Purification of native bone marrow megakaryocytes for studies of gene expression.

Megakaryocytes constitute less than 1% of all marrow cells, therefore purification of these giant platelet precursor cells represents a challenge. We describe two methods to ultra-purify mature megakaryocytes from murine marrow for the purpose of extracting RNA suitable for studies of gene expression. In the first approach, unit velocity gradients are used to enrich for megakaryocytes, which are then selected by fluorescence-activated cell sorting based upon size and high surface expression of CD41. In the second method, individual megakaryocytes, identified by their distinct morphology, are extracted using glass suction pipettes. Despite the small numbers of cells that can be isolated via the latter technique, recent studies have demonstrated how this pure population can be used to detect mRNA transcripts encoding ion channels and other proteins in the native megakaryocyte.

Expression profiling and electrophysiological studies suggest a major role for Orai1 in the store-operated Ca2+ influx pathway of platelets and megakaryocytes.

Store-operated Ca2+ influx represents a major route by which cytosolic Ca2+ can be elevated during platelet activation, yet its molecular identity in this cell type remains highly controversial. Using quantitative RT-PCR analysis of candidate receptor-operated cation entry pathways in human platelets, we show a >30-fold higher expression of message for the recently discovered Orai1 store-operated Ca2+ channel, and also the store Ca2+ sensor STIM1, when compared to the non-selective cation channels TRPC1, TRPC6 and TRPM2. Orai1 and STIM1 gene transcripts were also detected at higher levels than TRPC1, TRPC6 and TRPM2 in primary murine megakaryocytes and human megakaryocytic cell lines. In direct electrophysiological recordings from murine megakaryocytes, Ca2+ ionophore-induced store depletion stimulated CRAC currents, which are known to require Orai1, and these overlapped with TRPC6-like currents following P2Y receptor activation. Together with recent transgenic studies, these data provide evidence for STIM1:Orai1 as a primary pathway for agonist-evoked Ca2+ influx in the platelet and megakaryocyte.

Molecular and electrophysiological characterization of transient receptor potential ion channels in the primary murine megakaryocyte.

The molecular identity of platelet Ca(2+) entry pathways is controversial. Furthermore, the extent to which Ca(2+)-permeable ion channels are functional in these tiny, anucleate cells is difficult to assess by direct electrophysiological measurements. Recent work has highlighted how the primary megakaryocyte represents a bona fide surrogate for studies of platelet signalling, including patch clamp recordings of ionic conductances. We have now screened for all known members of the transient receptor potential (TRP) family of non-selective cation channels in murine megakaryocytes following individual selection of these rare marrow cells using glass micropipettes. RT-PCR detected messages for TRPC6 and TRPC1, which have been reported in platelets and megakaryocytic cell lines, and TRPM1, TRPM2 and TRPM7, which to date have not been demonstrated in cells of megakaryocytic/platelet lineage. Electrophysiological recordings demonstrated the presence of functional TRPM7, a constitutively active cation channel sensitive to intracellular Mg(2+), and TRPM2, an ADP-ribose-dependent cation channel activated by oxidative stress. In addition, the electrophysiological and pharmacological properties of the non-selective cation channels stimulated by the physiological agonist ADP are consistent with a major role for TRPC6 in this G-protein-coupled receptor-dependent Ca(2+) influx pathway. This study defines for the first time the principal TRP channels within the primary megakaryocyte, which represent candidates for Ca(2+) influx pathways activated by a diverse range of stimuli in the platelet and megakaryocyte.

Evaluation of relative yeast cell surface hydrophobicity measured by flow cytometry.

OBJECTIVE: To develop an efficient method for evaluating cell surface hydrophobicity and to apply the method to demonstrate the effects of fungal growth conditions on cell surface properties. METHODS: Yeast isolates were suspended in phosphate-buffered saline and mixed with deep blue-dyed polystyrene microspheres. Flow cytometry was used to detect the degree of microsphere binding to yeast cells. Different strains of yeast were compared for intrinsic microsphere binding activity and changes in growth conditions were invoked to modify the relative surface hydrophobicity. RESULTS: Commercially available blue-dyed polystyrene microspheres showed strong fluorescence in the FL3 channel, whereas yeast cells did not show appreciable FL3 fluorescence. Microspheres and yeast were generally distinguishable on the basis of size revealed by forward light scatter. This method showed a wide variation in intrinsic cell surface hydrophobicity among Candida albicans strains. Likewise, variation in hydrophobicity of non-albicans yeast species was observed. Growth on solid media, incubation at 25 degrees C, or 250 mg/dl glucose concentration increased hydrophobicity compared with growth in liquid media, incubation at 37 degrees C, or 50 mg/dl glucose, respectively. Growth in 1 x 10(-9) M estradiol had no appreciable effect on hydrophobicity. CONCLUSIONS: Stained latex microspheres fluoresced in the FL3 channel of the flow cytometer and bound to yeast cells to an extent related to the surface hydrophobicity of the yeast. Binding detected by flow cytometry showed that clinical yeast isolates varied in intrinsic binding capacity and this binding ability was altered by different growth conditions. The implications for virulence regulation among yeast isolates are discussed.