Shear Stress-Enhanced Internalization of Cell Membrane Proteins

Letter Cite This: Anal. Chem. 2018, 90, 5540−5545

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Shear Stress-Enhanced Internalization of Cell Membrane Proteins Indicated by a Hairpin-Type DNA Probe Ziyi He,† Wanling Zhang,† Sifeng Mao, Nan Li, Haifang Li, and Jin-Ming Lin* Beijing Key Laboratory of Microanalytical Methods and Instrumentation, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084, China S Supporting Information *

ABSTRACT: Shear stress is an important mechanical stimulus that plays a critical role in modulating cell functions. In this study, we investigated the regulating effects of shear stress on the internalization of cell membrane proteins in a microfluidic chip. A hairpin-type DNA probe was developed and indiscriminately anchored to the cell surface, acting as an indicator for the membrane proteins. When cells were exposed to shear stress generated from fluid cell medium containing external proteins, strong fluorescence was emanated from intracellular regions. With intensive investigation, results revealed that shear stress could enhance the specific cell endocytosis pathway and promote membrane protein internalization. This process was indicated by the enhanced intracellular fluorescence, generated from the internalized and mitochondria accumulated DNA probes. This study not only uncovered new cellular mechanotransduction mechanisms but also provided a versatile method that enabled in situ and dynamic indication of cell responses to mechanical stimuli.

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internalization from different aspects. However, they are generally confined to specific cell types and proteins, and dynamic monitoring of cell mechanical responses remains a vital issue. Recently, cell surface-anchored DNA sensors and hairpintype DNA probes have received much attention and have offered a robust way for real-time monitoring of dynamic cellular processes.17−19 On the basis of rational DNA design and cell surface engineering, real-time and in situ investigations of cellular microenvironments, 20,21 transmitter dynamics22 and transient membrane encounter events23 were achieved by fluorescence imaging. Here, we investigated the regulating effects of shear stress on the behavior of cell membrane proteins and the cell endocytosis process (Figure 1). We cultured cells in a microfluidic chip, where shear stress could be precisely modulated, and cell responses were monitored by fluorescence microscopy. A hairpin-type DNA probe was anchored to cell membrane proteins via a streptavidin−biotin linkage in an indiscriminate manner, acting as an indicator for the proteins. We found that, when the cells were exposed to shear stress generated by fluid cell medium containing external proteins, strong fluorescence was emanated from intracellular regions. This phenomenon was intensively investigated, and results revealed that shear stress

n living organisms, cells are located in complex microenvironments and surrounded by multiple cues that vary in time and space.1 Apart from physical and biochemical factors, cells are also subject to various mechanical stimuli. These mechanical processes are vital for cell growth, migration, differentiation, and apoptosis, and dysfunctional mechanotransduction can lead to numerous diseases.2−4 Among these mechanical cues, fluid shear stress (FSS) is one of the most important stimuli generated from blood flow or interstitial flow in cellular microenvironments. Cells sense shear stress via a series of receptors, which transmit mechanical signals through mechanosensitive pathways and lead to the regulation of cellular morphology, behavior, and functions.5,6 It has been reported that, under shear stress, endothelial cells were elongated and stress fibers were aligned with the direction of the flow.7 Shear stress can also regulate various cellular processes, including endothelial cell-dependent nitric oxide production, autophagy, cell adhesion, and microvilli formation.8−12 Recent research has illustrated that fluid shear stress can modulate the cell endocytosis process. Raghavan et al. reported that exposure of proximal tubule cells to physiologically relevant levels of fluid shear stress led to enhanced endocytosis of the megalin−cubilin receptor ligand albumin.13 Lawler et al. demonstrated venous shear induced internalization of E-cadherin in cells by a Src-dependent pathway.14 It was also reported that glycocalyx layers on the cell surface play an important role in the shear-dependent uptake of macromolecules into cells.15,16 These studies have indicated the important effects of shear stress on cell endocytosis and protein © 2018 American Chemical Society

