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Analysis of WASH complex member strumpellin
Pácalt, Ondřej ; Libusová, Lenka (advisor) ; Cvrčková, Fatima (referee)
Actin polymerization facilitated by the Arp2/3 complex plays a critical role in a wide range of cellular processes such as motility, endocytosis and cargo recycling. Activation and appropriate localization of the Arp2/3 complex is mediated by an interaction with the nucleation-promoting factor (NPF). WASH complex is the major endosomal NPF which plays a crucial role in the cargo recycling back to the trans-Golgi network (TGN) or plasma membrane. It is composed of five subunits: WASH1, SWIP, FAM21, CCDC53 and strumpellin. While WASH1 and FAM21 have been extensively studied, much less is known about strumpellin, a protein causally implicated in the onset of hereditary spastic paraplegia (HSP). This work focuses on strumpellin function in the cells, showing that only full-length protein incorporates into the WASH complex. In a strumpellin knock out cell line, we demonstrated that loss of strumpellin resulted in destabilization of the other WASH complex subunits. Still, an incomplete WASH complex without strumpellin was assembled. Cells also displayed enlarged endosomal subdomains and WASH complex nucleation activity on endosomes was largely diminished as assessed by loss of the actin patches. Finally, the absence of strumpellin was also accompanied by the accumulation of glucose transporter 1 (GLUT1)...
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Analysis of WASH complex member strumpellin
Pácalt, Ondřej ; Libusová, Lenka (advisor) ; Cvrčková, Fatima (referee)
Actin polymerization facilitated by the Arp2/3 complex plays a critical role in a wide range of cellular processes such as motility, endocytosis and cargo recycling. Activation and appropriate localization of the Arp2/3 complex is mediated by an interaction with the nucleation-promoting factor (NPF). WASH complex is the major endosomal NPF which plays a crucial role in the cargo recycling back to the trans-Golgi network (TGN) or plasma membrane. It is composed of five subunits: WASH1, SWIP, FAM21, CCDC53 and strumpellin. While WASH1 and FAM21 have been extensively studied, much less is known about strumpellin, a protein causally implicated in the onset of hereditary spastic paraplegia (HSP). This work focuses on strumpellin function in the cells, showing that only full-length protein incorporates into the WASH complex. In a strumpellin knock out cell line, we demonstrated that loss of strumpellin resulted in destabilization of the other WASH complex subunits. Still, an incomplete WASH complex without strumpellin was assembled. Cells also displayed enlarged endosomal subdomains and WASH complex nucleation activity on endosomes was largely diminished as assessed by loss of the actin patches. Finally, the absence of strumpellin was also accompanied by the accumulation of glucose transporter 1 (GLUT1)...
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