Maria Stumpf

Oligomerization, F-actin Interaction, and Membrane Association of the Ubiquitous Mammalian Coronin 3 Are Mediated by Its Carboxyl Terminus

Coronin 3 is a ubiquitously expressed member of the coronin protein family in mammals. In fibroblasts and HEK 293 cells, it is localized both in the cytosol and in the submembranous cytoskeleton, especially at lamellipodia and membrane ruffles. The carboxyl terminus of all coronins contains a coiled coil suggested to mediate dimerization. We show here that in contrast to other coronin homologues, the recombinant human coronin 3 carboxyl terminus forms oligomers rather than dimers, and that this part is sufficient to bind to and cross-link F-actin in vitro. The carboxyl terminus alone also conferred membrane association in vivo, and removal of the coiled coil abolished membrane localization but notin vitro F-actin binding. Coronin 3 is exclusively extracted as an oligomer from both the cytosol and the membrane fraction. Because oligomerization was not reported for other coronins, it might be a key feature governing coronin 3-specific functions. Cytosolic coronin 3 showed a high degree of phosphorylation, which is likely to regulate the subcellular localization of the protein.

Strumpellin is a novel valosin-containing protein binding partner linking hereditary spastic paraplegia to protein aggregation diseases

Mutations of the human valosin-containing protein gene cause autosomal-dominant inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia. We identified strumpellin as a novel valosin-containing protein binding partner. Strumpellin mutations have been shown to cause hereditary spastic paraplegia. We demonstrate that strumpellin is a ubiquitously expressed protein present in cytosolic and endoplasmic reticulum cell fractions. Overexpression or ablation of wild-type strumpellin caused significantly reduced wound closure velocities in wound healing assays, whereas overexpression of the disease-causing strumpellin N471D mutant showed no functional effect. Strumpellin knockdown experiments in human neuroblastoma cells resulted in a dramatic reduction of axonal outgrowth. Knockdown studies in zebrafish revealed severe cardiac contractile dysfunction, tail curvature and impaired motility. The latter phenotype is due to a loss of central and peripheral motoneuron formation. These data imply a strumpellin loss-of-function pathogenesis in hereditary spastic paraplegia. In the human central nervous system strumpellin shows a presynaptic localization. We further identified strumpellin in pathological protein aggregates in inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, various myofibrillar myopathies and in cortical neurons of a Huntingtons disease mouse model. Beyond hereditary spastic paraplegia, our findings imply that mutant forms of strumpellin and valosin-containing protein may have a concerted pathogenic role in various protein aggregate diseases.

Coronin 7, the mammalian POD-1 homologue, localizes to the Golgi apparatus

Coronins constitute an evolutionary conserved family of WD‐repeat actin‐binding proteins. Their primary function is thought to be regulating the actin cytoskeleton. Apart from that, several coronins were indirectly shown to participate in vesicular transport, establishment of cell polarity and cytokinesis. Here, we report a novel mammalian protein, coronin 7 (crn7), which is significantly different from other mammalian coronins in its domain architecture. Crn7 possesses two stretches of WD repeats in contrast to the other coronins only having one. The protein is expressed throughout the mouse embryogenesis and is strongly upregulated in brain and developing structures of the immune system in the course of development. In adult animals, both crn7 mRNA and protein are abundantly present in most organs, with significantly higher amounts in brain, kidney, thymus and spleen and lower amounts in muscle. At the subcellular level, the bulk of the protein appears to be present in the cytosol and in large cytosolic complexes. However, a significant portion of the protein is detected on vesicle‐like cytoplasmic structures as well as on the cis‐Golgi. In the Golgi region, crn7 staining appears broader than that of the cis‐Golgi markers Erd2p and β‐COP, still, the trans‐Golgi network appears predominantly crn7‐negative. Importantly, the membrane‐associated form of crn7 protein is phosphorylated on tyrosine residues, whereas the cytosolic form is not. Crn7 is the first coronin protein proven to localize to the Golgi membrane. We conclude that it plays a role in the organization of intracellular membrane compartments and vesicular trafficking rather than in remodeling the cytoskeleton.

