Microtubule-Targetable Fluorescent Probe: Site-Specific Detection

Publication Date (Web): April 28, 2015. Copyright © 2015 American ... To realize super-resolution fluorescence imaging, the spiropyran derivative (SP...
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Microtubule-Targetable Fluorescent Probe: Site-Specific Detection and Super-Resolution Imaging of Ultratrace Tubulin in Microtubules of Living Cancer Cells Hua Zhang,*,† Caixia Wang,† Tao Jiang,† Haiming Guo,*,† Ge Wang,†,‡ Xinhua Cai,‡ Lin Yang,† Yi Zhang,†,‡ Haichuan Yu,‡ Hui Wang,‡ and Kai Jiang† †

Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals; Key Laboratory of Green Chemical Media and Reactions, Ministry of Education; School of Chemistry and Chemical Engineering, Henan Normal University, 46 Jianshe Road, Muye Zone, Xinxiang, 453007, People’s Republic of China ‡ Xinxiang Medical University, 601 Jinsui Road, Hongqi Zone, Xinxiang, 453000, People’s Republic of China S Supporting Information *

ABSTRACT: Tubulins in microtubules have been recognized as potential targets in cancer chemotherapy for several years. However, their detection and imaging in living cells, especially following exposure to anticancer drugs, remains difficult to achieve. This difficulty is due to the very small cross section of microtubules and the very small changes in tubulin concentration involved. Photoswitchable fluorescent probes combined with the “super-resolution” fluorescence imaging technique present an exciting opportunity for site-specific detection and super-resolution imaging of specific microscopic populations, such as tubulin. In this study, a tubulin specific photoswitchable fluorescent probe (Tu-SP), that labels and detects ultratrace levels of tubulin in microtubules of living biosystems, was designed and evaluated. To realize super-resolution fluorescence imaging, the spiropyran derivative (SP), a classic photoswitch, was introduced to Tu-SP as a fluorophore. To detect ultratrace tubulin, Tu-SP employed the tubulin inhibitor, alkaloid colchicine (Tu), as a recognition unit. Tu-SP exhibited nearly nonintrinsic fluorescence before binding to tubulin, even if there were divalent metal ions and 375 nm lasers, respectively. After binding to tubulin, a dramatic increase in fluorescence was detected within milliseconds when irradiated at 375 nm, this increase is a result of the transformation of Tu-SP into a colored merocyanine (Tu-SP-1) with fluorescence. Tu-SP was successfully used for site-specific imaging of tubulin at a resolution of 20 ± 5 nm in microtubules of living cancer cells. More importantly, the probe was suitable for site-specific and quantitative detection of trace tubulin in microtubules of living biological samples. “Super-resolution” fluorescence imaging techniques combined with relevant probes have recently been developed to overcome diffraction limits,11−15 and the resultant probes appear to preserve the inherent noninvasive optical microscopy imaging capability. The reported probes, such as asFP595,16 Anti-Rabbit Alexa Fluor@647 (Cy5),16 EosFP,17 and PAmCherry,18 have been successfully used to image different proteins, including tubulin, at a super resolution (20 ± 5 nm). However, these probes are obtained by further modification of the macromolecule, fluorescent protein, or antibody, which is a time-consuming and tedious process. Exogenous macromolecular fluorescent proteins or antibodies could also greatly affect intracellular events compared with small molecules (molecular weight < 800). Moreover, these

T

ubulin is a protein that forms microtubules in eukaryotic cells, and can combine with the microtubule-associated proteins and other relevant proteins. Tubulin performs various functions in cell division, cell movement, intracellular material transportation, and so on.1,2 Clinical data suggest that the trace changes in tubulin are associated with cancer therapy. Thus, the control of these trace changes of tubulin in microtubules has become a new target of cancer therapy research.3 In comparison with other imaging modalities, fluorescence probes4−6 combined with confocal microscopy imaging technology present exciting opportunities for the selective imaging of tubulin, microtubules, and relevant events in living cells, which will benefit the cancer therapy research.7−10 These outstanding works have enriched current design strategies for probes. However, these probes exhibit poor spatial resolution when used to image and detect protein molecules in living cells. In fact, the resolution of such probes has been limited to 200 nm for several years. © 2015 American Chemical Society

Received: January 11, 2015 Accepted: April 28, 2015 Published: April 28, 2015 5216

