Microtubule dependent sorting of actin-binding proteins in mitosis.

The patterns of Formin B and of the Arp2/3 complex formed during mitosis were studied in a mutant of Dictyostelium discoideum that produces multinucleate cells, which divide by the ingression of unilateral cleavage furrows. During cytokinesis the cells of this mutant remain spread on a glass surface where they generate a planar pattern based on the sorting-out of actin-binding proteins. During anaphase, Formin B and Arp2/3 became localized to the regions of microtubule asters around the centrosomes; Formin B in particular in the form of round, quite uniformly covered areas. These areas have been shown to be depleted of myosin II and the actin-filament crosslinker cortexillin, and to be avoided by cleavage furrows on their path into the cell.

Fluctuations of formin binding in the generation of membrane patterns.

Circular actin waves that propagate on the substrate-attached membrane of Dictyostelium cells separate two distinct membrane domains from each other: an inner territory rich in phosphatidyl-(3,4,5) trisphosphate (PIP3) and an external area decorated with the PIP3-degrading 3-phosphatase PTEN. During wave propagation, the inner territory increases at the expense of the external area. Beyond a size limit, the inner territory becomes unstable, breaking into an inner and an external domain. The sharp boundary between these domains is demarcated by the insertion of an actin wave. During the conversion of inner territory to external area, the state of the membrane fluctuates, as visualized by dynamic landscapes of formin B binding. Here we analyze the formin B fluctuations in relation to three markers of the membrane state: activated Ras, PIP3, and PTEN.

Expanding ring-shaped cleavage furrows in multinucleate cells.

Multinucleate cells of Dictyostelium discoideum divide usually by unilateral cleavage furrows that ingress from the cell border. Along their path into the cell, they follow regions that are rich in myosin II and cortexillin and leave out the areas around the spindle poles that are populated with microtubule asters. In cells of a D. discoideum mutant that remain spread during mitosis we observed, as a rare event, cleavage by the expansion of a hole that is initiated in the middle of the cell area and has no connection with the cell's periphery. Here we show that these ring-shaped furrows develop in two phases, the first being reversible. During the first phase, the dorsal and ventral cell cortices come in close apposition and the cell membrane detaches locally from the substrate surface. The second phase comprises formation of the hole by membrane fusion and expansion of the opening toward the border of the cell, eventually cutting the multinucleate cell into pieces. We address the three-dimensional organization of ring-shaped furrows, their interaction with lateral furrows, and their association with filamentous myosin II and cortexillin. Thus, despite their geometrical divergence, similar molecular mechanisms might link the expanding hole to the standard contractile ring.

Patterning of the cell cortex and the localization of cleavage furrows in multi-nucleate cells.

In multi-nucleate cells of Dictyostelium, cytokinesis is performed by unilateral cleavage furrows that ingress the large cells from their border. We use a septase (sepA)-null mutant with delayed cytokinesis to show that in anaphase a pattern is generated in the cell cortex of cortexillin and myosin II. In multi-nucleate cells, these proteins decorate the entire cell cortex except circular zones around the centrosomes. Unilateral cleavage furrows are initiated at spaces free of microtubule asters and invade the cells along trails of cortexillin and myosin II accumulation. Where these areas widen, the cleavage furrow may branch or expand. When two furrows meet, they fuse, thus separating portions of the multi-nucleate cell from each other. Unilateral furrows are distinguished from the contractile ring of a normal furrow by their expansion rather than constriction. This is particularly evident for expanding ring-shaped furrows that are formed in the centre of a large multi-nucleate cell. Our data suggest that the myosin II-enriched area in multi-nucleate cells is a contractile sheet that pulls on the unilateral furrows and, in that way, expands them.

Genetic Instability Due to Spindle Anomalies Visualized in Mutants of Dictyostelium.

Aberrant centrosome activities in mutants of Dictyostelium discoideum result in anomalies of mitotic spindles that affect the reliability of chromosome segregation. Genetic instabilities caused by these deficiencies are tolerated in multinucleate cells, which can be produced by electric-pulse induced cell fusion as a source for aberrations in the mitotic apparatus of the mutant cells. Dual-color fluorescence labeling of the microtubule system and the chromosomes in live cells revealed the variability of spindle arrangements, of centrosome-nuclear interactions, and of chromosome segregation in the atypical mitoses observed.