Expression of coronin-3 (coronin-1C) in diffuse gliomas is related to malignancy†

Coronin‐3 (coronin‐1C), a homotrimeric F‐actin binding protein, has been shown to be important for cell migration and brain morphogenesis. Here, we present for the first time a detailed analysis of the expression pattern of coronin‐3 in human brain tumours and demonstrate that coronin‐3 expression correlates with malignant phenotype in diffuse gliomas. In general, the expression of coronin‐3 varies in different brain tumour entities. However, in diffuse gliomas, the number of coronin‐3 expressing tumour cells correlates with the degree of malignancy. High‐grade gliomas, such as anaplastic astrocytomas, anaplastic oligodendrogliomas, anaplastic oligoastrocytomas and glioblastomas, show high numbers of tumour cells positive for coronin‐3, while diffuse low‐grade gliomas, such as diffuse astrocytomas, oligodendrogliomas and oligoastrocytomas, exhibit low numbers of coronin‐3‐positive tumour cells. In order to explore and verify a contribution of coronin‐3 to the malignant phenotype of diffuse gliomas, we employed an efficient shRNA‐mediated coronin‐3 knockdown in U373 and A172 human glioblastoma cells. Coronin‐3 knockdown glioblastoma cells exhibited reduced levels of cell proliferation, cell motility and invasion into extracellular matrix compared to control cells. Together, our findings demonstrate evidence for a contribution of coronin‐3 expression in the malignant progression of diffuse gliomas. Copyright

Phosphorylation of CRN2 by CK2 regulates F-actin and Arp2/3 interaction and inhibits cell migration

CRN2 (synonyms: coronin 1C, coronin 3) functions in the re-organization of the actin network and is implicated in cellular processes like protrusion formation, secretion, migration and invasion. We demonstrate that CRN2 is a binding partner and substrate of protein kinase CK2, which phosphorylates CRN2 at S463 in its C-terminal coiled coil domain. Phosphomimetic S463D CRN2 loses the wild-type CRN2 ability to inhibit actin polymerization, to bundle F-actin, and to bind to the Arp2/3 complex. As a consequence, S463D mutant CRN2 changes the morphology of the F-actin network in the front of lamellipodia. Our data imply that CK2-dependent phosphorylation of CRN2 is involved in the modulation of the local morphology of complex actin structures and thereby inhibits cell migration.

Coronin 3 and its role in murine brain morphogenesis

Coronins belong to the fundamental WD40‐repeat proteins. They are mainly found at the submembraneous area, they bind F‐actin in vitro, and most of the seven mammalian coronins have unclear roles. Coronin 3 is abundantly expressed in the adult CNS. All murine brain areas express coronin 3 during embryogenesis and the first postnatal stages. Expression in grey matter decreases postnatally, except for hippocampal pyramidal and dentate gyrus neurons, and cerebellar Purkinje cells, while levels in white matter increase in the course of myelination. Consistently, coronin 3 is abundant in differentiating neuro‐2a and PC‐12 cells and in primary oligodendrocytes. Treatment with PKC activator PMA reduced coronin 3 protein levels. To address its functions, neuro‐2a and PC‐12 cells were transfected with GFP‐tagged coronin 3 versions. Full‐length coronin 3 among other areas localized to outgrowing neurites, whereas truncated proteins efficiently suppressed neurite formation. Our results favour a role for coronin 3 in neuron morphogenesis and possibly migration.

Heteromeric p97/p97R155C Complexes Induce Dominant Negative Changes in Wild-Type and Autophagy 9-Deficient Dictyostelium strains