DOI: 10.1021/acs.analchem.5b01089 Anal. Chem. 2015, 87, 5216−5222

Article

Analytical Chemistry Φx = Φs(Fx /Fs)(A s /A x )(λexs /λexx )(n x /ns)2

macromolecules cannot detect trace change of tubulin in microtubules of living cancer cells. Photoswitchable or photoactivable fluorescent probes19,20 as functional fluorescent small molecules can be used in photoactuated unimolecular logical switching-attained reconstruction to realize superresolution imaging. This type of probe not only offers high sensitivity, real-time imaging, and super resolution, but can also be very easy to modify. Consequently, designing this type of functional fluorescent, small molecules for site-specific detection and super-resolution imaging of ultratrace tubulin in microtubules, is of great importance. In the present study, we report a tubulin-specific photoswitchable fluorescent probe (Tu-SP) for site-specific detection and super-resolution imaging of ultratrace levels of tubulin in microtubules of living cells. A spiropyran derivative (SP) as the fluorophore and alkaloid colchicine (Tu) as the recognition unit was introduced into probe. After binding to ultratrace tubulin, Tu-SP showed gradual fluorescence changes within a linear concentration range of tubulin when alternately irradiated with 375 and 561 nm lasers. We successfully used Tu-SP probe for site-specific imaging of tubulin in microtubules of living cells at a super resolution of 20 ± 5 nm. More importantly, the probe was found to be suitable for site-specific quantitative detection of ultratrace tubulin in microtubules of living biological samples. To the best of our knowledge, Tu-SP presents better properties, such as accessibility to living cells, site-specificity for tubulin, and super-resolution imaging, among others, compared with existing probes.

(1)

where Φ represents quantum yield; F is the integrated area under the corrected emission spectrum; A is absorbance at the excitation wavelength; λex is the excitation wavelength; n is the refractive index of the solution (because of the low concentrations of the solutions (10−7−10−8 mol/L), the refractive indices of the solutions were replaced with those of the solvents); and the subscripts x and s refer to the unknown and the standard, respectively. Incubation and Staining of Living Cells. Hela cells were obtained from Institute of Basic Medical Sciences (IBMS) of the Chinese Academy of Medical Sciences. Phenol-red free Dulbecco’s Modified Eagle’s Medium (DMEM) from WelGene supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS) from Gibco was used to culture HeLa cells. Cells were cultured for 3 days in a CO2 incubator at 37 °C. One day before imaging, cells were seeded into a glassbottomed dish (MatTek, 35 mm dish with 20 mm well) and were incubated at 37 °C in a humidified incubator containing 5 wt %/vol CO2. The next day, 2.0 μL of Tu-SP was added into the cells and incubated for 30 min at 37 °C under 5% CO2 and washed three times with phosphate-buffered saline (PBS). Imaging of Microtubule by Confocal Microscopy. Fluorescence imaging of microtubule in cells was carried out with spectral confocal multiphoton microscopes (Olympus, FV1000). Numerical aperture (NA) was set at 1.42 (oil) and excitation wavelength was at 405 or 545 nm. The intensity images of Tubulin-Tracker Red, Tu-SP, and Hochest 33258 were recorded with the emission in the range of 605−615, 560−570, or 455−465 nm, respectively. Internal PMTs were used to collect the signals in an 8 bit unsigned 1024 × 1024 pixels at 400 Hz scan speed to obtain the images. Each measurement was carried out in five replicates (n = 5). Super-Resolution Imaging of Microtubule. The work that was reported by Hu was referenced in the imaging method.22 The inverted microscope (Olympus IX81), which equipped with a 100× oil immersion objective lens (NA = 1.45). Excitation light was imported into the back aperture of the objective by an array of appropriate filter sets. The EMCCD detector (ANDOR Ixon+) was used to collect the signals. For wide field super-resolution imaging, the 375 nm laser (duration time: 1−2 ms, power density: 0.06−0.7 W/cm2) and 561 nm laser (exposure rate: 1 s, power density: 848 W/cm2) were used. This imaging data were representative of many times replicate experiments process to collect enough frames for reconstructing the photoactuated unimolecular logical switching attained reconstruction images.22 Cytotoxicity. Hela cells were selected as cytotoxicity experimentation model cell. Cell viability was used as an index to evaluate the cytotoxicity of dyes for cells. Cells were seeded into 96-well plates, and 1 × 105 cells were accurately added into per well, which were incubated in 100 μL of DMEM. The dyes Tu-SP (10.0 μM) and Tu-SP-1 (10.0 μM) were added into cells, and cells were incubated for an additional 24 h. After then, 100 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was added into each well, followed by further incubation for 4 h at 37 °C in a humidified incubator containing 5 wt %/vol CO2. The DMEM was remove, then in order to dissolve the reddish-blue crystals, DMSO (200 μL/well) was added into each well. Optical density (OD) was determined at 570 and 630 nm, respectively.



