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BPM1 interacts with components of the DDR complex and stimulates DNA methylation at CHH sites, suggesting its involvement in the RdDM methylation pathway. The best-known function of MATH-BTB proteins, including Arabidopsis BPM proteins, is their role as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin-proteasome pathway. This paper reports a new CUL3-independent role of BPM1 in RNA-directed DNA methylation (RdDM). Using quantitative and qualitative Y2H, pull down, microscale thermophoresis and FRET-FLIM, we demonstrate that BPM1 interacts with DMS3 and RDM1, components of the chromatin remodeling DDR complex involved in the recruitment of the RdDM methylation machinery. All three proteins colocalized predominantly in the nucleus. The MATH domain, which specifically binds proteins destined for degradation, was not essential for interactions with DMS3 and RDM1. In plants overexpressing BPM1, endogenous DMS3 protein levels were stable, indicating that BPM1 does not induce proteasomal degradation. In RDM1-overexpressing plants, RDM1 was not ubiquitinated. Together, these results suggest that BPM1 does not mediate the degradation of DMS3 and RDM1. Additionally, overexpression of BPM1 caused increased global methylation levels as well as CHH methylation in promoters of two RdDM-regulated genes, FWA and CML41. Overall, BPM1 seems to have a stimulating effect on RdDM activity, and this role appears to be unrelated to its known function as a Cul3-based E3 ligase adaptor.

LrrkA is a Dictyostelium discoideum kinase with leucine-rich repeats. LrrkA stimulates Kil2 and intra-phagosomal killing of ingested bacteria in response to folate. In this study, we show that genetic inactivation of lrrkA also causes a previously unnoticed phenotype: lrrkA KO cells exhibit enhanced phagocytosis and cell motility compared to parental cells. This phenotype is cell autonomous, is reversible upon re-expression of LrrkA, and is not due to an abnormal response to inhibitory quorum-sensing factors secreted by D. discoideum in its medium. In addition, folate increases motility in parental D. discoideum cells, but not in lrrkA KO cells, suggesting that LrrkA plays a pivotal role in the cellular response to folate. On the contrary, lrrkA KO cells regulate gene transcription in response to folate in a manner indistinguishable from parental cells. Overall, based on analysis of mutant phenotypes, we identify gene products that participate in the control of intracellular killing, cell motility, and gene transcription in response to folate. These observations reveal a mechanism by which D. discoideum encountering bacterially-secreted folate can migrate, engulf, and kill bacteria more efficiently.

Both Dictyostelium amoebae and mammalian cells are endowed with an elaborate actin cytoskeleton that enables them to perform a multitude of tasks essential for survival. Although these organisms diverged more than a billion years ago, their cells share the capability of chemotactic migration, large-scale endocytosis, binary division effected by actomyosin contraction, and various types of adhesions to other cells and to the extracellular environment. The composition and dynamics of the transient actin-based structures that are engaged in these processes are also astonishingly similar in these evolutionary distant organisms. The question arises whether this remarkable resemblance in the cellular motility hardware is accompanied by a similar correspondence in matching software, the signalling networks that govern the assembly of the actin cytoskeleton. Small GTPases from the Rho family play pivotal roles in the control of the actin cytoskeleton dynamics. Indicatively, Dictyostelium matches mammals in the number of these proteins. We give an overview of the Rho signalling pathways that regulate the actin dynamics in Dictyostelium and compare them with similar signalling networks in mammals. We also provide a phylogeny of Rho GTPases in Amoebozoa, which shows a variability of the Rho inventories across different clades found also in Metazoa.

Macropinocytosis and phagocytosis are evolutionarily conserved forms of bulk endocytosis used by cells to ingest large volumes of fluid and solid particles, respectively. Both processes are regulated by Ras signaling, which is precisely controlled by mechanisms involving Ras GTPase activating proteins (RasGAPs) responsible for terminating Ras activity on early endosomes. While regulation of Ras signaling during large-scale endocytosis in WT Dictyostelium has been, for the most part, attributed to the Dictyostelium ortholog of human RasGAP NF1, in commonly used axenic laboratory strains, this gene is mutated and inactive. Moreover, none of the RasGAPs characterized so far have been implicated in the regulation of Ras signaling in large-scale endocytosis in axenic strains. In this study, we establish, using biochemical approaches and complementation assays in live cells, that Dictyostelium IQGAP-related protein IqgC interacts with active RasG and exhibits RasGAP activity toward this GTPase. Analyses of iqgC − and IqgC-overexpressing cells further revealed participation of this GAP in the regulation of both types of large-scale endocytosis and in cytokinesis. Moreover, given the localization of IqgC to phagosomes and, most prominently, to macropinosomes, we propose IqgC acting as a RasG-specific GAP in large-scale endocytosis. The data presented here functionally distinguish IqgC from other members of the Dictyostelium IQGAP family and call for repositioning of this genuine RasGAP outside of the IQGAP group.

