrical shape as a heat source. In such case, temperature distribution is formed around the heat source following the next equation [25];
where $r_i$s are the distance from the heat source [m], $T$($r_i$) [K] are the temperature at $r_i$, $Q$[W] is the power given by IR laser, $l$ [m] is the height of the cylindrical heat source, and $k$ [Wm-1K-1] is the thermal conductivity of solvent. The observed ratios were well approximated with this function; the close area is warmed but the effect does not reach farther than 150 μm [Fig. 5(C)].Fig. 5. Evaluation of the temperature detection system with w-view for quantum dot. (A) The system construction of w-view temperature detection system for Qdot. The mirror and filter combinations were changed from previously described for the temperature detection system for gTEMP ([19]). (B) Detected fluorescence ratio evaluation under on/off of IR laser. The plot shows each pair of fluorescence ratio of quantum dot under on/off condition of IR 1480 nm (orange: on; blue: off). The bigger value of ratio means higher temperature. (C) $\Delta$Ratio = Ratio$_ {on}$ - Ratio$_{off}$ distribution from IR laser spot. The origin (x, y = 0) is the IR laser spotted point. Blue dots indicate the ratio values of each observation and the solid blue line indicates the logarithmic estimated line($\Delta$Ratio$_{IR}$ = $-a\ln {r} + b$, a = 0.026, b = 0.180).
We exposed with IR laser at a close point of a targeted cell by the evaluated system, and analysed the temperature of the targeted cell (Fig. 6(A), upper panels). We confirmed that intracellular temperature was immediately increased by exposing with IR laser, and immediately returned to the baseline by stopping irradiation (Fig. 6(A), lower panel).
Fig. 6. IR laser effect of cells temperature and behaviour. (A) The images of cells (left) and quantum dots fluorescence (middle). The right panel shows the merged image of the cells and the quantum dots. The plot in the lower panel is the fluctuation of fluorescence ratio of quantum dots. IR laser switched on at 5 min and off at 15 min. (B) The images of a cell which warmed with IR laser spotting at a close point from the cell (yellow circle). The red lines were the boundary of the cell at each time point, and the yellow line showed the cell boundary at 0 min.
We also observed the cellular shape and their behaviour under irradiating IR laser (Fig. 6(B) and Supplement 1). The cell which irradiated close point with IR laser immediately started to move toward the irradiating spot, and stopped the movement immediately after stopping irradiation of IR [Fig. 6(B)]. The starting and stopping movement timing were synchronized with intracellular temperature increasing and returning at baseline [Fig. 6(A), lower panel and Fig. 6(B)].
This result suggests that temperature elevation may works as a trigger of enhancing cellular migration and elongation, which leader cells show frequently.
3. Discussion
We analysed intracellular temperature under wound healing assay with two different methods. One of the methods we used was FLIM imaging of thermosensitive polymer, the other was analysing wave length shift dependent on the temperature change of nano-scale semiconductor. These methods are based on the different principles to detect temperature, using different materials of the detectors, also using different physical indices to estimate temperature [14,18].
These two different methods indicated the same tendency of temperature difference between leader cells and follower cells in our experiments. Evaluating the result by the different methods which uses different principles is a standard tactics to check a systematic error of physical experiments. In this paper, we used two different methods for the same target, and the two experiments support the same results for a biological finding. The confirmed result is the existence of the temperature difference between leader cells and follower cells. The indicated range of difference were within the order of 100 ∼ 101°C among these two experiments. This range of temperature difference were frequently detected in previous studies [14–20] as the temperature difference among organelle, or the results of ion-pumping through channel proteins etc. 100 ∼ 101°C of temperature change around physiological temperature (37°C) is expected to have an impact to biochemical reactions in a cell, such as actin polymerization [26], which is undergoing at cellular peripheral during cellular migration and deformation.
On the other hand, we discovered the difference between these two methods on their precision. Temperature analysis using FPT is almost 7 times more precise than Qdot application of our case. We should recognise this difference and it is desired to improve the method with Qdot as a future work.
We examined if we can induce cellular movement/elongation by heating directly with infrared laser. This experiment showed that cells migrated/deformed only during heating. Similar results of applying IR laser on cell morphology changing were indicated in previous studies which used different cells [25,27–29]. Each study shows the potential roles of heating for cardiomyocytes pulsation, neuronal cells networking, any cells budding and embryogenesis control. Our results and these experiments are the first step to know the roles of temperature signals beyond species by controlling cellular behaviour with direct heating. The development of more physiological methods is desired for farther studies to confirm the resulting insight.
4. Conclusion
We confirmed that leader cells in wound healing assay were warmer than the follower cells. And when we warmed cells by IR laser, we can induce cellular deformation and migration dependent on the warming manipulation. These results suggest that temperature difference can be produced dependent on the cellar role in the belonging cellular population, also can work as the trigger to change their shape and behaviour to a suitable one for their role.
5. Materials and methods
5.1 Cell culture and the sample preparation of wound healing assay
NIH-3T3 cells were cultured with Dulbecco’s Modified Eagle Medium supplemented 10%v/v of fetal bovine serum, at 37°C, with 5% of CO2. Actin-GFP expressing NIH-3T3 was established with NIH-3T3 cells and pEGFP-C1 inserted Homo sapiens beta actin. Actin-GFP expressing cells were selected with 600 μg/mL G418 mixed culture medium. The cells were harvested on 35 mm-$\phi$ glass-bottom dishes (glass 12 mm-$\phi$) (Iwaki 3961-35) until the cells reach semi-confluent condition. Cells were scraped off on the observation day, 3 hrs before of the FPT or Qdot treatment, with yellow chips for micro-pipetters.
