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The role of the WASH complex in endolysosomal homeostasis
Beránková, Pavla ; Libusová, Lenka (advisor) ; Rösel, Daniel (referee)
The WASH (WASP and SCAR homologue) complex is an actin nucleation promoting factor essential for endosomal cargo sorting. Upon WASH complex depletion, endosomal cargoes are mislocalized and the endolysosomal system collapses. Here, we employed high-speed vesicle tracking and real-time rescue experiments to test the effect of WASH complex depletion on endolysosomal homeostasis. We found that large lysosome-like vacuoles emerge in knockout cell lines of individual WASH complex subunits, although the overall dynamics of the lysosomal network does not substantially change. Follow-up experiments revealed that the WASH complex does not act directly on the vacuoles during their rescue. Overall, the data indicate that the emergence of vacuoles in WASH complex knockouts is a secondary process that depends on the WASH complex indirectly.
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WASH complex and its interactome in human pathology
Pácalt, Ondřej ; Libusová, Lenka (advisor) ; Drobná Krejčí, Eliška (referee)
Efficient transport of cargo to its correct destination is required for the proper functioning of eukaryotic cells. Vesicular trafficking is one of the important means of intracellular transport. Impairment of this process often leads to serious pathologies. Sorting and recycling is the crucial part of vesicular trafficking as it enhances its efficiency. The WASH complex has a key role in the regulation of branched actin patches formation. If this occurs on the membrane of endosomes, then it affects sorting, recycling and cargo trafficking. Mutations in the WASH complex or its interacting partners cause diseases such as hereditary spastic paraplegia, Parkinson disease or light intellectual disability. Despite certain advance in the understanding of above-mentioned pathologies, mechanism of the pathogenesis is still elusive. Research in this field can reveal basic molecular mechanisms responsible for the complexity of cargo sorting, recycling and trafficking and thus provide better opportunities for treatment of affected individuals.
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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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Characterization of WASH complex member protein SWIP
Humhalová, Tereza ; Libusová, Lenka (advisor) ; Lánský, Zdeněk (referee)
WASH complex regulates actin dynamics on endosomes by activating the Arp2/3 complex, which subsequently induces generation of branched actin patches. WASH complex is required for proper recycling of many important transmembrane proteins. Although the general physiological function of WASH complex is known, the role of its single subunits have not yet been adequately specified. This work focuses on one of these subunits - protein SWIP. This protein maintains vesicular localization of some WASH complex subunits in the slime mold Dictyostelium discoideum and a point mutation in its sequence causes a severe neurodegenerative disease - autosomal recessive intellectual disorder (ARID). Our results show that SWIP truncation results in its inability to incorporate into WASH complex. However, the C-terminal part of SWIP is able to localize onto intracellular vesicles, which are not WASH complex positive. We have also studied the impact of ARID-causing SWIP mutation, and we show, that it does neither change the protein's ability to bind the complex nor the overall localization of WASH complex.
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The role of cytoskeleton in endosomal fusion and fission
Získalová, Tereza ; Libusová, Lenka (advisor) ; Tolde, Ondřej (referee)
Cytoskeleton plays a key role in endocytic process. Vesicules move along microtubules to target membranes. Microtubules also partake in the formation of endosomal tubules, from which recyclated vesicules are splitted off. Actin network has in endocytosis multi-ple effect as well. In the case of membrane fusion is its role both, positive and negative, for it creates mechanical force which facilitates the fusion in last stage. By contrast, in the first stage, it acts as a physical barrier, which needs to be removed. Actin also actively participates in fission of vesicules. Actin network and microtubules are thus interconnected with endocytic pathway in time and space. Right functional connection of the cytoskeleton with dynamics of endocytic vesicles is driven by many regulatory proteins. Among important regulators of actin network belong for example proteins of Arp2/3, WASH complex, WASP or Rab and Rho proteins. Powered by TCPDF (www.tcpdf.org)
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WASH complex and its interactome in human pathology
Pácalt, Ondřej ; Libusová, Lenka (advisor) ; Drobná Krejčí, Eliška (referee)
Efficient transport of cargo to its correct destination is required for the proper functioning of eukaryotic cells. Vesicular trafficking is one of the important means of intracellular transport. Impairment of this process often leads to serious pathologies. Sorting and recycling is the crucial part of vesicular trafficking as it enhances its efficiency. The WASH complex has a key role in the regulation of branched actin patches formation. If this occurs on the membrane of endosomes, then it affects sorting, recycling and cargo trafficking. Mutations in the WASH complex or its interacting partners cause diseases such as hereditary spastic paraplegia, Parkinson disease or light intellectual disability. Despite certain advance in the understanding of above-mentioned pathologies, mechanism of the pathogenesis is still elusive. Research in this field can reveal basic molecular mechanisms responsible for the complexity of cargo sorting, recycling and trafficking and thus provide better opportunities for treatment of affected individuals.
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Analysis of WASH complex component FAM21
Dostál, Vojtěch ; Libusová, Lenka (advisor) ; Žárský, Viktor (referee)
The dynamics and function of the actin cytoskeleton depends on polymerization and branching of actin filaments, an event that is stimulated by Arp2/3. Arp2/3-dependent branching is closely linked to the pentameric WASH complex which consists of WASH, strumpellin, SWIP, CCDC53 and FAM21. WASH complex is associated mainly with endosomes. It was traditionally localized to retromer-coated domains of early endosomes which enable sorting and recycling of endocytosed material. However, latest scientific data extend the role of WASH complex to other endosomal or even non-endosomal sites. Of all the subunits of the WASH complex, FAM21 is the most prominent hub for protein-protein interactions, thanks to its long unstructured C-terminal domain. In my diploma thesis FAM21 was localized to early and late endosomes and lysosomes of U2OS human cell line. Dictyostelium discoideum was then used as a model organism to investigate FAM21 protein interactions as well as the proteins associated specifically with the C terminal domain of FAM21. Results of the study shed new light on the complex network of FAM21 interactions and question the long-standing theories on the function of WASH complex in cells. Powered by TCPDF (www.tcpdf.org)
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