Nanoscale Imaging of an Unlabeled Secretory Protein in Living Cells

Feb 3, 2015 - low as 60 nm without any artifact. By acquiring the sequential SICM images of living cells, the protrusion and strings formation were ob...
0 downloads 0 Views 3MB Size
Download PDF
Letter pubs.acs.org/ac

Nanoscale Imaging of an Unlabeled Secretory Protein in Living Cells Using Scanning Ion Conductance Microscopy Yuji Nashimoto,† Yasufumi Takahashi,*,†,‡,§ Hiroki Ida,† Yoshiharu Matsumae,† Kosuke Ino,† Hitoshi Shiku,*,† and Tomokazu Matsue*,†,‡ †

Graduate School of Environmental Studies, Tohoku University, Sendai, Miyagi 980-8576, Japan WPI-Advanced Institute for Materials Research, Tohoku University, Sendai, Miyagi 980-8576, Japan § PRESTO, JST, Kawaguchi, Saitama 332-0012, Japan ‡

S Supporting Information *

ABSTRACT: Scanning ion conductance microscopy (SICM) was applied to evaluate an unlabeled secretory protein in living cells. The target protein, von Willebrand factor (vWF), was released from human endothelial cells by adding phorbol-12-myristate-13-acetate (PMA). We confirmed that SICM could be used to clearly visualize the complex network of vWF and to detect strings with widths as low as 60 nm without any artifact. By acquiring the sequential SICM images of living cells, the protrusion and strings formation were observed. We also detected the opening and closing motions of a small pore (∼500 nm), which is difficult to visualize with fluorescence methods. The results clearly demonstrate that SICM is a powerful tool to examine the changes in living cells during exocytosis.

T

feedback control of the pipet−sample distance. Because the feedback signal is not a mechanical force, SICM is particularly useful for soft and responsive cell surfaces.15,16 Korchev and coworkers improved SICM instrumentation17−19 and elucidated the mechanism of nanostructure formations on cellular membrane.17,20−24 Our group has reported an SICM-based cellular migration assay.25 In this study, we applied SICM to evaluate the vWF exocytosis in living endothelial cells without labeling. We were able to image the vWF and the pore during exocytosis beyond the diffraction limit. The present study clearly demonstrates that SICM is a powerful tool for the study of the dynamics of exocytosis.

he von Willebrand factor (vWF) is an adhesive multimeric glycoprotein that is mainly stored in the Weibel-Palade bodies (WPBs)1 of endothelial cells. When appropriately stimulated, the vWF is secreted into blood via the exocytosis of the WPBs. Fluorescence imaging techniques have been instrumental in advancing the knowledge of the dynamics of WPB exocytosis. Recently, studies using genetically labeled or immunostained vWF have elucidated the mechanism of WPB exocytosis and the extracellular behavior of the vWF.2−7 However, the interaction of fluorescent proteins with pores (WPBs) during exocytosis and the alteration of the behavior of the vWF by attaching antibodies were reported.4,5 Thus, nonlabeling methods for the evaluation of the vWF dynamics are needed to obtain complementary results for comparison. Atomic force microscopy (AFM) is a powerful tool for obtaining nanometer-scale structural changes in proteins without any labeling. AFM has revealed conformational changes in vWF under high shear stress8−10 and protein− protein interaction with coagulation factors.11 This knowledge has contributed to an understanding of the hemostasis maintained by the vWF. However, little is known about the dynamics of the vWF following its exocytosis through a living cell surface. The behavior of the vWF on the cellular membrane is important because a portion of the vWF remains anchored on the cell membrane and forms very long strings with high coagulability.12,13 Scanning ion conductance microscopy (SICM) can be also used to acquire the nanoscale topography of the sample, without labeling.14 SICM uses the ion current between an electrode in the nanopipette probe and a bath electrode for © XXXX American Chemical Society