Heterozygous mutations in the human VCP (p97) gene cause autosomal-dominant IBMPFD (inclusion body myopathy with early onset Paget’s disease of bone and frontotemporal dementia), ALS14 (amyotrophic lateral sclerosis with or without frontotemporal dementia) and HSP (hereditary spastic paraplegia). Most prevalent is the R155C point mutation. We studied the function of p97 in the social amoeba Dictyostelium discoideum and have generated strains that ectopically express wild-type (p97) or mutant p97 (p97R155C) fused to RFP in AX2 wild-type and autophagy 9 knock-out (ATG9KO) cells. Native gel electrophoresis showed that both p97 and p97R155C assemble into hexamers. Co-immunoprecipitation studies revealed that endogenous p97 and p97R155C-RFP form heteromers. The mutant strains displayed changes in cell growth, phototaxis, development, proteasomal activity, ubiquitinylated proteins, and ATG8(LC3) indicating mis-regulation of multiple essential cellular processes. Additionally, immunofluorescence analysis revealed an increase of protein aggregates in ATG9KO/p97R155C-RFP and ATG9KO cells. They were positive for ubiquitin in both strains, however, solely immunoreactive for p97 in the ATG9KO mutant. A major finding is that the expression of p97R155C-RFP in the ATG9KO strain partially or fully rescued the pleiotropic phenotype. We also observed dose-dependent effects of p97 on several cellular processes. Based on findings in the single versus the double mutants we propose a novel mode of p97 interaction with the core autophagy protein ATG9 which is based on mutual inhibition.

Molecular mechanism underlying the association of Coronin-7 with Golgi membranes

Abstract.Coronin-7 (Crn7) is a ubiquitous mammalian WD40-repeat protein that localizes to the Golgi complex, interacts with AP-1 adaptor complex via binding of a tyrosine-288-based sorting signal to the μ1-subunit of AP-1, and participates in the maintenance of the Golgi structure and function. Here, we define the requirements for the recruitment of Crn7 from the cytosol to the Golgi. We establish that Src activity is indispensable for the interaction of Crn7 with Golgi membranes. Crn7 binds Src in vivo and can be phosphorylated by recombinant Src in vitro. We demonstrate that tyrosine-758 is the major Src phosphorylation site. Further, to be targeted to membranes Crn7 requires the presence of cargo in the Golgi complex. Finally, downregulation of the μ1-subunit of AP-1 leads to the dispersal of Crn7 from the Golgi membranes. We propose a mechanism whereby sequential events of protein interaction and posttranslational modification result in the membrane targeting of Crn7.

Structural and Functional Diversity of Novel Coronin 1C (CRN2) Isoforms in Muscle

Coronin 1C (synonyms: coronin-3, CRN2), a WD40 repeat-containing protein involved in cellular actin dynamics, is ubiquitously expressed in human tissues. Here, we report on the identification and functional characterization of two novel coronin 1C isoforms, referred to as CRN2i2 and CRN2i3, which also associate with F-actin. Analyses of the coronin 1C gene disclosed a single promoter containing binding sites for myogenic regulatory factors and an alternative first exon 1b present in intron 1, which give rise to the novel isoforms. Chromatin immunoprecipitation studies demonstrate MyoD binding to a region of the CRN2 gene, which contains a highly conserved E-box element in exon 1a. Gel-filtration assays suggest that the largest isoform 3 exists as a monomer, in contrast to isoform 1 and isoform 2 appearing as trimers. CRN2i3, which can be induced by MyoD, is exclusively expressed in well-differentiated myoblasts as well as in mature skeletal muscle tissue. In human skeletal muscle, CRN2i3 is a novel component of postsynaptic neuromuscular junctions and thin filaments of myofibrils. Together, our findings postulate a role for CRN2 isoforms in the structural and functional organization of F-actin in highly ordered protein complexes.

Coronin7 regulates WASP and SCAR through CRIB mediated interaction with Rac proteins.

Coronin7 (CRN7) stabilizes F-actin and is a regulator of processes associated with the actin cytoskeleton. Its loss leads to defects in phagocytosis, motility and development. It harbors a CRIB (Cdc42- and Rac-interactive binding) domain in each of its WD repeat domains which bind to Rac GTPases preferably in their GDP-loaded forms. Expression of wild type CRN7 in CRN7 deficient cells rescued these defects, whereas proteins with mutations in the CRIB motifs which were associated with altered Rac binding were effective to varying degrees. The presence of one functional CRIB was sufficient to reestablish phagocytosis, cell motility and development. Furthermore, by molecular modeling and mutational analysis we identified the contact regions between CRN7 and the GTPases. We also identified WASP, SCAR and PAKa as downstream effectors in phagocytosis, development and cell surface adhesion, respectively, since ectopic expression rescued these functions.