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Rab8 and Rab8-interacting proteins as players in cell polarization

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Title: Rab8 and Rab8-interacting proteins as players in cell polarization
Author: Hattula, Katarina
Contributor: University of Helsinki, Faculty of Medicine, Haartman Institute, Department of pathologyInstitute of Biotechnology, University of Helsinki
Thesis level: Doctoral dissertation (article-based)
Abstract: Rab8 and its interacting proteins as regulators of cell polarization

During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer.

GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis.

The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive.

In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics.

Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6.

The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.Rab8 reglerar cellens form och utseende.

En avhandling vid medicinska fakulteten, Helsingfors universitet beskriver hur det GTP bindande proteinet Rab8 reglerar cellens form och utseende (cellpolarisering).

I en flercellig organism måste celler specialicera sig och röra på sig för att organismen skall utvecklas korrekt. För att detta skall kunna ske måste de först ändra form i en process kallad polarisering. Cellens komponenter organiseras då assymetriskt för att bilda funktionellt skilda delar.

Att en cell kan röra på sig har inte bara en essentiell funktion under utvecklingsskedet, det är också en process som behövs under hela organismens livstid. Det behövs t.ex. rörliga celler för att bekämpa infektioner och för att sår skall kunna läkas. Det är också viktigt att celler som inte skall röra på sig förblir stilla. Strikt kontroll av vilka celler som skall och inte skall röra på sig behövs kontinuerligt. Ett exempel på vad som kan hända om denna kontroll inte fungerar är cancer.

Små GTPaser är en stor grupp proteiner som fungerar som omkopplare eller strömbrytare i vidareföring av signaler inuti cellen. Deras funktioner i cellen är diversa och inkluderar bland annat kontroll av celltillväxt, celldelning, och membrantransport. Denna stora grupp (fler än 100 proteiner) kan delas upp i fem familjer: Ras, Rho, Rab, Arf och Ran. Ras var den första humana onkogenen att beskrivas 1981, och i uppskattningsvis en tredjedel av all cancer finns mutationer som leder till ett permanent aktiverat Ras-protein.

Rab-familjen av små GTPaser kontrollerar membrantransport i cellen och det är en medlem av denna familj, Rab8, som är fokuset av Katarina Hattulas avhandling. Tidigare forskning har inte klart kunnat visa vilken roll Rab8 har i cellen. I avhandlingen ges bevis på att Rab8 kraftigt påverkar cellens form och utseende. Resultatet av den forskning som presenteras visar en roll för Rab8 i återvinning av material som behövs för att en cell skall kunna bilda och upprätthålla en polariserad fenotyp. Något som bland annat behövs för att cellen skall kunna röra på sig.

Avhandlingen presenterar också i detalj hur man sökt efter proteiner som specifikt kan binda till och påverka Rab8s funktion. Tre nya Rab8 bindande protein beskrivs. Ett av dessa är optineurin, som på annat håll har visats vara knutet till glaukom, (en av de främsta anledningarna till blindhet i världen med 33 miljoner drabbade). En koppling mellan optineurin och Huntingtin, det protein vars gen är muterad i personer med Huntingtons sjukdom visas också. Ett annat Rab8 bindande protein som beskrivs är Rabin8. Det aktiverar Rab8 och behövs för att Rab8 ska kunna utföra sin uppgift vid cellpolartisering.

Sammantaget ger den forskning som presenteras en ny insikt i hur membrantransport reglerad av Rab8 aktivt bidrar till att ändra cellens form och utseende. Detta kan ge en ny insikt i cellulära processer som cellmigration såväl som en rad sjukdomsförlopp till exempel glaukom, Huntingtons sjukdom och cancer.
URI: URN:ISBN:978-952-10-4252-2
http://hdl.handle.net/10138/20432
Date: 2007-11-09
Copyright information: This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.
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