Nucleoside diphosphate kinases (NDPK/NME/Nm23) are housekeeping enzymes responsible for the maintenance of the cellular NTP pool by catalyzing the exchange of terminal phosphate from trinucleotides to dinucleotides through a high-energy phosphohistidine intermediate. At least 80 % of the cytoplasmic NDPK activity is being exerted by NME1/NDPK A and NME2/NDPK B which combine to form isoenzymes (A6, A5B1, …, B6). The NME1/NDPK A and NME2/NDPK B subunits have a variety of additional biological functions, from metastasis suppression and DNA damage repair to proliferation and development. Although eukaryotic NDPKs are active only as hexamers, it is unclear whether other NME functions require the hexameric form and how the isoenzyme composition varies in different cellular compartments. To investigate the effect of DNA damage on the intracellular localization of NME1 and NME2 and the composition of NME oligomers in the nucleus and cytoplasm, we used live-cell imaging and the FRET/FLIM technique. We demonstrated that exogenous NME1 and NME2 proteins colocalize in the cytoplasm of non-irradiated cells and move simultaneously to the nucleus after gamma irradiation. The FRET/FLIM experiments suggest that there is a slight shift in the homomer/heteromer balance between the nucleus and cytoplasm after DNA damage. Overall, our results suggest that NME1 and NME2 engage in mutual functions in the nucleus after irradiation, possibly performing specific functions in their homomeric state.

NME proteins were reported to influence signal transduction activity of small GTPases from the Ras superfamily by diverse mechanisms in addition to their generic NDP kinase activity, which replenishes the cytoplasmic pool of GTP. Comprehensive evidence shows that NME proteins modulate the activity of Ras GTPases, in particular members of the Rho family, via binding to their major activators GEFs. Direct interaction between several NMEs and Ras GTPases was also indicated in vitro and in vivo. These modes of regulation are mainly independent of the NME’s kinase activity. NMEs also modulate the Ras-mediated signal transduction by interfering with the formation of a Ras signaling complex at the plasma membrane. In several examples, NMEs were proposed to perform the role of GAP proteins by promoting hydrolysis of the bound GTP, but this activity still requires additional verification. Early suggestions that NMEs can activate small GTPases by direct phosphorylation of the bound GDP, or by high- rate loading of GTP onto a closely apposed GTPase, were largely dismissed. In this review article, we survey and put into perspective published examples of identified and hypothetical mechanisms of Ras signaling modulation by NME proteins. We also point out involvement of NMEs in the transcriptional regulation of components of Ras GTPases- mediated signal transduction pathways, and reciprocal regulation of NME function by small GTPases, particularly related to NME’s binding to membranes.

4-Amino-N-adamantylphthalimide (1) is a dye with moderate absorptivity (in CH3CN e363 = 4200 M−1 cm−1) and high quantum yield of fluorescence (ΦF = 0.15–0.80) that exhibits fluorosolvatochromic properties. The dye can be excited at 405 nm and the position of fluorescence maximum and the Stokes shift are well correlated with the ET(30) parameter. The excitation in the near-visible part of the spectrum and low cytotoxicity allow use of the dye in live cell microscopy. Due to its amphiphilic character, the dye stains artificial membranes in liposomes. Using confocal microscopy on two human cancer cell lines, we have shown that 1 stains primarily intracellular lipid droplets. Colocalization experiments with different organelle markers indicated that 1 additionally stains mitochondrial membranes. The fluorosolvatochromism of 1 allows the simultaneous visualization of mitochondria and intracellular lipid droplets in two separate emission channels, which has a potential use in cells and tissues exhibiting intense oxidative metabolism of lipids.

The RecA protein is a key bacterial recombination enzyme that catalyzes pairing and strand exchange between homologous DNA duplexes. In Escherichia coli, RecA protein assembly on DNA is mediated either by the RecBCD or RecFOR protein complexes. Correspondingly, two recombination pathways, RecBCD and RecF (or RecFOR), are distinguished in E. coli. Inactivation of both pathways in recB(CD) recF(OR) mutants results in severe recombination deficiency. Here we describe a novel, RecBCD- RecFOR-independent (RecBFI) recombination pathway that is active in ΔrecBCD sbcB15 sbcC(D) ΔrecF(OR) mutants of E. coli. In transductional crosses, these mutants show only four-fold decrease of recombination frequency relative to the wild-type strain. At the same time they recombine 40- to 90- fold better than their sbcB+ sbcC+ and ΔsbcB sbcC counterparts. The RecBFI pathway strongly depends on recA, recJ and recQ gene functions, and moderately depends on recG and lexA functions. Inactivation of dinI, helD, recX, recN, radA, ruvABC and uvrD genes has a slight effect on RecBFI recombination. After exposure to UV and gamma irradiation, the ΔrecBCD sbcB15 sbcC ΔrecF mutants show moderately increased DNA repair proficiency relative to their sbcB+ sbcC+ and ΔsbcB sbcC counterparts. However, introduction of recA730 allele (encoding RecA protein with enhanced DNA binding properties) completely restores repair proficiency to ΔrecBCD sbcB15 sbcC ΔrecF mutants, but not to their sbcB+ sbcC+ and ΔsbcB sbcC derivatives. Fluorescence microscopy with UV-irradiated recA-gfp fusion mutants suggests that the kinetics of RecA filament formation might be slowed down in the RecBFI pathway. Inactivation of 3'-5' exonucleases ExoVII, ExoIX and ExoX cannot activate the RecBFI pathway in ΔrecBCD ΔsbcB sbcC ΔrecF mutants. Taken together, our results show that the product of the sbcB15 allele is crucial for RecBFI pathway. Besides protecting 3' overhangs, SbcB15 protein might play an additional, more active role in formation of the RecA filament.