Received: February 14, 2018 Accepted: April 2, 2018 Published: April 2, 2018 5540

DOI: 10.1021/acs.analchem.8b00755 Anal. Chem. 2018, 90, 5540−5545

Letter

Analytical Chemistry

of the stem, making them in proximity and leading to fluorescence quenching. The loop sequence included a short PEG spacer, which could reduce the influence of negative charges of DNA. Compared with simple fluorophore labeled DNA probe (Figure S1), the original quenching state of probes on the cell membrane conduced to the recognition of the emerging intracellular fluorescence and facilitated quantitative analysis. The 3′ end of the DNA probe was modified with a biotin molecule. A three step modification procedure was utilized to indiscriminately anchor the DNA probes to cell membrane proteins. Aminos on proteins were first biotinylated and then connected to biotin-labeled DNA probes via streptavidin linkages. Flow cytometry analysis illustrated effective cell membrane modification (Figure S2). The live/ dead assay indicated that this kind of modification had no impact on cell viability (Figure S3), and the influence of DNA labeling on the cell endocytosis process was also negligible, confirmed by the similar fluorescence intensity of labeled cells and control cells when incubated with BSA-FITC under both static and flow conditions (Figure 2b). A microfluidic-based platform was developed for cell culture, shear stress stimulation, and in situ fluorescence monitoring (Figure S4). Cells cultured in the microchannels were first labeled by the DNA probes and exposed to fluid flow condition. The flow rate was increased every 15 min, from 1 μL/min to 10 μL/min, and cellular fluorescence excited at 561 nm (excitation wavelength of Cy3) was recorded during this whole process. As shown in Figure 3, negligible fluorescence could be observed at the original state, owing to the quenched fluorescence of the cell surface-anchored DNA probes. After being exposed to fluid cell medium, red fluorescence started to appear in the cytoplasm, showing punctate distribution, and the intensity gradually increased with the increase of flow rate (Figure 3a,d, Movie S1). Furthermore, when 100 nM human serum albumin (HSA) was added to the cell medium, the shear stress-induced intracellular fluorescence was significantly enhanced, showing strong signals in filamentous distribution (Figure 3b,d, Movie S2). With the increase of flow rates, the fluorescence intensity was continuously enhanced and this kind of fluorescence could be maintained in cells for as long as 8 h (Figure S5). In contrast, under static conditions, the fluorescence remained on the cell surface and the intensity was nearly invariable (Figure 3c). These phenomena could occur in both hepatocytes (HepG2) and endothelial cells (HUVEC, Figure S6). To investigate this cellular mechanical response, we first identified the intracellular localization of the red fluorescence. After being exposed to shear stress generated by fluid cell medium containing external proteins, cells were stained with mitochondria-specific dye mitotracker and lysosome-specific dye lysotracker, respectively. Cellular fluorescence was recorded (Figure 4), and the colocalization correlation was analyzed based on the measurement of Pearson’s correlation coefficient (PCC) (Figure S7; detailed explanation was demonstrated in the Supporting Information). Results indicated that there were two different intracellular localizations of the red fluorescence. One was mitochondria, which showed filamentous distribution, and the other was lysosomes, displaying spot-like signals. For cells with well-spread morphology, which illustrated good cell activity, the red fluorescence was predominantly accumulated in mitochondria, while for cells with rounding up morphology and increased lysosome activity, which always occurred during cell apoptosis, a large portion of red fluorescence was in the lysosomes, owing to the degradation of DNA probes by

Figure 1. Schematic illustration of shear stress regulating the internalization of cell membrane proteins indicated by a hairpin-type DNA probe. (Solid arrows indicated the predominant pathways of DNA probes in cells.)