EXPERIMENTAL SECTION Reagents and Apparatus. All chemicals were purchased from Sigma-Aldrich Reagent Company. The commercialization dye, Tubulin-Tracker Red, and Hochest 33258 were purchased from Invitrogen/Life Technologies Company. All solvents and reagents were of analytical grade and used without further purification. Silica gel (200−300 mesh) and aluminum oxide (100−200 mesh) was used for the column chromatographic purification. Doubly purified water was used in all experiments, which was prepared by Milli-Q system. LC/Q-TOF mass spectrometer was used to study mass spectral. An auto sampler operated in-line with a quantum triple quadrupole instrument in ESI positive or negative ion mode. A Bruker Avance (400 MHz) spectrometer was used to obtain the NMR spectra. The data was processed using squared sinebell window function, symmetrized, and displayed in magnitude mode. Optical density (OD) was determined by a microplate reader (Spectra Max M5, Molecular Devices). Spectra Measurements in Solution. A Lambd 950 spectrophotometer from PerkinElmer (U.S.A.) and a LS-55 spectrophotometer from PerkinElmer (U.S.A.) were used to obtain absorption spectra and fluorescence spectra, respectively. Each experiment was carried out in five replicates (n = 5). A 1.0 mM of Tu-SP stock solution was prepared in DMSO. The certain amount of Tu-SP stock solution was incrementally added into 3 mL of different solvents in separate quartz cuvette. The samples were stirred, and then the absorbance and fluorescence were detected. In all spectral experiments, the final solutions contained 0 and conjugated bond number (CBN) < 40 must be achieved for successful entry into living cells.27 The log P and CBN values of Tu-SP were 5.95 and 17, respectively. These results reveal that Tu-SP can enter living cells. Localization imaging experiments were performed to evaluate specific recognition of Tu-SP for tubulin in microtubules of living cancer cells.28 The images (Figure 3) of fluorescence staining by Tu-SP matched those of staining by TubulinTracker Red a commercial specific-microtubule dye, (Figure 3d, the Pearson’s correlation factor R2 = 0.96). This result demonstrates that Tu-SP could specifically recognize tubulin in microtubules of living cancer cells.27 Cytotoxicity experiment results revealed (Figure S8) that Tu-SP, Tu-SP-1, and Tu-SPM have low cell toxicity, and cells have high viability after it was incubated by Tu-SP.

Scheme 2. (a) Chemical Structures of Tu-SP, Tu-SP-1, and Tu-SP-M; (b) 1H NMR Spectra of Tu-SP, Tu-SP-1, and TuSP-M

problem, Zn2+ was selected as the divalent metal ion instance in this experiment. Tu-SP-1 chelated with Zn2+ to form Tu-SP-M (see Scheme 2, ε = 18470 M−1 cm−1, ΦTu‑SP‑M = 0.102, λem = 610 nm), the structure of which was further verified by 1H NMR (Figure S2, Tu-SP-M, trace). The stable chelation product Tu-SP-M (Figure S5) provided enough time to capture fluorescence signals by Electron-Multiplying CCD (EMCCD), which is significant for the super-resolution imaging of tubulin. Then, Tu-SP-M was transformed back into the nearly nonintrinsic fluorescent Tu-SP with irradiation by a 561 nm laser (Figure 1a, magenta line). The fluorescence of the probe remained a constant even when this “OFF-ONOFF-ON···” fluorescence process was cycled 20 times (Figure 1b) and was not affected by pH (3.7−11.7, Figure S6). These results reveal that Tu-SP is a satisfactory photoswitch for superresolution fluorescence imaging. More importantly, the fluorescence intensity of Tu-SP-M (ΦTu‑SP‑M = 0.102, Figure 1a, red line) was basic equal to that of Tu-SP-1 (ΦTu‑SP‑1 = 0.098, Figure 1a, violet line). So, there was 5219

DOI: 10.1021/acs.analchem.5b01089 Anal. Chem. 2015, 87, 5216−5222

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Analytical Chemistry

Figure 2. (a) Fluorescent change of Tu-SP (2.0 μM) upon different content of ultratrace tubulin increasing from 0 to 16 nM in in PBS buffer (pH 7.4). Exciting wavelength: 375 nm. (b) Fluorescence of TuSP (2.0 μM) linear related to ultratrace tubulin concentration (1.00− 13.6 nM).

Figure 4. Fluorescence intensity images in living HeLa cells. (a−d) Stained with Tu-SP (2.0 μM). (a-1), (b-1), (c-1), and (d-1) Stained with Tu-S-M (2.0 μM). In turn, the amount of va, which was added into cells (a, a-1, b, b-1, c, c-1, d, and d-1) was 1.0, 0.90, 0.60, and 0 mM. (a), (b), (c), (d), (a-1), (b-1), (c-1), and (d-1) Excitation wavelength = 405 nm, scan range = 605−615 nm. Scale bar, 15.0 μm. (e) and (e-1) The fluorescence intensity of Tu-SP and Tu-S-M (2.0 μM) indirectly linear related to tubulin. Images and data are representative of replicate experiments (n = 5).