Unilateral Cleavage Furrows in Multinucleate Cells.

Multinucleate cells can be produced in Dictyostelium by electric pulse-induced fusion. In these cells, unilateral cleavage furrows are formed at spaces between areas that are controlled by aster microtubules. A peculiarity of unilateral cleavage furrows is their propensity to join laterally with other furrows into rings to form constrictions. This means cytokinesis is biphasic in multinucleate cells, the final abscission of daughter cells being independent of the initial direction of furrow progression. Myosin-II and the actin filament cross-linking protein cortexillin accumulate in unilateral furrows, as they do in the normal cleavage furrows of mononucleate cells. In a myosin-II-null background, multinucleate or mononucleate cells were produced by cultivation either in suspension or on an adhesive substrate. Myosin-II is not essential for cytokinesis either in mononucleate or in multinucleate cells but stabilizes and confines the position of the cleavage furrows. In fused wild-type cells, unilateral furrows ingress with an average velocity of 1.7 µm × min-1, with no appreciable decrease of velocity in the course of ingression. In multinucleate myosin-II-null cells, some of the furrows stop growing, thus leaving space for the extensive broadening of the few remaining furrows.

Decision Making in Phagocytosis.

Dictyostelium cells are professional phagocytes that are capable of handling particles of variable shapes and sizes. Here we offer long bacteria that challenge the uptake mechanism to its limits and report on the responses of the phagocytes if they are unable to engulf the particle by closing the phagocytic cup. Reasons for failure may be a length of the particle much larger than the phagocyte's diameter, or competition with another phagocyte. A cell may simultaneously release a particle and engulf another one. The final phase of release can be fast, causing the phagosome membrane to turn inside-out and to form a bleb. Myosin-II may be involved in the release by generating tension at the plasma membrane, it does however not accumulate on the phagosome to act there directly in expelling the particle. Labeling with GFP-2FYVE indicates that processing of the phagosome with phosphatidylinositol 3-phosphate begins at the base of a long phagosome already before closure of the cup. The decision of releasing the particle can be made even at the stage of the processed phagosome.

Formins specify membrane patterns generated by propagating actin waves.

Circular actin waves separate two distinct areas on the substrate-attached cell surface from each other: an external area from an inner territory that is circumscribed by the wave. These areas differ in composition of actin-associated proteins and of phosphoinositides in the membrane. At the propagating wave, one area is converted into the other. By photo-conversion of Eos-actin and analysis of actin network structures we show that both in the inner territory and the external area the actin network is subject to continuous turnover. To address the question of whether areas in the wave pattern are specified by particular actin polymerizing machines, we locate five members of the formin family to specific regions of the wave landscape using TIRF microscopy and constitutively active formin constructs tagged with fluorescent protein. Formin ForB favors the actin wave and ForG the inner territory, whereas ForA, ForE, and ForH are more strongly recruited to the external area. Fluctuations of membrane binding peculiar to ForB indicate transient states in the specification of membrane domains before differentiation into ForB decorated and depleted ones. Annihilation of the patterns by 1 µM of the formin inhibitor SMIFH2 supports the implication of formins in their generation.

Actin Waves and Dynamic Patterning of the Plasma Membrane.

Plasma membrane and underlying actin network are connected to a functional unit that by non-linear interactions is capable of forming patterns. For instance, in cell motility and chemotaxis, cells polarize to form a protruding front and a retracting tail. Here we address dynamic patterns that are formed on a planar substrate surface and are therefore easily accessible to optical recording. In these patterns two distinct areas of the membrane and actin cortex are interconverted at the site of circular actin waves. The inner territory circumscribed by a wave is distinguished from the external area by a high PIP3 content and high Ras activity. In contrast, the external area is occupied with the PIP3-degrading phosphatase PTEN. In the underlying cortex, these areas differ in the proteins associated with the actin network. Actin waves can be formed at zones of increasing as well as decreasing Ras activity. Both types of waves are headed by myosin IB. When waves collide, they usually extinguish each other, and their decay is accompanied by the accumulation of coronin. No membrane patterns have been observed after efficient depolymerization of actin, suggesting that residual actin filaments are necessary for the pattern generating system to work. Where appropriate, we relate the experimental data obtained with Dictyostelium to human normal and malignant cell behavior, in particular to the role of Ras-GAP as an enhancer of macropinocytosis, to mutations in the tumor suppressor PTEN, to frustrated phagocytosis, and to the role of coronin in immune cells and neurons.