■

MATERIALS AND METHODS

Induction of vWF Exocytosis. Human umbilical vein endothelial cells (HUVECs, Lonza) were grown as previously described.26 Cells were cultured on plastic dishes in EGM-2 (Lonza). To induce vWF exocytosis, cells were preincubated in basal medium (EBM-2) (Lonza) for 30 min at 37 °C. Thereafter, they were stimulated with 40 nM phorbol-12myristate-13-acetate (PMA) (Wako) in EBM-2 for 20−30 min. PMA stimulates the influx of Ca2+ from the medium and elevates intracellular Ca2+ levels which induce the transportation of WPB containing vWF to extracellular space.5,27,28 Received: December 14, 2014 Accepted: January 31, 2015

A

DOI: 10.1021/ac5046388 Anal. Chem. XXXX, XXX, XXX−XXX

Letter

Analytical Chemistry

sample; thus, the vertical dimension of the sample can be measured without direct contact.24 The secreted vWF was observed in hopping mode in which the probe is moved vertically to first approach and then be retracted from the sample surface at each measurement point (Figure 1). Hopping mode is useful for capturing the complex surfaces of live cells.18,32 In this study, the width of the structure was given by the distance between points on the cross-sectional curve at which the value reached 10% of its maximum height from the baseline.

■

RESULTS AND DISCUSSION Imaging of vWF in Fixed Cells. First, we tested whether SICM could detect the fluorescently labeled vWF. The vWF was immunostained by generally used methods (Supporting Information). Figure 2 shows the optical, fluorescent, and SICM topographical images of the vWF on fixed HUVECs (100 × 100 μm). SICM successfully detected the vWFs that were seen at the same position of the fluorescence image (Figure 2i). At a smaller area (Figure 2ii, 25 × 25 μm; Figure 2iii, 2 × 2 μm), the complex network of the vWF could be visualized clearly. vWF strings as small as 60 nm in width could be detected without any artifact (Figure 2iv). Our result indicated that SICM allowed one to detect vWF structures by overcoming the diffraction limit (∼200 nm) in physiological conditions. Imaging of vWF in Living Cells. We acquired sequential SICM images of living HUVECs (Figure 3). All SICM measurements for live cells were performed without labeling. From 6 to 14 min, secretion materials with protrusion shapes were observed (Figure 3i). The protrusions became larger and reached their maximum height at 14 min with an average height

Figure 1. Schematic view of the vWF observed by SICM.

SICM Imaging. The basic arrangement of the SICM was described in previous papers25,29−31 and the Supporting Information. SICM nanopipettes were fabricated by pulling borosilicate glass pipettes (GC150F-10, Harvard Apparatus) using a CO2 laser puller (P-2000, Sutter Instruments). The tip diameter of the nanopipette was 20−100 nm. In our experiment, the bias potential of +0.2 V was applied between a working Ag/AgCl electrode located inside the nanopipette and a reference Ag/AgCl electrode immersed in the bath. The bias potential generates the ion flow across the nanopipette. When positive potential is applied inside the nanopipette, Cl− and other anions are transferred from the outside solution to the inside of the nanopipette, and cations (Na+, K+) are transferred from the nanopipette inside to the outside solution, through the aperture of the nanopipette. To maintain electroneutrality, chloride ion reacts at the Ag/AgCl electrode inside the nanopipette. Ion current drops if the pipet approaches the surface of the sample. This reduction of the current can be detected when the pipet is located near the

Figure 2. Imaging of vWF in fixed cells. (i) Optical, fluorescent, and SICM images of HUVECs fixed after PMA stimulation. HUVECs were immunostained with anti-vWF antibodies. Image size = 100 μm × 100 μm. HUVECs were fixed by 4% paraformaldehyde. (ii) Zoomed image shown in (i) as the red square. (iii) Zoomed image shown in (ii) as the red square. (iv) Cross-section of topography of the image shown in (iii) as the red line. B

