Formation of Cytoplasmic Actin-Cofilin Rods is Triggered by Metabolic Stress and Changes in Cellular pH.

Actin dynamics plays a crucial role in regulating essential cell functions and thereby is largely responsible to a considerable extent for cellular energy consumption. Certain pathological conditions in humans, like neurological disorders such as Alzheimer's disease or amyotrophic lateral sclerosis (ALS) as well as variants of nemaline myopathy are associated with cytoskeletal abnormalities, so-called actin-cofilin rods. Actin-cofilin rods are aggregates consisting mainly of actin and cofilin, which are formed as a result of cellular stress and thereby help to ensure the survival of cells under unfavorable conditions. We have used Dictyostelium discoideum, an established model system for cytoskeletal research to study formation and principles of cytoplasmic actin rod assembly in response to energy depletion. Experimentally, depletion of ATP was provoked by addition of either sodium azide, dinitrophenol, or 2-deoxy-glucose, and the formation of rod assembly was recorded by live-cell imaging. Furthermore, we show that hyperosmotic shock induces actin-cofilin rods, and that a drop in the intracellular pH accompanies this condition. Our data reveal that acidification of the cytoplasm can induce the formation of actin-cofilin rods to varying degrees and suggest that a local reduction in cellular pH may be a cause for the formation of cytoplasmic rods. We hypothesize that local phase separation mechanistically triggers the assembly of actin-cofilin rods and thereby influences the material properties of actin structures.

Actin assembly states in Dictyostelium discoideum at different stages of development and during cellular stress.

The actin cytoskeleton of non-muscle cells is essential for cellular structure and subcellular organization, and the dynamic regulation of actin assembly and disassembly is a prerequisite for motility. Pioneering work using Dictyostelium discoideum focused on the biochemical analysis of non-muscle actin, the identification of actin-regulating proteins and their specific functions during processes like cell migration, cytokinesis, phagocytosis, and morphogenesis. Although subsequent work in higher eukaryotes revealed that the processes regulating actin dynamics are often much more complex, results obtained by using Dictyostelium have been of fundamental importance because they have contributed significantly to our understanding of the actin cytoskeleton in higher eukaryotes. Dictyostelium is an accepted model system for studying fast moving cells, because the single cells of the organism share many similarities with cells of the immune system such as human neutrophils. Here we provide a brief overview on the milestones of research of the actin cytoskeleton taking advantage of Dictyostelium. Furthermore, we summarize how actin structures and cytoskeletal dynamics at different stages of development have been visualized, and give an overview on the current focus of research. In addition, we discuss results showing actin assembly states during phases of cellular stress and how stress-induced actin assembly states may contribute to our understanding of certain diseases.

Ate1-mediated posttranslational arginylation affects substrate adhesion and cell migration in Dictyostelium discoideum.

The highly conserved enzyme arginyl-tRNA-protein transferase (Ate1) mediates arginylation, a posttranslational modification that is only incompletely understood at its molecular level. To investigate whether arginylation affects actin-dependent processes in a simple model organism, Dictyostelium discoideum, we knocked out the gene encoding Ate1 and characterized the phenotype of ate1-null cells. Visualization of actin cytoskeleton dynamics by live-cell microscopy indicated significant changes in comparison to wild-type cells. Ate1-null cells were almost completely lacking focal actin adhesion sites at the substrate-attached surface and were only weakly adhesive. In two-dimensional chemotaxis assays toward folate or cAMP, the motility of ate1-null cells was increased. However, in three-dimensional chemotaxis involving more confined conditions, the motility of ate1-null cells was significantly reduced. Live-cell imaging showed that GFP-tagged Ate1 rapidly relocates to sites of newly formed actin-rich protrusions. By mass spectrometric analysis, we identified four arginylation sites in the most abundant actin isoform of Dictyostelium, in addition to arginylation sites in other actin isoforms and several actin-binding proteins. In vitro polymerization assays with actin purified from ate1-null cells revealed a diminished polymerization capacity in comparison to wild-type actin. Our data indicate that arginylation plays a crucial role in the regulation of cytoskeletal activities.

Actin-Interacting Protein 1 Contributes to Intranuclear Rod Assembly in Dictyostelium discoideum.

Intranuclear rods are aggregates consisting of actin and cofilin that are formed in the nucleus in consequence of chemical or mechanical stress conditions. The formation of rods is implicated in a variety of pathological conditions, such as certain myopathies and some neurological disorders. It is still not well understood what exactly triggers the formation of intranuclear rods, whether other proteins are involved, and what the underlying mechanisms of rod assembly or disassembly are. In this study, Dictyostelium discoideum was used to examine appearance, stages of assembly, composition, stability, and dismantling of rods. Our data show that intranuclear rods, in addition to actin and cofilin, are composed of a distinct set of other proteins comprising actin-interacting protein 1 (Aip1), coronin (CorA), filactin (Fia), and the 34 kDa actin-bundling protein B (AbpB). A finely tuned spatio-temporal pattern of protein recruitment was found during formation of rods. Aip1 is important for the final state of rod compaction indicating that Aip1 plays a major role in shaping the intranuclear rods. In the absence of both Aip1 and CorA, rods are not formed in the nucleus, suggesting that a sufficient supply of monomeric actin is a prerequisite for rod formation.

Fluorescent reporters and methods to analyze fluorescent signals.

The use of fluorescent reporters and the development of new imaging technologies have revolutionized studies in cell biology. During recent years the number of fluorescent proteins offering the ability to visualize the distribution of proteins, organelles, and cells has increased tremendously. In parallel, the imaging tools available were refined rapidly enabling now the use of a huge spectrum of specialized methods to explore the cellular and subcellular localization and dynamics of fluorescently tagged markers. This chapter presents an overview of fluorescent reporters and methods available, and describes a selection of those that are routinely applicable in imaging studies using Dictyostelium discoideum.

