Linear podosomes display low Cdc42 activity for proplatelet elongation by megakaryocytes.

Blood platelets result from differentiation of megakaryocytes (MKs) into the bone marrow. It culminates with the extension of proplatelets (PPT) through medullar sinusoids and release of platelets in the blood stream. Those processes are regulated by contact with the microenvironment mediated by podosomes.

Megakaryocytes form linear podosomes devoid of digestive properties to remodel medullar matrix.

Bone marrow megakaryocytes (MKs) undergo a maturation involving contacts with the microenvironment before extending proplatelets through sinusoids to deliver platelets in the bloodstream. We demonstrated that MKs assemble linear F-actin-enriched podosomes on collagen I fibers. Microscopy analysis evidenced an inverse correlation between the number of dot-like versus linear podosomes over time.

Liposome-Based Methods to Study GTPase Activation by Phosphoinositides.

Phosphoinositides (PIPs) are lipid messengers with different functions according to their localization. After their local production by the action of lipid kinases or phosphatases, PIPs regulate various biological processes such as cytoskeleton rearrangement, membrane remodeling/trafficking, or gene expression through binding of their phosphorylated inositol head group with different protein domains such as PH, PX, and FYVE. It is well known that PIPs regulate the activity of small GTPases by interacting with and activating Guanyl-nucleotide Exchange Factor (GEF) proteins through specific domains such as the ones mentioned above.

Liposome-Based Methods to Study Protein-Phosphoinositide Interaction.

Following their generation by lipid kinases and phosphatases, phosphoinositides regulate important biological processes such as cytoskeleton rearrangement, membrane remodeling/trafficking, and gene expression through the interaction of their phosphorylated inositol head group with a variety of protein domains such as PH, PX, and FYVE. Therefore, it is important to determine the specificity of phosphoinositides toward effector proteins to understand their impact on cellular physiology. Several methods have been developed to identify and characterize phosphoinositide effectors, and liposomes-based methods are preferred because the phosphoinositides are incorporated in a membrane, the composition of which can mimic cellular membranes.

Megakaryocytes use in vivo podosome-like structures working collectively to penetrate the endothelial barrier of bone marrow sinusoids.

Background: Blood platelets are anucleate cell fragments that prevent bleeding and minimize blood vessel injury. They are formed from the cytoplasm of megakaryocytes located in the bone marrow. For successful platelet production, megakaryocyte fragments must pass through the sinusoid endothelial barrier by a cell biology process unique to these giant cells as compared with erythrocytes and leukocytes.