Megakaryocytes (MKs) are large myeloid cells that manufacture blood platelets. During megakaryo-cytopoiesis a unique membrane complex, the demarcation membrane system (DMS), develops to provide additional membrane for the generation of future platelets. The mechanisms by which the DMS is remodelled during thrombocytopoiesis are unclear and, to date, investigations of the DMS have relied upon electron microscopy of fixed tissue. We have now studied this membrane cytoarchitecture in living MKs using patch-clamp membrane capacitance measurements and single- or dual-photon confocal fluorescence microscopy. Male Wistar rats were killed by exposure to a rising concentration of CO₂ followed by cervical dislocation and MKs isolated as described elsewhere (Hussain & Mahaut-Smith, 1998). Membrane capacitance was measured using the whole-cell patch-clamp configuration by integration or electronic compensation of capacitative transients following ± 5 or ± 10 mV voltage steps. Bath-applied di-8-ANEPPS (10 µM) was studied using 488 nm excitation and > 505 or > 515 nm emission on either a Zeiss LSM 510 or Leica TCS-NT confocal microscope. Extracellularly applied membrane-impermeant Oregon Green 488 BAPTA-1 (50 µM) was monitored using a Leica TCS-SP-MP with excitation from a solid-state Millenia V-pumped Tsunami TI/Sapphire laser. The laser was tuned to 797 nm and emitted light captured at 505-640 nm. Di-8-ANEPPS stained small myeloid cells with a halo pattern yet rapidly penetrated deep into the cytoplasm of the MKs, leaving an eccentric or central unstained area presumed to be the nucleus. This pattern of staining indicates that the MK plasma membrane displays extensive invaginations, as originally suggested using electron microscopy of extracellular electron-dense markers (Behnke, 1968). The pattern of MK staining with extracellular Oregon Green 488 BAPTA-1 confirmed the presence of the membrane invaginations (Fig. 1). MK membrane capacitance measurements were 8.2 ± 2.1 (mean ± S.D., n = 122) times that expected, assuming a spherical geometry and specific membrane capacitance of 1 µF cm^-2. This high membrane capacitance suggests the electrical continuity of a significant proportion of the MK DMS with the cell surface membrane. We conclude that biophysical capacitative measurements and confocal fluorescence microscopy of membrane-impermeant indicators represent a novel approach to the study of the MK DMS during development and thrombocytopoiesis.This work was funded by the British Heart Foundation, Medical Research Council and Wellcome Trust.

Figure 1. Dual-photon confocal fluorescence of marrow cells immersed in Oregon Green 488 BAPTA-1. Extracellular-continuous areas due to membrane invaginations are visible in the MK and absent from other cells.

Behnke, O. (1968). J. Ultrastruct. Res. 24, 412-433.

Hussain, J.F. & Mahaut-Smith, M.P. (1998). J. Physiol. 511, 797-801. abstract