engleski

Characterization of SACM1L phosphatase and PI4P in Dami cell line and primary megakaryocytes

Phosphoinositides are phosphorylated membrane lipids that control the function of secretory organelles, act as signal transducers and have a role in actin reorganization, cell growth and proliferation. Phosphatidylinositol-4-phosphate (PI4P) is highly abundant in cells and is critical for lipid transport and delivery of cargo from the Golgi apparatus to the plasma membrane. SACM1L is a major phosphatase that metabolizes PI4P at the endoplasmic reticulum and allows accumulation of PI4P at the Golgi apparatus. In megakaryocytes, precursors of blood platelets, there is an active transport between the Golgi apparatus and the growing intracellular membranes, required for platelet production. In this thesis, we investigated the expression, localization and function of SACM1L and PI4P in human megakaryoblastic leukemia cell line (Dami), and in primary megakaryocytes derived from mouse bone marrow. We used Western blot analysis to examine the level of SACM1L expression and we found that it slightly increases during maturation of Dami cell line and primary megakaryocytes. We examined SACM1L localization by immunofluorescence, and we found that it is localized mostly at the endoplasmic reticulum and partially to the Golgi in both Dami cells and in primary megakaryocytes. Furthermore, exogenous expression of wild-type SACM1L in Dami cells decreased PI4P levels in comparison to the expression of catalytically dead form. SACM1L functionality in primary megakaryocytes was proven by its inhibition with hydrogen peroxide and subsequent increase in PI4P levels. Next, we observed that PI4P is localized at the Golgi in all maturation states in Dami cells which was confirmed with the exogenous expression of a PI4P binding probe. On the other hand, in primary immature megakaryocytes, PI4P is mostly localized at the Golgi, while in mature megakaryocytes it translocates to the PM. Finally, inhibition of kinases that produce PI4P (PI4K) caused a significant increase in the number of megakaryocytes forming proplatelets. These data indicate that SACM1L and PI4P could have a role in megakaryocyte maturation and platelet formation. Also, these results open new questions about the role of PI4P in megakaryocyte maturation, which is a subject of future research.

Phosphatidylinositol-4-phosphate, SACM1L phosphatase, megakaryocytes, proplatelets

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