enhanced the specific cell endocytosis pathway and promoted membrane protein internalization. This process thereby led to the cellular entry, mitochondria accumulation, and fluorescence recovery of the DNA probes, inducing the strong fluorescence. This study not only uncovered new regulating effects of shear stress on cell endocytosis but also provided a versatile method that enabled one to dynamically indicate the extent of membrane protein internalization. The DNA probe was designed as a hairpin type consisting of a stem and a loop sequence (Figure 2a). A pair of fluorophore (Cy3) and quencher (BHQ-2) was attached at either terminus

Figure 2. DNA probe design and cell surface labeling. (a) The sequence and structure of the DNA probe. (b) The influence of DNA labeling on the cell endocytosis process. Labeled cells and control cells were incubated with BSA-FITC (1 μM) in either static or flow condition, and cellular fluorescence at 488 nm was recorded and plotted. 5541

DOI: 10.1021/acs.analchem.8b00755 Anal. Chem. 2018, 90, 5540−5545

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Analytical Chemistry

Figure 3. Real-time fluorescence monitoring of HepG2 cells exposed to fluid flow or static condition. (a) Fluorescence images of cells exposed to fluid cell medium at different flow rates. The flow rate was increased every 15 min, from 1 μL/min to 10 μL/min, and cellular fluorescence excited at 561 nm (red) was recorded. (b) Fluorescence images of cells exposed to fluid cell medium containing 100 nM HSA at different flow rates. (c) Fluorescence images of cells exposed to cell medium containing 100 nM HSA under static condition. (d) High magnified fluorescence images of cells exposed to flow or static condition. Hochest 33342 was used as the nuclear stain (blue color).

or the fluorophores dissociation when trapped in the intermembrane space of mitochondria. Red fluorescence signals were shown in mitochondria after long-time (>5 h) cell incubation with dissociative oligonucleotides.24,25 However, in our experiments, under the effect of shear stress, red fluorescence appeared in mitochondria in as little as 10 min. Because of the strong linkage between the cell membrane proteins and DNA probes, it could be deduced that shear stress enhanced cellular internalization of membrane proteins, thereby leading to the cellular entry of the membrane protein-bound DNA probes. The hairpin-type DNA probes were then accumulated in mitochondria driven by the interactions between Cy3 fluorophore and mitochondrial membrane potential, and subsequent fluorescence recovery induced the strong intracellular red fluorescence. As mentioned above, shear stress and external proteins were both essential for promoting membrane protein internalization. To evaluate the relationship between shear stress and cellular responses, cell medium containing external proteins (100 nM insulin) was injected into the microchannel at different flow rates, and the alterations of average cell fluorescence with time was recorded and analyzed. As shown in Figure 5a, at the flow rate of 1 μL/min (FSS: 0.078 dyn/cm2), the cell fluorescence slightly decreased along with the time. When the flow rate was 2 μL/min, the fluorescence first increased and then gradually decreased. However, when the flow rate reached 5 μL/min, the intracellular fluorescence intensity continually increased throughout time. These results illustrated the important role of shear stress, which enhanced membrane protein internalization until it exceeded a certain threshold. To elucidate the roles of external proteins, we profiled cellular responses to a series of proteins under shear stress. As shown in Figure 5b, except goat antimouse IgG, the other three proteins (BSA, IL-8, TGF-β) all led to the enhanced

Figure 4. Co-localization of DNA probes (red) with mitochondria or lysosomes. Fluorescence images of cells stained with Mitotracker (top, green) or Lysotracker (bottom, green) after being exposed to shear stress.

endonucleases. Since most cells had good activity and showed well-spread morphology during the shear stress stimulation, the mitochondria were the major location of the red fluorescence. Previous research reported that cyanine-dye (Cy3) labeled oligonucleotides could nonspecifically accumulate in mitochondria, driven by the interaction of positively charged cyanine and negative mitochondrial membrane potential. For hairpin-type molecule beacons, both mitochondria accumulation and fluorescence recovery were observed when they were internalized in cells. The fluorescence recovery might be due to alteration of the DNA conformation during the interaction 5542

