Lentiviral Short Hairpin RNA Silencing of DNM3Decreases Megakaryoycte Progenitor Proliferation and Inhibits the Formation of the Demarcation Membrane System.

Abstract 4594

Dynamin 3 (DNM3) is a mechanochemical enzyme, which participates in membrane dynamics by hydrolyzing nucleotides to link cell membranes to the actin cytoskeleton. Recently, we identified DNM3 to be differentially up-regulated during megakaryocyte (MK) development and reported that DNM3 participates in the proliferation of MK progenitors. We report here that dynamin 3 not only participates in MK progenitor amplification, but is also involved in the formation of the demarcation membrane system (DMS). To explore the role of dynamin 3 during MK development, we used three different lentiviral DNM3 shRNA constructs to infect human umbilical cord blood (UCB) CD34⁺ cells that were then cultured to differentiate along the MK lineage. Relative to controls, CD34+ cells transfected with DNM3 shRNAs significantly decreased the total number of cells produced in culture as well as the number of colony forming unit-megakaryocytes (CFU-MKs). As a result, the total number of CD41+ and CD61+ progeny cells was also reduced. To investigate the role dynamin 3 plays during the maturation of MKs, a Src family kinase inhibitor, SU6656, was used to induce high polyploidization and the development of a complex DMS in the UT-7/mpl cell line. The styryl membrane dye di-8-ANEPPS was used to monitor the formation of the DMS. In the absence of SU6656 treatment, UT-7/mpl cells showed a peripheral staining pattern that was indicative of cells that did not have extensive plasma membrane invaginations. In contrast, the staining pattern of UT-7/mpl cells treated with SU6656 clearly showed a well-developed plasma membrane system with invaginations. When DNM3 was silenced in SU6656 treated UT-7/mpl cells the results showed a significant reduction in di-8-ANEPPS staining relative to empty vector and non-specific negative controls. These findings are consistent with the conclusion that dynamin 3 participates in the morphogenesis of the DMS.