Figure 3. Fluorescence localization images in living HeLa cells. (a) Stained with Tu-SP (2.0 μM); (b) Stained with Tubulin-Tracker Red (5.0 μM); (c) Stained with Hochest 33258 (5.0 μM); (d) Merged image of a, b, and c: (a) excitation wavelength = 405 nm, scan range = 605−615 nm (false-color: green); (b) excitation wavelength = 545 nm, scan range = 560−570 nm (false-color: red); (c) excitation wavelength = 405 nm, scan range = 455−465 nm (false-color: blue); scale bar, 15.0 μm. Images and data are representative of replicate experiments (n = 5).

To verify the capacity of Tu-SP for detecting, recognizing and quantifying ultratrace tubulin, the concentration of tubulin in living cells was controlled using the inhibitor vinca alkaloid (va). Figure 4a shows no evident fluorescence signals form TuSP in cells after tubulin was completely inhibited by va (1.0 mM); this result indicates that Tu-SP could specifically recognize tubulin in living cells. Furthermore, the fluorescence intensity in living cells decreased with decreasing tubulin concentration at ultratrace levels; this activity was gradually inhibited by va (Figure 4b, c, and d, respectively). An indirect linear relationship between Tu-SP and tubulin concentration (Figure 4e) was further observed. The results obtained thus far demonstrate that Tu-SP can be used to specifically label and quantitatively detect ultratrace tubulin in microtubules of living cancer cells. Similar conclusions may be derived from the results of Tu-SP-M detection (Figure 4a-1 to e-1). Super-Resolution Imaging of Tubulin in Microtubules. Tu-SP was further employed for super-resolution

imaging of tubulin in microtubules of living cells by photoactuated unimolecular logical switching attained reconstruction (Figure 5). When a laser with increased power was used to illuminate the cells, a rapid decay in the fluorescence intensity of the cells was observed until Tu-SP reached a steady state. Blinking of individual Tu-SP molecules was also detected. Under this condition, localization exhibited a mildly decreasing trend (Figure 5a,b), that is, the amount of molecules/μm2 within 10000 frames decreased and the average localization precision was 20 ± 5 nm (Figure 5c). Living cells tubulin concentrations determined by photoactuated unimolecular logical switching attained reconstruction are shown in Figure 5a. In comparison to Figure 3a and 4a−d, significantly higher resolution was obtained (Figure 5a) and submicroscopic filaments with diameters below 72 nm were detected (Figure 5d). 5220

DOI: 10.1021/acs.analchem.5b01089 Anal. Chem. 2015, 87, 5216−5222

Analytical Chemistry



Article

ASSOCIATED CONTENT

S Supporting Information *

Experimental and analytical details. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01089.



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (21402043, 21171051, and 61176004). Basic and frontier research programs of the Henan province (142300410421). Key scientific research project of higher education of the Henan province (15A150017). Dr. start-up project funding of Henan Normal University (qd14119).



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Figure 5. Images and data are representative of replicate experiments (n = 5). (a) Imaging of Tubulin by confocal microscopic imaging (green) and photoactuated unimolecular logical switching attained reconstruction (red) after staining with Tu-SP (2.0 μM); scale bar: 200 nm. (b) Images of the boxed regions (scale bar: 300 nm). (c) Localization precision histogram. (d) The relative fluorescence of scale bar region in panels b, respectively (diffraction-limited: black line; super-resolved: red line).



CONCLUSIONS In this work, we designed and synthesized a photoswitchable tubulin-specific fluorescent probe (Tu-SP) based on a spiropyran derivative; the fluorescence signals of this probe were gathered by specific interactions with tubulin in microtubules of living cells. The specificity of Tu-SP for tubulin results from the introduction of a tubulin inhibitor (colchicine) to the molecule. An “OFF-ON-OFF-ON···” fluorescence cyclic process, which occurs as a fluorescence switch phenomenon between Tu-SP and a classic cyanine dye (Tu-SP-1), is brought about by alternate irradiation by 375 and 561 nm lasers after binding to tubulin. Therefore, Tu-SP was used for site-specific imaging of tubulin in microtubules at a super resolution of 20 ± 5 nm in living cancer cells. The fluorescence intensity generated by Tu-SP-1 presented a linear relationship with increasing amounts of tubulin (1.00−13.6 nM) at trace levels in microtubules of living cancer cells. Thus, Tu-SP may be suitable for use during in situ specific fluorescence imaging and quantitative detection of trace tubulin in living biological samples. We believe that Tu-SP could serve as a practical tool for observing and detecting trace change of tubulin in microtubules during cancer therapy. 5221

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