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The role of phosphoinositides in macropinocytosis
Hájek, Tomáš ; Doubravská, Lenka (advisor) ; Španielová, Hana (referee)
Macropinocytosis is non-selective actin-dependent type of endocytosis. It is important for the normal physiology of some cell types. However, it is used by intracellular parasites which internalise themselves into host cells in this way and also play a role in the nutritional supply in some type of cancer cells. During macropinocytosis a self-organized subdomain of plasma membrane is separated by a diffusion barrier named macropinocytic cup. RAC1-driven actin polymerization is required for membrane protrusion at the cup periphery, where a narrow ring of the actin nucleating factors is present. In contrast, actin dissociation occurs at the base of the cup due to RAS-controlled formation of phosphatidylinositol trisphosphates (PIP3). During cup closure sequential breakdown of PIP3 to phosphatidylinositol and acquisition of the endosomal identity of the newly formed vesicle is necessary. As a result of tubulation in the early stages of macropinocytosome maturation the vesicle decreases in diameter and stabilizes. At late stages the macropinocytic vesicle may fuse with the lysosome, allowing internalized material to enter this degradative organelle. Throughout the process specific types of phosphatidylinositols are part of the membrane providing signal transduction and membrane identity. These phospholipids...
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Signalization of adenylate cyclase toxin of Bordetella pertussis in macrophages.
Černý, Ondřej ; Kamanová, Jana (advisor) ; Kuthan, Martin (referee)
Adenylate cyclase toxin (CyaA) is a key virulence factor of Bordetella pertussis, the causative agent of whooping cough. The toxin targets primarily myeloid phagocytes expressing CD11b/CD18 (αMβ2, CR3, Mac-1) and by elevation of cytosolic cAMP levels it paralyses their macropinocytic and opsono-phagocytic functions. Here, we dissected the cAMP-regulated pathway responsible for the block of macrophage macropinocytosis and characterized the capacity of CyaA-treated macrophages to shut- down Akt (protein kinase B, PKB) signaling; that controls nitric oxide (NO) production by macrophages. By using specific activators of protein kinase A (PKA) and for the exchange protein activated by cAMP (Epac), we show that activation of the cAMP effector Epac inhibits macropinocytosis in macrophages. Moreover, upon transfection of macrophages by the constitutively active and dominant negative variants of a downstream effector of Epac, the small GTPase Rap1, inhibition or upregulation of macrophage macropinocytosis was observed, respectively. It was reported previously that the Epac/Rap1 pathway regulates activity of tyrosin phosphatase SHP-1 as well as of protein phosphatase 2 A (PP2A). We show that inhibition of both tyrosin phosphatases and PP2A interferes with CyaA-mediated block of macropinocytosis. These...
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Signalization of adenylate cyclase toxin of Bordetella pertussis in macrophages.
Černý, Ondřej ; Kuthan, Martin (referee) ; Kamanová, Jana (advisor)
Adenylate cyclase toxin (CyaA) is a key virulence factor of Bordetella pertussis, the causative agent of whooping cough. The toxin targets primarily myeloid phagocytes expressing CD11b/CD18 (αMβ2, CR3, Mac-1) and by elevation of cytosolic cAMP levels it paralyses their macropinocytic and opsono-phagocytic functions. Here, we dissected the cAMP-regulated pathway responsible for the block of macrophage macropinocytosis and characterized the capacity of CyaA-treated macrophages to shut- down Akt (protein kinase B, PKB) signaling; that controls nitric oxide (NO) production by macrophages. By using specific activators of protein kinase A (PKA) and for the exchange protein activated by cAMP (Epac), we show that activation of the cAMP effector Epac inhibits macropinocytosis in macrophages. Moreover, upon transfection of macrophages by the constitutively active and dominant negative variants of a downstream effector of Epac, the small GTPase Rap1, inhibition or upregulation of macrophage macropinocytosis was observed, respectively. It was reported previously that the Epac/Rap1 pathway regulates activity of tyrosin phosphatase SHP-1 as well as of protein phosphatase 2 A (PP2A). We show that inhibition of both tyrosin phosphatases and PP2A interferes with CyaA-mediated block of macropinocytosis. These...
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