Cyclase associated protein CAP in the regulation of the actin cytoskeleton and cell polarity in Dictyostelium discoideum

Sultana, Hameeda (2004). Cyclase associated protein CAP in the regulation of the actin cytoskeleton and cell polarity in Dictyostelium discoideum. PhD thesis, Universität zu Köln. Open Access

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Abstract

Dictyostelium discoideum amoebae offer great opportunities to elucidate the signalling pathways involved in the generation of cell polarity. They constantly change their shape and form new ends in response to a plethora of environmental signals. This process requires distinct signalling molecules, the chemotactic machinery and components of the cytoskeleton such as CAP, which in Dictyostelium is involved in actin cytoskeleton rearrangements. Dictyostelium cells deficient for CAP show a defect in cell polarization during development and an altered cAMP relay response. In this work we wanted to study the position of CAP in the signalling network and made use of mutants defective in components of cAMP signalling (ACA, cAR1/3, G-alpha2, G-beta, PI3-kinase, Pianissimo and PKA) in which we studied CAP and CAP associated responses. Our studies revealed that CAP functions independent of ACA, cAR1/3, G-alpha2, G-beta, PI3-kinase and Pianissimo whereas the cAMP dependent protein kinase A (PKA) is necessary for targeting CAP to the sites of its cellular action. The localization data suggest that CAP and PKA may functionally co-operate to control actin organization and cell polarity. We further observed that CAP functions downstream of PI3-kinases or in parallel pathways because overexpression of CAP rescued the severe impairment in pinocytosis in the pik1-/2- cells, their altered distribution of F-actin and also the abnormal developmental phenotypes. In line with this proposal CAP could partially rescue the phagocytosis defect of the g-beta cells. CAP expression also improved the development of aggregation deficient aca- cells but did not lead to complete restoration. The cell polarity and streaming defects of aca- cells were also rescued by the moderate expression of CAP making CAP a molecule downstream in the hierarchy of the signal transduction cascade. On the other hand, CAP regulates LimD, a component of the actin cytoskeleton which in Dictyostelium is involved in generation of cell polarity as shown by an altered behaviour of GFP-LimD in CAP bsr cells in comparison to wild type. To gain more insight into the in vivo functioning of CAP, we performed a search for binding partners which suggested that CAP associates and interacts with the vacuolar ATPases complex, and that the absence of CAP disrupts the vacuolar network. This underlines the findings obtained in the PI3-kinase mutants that CAP is a general regulator of endocytosis. We also identified ARP2/3 complex p34-ARC subunit, Rab4, Rab11 and porin as potential interacting partners of CAP by biochemical methods. The significance of this interaction is under investigation. Finally, our microarray analysis suggested that in the absence of CAP the actin cytoskeleton and signalling machinery is strongly affected. The up-regulation of regA (negative regulator of intracellular cAMP), and kinases like erk1, statA and pakA might be of relevance for the altered cAMP relay of CAP bsr cells. The repression of the expression of cytoskeletal components like profilins I and II, villidin, coactosin, myosin and signalling molecules like NDP kinases

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Thesis (PhD thesis)

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CAP in Dictyostelium discoideum ist in die Regulation des Zytoskeletts und in Signaltransduktionsprozesse involviert. Ein Verlust des Proteins führt zu Defekten in Wachstum und Entwicklung und einem Verlust der Zellpolarität. Zellpolarität ist ein herausragendes Merkmal der cAMP-abhängigen Chemotaxis in Dictyostelium. Das Vorhandensein einer Reihe von definierten Mutanten mit Defekten in der Chemotaxis, hat es erlaubt, in dieser Arbeit die Einordnung von CAP in die caMP Signaltransduktionskaskade zu untersuchen. Zur Verfügung standen Mutanten für den cAMP Rezeptor, die assoziierten G-Proteine, für die Adenylatzyklase, PI3-Kinasen und die cAMP-abhängige Proteinkinase A, die für die intrazelluläre Wirkung des cAMP notwendig ist. Untersucht wurden die Lokalisation von CAP und die Umverteilung von CAP während Aktin-abhängiger Prozesse. Dies hat gezeigt, dass CAP unabhängig von diesen Molekülen funktioniert. Im Gegenteil, es wurde beobachtet, dass CAP-Überexpression bestimmte Defekte wie den Endozytosedefekt in der PI3-Kinase Mutante oder den Entwicklungsdefekt in der ACA-Mutante zum Teil aufheben kann. CAP liegt also weiter stromaufwärts in der cAMP Signaltransduktionskette. Für LimD, eine Komponente des Aktinnetzwerks, deren Ausfall in Dictyostelium zu einem Polaritätsverlust führt, wurde beobachtet, dass CAP essentiell für seine korrekte Lokalisation und Umverlagerung in der Zelle ist. Ähnlich abhängig von CAP sind Komponenten der vakuolären ATPase, einem Proteinkomplex von endozytotischen und vakuolären Membranen in Dictyostelium. Immunpräzipitationsexperimente belegen eine direkte Verbindung mit der v-ATPase. Weitere Bindeproteine von CAP sind Komponenten des Zytoskeletts. Diese Ergebnisse werden auch durch unsere Microarray-Analysen unterstützt, die in der CAP-Mutante Veränderungen bei Signalmolekülen und in Zytoskelettkomponenten zeigen.

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