5.2 FLIM for temperature detection
NIH-3T3 cells were labeled with a cell-permeable fluorescent polymeric thermometer (FPT, Funakoshi), and time-correlated single-photon counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) was performed on a TCS SP8-FALCON confocal laser-scanning microscope (Leica Microsystems), as previously described with modification ( [14,17]). In this paper, we used cell-permeable FPT, which was cationic FPT prepared with APTMA instead of SPA ( [17]). FPTs were introduced into NIH-3T3 cells by replacing the culture medium to 5% glucose solution containing 0.03 w/v% of FPTs after rinsed the cells cultured in 35-mm glass bottom dish (glass 12 $\phi$) with 2 mL of PBS(-). After the cells were placed at room temperature in light-shading condition for 10 min, FPT-glucose solution was removed. Cells were washed twice with 2 mL of PBS(-) and added observation medium (phenol red-free). To calibrate the temperature dependency line of FPT, FPT in NIH-3T3 cells was excited with 470 nm pulsed diode laser at the repetition rate of 20MHz, and the fluorescence over 500 nm was measured using Leica SP8 FALCON system equipped 60 magnitude objective. To obtain the conventional fluorescence lifetime (tf), the fluorescence decay curves of FPT in single cells were fitted with a double exponential function, as previously reported [14].
The calibration curve for the temperature imaging of NIH-3T3 cells with FPT indicated in Supplement 1 was obtained by approximating the relationship between the fluorescence lifetime of the FPT in NIH-3T3 cells. The experiments were replicated 3 times independently.
5.3 Intracellular temperature detection with quantum dot
We analysed intracellular temperature with quantum dot 655 by the modified method from the previous work [18]. Briefly except the modified parts, Qtracker nanocrystals and Qtracker carrier were mixed and incubated for 5 min at room temperature to prepare the 1 μM solution. The solution was diluted with 37°C cell culture medium for the concentration of 10 nM or 50 nM and vortexed for 30 seconds. We used a confocal laser microscope FV1000 (Olympus), equipped 405 and 488 nm laser and spectrograph which has 2 nm resolution of wavelength. The temperature of cells and the other samples for observation was maintained during the observation in a stagetop culturing system (STRF-WELSX, Tokai Hit). We modified the calibration method from the previous study as described in the following subsection.
5.4 Fluorescence intensity ratio calculation
In order to use multiple quantum dots for temperature detection of multiple cells, we prepared a new regression function based on the data of multiple Qdots. We defined the Fluorescence Intensity Ratio as;
After that, we used 2D Gaussian fitting to define the area of Qdot fluorescence as a region of interest (ROI). The
5.5 Optimization of the range of the Qdot fluorescence wavelength for a one dimensional Gaussian fitting
In order to obtain the maximum correlation coefficient, we also optimized the range of the Qdot fluorescence wavelength for one dimensional Gaussian fitting. After once calculated the fluorescence intensity ratio following Eq. (2), performed one dimensional Gaussian fitting to calculate $f(\lambda )$ of Eq. (5). The value of fluorescence intensity ratio was calculated by integration $f(\lambda )$ in the range of $\lambda _{rmin}$ to $\lambda _{rmid}$ and $\lambda _{rmid}$ to $\lambda _{rmax}$. Pearson’s correlation coefficient between temperature detected by thermistor and the above ratio was calculated for all the obtained fluorescence spectrum, and performed Bayesian optimization among scanning wavelength range ($\lambda _{fmin}$ and $\lambda _{fmax}$) and ratio defining range ($\lambda _{rmin}$, $\lambda _{rmid}$, $\lambda _{rmax}$) until the correlation coefficient get maximum value. We produced regression line of temperature and the ratio based on this optimized fluorescence ratio ($Ration_{optimal}$).
5.6 Intracellular temperature measurement and cellular behavior observation with w-view system
Local heating inside the culture dish was used IR-LEGO 1000 microscope system equipped with 1,460 nm CW laser diode ( [31]). This system enables to focus IR laser beam on the glass bottom of culture dish and produces a heat spot. W-VIEW GEMINI-2C image splitting system, A1280-10 (Hamamatsu Photonics), equipped two CMOS cameras (Orca Flash 4, Hamamatsu photonics) onto the IR-LEGO was used for measuring temperature of the cells with Qdots by exchanging the mirror set for Qdots ( [19]. For Qdots experiments we selected a short wavelength side band-path filter (FF02-655/40-25, Semrock), and a long wavelength side band-path filter (FF01-650/60-25, Semrock) as shown in Fig. 5(A). In this experiments, we defined fluorescence intensity ratio as follows:
where $Intensity_{long}$ is the integral of fluorescence intensity between 654~683 nm, $Intensity_{short}$ is the integral of fluorescence intensity between 631~654 nm. This system was also used to observe the change of cellular shape and behaviour in Fig. 6 additionally to warm up cells with IR and to analyse cellular temperature.Funding
Japan Society for the Promotion of Science (17H06258, 20H02586); Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences (18-205, 19-205, 20-205); National Institute of Natural Science, National Institute for Basic Biology (17-524, 18-509, 19-346).
Acknowledgments
This study was supported by NIBB Collaborative Research Program for integrative imaging (17-524; 18-509; 19-346) and by the Cooperative Study Program of Exploratory Research Center on Life and Living Systems (ExCELLS) (ExCELLS program No.18-205; 19-205; 20-205) to NH, and partially supported by JSPS grants 17H06258 and 20H02586 to YK. Bayesian optimisation was performed by using SigOpt.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
Supplemental document
See Supplement 1 for supporting content.
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