Shear stress-enhanced internalization of cell membrane proteins

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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 Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00755 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 2, 2018

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Analytical Chemistry

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 † These authors contributed equally 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 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 specific cell endocytosis pathway, 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 indicating of cell responses to mechanical stimuli.

In 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, 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 stimulus 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 researches have illustrated that fluid shear stress can modulate 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 the internalization of E-cadherin in cells, by a Src-dependent pathway.14 It was also reported that glycocalyx layers on cell surface play an important role in the shear-dependent uptake of macromolecules into cells.15,16 These studies have indicated the im-

portant effects of shear stress on cell endocytosis and protein 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 offered a robust way for real-time monitoring of dynamic cellular processes.17-19 Base on rational DNA design and cell surface engineering, real-time and in-situ investigations of cellular microenvironments,20,21 transmitter dynamics,22 as well as 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 cell endocytosis process (Figure 1). We cultured cells in a microfluidic chip, where shear stress could be precisely modulated, and cell responses were

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).

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monitored by fluorescence microscopy. A hairpin-type DNA probe was anchored to cell membrane proteins via a streptavidinbiotin 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 enhanced specific cell endocytosis pathway and promoted membrane protein internalization. This process thereby led to the cellular entering, 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 dynamically indicating 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) were attached at either terminus 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 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 steps 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). Live/dead assay indicated that this kind of modification had no impact on cell viability (Figure S3). And the influence of DNA labeling on cell

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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 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.

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 surfaceanchored DNA probes. After exposed to fluid cell medium, red

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).


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Analytical Chemistry mitochondria after long-time (>5 h) cell incubation with dissociative oligo-

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

fluorescence started to appear in the cytoplasm, showing punctate distribution, and the intensity gradually increased with the increase of flow rate (Figure 3a, 3d, 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, 3d, Movie S2). With the increase of flow rates, the fluorescence intensity was continuously enhanced and this kind of fluorescence could maintain in cells for as long as 8 h (Figure S5). In contrast, under static conditions, the fluorescence was remained on 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 exposed to shear stress generated by fluid cell medium containing external proteins, cells were stained with mitochondriaspecific 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 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-spreading 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 endonucleases. Since most cells were in good activity and showed well-spreading morphology during the shear stress stimulation, the mitochondria were the major location of the red fluorescence. Previous researches reported that cyanine-dye (Cy3) labeled oligonucleotides could non-specifically accumulate in mitochondria, driven by the interaction of positivecharged cyanine and negative mitochondrial membrane potential. And 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, or the fluorophores dissociation when trapped in the intermembrane space of mitochondria. Red fluorescence signals were shown in

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 anti-mouse IgG, TGF-β: transforming growth factor-β); (c) Fluorescence imaging of cells exposed to fluid cell medium containing 100 nM transferrin (up) or 100 nM insulin (below). (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).

nucleotides.24,25 However, in our experiments, under the effect of shear stress, red fluorescence appeared in mitochondria as short 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 entering 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 analysed. 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 to 5 µL/min, the intracellular fluorescence intensity continually increased throughout the 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 anti-mouse IgG, other three proteins (BSA, IL-8, TGF-β) all led to the enhanced intracellular fluorescence in flow condition. The inaction of IgG might be due to the lack of targeting epitopes on cell surface. Previous researches 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 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 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 enhanced the caveolin-dependent endocytosis pathway, while the clathrin-dependent endocytosis was less affected. Recent studies have elucidated that caveolae was not only important for cell endocytosis, but also 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 methylβ-cyclodextrin) and 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 caveolindependent 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 were 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 by cells, along with the internalization of cell membrane proteins. This process was dynamically indicated by the membrane-anchored hairpin-type DNA probes, who 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

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phenomenon could occur in various cell types. This research allowed intuitionistic investigation of 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 cell endocytosis process. Combined 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 researches. For further investigation, the DNA probe as well as the labeling process would be optimized, to reduce the impact on intrinsic cellular processes and improve the indicating performance.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental methods; Figures S1 to S8 (PDF); Movie S1, S2

AUTHOR INFORMATION Corresponding Author [email protected].

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21435002, 21621003, 21727814, 21775086).

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(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. International Journal of Nanomedicine 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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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) 185x225mm (300 x 300 DPI)



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 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. 77x44mm (300 x 300 DPI)


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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). 269x166mm (300 x 300 DPI)



Figure 4. Co-localization of DNA probes (red) with mitochondria or lysosomes. Fluorescence images of cells stained with Mitotracker (up, green) or Lysotracker (below, green) after exposed to shear stress. 205x157mm (300 x 300 DPI)


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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 anti-mouse IgG, TGF-β: transforming growth factor-β); (c) Fluorescence imaging of cells exposed to fluid cell medium containing 100 nM transferrin (up) or 100 nM insulin (below). (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). 272x404mm (300 x 300 DPI)



The regulating effects of shear stress on cell membrane proteins were real-timely monitored based on the cell surface-anchored DNA probes. 277x147mm (300 x 300 DPI)


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Shear stress-enhanced internalization of cell membrane proteins

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