Expression of MTPG-24, a Peptide Derived from a Protein Regulating Dynamic Changes in Cortical Actin, Induces Endomitotic Replication in Megakaryocytic Cells.

Prior to the fragmentation of their cytoplasm leading to platelet formation, differentiating megakaryocytes (MKs) increase their ploidy and cellular volume by undergoing repeated rounds of DNA replication without concomitant cell division, a process known as endomitosis. There is now accumulating evidence that endomitotic MKs develop as a result of aberrant regulation of the cleavage furrow formation, the site where dynamic changes in actin, myosin and membrane structure mediate the process of cytokinesis during animal cell division. Here we report that transfection of human megakaryocytic cell lines with vectors expressing a 24mer-peptide (MTPG-24) resulted in remarkable changes in morphology, resulting in cells with the appearance of mature polyploid megakaryocytes. MTPG-24 is derived from a protein involved in regulating actin-based structures and motility which possesses a putative site for PKA phosphorylation. Despite its hydrophilic primary structure, MTPG-24 fused to a fluorescent tag was detected closely associated to the plasma membrane where it accumulated in a punctuate pattern, in addition to co-localizing with a Golgi marker. Interestingly, in each recombinant clone, cells with different ploidy levels developed among normally dividing ones, suggesting that transit from a mitotic cycle to an endomitotic cycle seemed to only occur in cells having completed a given number of cell cycles. These effects were obtained independently of the nature of the tag fused to the peptide (Hemagglutinin, Orange protein, GFP) but were not observed with a control peptide in which the amino acids of MTPG-24 were disposed in an arbitrarily sequence. Because it possibly interferes with some effector molecules required for cleavage furrow membrane assembly in late anaphase, MTPG-24 provides thus a new tool for understanding the molecular basis of endomitosis. Furthermore, since there is a correlation between DNA content and the numbers of platelets released by MK, we are currently testing whether this peptide could be used to increase the ploidy level of CD34+ human cord blood-derived MKs which have a low propensity to become highly polyploid.