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Regulation of GRAF1 membrane sculpting function during cell movement
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Group Richard Lundmark)
2015 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Reglering av den membranskulpterande funktionen hos GRAF1 vid cellrörelse (Swedish)
Abstract [en]

All eukaryotic cells rely on endocytic events to satisfy a constant need for nutrient and fluid uptake from their surroundings. Endocytosis-dependent turnover of cell surface constituents also serves to control signal transduction and establish morphological changes in response to extracellular stimuli. During endocytosis, distinct protein machineries re-sculpt the plasma membrane into vesicular carriers that enclose molecules that are to be taken up into the cell. Besides those produced from the canonical clathrin-mediated endocytic machinery, it is becoming increasingly clear that other membrane carriers exist. The indisputable connection between the function of these uptake systems and various disease states, highlights why it is so important to increase our knowledge about the underlying molecular machineries.

The aim of this thesis was therefore to characterise the function of GRAF1, a protein suggested to be a tumour suppressor due to that the gene has been found to be mutated in certain cancer patients. My work focused on understanding how this protein operates during formation of clathrin-independent carriers, with possible implications for disease development.

Previous in vitro studies showed that GRAF1 harbours a GTPase activating domain to inactivate Rho GTPase Cdc42, a major actin cytoskeleton regulator. Herein, microscopy based approaches used to analyse HeLa cells demonstrated the importance of a transient interaction between GRAF1 and Cdc42 for proper processing of GRAF1-decorated carriers. Although GRAF1-mediated inactivation of Cdc42 was not vital for the budding of carriers from the plasma membrane, it was important for carrier maturation. In addition, studies of purified GRAF1 and its association with lipid bilayers identified a membrane scaffolding-dependent oligomerisation mechanism, with the ability to sculpt membranes. This was consistent with the assumption that GRAF1 possesses an inherent banana shaped membrane binding domain. Remarkably, this function was autoinhibited and in direct competition with the Cdc42 interaction domain.

Finally, other novel GRAF1 interaction partners were identified in this study. Interestingly, many of these partners are known to be associated with protein complexes involved in cell adherence, spreading and migration. Although never actually seen localising to mature focal adhesions that anchor cells to their growth surface, dynamic GRAF1 carriers were captured travelling to and from such locations. Moreover, GRAF1 was recruited specifically to smaller podosome-like structures. Consistent with this, the tracking of GRAF1 in live cells uncovered a clear pattern of dynamic carrier formation at sites of active membrane turnover – notably protrusions at the cell periphery. Furthermore, the silencing of GRAF1 gave rise to cells defective in spreading and migration, indicating a targeting of GRAF1-mediated endocytosis to aid in rapid plasma membrane turnover needed for morphological changes that are a prerequisite for cell movement. Since these cells exhibited an increase in active Rab8, a GTPase responsible for polarised vesicle transport, the phenotype could also be explained by a defect in Rab8 trafficking that results in hyperpolarisation.

Taken together, the spatial and temporal regulation of GRAF1 membrane sculpting function is likely to be accomplished via its membrane binding propensity, in concert with various protein interactions. The importance of GRAF1 in aiding membrane turnover during cell movement spans different functional levels – from its local coordination of membrane and actin dynamics by interacting with Cdc42, to its global role in membrane lipid trafficking.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2015. , 62 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1761
Keyword [en]
Endocytosis, migration, polarisation, tension, CLIC/GEEC, GRAF1, Rho GTPase, Cdc42, Rab8
National Category
Cell and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-111213ISBN: 978-91-7601-377-9 (print)OAI: oai:DiVA.org:umu-111213DiVA: diva2:868502
Public defence
2015-12-04, N300, Naturvetarhuset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2015-11-13 Created: 2015-11-10 Last updated: 2015-11-13Bibliographically approved
List of papers
1. Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF1
Open this publication in new window or tab >>Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF1
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2015 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, no 22, 4183-4195 p.Article in journal (Refereed) Published
Abstract [en]

Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics, processes that are regulated by Rho GTPases. Here, we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase activating protein (GAP) GRAF1, facilitate rapid cell surface turnover at the leading edge. Both Cdc42 and GRAF1 were required for fluid phase uptake and regulated the generation of transient GRAF1-coated endocytic carriers, distinct from clathrin coated vesicles. GRAF1 was found to transiently assemble at discrete Cdc42-enriched punctae at the plasma membrane resulting in a corresponding decrease in Cdc42 microdomain association. However, Cdc42 captured in its active state was, via a GAP domain mediated interaction, localised together with GRAF1 on accumulated internal structures derived from the cell surface. Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers affected in their endosomal processing. We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism.

National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-111228 (URN)10.1242/jcs.174417 (DOI)000366314900017 ()26446261 (PubMedID)
Available from: 2015-11-11 Created: 2015-11-10 Last updated: 2017-12-01Bibliographically approved
2. GRAF1 sculpts membrane through a regulated oligomerisation reaction
Open this publication in new window or tab >>GRAF1 sculpts membrane through a regulated oligomerisation reaction
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-111219 (URN)
Available from: 2015-11-11 Created: 2015-11-10 Last updated: 2015-11-13
3. The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading
Open this publication in new window or tab >>The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading
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2011 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 22, no 22, 4380-4389 p.Article in journal (Refereed) Published
Abstract [en]

The rho GTPase-activating protein GTPase regulator associated with focal adhesion kinase-1 (GRAF1) remodels membranes into tubulovesicular clathrin-independent carriers (CLICs) mediating lipid-anchored receptor endocytosis. However, the cell biological functions of this highly prevalent endocytic pathway are unclear. In this article, we present biochemical and cell biological evidence that GRAF1 interacted with a network of endocytic and adhesion proteins and was found enriched at podosome-like adhesions and src-induced podosomes. We further demonstrate that these sites comprise microdomains of highly ordered lipid enriched in GRAF1 endocytic cargo. GRAF1 activity was upregulated in spreading cells and uptake via CLICs was concentrated at the leading edge of migrating cells. Depletion of GRAF1, which inhibits CLIC generation, resulted in profound defects in cell spreading and migration. We propose that GRAF1 remodels membrane microdomains at adhesion sites into endocytic carriers, facilitating membrane turnover during cell morphological changes.

Place, publisher, year, edition, pages
Bethesda: American Society for Cell Biology, 2011
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-50518 (URN)10.1091/mbc.E10-12-0936 (DOI)000297164200017 ()
Available from: 2011-12-13 Created: 2011-12-13 Last updated: 2017-12-08Bibliographically approved
4. Endocytic turnover of Rab8 controls cell polarisation
Open this publication in new window or tab >>Endocytic turnover of Rab8 controls cell polarisation
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-111222 (URN)
Available from: 2015-11-11 Created: 2015-11-10 Last updated: 2015-11-13

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Francis, Monika K.

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