The model organism D. discoideum is well suited to investigate basic questions of molecular and cell biology, particularly those related to the structure, regulation, and dynamics of the cytoskeleton, signal transduction, cell-cell adhesion, and development. D. discoideum cells make use of Rho-regulated signaling pathways to reorganize the actin cytoskeleton during chemotaxis, endocytosis, and cytokinesis. In this organism the Rho family encompasses 20 members, several belonging to the Rac subfamily, but there are no representatives of the Cdc42 and Rho subfamilies. Here we present protocols suitable for monitoring the actin polymerization response and the activation of Rac upon stimulation of aggregation-competent cells with the chemoattractant cAMP, and for monitoring the localization and dynamics of Rac activity in live cells.

Proteins are typically categorized into protein families based on their domain organization. Yet, evolutionarily unrelated proteins can also be grouped together according to their common functional roles. Sequestering proteins constitute one such functional class, acting as macromolecular buffers and serving as an intracellular reservoir ready to release large quantities of bound proteins or other molecules upon appropriate stimulation. Another functional protein class comprises effector proteins, which constitute essential components of many intracellular signal transduction pathways. For instance, effectors of small GTP-hydrolases are activated upon binding a GTP-bound GTPase and thereupon participate in downstream interactions. Here we describe a member of the IQGAP family of scaffolding proteins, DGAP1 from Dictyostelium, which unifies the roles of an effector and a sequestrator in regard to the small GTPase Rac1. Unlike classical effectors, which bind their activators transiently leading to short-lived signaling complexes, interaction between DGAP1 and Rac1-GTP is stable and induces formation of a complex with actin-bundling proteins cortexillins at the back end of the cell. An oppositely localized Rac1 effector, the Scar/WAVE complex, promotes actin polymerization at the cell front. Competition between DGAP1 and Scar/WAVE for the common activator Rac1-GTP might provide the basis for the oscillatory re-polarization typically seen in randomly migrating Dictyostelium cells. We discuss the consequences of the dual roles exerted by DGAP1 and Rac1 in the regulation of cell motility and polarity, and propose that similar signaling mechanisms may be of general importance in regulating spatiotemporal dynamics of the actin cytoskeleton by small GTPases.

Over time we have come to appreciate that the complex regulation of Rho GTPases involves additional mechanisms beyond the activating role of RhoGEFs, the inactivating function of RhoGAPs, and the sequestering activity of RhoGDIs. One class of regulatory mechanisms includes direct modifications of Rho proteins such as isoprenylation, phosphorylation, and SUMOylation. Rho GTPases can also regulate each other by means of crosstalk signaling, which is again mostly mediated by GEFs, GAPs, and GDIs. More complex mutual regulation ensues when and where two or more Rho proteins activate a common molecular target, i.e. share a common effector. We have recently unraveled a reciprocal mechanism wherein spatiotemporal dynamics of Rac1 activity during migration of Dictyostelium cells is apparently regulated by antagonizing interactions of Rac1-GTP with two distinct effectors. By monitoring specific fluorescent probes, activated Rac1 is simultaneously present at the leading edge, where it participates in Scar/WAVE-mediated actin polymerization, and at the trailing edge, where it induces formation of a DGAP1/cortexillin actin- bundling complex. Strikingly, in addition to their opposed localization, the two populations of activated Rac1 also display opposite kinetics of recruitment to the plasma membrane upon stimulation by chemoattractants. These findings with respect to Rac1 in Dictyostelium suggest a novel principle for regulation of Rho GTPase activity that might also play a role in other cell types and for other Rho family members.

