Fluorescence Approaches to Image and Quantify the Demarcation Membrane System in Living Megakaryocytes
The demarcation membrane system (DMS) develops to provide additional surface membrane for the process of platelet production. The DMS is an invagination of the plasma membrane that can extend throughout the extranuclear volume of mature megakaryocytes and its lumen is continuous with the extracellular solution. DMS ultrastructure in fixed samples has been extensively studied using transmission electron microscopy (TEM) and more recently with focused ion beam scanning EM. In addition, whole cell patch clamp membrane capacitance provides a direct measurement of DMS content in living megakaryocytes. However, fluorescence methods to image and quantify the DMS in living megakaryocytes provide several advantages. For example, confocal fluorescence microscopy is easier to use compared to EM or electrophysiological methods and the required equipment is more readily available. In addition, use of living cells avoids artifacts known to occur during the fixation, dehydration, or embedding steps used to prepare EM samples. Here we describe the use of styryl dyes such as FM 1–43 or di-8-ANEPPS and impermeant fluorescent indicators of the extracellular space as simple approaches for imaging and quantification of the DMS.
Key wordsMegakaryocytes Demarcation membrane system Styryl dyes Megakaryopoiesis
We are grateful to Dr. Kees Straatman of the Advanced Imaging Facility, Core Biotechnology Services, University of Leicester for discussion on imaging and microscopy. Work in the authors’ laboratory that led to the methods described in this chapter was funded by the Medical Research Council and the British Heart Foundation. S.O. was supported by the Kurdistan Regional Government Ministry of Higher Education and Scientific Research.
A video showing the rapid time course of staining of the demarcation membrane system in a rat megakaryocyte following perfusion with FM 2-10 and rapid destaining following wash. (MP4 2731 kb)
- 1.De Marsh QB, Kautz J, Motulsky AG (1955) An electron microscope study of platelets and megakaryocytes. J Clin Investig 34:929–930Google Scholar
- 4.Yamada E (1955) Some features of the fine structure of megakaryocytes in mouse spleen. Anat Rec 121:458Google Scholar
- 10.Becker RP, De Bruyn PP (1976) The transmural passage of blood cells into myeloid sinusoids and the entry of platelets into the sinusoidal circulation; a scanning electron microscopic investigation. AmJAnat 145(2):183–205Google Scholar
- 16.Italiano JE (2017) Megakaryocyte development and platelet production. In: Gresele P, Kleiman NS, Lopez JA, Page CP (eds) Platelets in thrombotic and non-thrombotic disorders: pathophysiology, pharmacology and therapeutics. Springer International Publishing AGGoogle Scholar
- 17.Nishimura S, Nagasaki M, Kunishima S, Sawaguchi A, Sakata A, Sakaguchi H, Ohmori T, Manabe I, Italiano JE Jr, Ryu T, Takayama N, Komuro I, Kadowaki T, Eto K, Nagai R (2015) IL-1alpha induces thrombopoiesis through megakaryocyte rupture in response to acute platelet needs. J Cell Biol 209(3):453–466. https://doi.org/10.1083/jcb.201410052 CrossRefPubMedPubMedCentralGoogle Scholar
- 26.Mahaut-Smith MP, Thomas D, Higham AB, Usher-Smith JA, Hussain JF, Martinez-Pinna J, Skepper JN, Mason MJ (2003) Properties of the demarcation membrane system in living rat megakaryocytes. Biophys J 84(4):2646–2654. https://doi.org/10.1016/S0006-3495(03)75070-X CrossRefPubMedPubMedCentralGoogle Scholar
- 36.Hirose K, Kadowaki S, Tanabe M, Takeshima H, Iino M (1999) Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns. Science 284 (5419):1527–1530Google Scholar
- 38.Carter RN, Tolhurst G, Walmsley G, Vizuete-Forster M, Miller N, Mahaut-Smith MP (2006) Molecular and electrophysiological characterization of transient receptor potential ion channels in the primary murine megakaryocyte. J Physiol 576(Pt 1):151–162. https://doi.org/10.1113/jphysiol.2006.113886 CrossRefPubMedPubMedCentralGoogle Scholar
- 41.Jurak Begonja A, Pluthero FG, Suphamungmee W, Giannini S, Christensen H, Leung R, Lo RW, Nakamura F, Lehman W, Plomann M, Hoffmeister KM, Kahr WH, Hartwig JH, Falet H (2015) FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets. Blood 126:80. https://doi.org/10.1182/blood-2014-07-587600 CrossRefGoogle Scholar
- 42.Antkowiak A, Viaud J, Severin S, Zanoun M, Ceccato L, Chicanne G, Strassel C, Eckly A, Leon C, Gachet C, Payrastre B, Gaits-Iacovoni F (2016) Cdc42-dependent F-actin dynamics drive structuration of the demarcation membrane system in megakaryocytes. J Thromb Haemost: JTH 14(6):1268–1284. https://doi.org/10.1111/jth.13318 CrossRefPubMedGoogle Scholar