Local Ras activation, PTEN pattern, and global actin flow in the chemotactic responses of oversized cells.

Chemotactic responses of eukaryotic cells require a signal processing system that translates an external gradient of attractant into directed motion. To challenge the response system to its limits, we increased the size of Dictyostelium discoideum cells by using electric-pulse-induced fusion. Large cells formed multiple protrusions at different sites along the gradient of chemoattractant, independently turned towards the gradient and competed with each other. Finally, these cells succeeded to re-establish polarity by coordinating front and tail activities. To analyse the responses, we combined two approaches, one aimed at local responses by visualising the dynamics of Ras activation at the front regions of reorientating cells, the other at global changes of polarity by monitoring front-to-tail-directed actin flow. Asymmetric Ras activation in turning protrusions underscores that gradients can be sensed locally and translated into orientation. Different to cells of normal size, the polarity of large cells is not linked to an increasing front-to-tail gradient of the PIP3-phosphatase PTEN. But even in large cells, the front communicates with the tail through an actin flow that might act as carrier of a protrusion inhibitor.

Membrane and actin reorganization in electropulse-induced cell fusion.

When cells of Dictyostelium discoideum are exposed to electric pulses they are induced to fuse, yielding motile polykaryotic cells. By combining electron microscopy and direct recording of fluorescent cells, we have studied the emergence of fusion pores in the membranes and the localization of actin to the cell cortex. In response to electric pulsing, the plasma membranes of two contiguous cells are turned into tangles of highly bent and interdigitated membranes. Live-imaging of cells double-labeled for membranes and filamentous actin revealed that actin is induced to polymerize in the fusion zone to temporarily bridge the gaps in the vesiculating membrane. The diffusion of green fluorescent protein (GFP) from one fusion partner to the other was scored using spinning disc confocal microscopy. Fusion pores that allowed intercellular exchange of GFP were formed after a delay, which lasted up to 24 seconds after exposure of the cells to the electric field. These data indicate that the membranes persist in a fusogenic state before pores of about 3 nm diameter are formed.

Curvature recognition and force generation in phagocytosis.

BACKGROUND: The uptake of particles by actin-powered invagination of the plasma membrane is common to protozoa and to phagocytes involved in the immune response of higher organisms. The question addressed here is how a phagocyte may use geometric cues to optimize force generation for the uptake of a particle. We survey mechanisms that enable a phagocyte to remodel actin organization in response to particles of complex shape. RESULTS: Using particles that consist of two lobes separated by a neck, we found that Dictyostelium cells transmit signals concerning the curvature of a surface to the actin system underlying the plasma membrane. Force applied to a concave region can divide a particle in two, allowing engulfment of the portion first encountered. The phagosome membrane that is bent around the concave region is marked by a protein containing an inverse Bin-Amphiphysin-Rvs (I-BAR) domain in combination with an Src homology (SH3) domain, similar to mammalian insulin receptor tyrosine kinase substrate p53. Regulatory proteins enable the phagocyte to switch activities within seconds in response to particle shape. Ras, an inducer of actin polymerization, is activated along the cup surface. Coronin, which limits the lifetime of actin structures, is reversibly recruited to the cup, reflecting a program of actin depolymerization. The various forms of myosin-I are candidate motor proteins for force generation in particle uptake, whereas myosin-II is engaged only in retracting a phagocytic cup after a switch to particle release. Thus, the constriction of a phagocytic cup differs from the contraction of a cleavage furrow in mitosis. CONCLUSIONS: Phagocytes scan a particle surface for convex and concave regions. By modulating the spatiotemporal pattern of actin organization, they are capable of switching between different modes of interaction with a particle, either arresting at a concave region and applying force in an attempt to sever the particle there, or extending the cup along the particle surface to identify the very end of the object to be ingested. Our data illustrate the flexibility of regulatory mechanisms that are at the phagocyte's disposal in exploring an environment of irregular geometry.