DOI: 10.1021/acs.analchem.8b00755 Anal. Chem. 2018, 90, 5540−5545

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Analytical Chemistry

Figure 5. Profiling cellular responses to different shear stress or proteins and endocytosis inhibitor treatments for mechanism investigation. (a) Fluorescence responses of HepG2 cells exposed to different flow rates. (b) Profiling cellular responses to different proteins under flow condition (BSA: bovine serum albumin; IL-8: interleukin-8; IgG: goat antimouse IgG; TGF-β: transforming growth factor-β). (c) Fluorescence imaging of cells exposed to fluid cell medium containing 100 nM transferrin (top) or 100 nM insulin (bottom). (d) Fluorescence responses of HepG2 cells pretreated with caveolin-dependent endocytosis inhibitors genistein and methyl-β-cyclodextrin. (Flow rate was set as 5 μL/min.) (e) Cellular fluorescence responses of cells pretreated with H+-ATPase inhibitor bafilomycin A1. (Flow rate was set as 5 μL/min.)

intracellular fluorescence in the flow condition. The inaction of IgG might be due to the lack of targeting epitopes on the cell surface. Previous research demonstrated that shear stress increased protein endocytosis in cells,13 and similar phenomena could also be observed in our former experiment (Figure 2b). Therefore, we deduced that cells endocytosed external proteins which had corresponding receptors on the cell membrane, and fluid shear stress could remarkably enhance this endocytosis process. Proteins were taken in by forming vesicles from plasma membrane, leading to the internalization of cell membrane

proteins, together with the anchored DNA probes. To investigate whether shear stress promoted the specific endocytosis pathway, we monitored the cell responses to two kinds of proteins under shear stress: insulin and transferrin, which were taken by cells via caveolin-dependent endocytosis and clathrin-dependent endocytosis, respectively.26,27 As shown in Figure 5c, under the same condition, insulin led to obvious fluorescence recovery in cells, while transferrin resulted in attenuated fluorescence responses. This phenomenon indicated that shear stress might mainly enhance the caveolin-dependent 5543

DOI: 10.1021/acs.analchem.8b00755 Anal. Chem. 2018, 90, 5540−5545

Letter

Analytical Chemistry endocytosis pathway, while the clathrin-dependent endocytosis was less affected. Recent studies have elucidated that caveolae not only was important for cell endocytosis but also was a crucial mechanotransducer in cells.28 Chronic exposure of cells to laminar shear increased the total number of caveolae by 45− 48% above static control.29 Hence, the mechanical sensitivity of caveolae might result in the influence of caveolin-dependent endocytosis by shear stress. To further confirm the mechanism, cells were pretreated with caveolin-dependent endocytosis inhibitors (genistein and methyl-β-cyclodextrin) and a clathrin-dependent endocytosis inhibitor (chlorpromazine hydrochloride), and their fluorescence responses to shear stress were monitored. As shown in Figure 5d, both caveolin-dependent endocytosis inhibitors suppressed the intracellular fluorescence responses, revealing the important role of caveolin-dependent endocytosis in cellular mechanotransduction under shear stress. However, when cells were treated with chlorpromazine, the fluorescence response was instead enhanced (Figure S8). This might be due to the inhibition mechanism of chlorpromazine, which accelerated the transfer of clathrin protein and its helper protein AP2 to endosomes to inhibit clathrin-dependent endocytosis, and this process would lead to the internalization of indiscriminately labeled DNA probes, thus intensifying the fluorescence. We also treated cells with H+-ATPase inhibitor bafilomycin A1,30 and attenuated fluorescence recovery was observed (Figure 5e). This result indicated that the stability of acidic environments in intracellular vesicles was also essential for fluorescence recovery. Therefore, we concluded that fluid shear stress could enhance the caveolin-dependent cell endocytosis, in which external proteins were taken up by the cells, along with the internalization of cell membrane proteins. This process was dynamically indicated by the membrane-anchored hairpin-type DNA probes, that entered cytoplasm with the proteins, accumulated in mitochondria, and led to enhanced intracellular fluorescence. The cellular responses were highly relevant to the magnitude of shear stress, and this phenomenon could occur in various cell types. This research allowed intuitionistic investigation of the cell endocytosis process under mechanical stimuli and was quite suitable to profile responses of different cell types to different proteins. In summary, this study investigated the regulating effects of fluid shear stress on membrane protein internalization and the cell endocytosis process. Combining cell surface-anchored DNA probe, microfluidic chip, and fluorescence imaging, in situ and dynamic monitoring of cellular mechanical responses could be achieved. This work not only uncovered new cellular mechanotransduction mechanisms but also provided a versatile approach for dynamic cell monitoring, which was feasible to extend to diverse cell mechanic and endocytosis related research. For further investigation, the DNA probe as well as the labeling process should be optimized to reduce the impact on intrinsic cellular processes and improve the indicated performance.