Advanced fluorescence microscopy techniques--FRAP, FLIP, FLAP, FRET and FLIM.

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Förster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research.

Genetic evidence for concerted control of actin dynamics in cytokinesis, endocytic traffic, and cell motility by coronin and Aip1.

Coronin and actin-interacting protein 1 (Aip1) are actin-binding proteins that by different mechanisms inhibit actin polymerization or enhance the disassembly of actin filaments. Cells of Dictyostelium discoideum lacking both proteins are retarded in growth and early development and often fail to proceed to fruiting body formation. Coronin/Aip1-null cells show numerous surface protrusions enriched in filamentous actin and cofilin. We show that the double-null cells are characterized by an increase in filamentous actin that causes a thickening of the cell cortex. This imbalance has severe consequences for processes that rely on the dynamic reorganization of the actin cytoskeleton, such as cell motility, cytokinesis and endocytosis. Although cell motility is considerably slowed down, the double-mutant cells are still capable of orientating in a gradient of chemoattractant. The cytokinesis defect is caused by the lack of proper cleavage furrow formation, a defect that is partially rescued by low concentrations of latrunculin A, an inhibitor of actin polymerization. Furthermore, we demonstrate that the disassembly of the actin coat after phagocytic or macropinocytic uptake is significantly delayed in the double-mutant cells. Our results prove that coronin and Aip1 are important effectors that act together in maintaining the balance of actin polymerization and depolymerization in living cells.

The STE group kinase SepA controls cleavage furrow formation in Dictyostelium.

During a REMI screen for proteins regulating cytokinesis in Dictyostelium discoideum we isolated a mutant forming multinucleate cells. The gene affected in this mutant encoded a kinase, SepA, which is an ortholog of Cdc7, a serine-threonine kinase essential for septum formation in Schizosaccharomyces pombe. Localization of SepA-GFP in live cells and its presence in isolated centrosomes indicated that SepA, like its upstream regulator Spg1, is associated with centrosomes. Knockout mutants of SepA showed a severe cytokinesis defect and a delay in development. In multinucleate SepA-null cells nuclear division proceeded normally and synchronously. However, often cleavage furrows were either missing or atypical: they were extremely asymmetric and constriction was impaired. Cortexillin-I, a marker localizing strictly to the furrow in wild-type cells, demonstrated that large, crescent-shaped furrows expanded and persisted long after the spindle regressed and nuclei returned to the interphase state. Outside the furrow the filamentous actin system of the cell cortex showed strong ruffling activity. These data suggest that SepA is involved in the spatial and temporal control system organizing cortical activities in mitotic and postmitotic cells.

The three-dimensional dynamics of actin waves, a model of cytoskeletal self-organization.

Actin polymerization is typically initiated at specific sites in a cell by membrane-bound protein complexes, and the resulting structures are involved in specialized cellular functions, such as migration, particle uptake, or mitotic division. Here we analyze the potential of the actin system to self-organize into waves that propagate on the planar, substrate-attached membrane of a cell. We show that self-assembly involves the ordered recruitment of proteins from the cytoplasmic pool and relate the organization of actin waves to their capacity for applying force. Three proteins are shown to form distinct three-dimensional patterns in the actin waves. Myosin-IB is enriched at the wave front and close to the plasma membrane, the Arp2/3 complex is distributed throughout the waves, and coronin forms a sloping layer on top of them. CARMIL, a protein that links myosin-IB to the Arp2/3 complex, is also recruited to the waves. Wave formation does not depend on signals transmitted by heterotrimeric G-proteins, nor does their propagation require SCAR, a regulator upstream of the Arp2/3 complex. Propagation of the waves is based on an actin treadmilling mechanism, indicating a program that couples actin assembly to disassembly in a three-dimensional pattern. When waves impinge on the cell perimeter, they push the edge forward; when they reverse direction, the cell border is paralyzed. These data show that force-generating, highly organized supramolecular networks are autonomously formed in live cells from molecular motors and proteins controlling actin polymerization and depolymerization.

Myosin Va phosphorylated on Ser1650 is found in nuclear speckles and redistributes to nucleoli upon inhibition of transcription.

Nuclear actin and nuclear myosins have been implicated in the regulation of gene expression in vertebrate cells. Myosin V is a class of actin-based motor proteins involved in cytoplasmic vesicle transport and anchorage, spindle-pole alignment and mRNA translocation. In this study, myosin-Va, phosphorylated on a conserved serine in the tail domain (phospho-ser(1650) MVa), was localized to subnuclear compartments. A monoclonal antibody, 9E6, raised against a peptide corresponding to phosphoserine(1650) and flanking regions of the murine myosin Va sequence, was immunoreactive to myosin Va heavy chain in cellular and nuclear extracts of HeLa cells, PC12 cells and B16-F10 melanocytes. Immunofluorescence microscopy with this antibody revealed discrete irregular spots within the nucleoplasm that colocalized with SC35, a splicing factor that earmarks nuclear speckles. Phospho-ser(1650) MVa was not detected in other nuclear compartments, such as condensed chromatin, Cajal bodies, gems and perinucleolar caps. Although nucleoli also were not labeled by 9E6 under normal conditions, inhibition of transcription in HeLa cells by actinomycin D caused the redistribution of phospho-ser(1650) MVa to nucleoli, as well as separating a fraction of phospho-ser(1650) MVa from SC35 into near-neighboring particles. These observations indicate a novel role for myosin Va in nuclear compartmentalization and offer a new lead towards the understanding of actomyosin-based gene regulation.