DOI: 10.1021/ac5046388 Anal. Chem. XXXX, XXX, XXX−XXX

Letter

Analytical Chemistry

Figure 3. Imaging of the vWF in living cells. Sequential images of living HUVECs after PMA stimulation. Arrow head indicated the protrusion. (i) Protrusion formation and (ii) the time course of the cross-sectional view of the white line in the right image represented from (i) at 14 min. (iii) The extension of the protrusion. (iv) The opening and closing motions of the small pore (arrow).

of 252.0 ± 125.2 nm (n = 17) and then gradually disappeared until 30 min (Figure 3ii, Figure S1, Supporting Information). We performed immunostaining on the protrusions. As a result, the same secretion materials as those revealed by SICM could not be observed after immunostaining. This was because of the fast dispersal and structural changes associated with the fixation. However, we concluded that the observed protrusions were from the vWF because no surface change was observed in HUVECs without PMA stimulation (Figure S2, Supporting Information). The timescale of the disappearance of the protrusions by diffusion was consistent with the previously reported dispersal dynamics of the vWF.2,3 Interestingly, we were not able to detect the nondiffusional large protrusions observed in the fixed cells. Thus, it was likely that the fixation process promoted the release of vWF for the protrusion size to grow. Strings formation on the cellular surface was also observed (Figure 3iii). In SICM images of 6−14 min, the string gradually became clear. At 14 min, the string that had a maximum height of 382 nm and length of 7.00 μm could be observed. Previously, the strings formation induced by shear stress was fluorescently observed by very diluted vWF antibodies.7 Our results were slightly different from these previous results. This might be because SICM imaging was performed in static conditions. We also detected the opening and closing motions of pores at 18 min (Figure 3iv, arrow). The pore diameters were ∼500 nm, which corresponded to the previous report in which fixed HUVECs were observed by AFM.33 Until now, three modes of exocytosis of WPBs, namely “conventional”, “lingering-kiss”, and “multigranular”, have been reported.6 The pore size we observed was consistent with that observed in multigranular exocytosis and exhibited the biggest pore size (approximately

300−1000 nm in diameter) among the three modes. Our results indicated the possibility that the pore remained in the opening state at the cellular membrane during several minutes after multigranular exocytosis.

■

CONCLUSION

■

ASSOCIATED CONTENT

In this study, vWF exocytosis was successfully evaluated in living cells using SICM without labeling. The timescale of the vWF dynamics was almost equal to that observed by fluorescence techniques. This indicated that the fluorescent labeling of the vWF brought only a small effect in its dynamics. SICM could also detect vWF strings and pore formation, which is difficult to observe using fluorescence methods. In addition, SICM successfully visualized the vWF strings as small as 60 nm. Furthermore, SICM maintained enough resolution to visualize the dynamic cell membrane surface topography change of the pore during exocytosis (∼500 nm) in time-lapse imaging. The present study clearly demonstrates that SICM is a powerful tool for the study of the dynamics of exocytosis in living cells. S Supporting Information *

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

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel/Fax: +81-22-7957281. *E-mail: [email protected] *E-mail: [email protected]. C