Formins and Arp2/3 complex are major actin assembly factors in eukaryotic cells. While Arp2/3 complex induces branching of the existing filaments, thus creating a dense actin meshwork at the cell's leading edge, formins promote nucleation and elongation of linear actin filaments, mostly in filopodia, stress fibers and cytokinetic rings1. Formins are defined by the presence of a highly conserved C-terminal formin homology 2 (FH2) domain preceded by an FH1 domain, which are necessary and sufficient to nucleate actin polymerisation and elongate filaments2. Diaphanous-related formins (DRFs) constitute a conserved subfamily in which core FH1 and FH2 domains are flanked by regulatory domains: a GTPase binding domain (GBD), a Diaphanous-inhibitory domain (DID) and a dimerization domain (DD) at the N terminus, and a C-terminal Diaphanous-autoregulatory domain (DAD). DRFs are autoinhibited through interaction between DID and DAD and binding of a Rho GTPase to GBD releases this inhibition. We have characterized formin G (ForG) from the professional phagocyte Dictyostelium discoideum as a Diaphanous-related formin that fully conforms to the canonical domain organization of DRFs, and described its role in large-scale endocytosis3. Biochemical assays identified ForG as a rather week actin nucleator that efficiently elongates actin filaments. Live cell imaging demonstrated that ForG was enriched at the plasma membrane during the formation and maturation of macropinocytic cups. Analysis of phagocytosis also revealed localization to nascent phagocytic cups and ForG remained associated with the membrane over the entire course of engulfment and internalization of yeast particle (Fig. 1). Real-time assays of the nutrient uptake showed markedly decreased fluid-phase uptake of TRITC-dextran and phagocytosis of TRITC-labeled yeast particles in forG null cells compared with control cells. Interestingly, yeast-two-hybrid (Y2H) assay failed to identify any interaction of ForG with Rho family GTPases, although DRFs have been previously shown to act exclusively as effectors of Rho GTPases1. Strikingly, another Y2H screen and bimolecular fluorescence complementation (BiFC) in vivo identified specifically activated forms of RasB and RasG, but not their dominant negative variants, as ForG interactors (Fig. 2). The role of RasG in macropinocytosis has already been well established. To assess the role of RasB in this process, we have created rasB null cells and tested their nutrient uptake. The uptake of large particles was substantially impaired in rasB deficient cells, while pinocytosis rates were only slightly diminished. Taken together, these data demonstrate the significance of ForG for actin assembly in endocytic structures, with RasB being mainly responsible for its regulation during phagocytosis of large particles, and RasG being the main regulator of ForG in macropinocytosis.

Small Rho GTPases are major regulators of the actin cytoskeleton dynamics in eukaryotic cells. Sophisticated tools used to investigate their activity in living cells include probes based on fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC), and photoactivation. However, such methods are of limited use in quickly migrating cells due to a short time available for image acquisition leading to a low signal-to-noise ratio. Attempts to remedy this effect by increasing the intensity of illumination are restricted by photobleaching of probes and the cell photosensitivity. Here we present design and characterization of a new fluorescent probe that selectively binds to active form of Rac1 GTPases, and demonstrate its superior properties for imaging in highly motile Dictyostelium cells. The probe is based on the GTPase-binding domain (GBD) from DPAKa kinase, and was selected on the basis of yeast two-hybrid screen, GST pull-down assay, and FRET measurements by fluorescence lifetime imaging microscopy (FLIM). DPAKa(GBD) probe binds specifically to GTP-bound Rac1 at the cell membrane and features a low cytoplasmic background. The main advantage of DPAKa(GBD) in comparison to similar probes is its finely graded intensity distribution along the entire plasma membrane, which enables quantitative measurements of the Rac1 activity in different parts of the membrane. Finally, expression of DPAKa(GBD) induces no adverse effects on cell growth, motility and cytokinesis.

The Hedgehog-Gli (Hh-Gli) signaling pathway was examined in ovarian dermoids, which show characteristics of both tumors and developmental malformations. Dermoids are classified as mature teratomas that present differentiation into various tissues, mostly epidermal elements such as glands, multilayered epithelium, hair follicles and occasionally bone and cartilage. Their development is attributed to aberrant meiosis of germinal cells within the ovary. We showed activation of the Hh-Gli signaling in ovarian dermoid primary cultures. Cyclopamine treatment slows down cell proliferation, while the Sonic Hedgehog (Shh) protein stimulates cell proliferation and induces internalization of the Patched (Ptch) protein, which accumulates in the form of granules in the cytoplasm, colocalized with the Shh protein. Cyclopamine treatment decreases Gli1 localization in the nucleus compared to non-treated cells. Based on our observations, the mechanism of Hedgehog activation in the ovarian dermoids could be the ligand-dependent autocrine pathway, which can also be stimulated by paracrine signals.

We describe a simple optical configuration for dark-field microscopy at low magnification, realized with the use of standard microscope components. An inherent high contrast makes this method attractive for computer-assisted tracking and counting of microorganisms. We applied this setup for dark-field microscopy to measure the speed of migrating Dictyostelium amoebae.

