Proefschrift Universiteit van Amsterdam. / Auteursnaam op omslag: Quirine Lisman. Met bibliogr., lit. opg.-Met samenvatting in het Nederlands.

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Abstract Anion exchanger 2 (AE2) is a member of the anion exchanger gene family, which includes three additional members, AE1, AE3, and AE4. They are also known as Na+-independent Cl-/HCO3- exchangers, and their major function is to regulate intracellular pH and chloride concentration. All four isoforms have several N-terminally truncated variants that are often expressed cell type specifically. Red blood cells express the full-length AE1 isoform that interacts with ankyrin, an adapter protein linking plasma membrane to the spectrin-based membrane skeleton. This membrane skeleton association is essential for maintaining the membrane integrity of red blood cells. AE3 variants are mainly found in the brain and heart, whereas AE4 is localized in the kidney. Anion exchanger 2 is expressed in every cell line and tissue studied thus far, and it has been mainly localized to the plasma membrane. However, we found two types of localization/targeting of the AE2 protein in several of the cell lines studied. The protein was localized to either the plasma membrane or the Golgi complex, depending on the cell type. The AE2 variant expressed in these cells was identified as the full-length AE2 protein. The determinants of differential intracellular targeting were assessed. We hypothesized that Golgi-AE2 is anchored to the Golgi membranes via its association with the Golgi membrane skeleton. We were able to show that the Golgi localization of AE2 correlated with the cell type specific expression of Ank195, a Golgi membrane skeletal protein. In cells where AE2 was targeted to the plasma membrane, Ank195 was not expressed. In addition, the detergent insolubility and co-redistribution properties of AE2 and Ank195 strongly suggested that these proteins interact with each other. The Golgi membrane skeleton has been shown to be necessary for maintaining the Golgi structure. Our studies were consistent with these findings, showing that in cells in which AE2 expression was reduced by using AE2-specific antisense oligonucleotides, the Golgi complex was dispersed. The spectrin-based membrane skeleton was probably partially detached from the Golgi membranes leading to breakdown of the Golgi structure and disorganization of the microtubules associated with it. The present findings suggest that the targeting of AE2 is cell type specific, and that Golgi-localized AE2 serves as a membrane association site for the spectrin-based Golgi membrane skeleton, thereby participating in the maintenance of the Golgi structure.

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Yes / The contribution of the actin cytoskeleton to the unique architecture of the Golgi complex is manifold. An important player in this process is Coronin7 (CRN7), a Golgi-resident protein that stabilizes F-actin assembly at the trans-Golgi network (TGN) thereby facilitating anterograde trafficking. Here, we establish that CRN7-mediated association of F-actin with the Golgi apparatus is distinctly modulated via the small Rho GTPase Cdc42 and N-WASP. We identify N-WASP as a novel interaction partner of CRN7 and demonstrate that CRN7 restricts spurious F-actin reorganizations by repressing N-WASP ‘hyperactivity’ upon constitutive Cdc42 activation. Loss of CRN7 leads to increased cellular F-actin content and causes a concomitant disruption of the Golgi structure. CRN7 harbours a Cdc42- and Rac-interactive binding (CRIB) motif in its tandem β-propellers and binds selectively to GDP-bound Cdc42N17 mutant. We speculate that CRN7 can act as a cofactor for active Cdc42 generation. Mutation of CRIB motif residues that abrogate Cdc42 binding to CRN7 also fail to rescue the cellular defects in fibroblasts derived from CRN7 KO mice. Cdc42N17 overexpression partially rescued the KO phenotypes whereas N-WASP overexpression failed to do so. We conclude that CRN7 spatiotemporally influences F-actin organization and Golgi integrity in a Cdc42- and N-WASP-dependent manner. / This work was supported by SFB 670 and DFG NO 113/22. K.B. was supported by a fellowship from the NRW International Graduate School “From Embryo to Old Age: the Cell Biology and Genetics of Health and Disease” (IGSDHD), Cologne.

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Protein trafficking between the endoplasmic reticulum (ER) and Golgi complex is regulated by the activity of ADP-ribosylation factors (Arfs). Arf activation by guanine nucleotide exchange factors (GEFs) leads to the recruitment of the coatomer protein COPI and vesicle formation. By using fluorescently-tagged proteins in live cells, we have been able to identify novel functions for Arfs and the Arf-GEF GBF1 at the ER-Golgi intermediate compartment (ERGIC) and mitotic Golgi clusters. We first focused on Arf function at the ERGIC after observing both class I (Arf1) and class II (Arfs 4 and 5) Arfs at this structure. We discovered that class II Arfs remain bound to ERGIC membranes independently of GBF1 activity following treatment with brefeldin A (BFA). Further characterization of the class II Arfs using additional pharmacological agents such as Exo1 and inactive mutant forms of Arf4 demonstrated that the class II Arfs associate with the ERGIC membrane via receptors distinct from GBF1. Our work suggests that GBF1 accumulation on membranes in the presence of BFA is due to loss of Arfs from the membrane rather than the formation of an abortive complex with Arf and GBF1. Next, while studying GBF1 in live cells, we unexpectedly observed GBF1 localizing to large fragmented structures during mitosis. We identified these structures as mitotic Golgi fragments that are positive for GBF1 and COPI throughout mitosis. Again using live cells treated with BFA and Exo1, we demonstrated that GBF1 concentrates on these mitotic fragments suggesting that they are derived from Golgi membranes. By colocalization studies and fluorescence recovery after photobleaching, we demonstrated that these mitotic fragments maintain a cis-to-trans subcompartmental Golgi polarization and membrane dynamics of GBF1 similar to interphase cells. Interestingly, inactivation of GBF1 and loss of COPI from the membranes of the mitotic Golgi fragments did not delay progressing through mitosis. Our results from our second project indicate for the first time that the mitotic Golgi clusters are bona fide Golgi structures that exist throughout mitosis with a functional COPI machinery.