DOI: 10.1021/ac5046388 Anal. Chem. XXXX, XXX, XXX−XXX

Letter

Analytical Chemistry Author Contributions

(20) Gorelik, J.; Shevchuk, A. I.; Frolenkov, G. I.; Diakonov, I. A.; Lab, M. J.; Kros, C. J.; Richardson, G. P.; Vodyanoy, I.; Edwards, C. R.; Klenerman, D.; Korchev, Y. E. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 5819−5822. (21) Zhang, Y. J.; Gorelik, J.; Sanchez, D.; Shevchuk, A.; Lab, M.; Vodyanoy, I.; Klenerman, D.; Edwards, C.; Korchev, Y. Kidney Int. 2005, 68, 1071−1077. (22) Shin, W.; Gillis, K. D. Biophys. J. 2006, 91, L63−L65. (23) Shevchuk, A. I.; Hobson, P.; Lab, M. J.; Klenerman, D.; Krauzewicz, N.; Korchev, Y. E. Pfluegers Arch. 2008, 456, 227−235. (24) Shevchuk, A. I.; Novak, P.; Taylor, M.; Diakonov, I. A.; ZiyadehIsleem, A.; Bitoun, M.; Guicheney, P.; Lab, M. J.; Gorelik, J.; Merrifield, C. J.; Klenerman, D.; Korchev, Y. E. J. Cell Biol. 2012, 197, 499−508. (25) Takahashi, Y.; Ito, K.; Wang, X. W.; Matsumae, Y.; Komaki, H.; Kumatani, A.; Ino, K.; Shiku, H.; Matsue, T. Electrochemistry 2014, 82, 331−334. (26) Nashimoto, Y.; Takahashi, Y.; Takano, R.; Miyashita, K.; Yamada, S.; Ino, K.; Shiku, H.; Matsue, T. Anal. Bioanal. Chem. 2014, 406, 275−282. (27) Loesberg, C.; Gonsalves, M. D.; Zandbergen, J.; Willems, C.; Vanaken, W. G.; Stel, H. V.; Vanmourik, J. A.; Degroot, P. G. Biochim. Biophys. Acta 1983, 763, 160−168. (28) Rondaij, M. G.; Bierings, R.; Kragt, A.; van Mourik, J. A.; Voorberg, J. Arterioscler., Thromb., Vasc. Biol. 2006, 26, 1002−1007. (29) Takahashi, Y.; Shevchuk, A. I.; Novak, P.; Murakami, Y.; Shiku, H.; Korchev, Y. E.; Matsue, T. J. Am. Chem. Soc. 2010, 132, 10118− 10126. (30) Takahashi, Y.; Shevchuk, A. I.; Novak, P.; Zhang, Y.; Ebejer, N.; Macpherson, J. V.; Unwin, P. R.; Pollard, A. J.; Roy, D.; Clifford, C. A.; Shiku, H.; Matsue, T.; Klenerman, D.; Korchev, Y. E. Angew. Chem., Int. Ed. 2011, 50, 9638−9642. (31) Takahashi, Y.; Shevchuk, A. I.; Novak, P.; Babakinejad, B.; Macpherson, J.; Unwin, P. R.; Shiku, H.; Gorelik, J.; Klenerman, D.; Korchev, Y. E.; Matsue, T. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 11540−11545. (32) Takahashi, Y.; Murakami, Y.; Nagamine, K.; Shiku, H.; Aoyagi, S.; Yasukawa, T.; Kanzaki, M.; Matsue, T. Phys. Chem. Chem. Phys. 2010, 12, 10012−10017. (33) Goerge, T.; Niemeyer, A.; Rogge, P.; Ossig, R.; Oberleithner, H.; Schneider, S. W. J. Membr. Biol. 2002, 187, 203−211.

Y.N., Y.T., H.I., H.S., and T.M. planned and performed experiments and analyzed data; Y.N., Y.T., Y.M., K.I., H.S., and T.M wrote the manuscript; Y.N., Y.T., H.S., and T.M conceived the project. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The fluorescence microscope was kindly lent by Ali Khademhosseini’s lab (WPI-Advanced Institute for Materials Research, Tohoku University, Japan). This work was supported by the Cabinet Office, Government of Japan, through its “Funding Program for Next Generation World-Leading Researchers” (to H.S.), Technology for Advanced Measurement and Analysis from the Japan Science and Technology Agency (JST). Y.N. acknowledges the support received from Research Fellow of Japan Society for the Promotion of Science. Y.T. was supported by PREST from JST.