Introduction Integrins are heterodimeric glycoproteins that bind cells to extracellular matrix proteins. Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) form that facilitate the linkage between integrins and the actin cytoskeleton and permit bidirectional signalling. The αV integrin is expressed in most tumour cells where it regulates an array of cellular functions and plays a role in anti-tumour drug resistance. The aim of our work was to assess αV-dependent changes in IAC composition in MDA-MB-435S melanoma cells in order to better understand the increased sensitivity to paclitaxel and vincristine upon integrin αV knockdown. Material and methods Integrin αV-specific shRNA was cloned into pSUPER.puro, transfected into MDA-MB-435S cells using Lipofectamine, and cell clones were selected using puromycin. The sensitivity of cells to antitumor drugs was determined using an MTT assay. Cell migration was monitored using a Transwell assay. IACs were isolated following crosslinking and their molecular composition analysed using mass spectrometry (MS)–based proteomics. Results and discussions In two MDA-MB-435S-derived cell clones with decreased expression of integrin αV, expressing 15% (2αV) or 5% (3αV) of the control cells amount, increased sensitivity to paclitaxel and vincristine, decreased sensitivity to cisplatin, and decreased migration were observed in line with previous results obtained following transient transfection with integrin αV siRNA. In cell clones 2αV and 3αV, which were smaller than control cells and lacked stress fibres, the number of focal adhesions was shown to be significantly lower as observed by interference reflection microscopy and immunofluorescence detection of phospho-paxillin, phospho-FAK and phospho-Src. MS analysis of isolated IACs from control MDA-MB-435S, 2αV and 3αV cells identified 282 proteins, including 36 out of 60 consensus adhesome proteins. As expected, in clones 2αV and 3αV, integrins αV, β3 and β5 were detected at much lower levels compared with control cells. In addition, lower levels of talin-1 and −2, vinculin, alpha-actinin-4, tensin-3, filamin-A and -B, liprin β1 and plectin were detected. Conclusion These data will enable follow-up analyses of the mechanisms of signalling by integrins αVβ3/β5 and therefore represent a valuable resource to improve our understanding of the mechanisms involved in adhesion control of cell sensitivity to antitumor drugs and metastatic potential.

The concept of pilot pseudopodia is reconsidered 30 years after its inauguration (Gerisch, G., Hu¨ lser, D., Malchow, D., Wick, U., 1975. Cell communication by periodic cyclic-AMP pulses. Phil. Trans. R. Soc. Lond. B 272, 181– 192). The original hypothesis stated that protruding pseudopodia serve as dynamic sensory organelles that aid a cell in perceiving variations of chemoattractant concentration and, consequently, in navigation during chemotaxis. This influential idea is reevaluated in the light of recent findings about the mechanisms governing chemotactic cell motility, morphology and dynamics of pseudopodia, and about molecular constituents and regulators of pseudopod extension and retraction. It is proposed that stimulation by a chemoattractant modulates speed of pseudopod protrusion and thereby increases cell elongation. Elongation further enhances chemotactic sensitivity of the cell to shallow chemoattractant gradients, reinforces cell polarization, and finally leads to suppression of lateral pseudopodia and continuation of cell migration in the gradient direction.

Many living organisms form mineral phases through biologically controlled processes, known as biomineralization. Thus created materials are composites of both, mineral and organic components. The shell of the gastropod mollusc red abalone (Haliotis rufescens) consists of calcite and aragonite layers, each of them containing characteristic biopolymers responsible for biomineralization. In this work, the effect of interstitial green sheet polypeptide, GP, extracted from the green layer of the mollusc red abalone shell, on the process of spontaneous precipitation of calcium carbonate polymorphs, was investigated. Three precipitation systems, in which the initial mineralogical composition of the precipitate was different, have been studied. Thus, in system (1) calcite appeared, in system (2) a mixture of calcite and vaterite was found, while in system (3), ASW, only aragonite precipitated. However, the precipitation kinetic measurements, X-ray diffraction, FT-IR spectroscopy, and light and scanning electron microscopy indicated that the addition of GP in the model systems caused the inhibition of precipitation and change of morphology of crystals as a consequence of GP adsorption on the crystal surfaces and its entrapment into the mineral structure. (doi: 10.5562/cca2197)

Dictyostelium cells are professional phagocytes that are optimally suited for the imaging of phagosome processing from particle uptake to exocytosis. In order to design fluorescent probes for monitoring membrane trafficking in the endocytic pathway, we have dissected a membrane protein, golvesin, and have linked fragments of its sequence to GFP. Endogenous golvesin is partitioned between the ER, the Golgi apparatus, endosomes, and the contractile vacuole complex. We have localized signals that are required for exit from the Golgi to post-Golgi compartments to the C-terminal region of the golvesin sequence. One GFP-tagged fragment turned out to be a highly specific Golgi marker and was used to demonstrate the interaction of Golgi tubules with phagosomes. Signals essential for the retrieval of golvesin at the end of phagosome processing were localized to the N-terminal region. A truncated golvesin construct escaping retrieval was employed in recording the delivery of a phagosomal protein to the plasma membrane. Applying this construct to a phagosome filled with multiple particles, we observed that the phagosome is segmented during exocytosis, meaning that sequential release of particles alternates with membrane fusion.

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. Cell Motil. Cytoskeleton 56:130 –139, 2003. © 2003 Wiley-Liss, Inc.