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Experimental methods; responses of the cells anchored with simple fluorophore labeled DNA probes; cell surface modification procedure and its evaluation by flow cytometry; influence of DNA labeling on cell viability and endocytosis process; photos of microfluidic platform; fluorescence alterations of DNA probes after internalized in cells; fluorescence monitoring of HUVEC cells under flow condition; explanation for the DNA probe colocalization analysis; response of cells pretreated with clathrin-dependent endocytosis inhibitor (PDF) Real-time fluorescence monitoring of HepG2 cells exposed to fluid cell medium at different flow rates (AVI) Real-time fluorescence monitoring of HepG2 cells exposed to fluid cell medium containing 100 nM HSA at different flow rates (AVI)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ziyi He: 0000-0002-9817-4791 Jin-Ming Lin: 0000-0001-8891-0655 Author Contributions †

Z.H. and W.Z. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 21435002, 21621003, 21727814, and 21775086).

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REFERENCES

(1) Kise, K.; Kinugasa-Katayama, Y.; Takakura, N. Adv. Drug Delivery Rev. 2016, 99, 197. (2) Isermann, P.; Lammerding, J. Curr. Biol. 2013, 23, R1113. (3) Humphrey, J. D.; Dufresne, E. R.; Schwartz, M. A. Nat. Rev. Mol. Cell Biol. 2014, 15, 802. (4) Ehrlicher, A. J.; Nakamura, F.; Hartwig, J. H.; Weitz, D. A.; Stossel, T. P. Nature 2011, 478, 260. (5) Tzima, E.; Irani-Tehrani, M.; Kiosses, W. B.; Dejana, E.; Schultz, D. A.; Engelhardt, B.; Cao, G.; DeLisser, H.; Schwartz, M. A. Nature 2005, 437, 426. (6) White, C. R.; Frangos, J. A. Philos. Trans. R. Soc., B 2007, 362, 1459. (7) Potter, C. M.; Lundberg, M. H.; Harrington, L. S.; Warboys, C. M.; Warner, T. D.; Berson, R. E.; Moshkov, A. V.; Gorelik, J.; Weinberg, P. D.; Mitchell, J. A. Arterioscler., Thromb., Vasc. Biol. 2011, 31, 384. (8) Castro-Nunez, L.; Dienava-Verdoold, I.; Herczenik, E.; Mertens, K.; Meijer, A. B. J. J. Thromb. Haemostasis 2012, 10, 1929. (9) Teichmann, J.; Morgenstern, A.; Seebach, J.; Schnittler, H. J.; Werner, C.; Pompe, T. Biomaterials 2012, 33, 1959. (10) Liu, J.; Bi, X.; Chen, T.; Zhang, Q.; Wang, S. X.; Chiu, J. J.; Liu, G. S.; Zhang, Y.; Bu, P.; Jiang, F. Cell Death Dis. 2015, 6, e1827. (11) Miura, S.; Sato, K.; Kato-Negishi, M.; Teshima, T.; Takeuchi, S. Nat. Commun. 2015, 6, 8871. (12) Chistiakov, D. A.; Orekhov, A. N.; Bobryshev, Y. V. Acta Physiol. 2017, 219, 382. (13) Raghavan, V.; Rbaibi, Y.; Pastor-Soler, N. M.; Carattino, M. D.; Weisz, O. A. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 8506. (14) Lawler, K.; O’Sullivan, G.; Long, A.; Kenny, D. Cancer Sci. 2009, 100, 1082.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b00755. 5544