■

REFERENCES

(1) Weibel, E. R.; Palade, G. E. J. Cell Biol. 1964, 23, 101−112. (2) Romani de Wit, T.; Rondaij, M. G.; Hordijk, P. L.; Voorberg, J.; van Mourik, J. A. Arterioscler., Thromb., Vasc. Biol. 2003, 23, 755−761. (3) Hannah, M. J.; Skehel, P.; Erent, M.; Knipe, L.; Ogden, D.; Carter, T. J. Biol. Chem. 2005, 280, 22827−22830. (4) Babich, V.; Meli, A.; Knipe, L.; Dempster, J. E.; Skehel, P.; Hannah, M. J.; Carter, T. Blood 2008, 111, 5282−5290. (5) Valentijn, K. M.; van Driel, L. F.; Mourik, M. J.; Hendriks, G. J.; Arends, T. J.; Koster, A. J.; Valentijn, J. A. Blood 2010, 116, 1807− 1816. (6) Valentijn, K. M.; Sadler, J. E.; Valentijn, J. A.; Voorberg, J.; Eikenboom, J. Blood 2011, 117, 5033−5043. (7) Mourik, M. J.; Valentijn, J. A.; Voorberg, J.; Koster, A. J.; Valentijn, K. M.; Eikenboom, J. J. Thromb. Haemostasis 2013, 11, 2009−2019. (8) Siedlecki, C. A.; Lestini, B. J.; Kottke-Marchant, K.; Eppell, S. J.; Wilson, D. L.; Marchant, R. E. Blood 1996, 88, 2939−2950. (9) Novak, L.; Deckmyn, H.; Damjanovich, S.; Harsfalvi, J. Blood 2002, 99, 2070−2076. (10) Steppich, D. M.; Angerer, J. I.; Opfer, J.; Sritharan, K.; Schneider, S. W.; Thalhammer, S.; Wixforth, A.; Alexander-Katz, A.; Schneider, M. F. Biochem. Biophys. Res. Commun. 2008, 369, 507−512. (11) Bonazza, K.; Rottensteiner, H.; Seyfried, B. K.; Schrenk, G.; Allmaier, G.; Turecek, P. L.; Friedbacher, G. Anal. Bioanal. Chem. 2014, 406, 1411−1421. (12) Dong, J. F.; Moake, J. L.; Nolasco, L.; Bernardo, A.; Arceneaux, W.; Shrimpton, C. N.; Schade, A. J.; McIntire, L. V.; Fujikawa, K.; Lopez, J. A. Blood 2002, 100, 4033−4039. (13) De Ceunynck, K.; De Meyer, S. F.; Vanhoorelbeke, K. Blood 2013, 121, 270−277. (14) Hansma, P. K.; Drake, B.; Marti, O.; Gould, S. A. C.; Prater, C. B. Science 1989, 243, 641−643. (15) Korchev, Y. E.; Bashford, C. L.; Milovanovic, M.; Vodyanoy, I.; Lab, M. J. Biophys. J. 1997, 73, 653−658. (16) Rheinlaender, J.; Geisse, N. A.; Proksch, R.; Schaffer, T. E. Langmuir 2011, 27, 697−704. (17) Shevchuk, A. I.; Frolenkov, G. I.; Sanchez, D.; James, P. S.; Freedman, N.; Lab, M. J.; Jones, R.; Klenerman, D.; Korchev, Y. E. Angew. Chem., Int. Ed. 2006, 45, 2212−2216. (18) Novak, P.; Li, C.; Shevchuk, A. I.; Stepanyan, R.; Caldwell, M.; Hughes, S.; Smart, T. G.; Gorelik, J.; Ostanin, V. P.; Lab, M. J.; Moss, G. W.; Frolenkov, G. I.; Klenerman, D.; Korchev, Y. E. Nat. Methods 2009, 6, 279−281. (19) Novak, P.; Shevchuk, A.; Ruenraroengsak, P.; Miragoli, M.; Thorley, A. J.; Klenerman, D.; Lab, M. J.; Tetley, T. D.; Gorelik, J.; Korchev, Y. E. Nano Lett. 2014, 14, 1202−1207. D

DOI: 10.1021/ac5046388 Anal. Chem. XXXX, XXX, XXX−XXX