A C-terminal 63-kDa fragment of talin A from Dictyostelium discoideum forms a slowly dissociating complex with F-actin in vitro. This talin fragment (TalC63) has been tagged with GFP and used as a trap for actin filaments in chemotactic cell movement, endocytosis, and mitotic cell division. TalC63 efficiently sequesters actin filaments in vivo. Its translocation reflects the direction and efficiency of an actin flow. Along the body of a migrating Dictyostelium cell, this flow is directed from the front to the tail. If during chemotaxis one or two new fronts are induced, the flow is always directed away from these fronts. The flow thus reflects the re-programming of cell polarity in response to changing gradients of chemoattractant. In endocytosis, the fluorescent complexes are translocated to the base of a phagocytic or macropinocytic cup. During mitosis, the complexes of F-actin with TalC63 accumulate within the midzone of anaphase cells. If TalC63 is strongly expressed, the entire cleavage furrow is filled out by sequestered actin filaments, and cytokinesis is severely impaired. These cells are considered to mimic the phenotype of mutants deficient in the shredding of actin filaments that normally occurs in the mid-zone of a dividing cell. Cell Motil. Cytoskeleton 53:136–149, 2002. © 2002 Wiley-Liss, Inc.

The Dictyostelium kinase DPAKa is a member of the p21-activated kinase (PAK) family, consisting of an N-terminal domain characterized by a coiled-coil region and proline-rich motifs, a Rac-binding CRIB-domain, and a highly conserved C-terminal kinase domain. In this study we show that cells overexpressing a C-terminal DPAKa fragment comprising the kinase domain are significantly impaired in motility and phagocytosis, whereas DPAKa-null cells display no obvious phenotypic change. We analyzed the in vivo localization of full-length and truncated DPAKa tagged with green fluorescent protein (GFP). The N-terminal fragments show a highly dynamic cortical localization without a permanent polarized enrichment, whereas the C-terminal fragment is homogenously distributed throughout the cell. The localization of full-length DPAKa is similar to that of myosin II at the rear end of locomoting cells and at the base of phagocytic cups. During mitosis DPAKa is gradually recruited to the cell cortex starting at metaphase, which also parallels the dynamics of myosin II cortical recruitment. However, in contrast to myosin II, DPAKa does not accumulate in the cleavage furrow but stays uniformly distributed throughout the cell cortex. This finding contrasts with previous work claiming accumulation of DPAKa in the cleavage furrow of dividing cells. Our results suggest that the N-terminus directs DPAKa to the cortex, and the C-terminus is necessary for restricting its localization to the rear of moving cells during chemotaxis. Therefore, DPAKa may play distinct roles in myosin II regulation during cell movement and cell division.

The actin system forms a supramolecular, membrane-associated network that serves multiple functions in Dictyostelium cells, including cell motility controlled by chemoattractant, phagocytosis, macropinocytosis, and cytokinesis. In executing these functions the monomeric G-actin polymerizes reversibly, and the actin filaments are assembled into membrane-anchored networks together with other proteins involved in shaping the networks and controlling their dynamics. Most impressive is the speed at which actin-based structures are built, reorganized, or disassembled. We used GFP-tagged coronin and Arp3, an intrinsic constituent of the Arp2/3 complex, as examples of proteins that are recruited to highly dynamic actin-filament networks. By fluorescence recovery after photobleaching (FRAP), average exchange rates of cell-cortex bound coronin were estimated. A nominal value of 5 s for half-maximal incorporation of coronin into the cortex, and a value of 7 s for half-maximal dissociation from cortical binding sites has been obtained. Actin dynamics implies also flow of F-actin from sites of polymerization to sites of depolymerization, i.e. to the tail of a migrating cell, the base of a phagocytic cup, and the cleavage furrow in a mitotic cell. To monitor this flow, we expressed in Dictyostelium cells a GFP-tagged actin-binding fragment of talin. This fragment (GFP-TalC63) translocates from the front to the tail during cell migration and from the polar regions to the cleavage furrow during mitotic cell division. The intrinsic dynamics of the actin system can be manipulated in vivo by drugs or other probes that act either as inhibitors of actin polymerization or as stabilizers of filamentous actin. In order to investigate structure–function relationships in the actin system, a technique of reliably arresting transient network structures is in demand. We discuss the potential of electron tomography of vitrified cells to visualize actin networks in their native association with membranes.

The Arp2/3 complex is a ubiquitous and important regulator of the actin cytoskeleton. Here we identify this complex from Dictyostelium and investigate its dynamics in live cells. The predicted sequences of the subunits show a strong homology to the members of the mammalian complex, with the larger subunits generally better conserved than the smaller ones. In the highly motile cells of Dictyostelium, the Arp2/3 complex is rapidly re-distributed to the cytoskeleton in response to external stimuli. Fusions of Arp3 and p41-Arc with GFP reveal that in phagocytosis, macropinocytosis, and chemotaxis the complex is recruited within seconds to sites where actin polymerization is induced. In contrast, there is little or no localization to the cleavage furrow during cytokinesis. Rather the Arp2/3 complex is enriched in ruffles at the polar regions of mitotic cells, which suggests a role in actin polymerization in these ruffles.

