Search

p97 (VCP or valosin containing protein in mammals and Cdc48p in yeast) is a ubiquitously expressed and evolutionarily highly conserved hexameric member of the magnesium-dependent Walker P-loop AAA-ATPases. p97 has been associated with various essential cellular processes including ubiquitin-proteasome[for full text, please go to the a.m. URL], 57th Annual Meeting of the German Society for Neuropathology and Neuroanatomy (DGNN)

Rac proteins are members of a broad family of monomeric Rho GTPases that act as key regulators of the actin cytoskeleton. Rac1A from highly motile Dictyostelium cells binds to the IQGAP-related protein DGAP1, which forms a cortical complex with actin-binding proteins cortexillins. This complex localizes to the rear end of moving cells and to the cleavage furrow of dividing cells, where it supports efficient cytokinesis. To gain further insight into the role of Rac1A in Dictyostelium cells, we created a fluorescent probe that specifically binds its GTP-bound form. A screen based on yeast two-hybrid and GST pull-down assays resulted in the selection of an interaction partner specific for the active form of Rac1A, a GBD (GTPase-binding domain) from rat PAK1 kinase. PAK1_GBD was fused N-terminally to YFP and expressed in Dictyostelium cells. In non-motile cells, our probe was strongly enriched throughout the cortex, while in motile cells it always localized to the leading edge (Fig. 1). During phagocytosis and macropinocytosis, the probe localized to endocytotic cups. During cytokinesis and chemotaxis the probe didn’t show any prominent localization. In order to demonstrate an interaction between PAK1_GBD and GTP-Rac1A in living cells, we employed fluorescence resonance energy transfer (FRET) approach. A unimolecular probe was constructed, where PAK1_GBD and Rac1A were sandwiched between florescent proteins YFP and mRFP. This FRET probe has a prominent cortical localization and measurements by sensitized emission of the acceptor indicate that PAK1_GBD and GTP-Rac1A interact in the cortex of living cells. Altogether, our results demonstrate that localization of active Rac1A, as reported by our probe PAK1_GBD-YFP, does not correspond to localization of GFP-DGAP1 and GFP-cortexillin, the other components of the aforementioned cortical complex that contains Rac1A. Based on these results, we propose that Rac1A has a dual role in regulation of the actin cytoskeleton. In addition to its established role in recruitment of the DGAP1-cortexillin complex to the rear parts of a polarized cell, it also participates in signaling pathways that control de novo actin polymerization at the protruding regions of the cell.

Monitoring the dynamics of Rac proteins in Dictyostelium discoideum

Rac proteins are members of a broad family of monomeric Rho GTPases that act as key regulators of the actin cytoskeleton. We are using genetically tractable and highly motile Dictyostelium discoideum cells to study localization, dynamics and activity of Rac1A in processes that involve actin remodeling. To gain insight into its role in D. discoideum, we created a fluorescent probe that specifically binds the active form of Rac1A. Since Dictyostelium Rac GTPases are strongly related to each other, we employed yeast two hybrid (Y2H) assay to test the interaction between 4 potential binding partners and 11 Rac proteins. In this way, we could pinpoint the interactor specific for active Rac1A. We tested an IQGAP-related protein DGAP1, which binds active Rac1A within a tetrameric cortical complex, and its GTPase-binding domain (GBD). We also tested regulatory domains of two other Rac downstream effectors that contain CRIB (Cdc/Rac interactive binding) motif: PBD (p21-binding domain) from Dictyostelium PAKa kinase and GBD from rat PAK1 kinase. Only GBD from rat Pak1 interacted under stringent conditions in Y2H assay with Rac1A. Moreover, it was the only partner that did not interact with the constitutively inactive form of Rac1A. This interaction was confirmed by GST pull-down assay. Therefore, PAK1_GBD was fused N-terminally to YFP and expressed in wild-type cells. In non-motile cells, our probe was strongly enriched throughout the cortex, while in motile cells it always localized to the leading edge. In phagocytosis and macropinocytosis, it localized to the endocytotic cup. During cytokinesis the probe didn’ t show any prominent localization.

