| Summary: | The demarcation membrane system (DMS) is an extensive plasma membrane invagination system of the megakaryocyte (MK) that provides a membrane reserve for platelet generation. The properties of the DMS are poorly understood, particularly in living MKs. In this study, advanced electron microscopy, live cell confocal imaging and pharmacological tools were used to study the DMS and its connections with the extracellular environment. Confocal imaging of membrane-impermeant extracellular fluorescent indicators (HPTS, FITC-dextrans between 4 and 2000 kDa, and quantum dots) provided evidence for a discrete molecular cut-off (≈110kDa, estimated to be ≈11.0 nm by dynamic light scatter (DLS) measurements) for access to the DMS, which will prevent entry of large adhesion molecules (e.g. fibrinogen). Using extra-high resolution scanning electron microscopy (SEM) of fixed MKs, membrane invagination pores (MIPs) were observed on the MK surface that were variable in size but generally larger than predicted from live cell imaging experiments. A neck constriction was observed beneath the surface opening of the DMS using Gatan 3-view serial block face EM and may explain the rejection of molecules smaller than the surface pore. After inhibition of Cdc42, the DMS allowed entry of all indicators tested (up to 2000 kDa = reported to be ≈ 54.0 nm diameter). Using membrane-impermeant fluorescent indicators, electrophysiological capacitance measurements and transmission EM, multiple cationic amphiphilic drugs (CADs) were shown to cause complete surface detachment of the DMS. These compounds included the calmodulin inhibitor W-7, the phospholipase-C inhibitor U-73122 and anti-psychotic phenothiazines (trifluoperazine and chlorpromazine). CADs also caused loss of T tubules in cardiac ventricular myocytes and the open canalicular system of platelets. A possible explanation for this action is the ability of CADs to interfere with the interaction between PIP2 and binding of BAR domain containing proteins or the cytoskeleton. This work provides new insights into plasma membrane invagination systems.
|
|---|