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.

The ability of Dictyostelium cells to divide without myosin II in a cell cycle-coupled manner has opened two questions about the mechanism of cleavage furrow ingression. First, are there other possible functions for myosin II in this process except for generating contraction of the furrow by a sliding filament mechanism? Second, what could be an alternative mechanical basis for the furrowing? Using aberrant changes of the cell shape and anomalous localization of the actin-binding protein cortexillin I during asymmetric cytokinesis in myosin II-deficient cells as clues, it is proposed that myosin II filaments act as a mechanical lens in cytokinesis. The mechanical lens serves to focus the forces that induce the furrowing to the center of the midzone, a cortical region where cortexillins are enriched in dividing cells. Additionally, continual disassembly of a filamentous actin meshwork at the midzone is a prerequisite for normal ingression of the cleavage furrow and a successful cytokinesis. If this process is interrupted, as it occurs in cells that lack cortexillins, an overassembly of filamentous actin at the midzone obstructs the normal cleavage. Disassembly of the crosslinked actin network can generate entropic contractile forces in the cortex, and may be considered as an alternative mechanism for driving ingression of the cleavage furrow. Instead of invoking different types of cytokinesis that operate under attached and unattached conditions in Dictyostelium, it is anticipated that these cells use a universal multifaceted mechanism to divide, which is only moderately sensitive to elimination of its constituent mechanical processes.

Background: Myosin II, a conventional myosin, is dispensable for mitotic division in Dictyostelium if the cells are attached to a substrate, but is required when the cells are growing in suspension. Only a small fraction of myosin II-null cells fail to divide when attached to a substrate. Cortexillins are actin-bundling proteins that translocate to the midzone of mitotic cells and are important for the formation of a cleavage furrow, even in attached cells. Here, we investigated how myosin II and cortexillin I cooperate to determine the position of a cleavage furrow. Results: Using a green fluorescent protein (GFP)–cortexillin I fusion protein as a marker for priming of a cleavage furrow, we found that positioning of a cleavage furrow occurred in two steps. In the first step, which was independent of myosin II and substrate, cortexillin I delineated a zone around the equatorial region of the cell. Myosin II then focused the cleavage furrow to the middle of this cortexillin I zone. If asymmetric cleavage in the absence of myosin II partitioned a cell into a binucleate and an anucleate portion, cell-surface ruffles were induced along the cleavage furrow, which led to movement of the anucleate portion along the connecting strand towards the binucleate one. Conclusions: In myosin II-null cells, cleavage furrow positioning occurs in two steps: priming of the furrow region and actual cleavage, which may proceed in the middle or at one border of the cortexillin ring. A control mechanism acting at late cytokinesis prevents cell division into an anucleate and a binucleate portion, causing a displaced furrow to regress if it becomes aberrantly located on top of polar microtubule asters.

Dictyostelium discoideum contains a full-length homologue of talin, a protein implicated in linkage of the actin system to sites of cell-to-substrate adhesion in fibroblasts and neuronal growth cones. Gene replacement eliminated the talin homologue in Dictyostelium and led to defects in phagocytosis and cell-to-substrate interaction of moving cells, two processes dependent on a continuous cross talk between the cell surface and underlying cytoskeleton. The uptake rate of yeast particles was reduced, and only bacteria devoid of the carbohydrate moiety of cell surface lipopolysaccharides were adhesive enough to be recruited by talin-null cells in suspension and phagocytosed. Cell-to-cell adhesion of undeveloped cells was strongly impaired in the absence of talin, in contrast with the cohesion of aggregating cells mediated by the phospholipid-anchored contact site A glycoprotein, which proved to be less talin dependent. The mutant cells were still capable of moving and responding to a chemoattractant, although they attached only loosely to a substrate via small areas of their surface. With their high proportion of binucleated cells, the talin-null mutants revealed interactions of the mitotic apparatus with the cell cortex that were not obvious in mononucleated cells.

Over time we have come to appreciate that the complex regulation of Rho GTPases involves additional mechanisms beyond the activating role of RhoGEFs, the inactivating function of RhoGAPs and the sequestering activity of RhoGDIs. One class of regulatory mechanisms includes direct modifications of Rho proteins such as isoprenylation, phosphorylation and SUMOylation. Rho GTPases can also regulate each other by means of crosstalk signaling, which is again mostly mediated by GEFs, GAPs and GDIs. More complex mutual regulation ensues when and where two or more Rho proteins activate a common molecular target, i.e., share a common effector. We have recently unraveled a reciprocal mechanism wherein spatiotemporal dynamics of Rac1 activity during migration of Dictyostelium cells is apparently regulated by antagonizing interactions of Rac1-GTP with two distinct effectors. By monitoring specific fluorescent probes, activated Rac1 is simultaneously present at the leading edge, where it participates in Scar/WAVE-mediated actin polymerization, and at the trailing edge, where it induces formation of a DGAP1/cortexillin actin-bundling complex. Strikingly, in addition to their opposed localization, the two populations of activated Rac1 also display opposite kinetics of recruitment to the plasma membrane upon stimulation by chemoattractants. These findings with respect to Rac1 in Dictyostelium suggest a novel principle for regulation of Rho GTPase activity that might also play a role in other cell types and for other Rho family members.

