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.

Cells responding to chemoattractant on a structured substrate.

Cell migration on an adhesive substrate surface comprises actin-based protrusion at the front and retraction of the tail in combination with coordinated adhesion to, and detachment from, the substrate. To study the effect of cell-to-substrate adhesion on the chemotactic response of Dictyostelium discoideum cells, we exposed the cells to patterned substrate surfaces consisting of adhesive and inert areas, and forced them by a gradient of chemoattractant to enter the border between the two areas. Wild-type as well as myosin II-deficient cells stop at the border of an adhesive area. They do not detach with their rear part, while on the nonadhesive area they protrude pseudopods at their front toward the source of chemoattractant. Avoidance of the nonadhesive area may cause a cell to move in tangential direction relative to the attractant gradient, keeping its tail at the border of the adhesive surface.

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.

The Architecture of Traveling Actin Waves Revealed by Cryo-Electron Tomography.

Actin waves are dynamic supramolecular structures involved in cell migration, cytokinesis, adhesion, and neurogenesis. Although wave-like propagation of actin networks is a widespread phenomenon, the actin architecture underlying wave propagation remained unknown. In situ cryo-electron tomography of Dictyostelium cells unveils the wave architecture and provides evidence for wave progression by de novo actin nucleation. Subtomogram averaging reveals the structure of Arp2/3 complex-mediated branch junctions in their native state, and enables quantitative analysis of the 3D organization of branching within the waves. We find an excess of branches directed toward the substrate-attached membrane, and tent-like structures at sites of branch clustering. Fluorescence imaging shows that Arp2/3 clusters follow accumulation of the elongation factor VASP. We propose that filament growth toward the membrane lifts up the actin network as the wave propagates, until depolymerization of oblique filaments at the back causes the collapse of horizontal filaments into a compact layer.

Co-existence of Ras activation in a chemotactic signal transduction pathway and in an autonomous wave - forming system.

The activation of Ras is common to two activities in cells of Dictyostelium discoideum: the directed movement in a gradient of chemoattractant and the autonomous generation of propagating waves of actin polymerization on the substrate-attached cell surface. We produced large cells by electric-pulse induced fusion to simultaneously study both activities in one cell. For imaging, a fluorescent label for activated Ras was combined with labels for filamentous actin, PIP3, or PTEN. Chemotactic responses were elicited in a diffusion gradient of cyclic AMP. Waves initiated at sites separate from the front of the cell propagated in all directions. Nevertheless, the wave-forming cells were capable of recognizing the attractant gradient and managed to migrate in its direction.

Oscillatory Switches of Dorso-Ventral Polarity in Cells Confined between Two Surfaces.

To maneuver in a three-dimensional space, migrating cells need to accommodate to multiple surfaces. In particular, phagocytes have to explore their environment in the search for particles to be ingested. To examine how cells decide between competing surfaces, we exposed single cells of Dictyostelium to a defined three-dimensional space by confining them between two planar surfaces: those of a cover glass and of a wedged microcantilever. These cells form propagating waves of filamentous actin and PIP3 on their ventral substrate-attached surface. The dynamics of wave formation in the confined cells was explored using two-focus fluorescence imaging. When waves formed on one substrate, wave formation on the other substrate was efficiently suppressed. The propensity for wave formation switched between the opposing cell surfaces with periods of 2-5 min by one of two modes: 1) a rolling mode involving the slipping of a wave along the nonattached plasma membrane and 2) de novo initiation of waves on the previously blank cell surface. These data provide evidence for a cell-autonomous oscillator that switches dorso-ventral polarity in a cell simultaneously exposed to multiple substrate surfaces.

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.

Actin Organization in Cells Responding to a Perforated Surface, Revealed by Live Imaging and Cryo-Electron Tomography.

In a 3D environment, motile cells accommodate their protruding and retracting activities to geometrical cues. Dictyostelium cells migrating on a perforated film explored its holes by forming actin rings around their border and extending protrusions through the free space. The response was initiated when an actin wave passed a hole, and the rings persisted only in the PIP3-rich territories surrounded by a wave. To reconstruct actin structures from cryo-electron tomograms, actin rings were identified by cryo-correlative light and electron microscopy, and thin wedges of relevant regions were obtained by cryo-focused ion-beam milling. Retracting stages were distinguished from protruding ones by the accumulation of myosin-II. Early actin rings consisted of filaments pointing upright from the membrane, entangled with a meshwork of filaments close to the membrane. Branches identified at later stages suggested that formin-based nucleation of filaments was followed by Arp2/3-mediated network stabilization, which prevented buckling of the force-generating filaments.