A novel RacGEF is involved in regulation of cytokinesis, actin organization and cell motility in Dictyostelium.

DdLimE regulates cell motility and cytokinesis in Dictyostelium. To specify its function, we generated knock-out mutants and analyzed mitosis by marking the mitotic apparatus with GFP--tubulin. Characteristic of DdLimE-null cells is a late reversal of cytokinesis caused by backward movement of the incipient daughter cells. This process of retro-cytokinesis is accompanied by a delay in disassembly of the mitotic spindle. The length of interphase microtubules is increased and their depolymerization at prophase is impaired. These data indicate that DdLimE links the cortical actin network, where it is located, to the microtubule system, whose dynamics it regulates.

Signaling molecules as regulators of cytokinesis and cell motility.

Cytokinesis in eukaryotic organisms is under the control of small GTP-binding proteins, although the underlying molecular mechanisms are not fully understood. Cortexillins are actin-binding proteins whose activity is crucial for cytokinesis in Dictyostelium. Here we show that the IQGAP-related and Rac1-binding protein DGAP1 specifically interacts with the C-terminal, actin-bundling domain of cortexillin I. Like cortexillin I, DGAP1 is enriched in the cortex of interphase cells and translocates to the cleavage furrow during cytokinesis. The activated form of the small GTPase Rac1A recruits DGAP1 into a quaternary complex with cortexillin I and II. In DGAP1(-) mutants, a complex can still be formed with a second IQGAP-related protein, GAPA. The simultaneous elimination of DGAP1 and GAPA, however, prevents complex formation and localization of the cortexillins to the cleavage furrow. This leads to a severe defect in cytokinesis, which is similar to that found in cortexillin I/II double-null mutants. Our observations define a novel and functionally significant signaling pathway that is required for cytokinesis.

Cortexillins are actin-bundling proteins that form a parallel two-stranded coiled-coil rod. Actin-binding domains of the alpha-actinin/spectrin type are located N-terminal to the rod and unique sequence elements are found in the C-terminal region. Domain analysis ill vitro revealed that the N-terminal domains are not responsible for the strong actin-filament bundling activity of cortexillin I. The strongest activity resides in the C-terminal region. Phosphatidylinositol 4, 5-bisphosphate (PIP2) suppresses this bundling activity by binding to a C-terminal nonapeptide sequence. These data define a new PIP2-regulated actin-bundling site. In vivo the PIP2-binding motif enhances localization of a C-terminal cortexillin I fragment to the cell cortex and improves the rescue of cytokinesis. This motif is not required, however, for translocation to the cleavage furrow. A model is presented proposing that cortexillin translocation is based on a mitotic cycle of polar actin polymerization and midzone depolymerization.

Dictyostelium is a suitable experimental system in which to study the effects of mutations in actin-binding proteins on cell motility. Three cytoskeletal mutants that show distinct alterations in cell shape, chemotactic movement and cytokinesis serve to illustrate the diversity of phenotypes. Cells lacking talin, a protein which in many mammalian cell types is a constituent of focal complexes that link the actin cytoskeleton to the plasma membrane, are strongly impaired in adhesion to external surfaces. Coronin is an actin-associated protein that belongs to the WD-repeat family of proteins, which are engaged in protein-protein interactions involved in signalling pathways. Cells lacking coronin build large hyaline protrusions at their leading edge, diagnostic of an imbalance in the actin polymerization/depolymerization cycle. Cells devoid of a pair of cortexillins, which are novel members of the spectrin/alpha-actinin superfamily of actin-binding proteins, form an atypical cleavage furrow on a solid surface and fail to divide in suspension. Other mutants in which one or more actin-binding proteins have been knocked out have weaker phenotypes. With these mutants, cells need to be subjected to special conditions in order to reveal an effect on cell motility. For instance, only on weakly adhesive surfaces is a disturbance in the spatio-temporal co-ordination of protrusion and retraction of the cell body, and of the attachment to and detachment from a substratum, observed in a mutant that lacks three actin-binding proteins: alpha-actinin, 120 kDa F-actin gelation factor and severin.