Interference reflection microscopy (IRM) utilises interference of light reflected from closely apposed surfaces to provide an image containing information about the separation of those surfaces. In cell biology, IRM is used to image structures at the base of adherent cells and to measure cell–substratum distances, as well as to investigate mechanisms of cell–substratum adhesion. IRM is also used to study topology and dynamics of biomimetic systems such as vesicles, supported membranes and other multilayered structures. Basic IRM optical configuration is relatively easy to set up, and image analysis can provide information about interfacial distances with nanometer precision and millisecond time resolution. IRM can be readily combined with other microscopic techniques, and with force transducing devices such as optical tweezers, micropipettes and microcantilevers. New advancements in the field include dual-wavelength IRM and fluctuation contrast IRM.

Background information Nucleoside diphosphate kinases (NDPKs) are ubiquitous phosphotransfer enzymes responsible for producing most of the nucleoside triphosphates except for ATP. This role is important for the synthesis of nucleic acids and proteins and the metabolism of sugars and lipids. Apart from this housekeeping role NDPKs have been shown to have many diverse functions such as roles in proliferation and endocytosis. These additional roles are independent of enzyme activity but often are dependent on the expression level of the protein. Results In this study we altered the expression level of NDPK in the model eukaryotic organism Dictyostelium discoideum and demonstrated that NDPK plays a role in growth, endocytosis and exocytosis. In Dictyostelium NDPK negatively regulates endocytosis in contrast to the positive regulatory role identified in higher eukaryotes and this may be explained by the differences in types of endocytosis that have been studied in the different systems – phagocytosis and macropinocytosis in Dictyostelium compared with micropinocytosis in mammalian cells. The biological functions identified in these experiments are consistent with NDPK’s function as a metastatic suppressor and suggest that the metastatic suppressor roles may have evolved from an ancestral eukaryotic function in growth and endocytic vesicle trafficking. Conclusions NDPK in the model eukaryote Dictyostelium discoideum plays a role in growth, exocytosis and endocytosis. This is the first report of NDPK negatively regulating macropinocytosis which is the major fluid phase uptake mechanism for macrophage and dendritic cells and non dendritic cells exposed to growth factors.

The formation of mixed aggregates has been investigated in the mixture of oppositely charged surfactants vastly differing in molecular geometry and size. The systems considered is mixture of the cationic gemini surfactant, ethanediyl-1, 2-bis(dodecyldimethylammonium bromide), and anionic surfactant, sodium dodecyl sulfate. Various mixed nano- and microaggregates (micelles, vesicles, thin lamellar sheets, and tubules) were formed depending on bulk composition and total surfactant concentration. Two types of aggregates were found in precipitate, the tubules as prevailing aggregates on the gemini-rich side, and vesicles as prevailing aggregates on the SDS-rich side. The tubules formation was ascribed to mutual influence of specific structure of cationic dimeric surfactant and electrostatic interactions at the bilayer/solution interface. The proposed mechanism involved the formation of lamellar sheets, which rolled-up into tubules.

BACKGROUND AND PURPOSE: The evidence for traumatic brain injury in amateur boxers is controversial. Hypothetically, sudden acceleration of the head due to the impact of the blow during the boxing fight might result in diffuse axonal injury or contusion. We wanted to determine whether cerebral microhemorrhages occur more often in amateur boxers than in nonboxers. MATERIALS AND METHODS: In 42 male, classical amateur boxers and in 37 healthy, nonboxing male volunteers we performed cranial MR imaging at 3T. The study protocol included a transverse dual spin-echo MR imaging sequence, a 3D sagittal magnetization-prepared rapid acquisition of gradient echo sequence, a coronal T2*-weighted sequence, and an axial time-of-flight MR angiography sequence. MR imaging data were made anonymous before 2 neuroradiologists independently evaluated the images. In addition, the following risk factors were assessed: total numbers of fights and knockouts, weight division, and duration of boxing. We compared the group proportions of microhemorrhages with Fisher test of exact probability. RESULTS: There was a statistically higher prevalence of cerebral microhemorrhages in the group of boxers (3 of 42; 7.1%) than in nonboxing persons (0 of 37; 0%). This difference was not statistically significant, however ( P = .2479; Fisher exact test). CONCLUSION: Although we detected more microhemorrhages in amateur boxers than in nonboxing persons, this difference did not prove to be significant.