Wave Patterns in Cell Membrane and Actin Cortex Uncoupled from Chemotactic Signals.

When cells of Dictyostelium discoideum orientate in a gradient of chemoattractant, they are polarized into a protruding front pointing toward the source of attractant, and into a retracting tail. Under the control of chemotactic signal inputs, Ras is activated and PIP3 is synthesized at the front, while the PIP3-degrading phosphatase PTEN decorates the tail region. As a result of signal transduction, actin filaments assemble at the front into dendritic structures associated with the Arp2/3 complex, in contrast to the tail region where a loose actin meshwork is associated with myosin-II and cortexillin, an antiparallel actin-bundling protein. In axenically growing strains of D. discoideum, wave patterns built by the same components evolve in the absence of any external signal input. Since these autonomously generated patterns are constrained to the plane of the substrate-attached cell surface, they are optimally suited to the optical analysis of state transitions between front-like and tail-like states of the membrane and the actin cortex. Here, we describe imaging techniques using fluorescent proteins to probe for the state of the membrane, the reorganization of the actin network, and the dynamics of wave patterns.

Actin and PIP3 waves in giant cells reveal the inherent length scale of an excited state.

The membrane and actin cortex of a motile cell can autonomously differentiate into two states, one typical of the front, the other of the tail. On the substrate-attached surface of Dictyostelium discoideum cells, dynamic patterns of front-like and tail-like states are generated that are well suited to monitor transitions between these states. To image large-scale pattern dynamics independently of boundary effects, we produced giant cells by electric-pulse-induced cell fusion. In these cells, actin waves are coupled to the front and back of phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-rich bands that have a finite width. These composite waves propagate across the plasma membrane of the giant cells with undiminished velocity. After any disturbance, the bands of PIP3 return to their intrinsic width. Upon collision, the waves locally annihilate each other and change direction; at the cell border they are either extinguished or reflected. Accordingly, expanding areas of progressing PIP3 synthesis become unstable beyond a critical radius, their center switching from a front-like to a tail-like state. Our data suggest that PIP3 patterns in normal-sized cells are segments of the self-organizing patterns that evolve in giant cells.

Reversible membrane pearling in live cells upon destruction of the actin cortex.

Membrane pearling in live cells is observed when the plasma membrane is depleted of its support, the cortical actin network. Upon efficient depolymerization of actin, pearls of variable size are formed, which are connected by nanotubes of ~40 nm diameter. We show that formation of the membrane tubes and their transition into chains of pearls do not require external tension, and that they neither depend on microtubule-based molecular motors nor pressure generated by myosin-II. Pearling thus differs from blebbing. The pearling state is stable as long as actin is prevented from polymerizing. When polymerization is restored, the pearls are retracted into the cell, indicating continuity of the membrane. Our data suggest that the alternation of pearls and strings is an energetically favored state of the unsupported plasma membrane, and that one of the functions of the actin cortex is to prevent the membrane from spontaneously assuming this configuration.

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.

PIP3 waves and PTEN dynamics in the emergence of cell polarity.

In a motile eukaryotic cell, front protrusion and tail retraction are superimposed on each other. To single out mechanisms that result in front to tail or in tail to front transition, we separated the two processes in time using cells that oscillate between a full front and a full tail state. State transitions were visualized by total internal reflection fluorescence microscopy using as a front marker PIP3 (phosphatidylinositol [3,4,5] tris-phosphate), and as a tail marker the tumor-suppressor PTEN (phosphatase tensin homolog) that degrades PIP3. Negative fluctuations in the PTEN layer of the membrane gated a local increase in PIP3. In a subset of areas lacking PTEN (PTEN holes), PIP3 was amplified until a propagated wave was initiated. Wave propagation implies that a PIP3 signal is transmitted by a self-sustained process, such that the temporal and spatial profiles of the signal are maintained during passage of the wave across the entire expanse of the cell membrane. Actin clusters were remodeled into a ring along the perimeter of the expanding PIP3 wave. The reverse transition of PIP3 to PTEN was linked to the previous site of wave initiation: where PIP3 decayed first, the entry of PTEN was primed.