DOI: 10.1021/acs.analchem.8b00755 Anal. Chem. 2018, 90, 5540−5545

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Analytical Chemistry (15) Ueda, A.; Shimomura, M.; Ikeda, M.; Yamaguchi, R.; Tanishita, K. Am. J. Physiol Heart Circ Physiol 2004, 287, H2287. (16) Barkefors, I.; Aidun, C. K.; Ulrika Egertsdotter, E. M. J. Biomed. Biotechnol. 2007, 2007, 65136. (17) Pan, W.; Zhang, T. T.; Yang, H. J.; Diao, W.; Li, N.; Tang, B. Anal. Chem. 2013, 85, 10581. (18) Li, N.; Yang, H. J.; Pan, W.; Diao, W.; Tang, B. Chem. Commun. 2014, 50, 7473. (19) Luan, M. M.; Li, N.; Pan, W.; Yang, L. M.; Yu, Z. Z.; Tang, B. Chem. Commun. 2017, 53, 356. (20) Zhao, W.; Schafer, S.; Choi, J.; Yamanaka, Y. J.; Lombardi, M. L.; Bose, S.; Carlson, A. L.; Phillips, J. A.; Teo, W.; Droujinine, I. A.; Cui, C. H.; Jain, R. K.; Lammerding, J.; Love, J. C.; Lin, C. P.; Sarkar, D.; Karnik, R.; Karp, J. M. Nat. Nanotechnol. 2011, 6, 524. (21) Qiu, L.; Zhang, T.; Jiang, J.; Wu, C.; Zhu, G.; You, M.; Chen, X.; Zhang, L.; Cui, C.; Yu, R.; Tan, W. J. Am. Chem. Soc. 2014, 136, 13090. (22) Tokunaga, T.; Namiki, S.; Yamada, K.; Imaishi, T.; Nonaka, H.; Hirose, K.; Sando, S. J. Am. Chem. Soc. 2012, 134, 9561. (23) You, M.; Lyu, Y.; Han, D.; Qiu, L.; Liu, Q.; Chen, T.; Sam Wu, C.; Peng, L.; Zhang, L.; Bao, G.; Tan, W. Nat. Nanotechnol. 2017, 12, 453. (24) Rhee, W. J.; Bao, G. Nucleic Acids Res. 2010, 38, e109. (25) Mailänder, V.; Tomcin, S.; Baier, G.; Landfester, K. Int. J. Nanomed. 2014, 5471. (26) McMahon, H. T.; Boucrot, E. Nat. Rev. Mol. Cell Biol. 2011, 12, 517. (27) Stralfors, P. Adv. Exp. Med. Biol. 2012, 729, 111. (28) Nassoy, P.; Lamaze, C. Trends Cell Biol. 2012, 22, 381. (29) Boyd, N. L.; Park, H.; Yi, H.; Boo, Y. C.; Sorescu, G. P.; Sykes, M.; Jo, H. Am. J. Physiol Heart Circ Physiol 2003, 285, H1113. (30) Lafourcade, C.; Sobo, K.; Kieffer-Jaquinod, S.; Garin, J.; van der Goot, F. G. PLoS One 2008, 3, e2758.

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DOI: 10.1021/acs.analchem.8b00755 Anal. Chem. 2018, 90, 5540−5545