ABSTRACT
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.
Subject(s)
Dictyostelium , Anaphase , Centrosome/metabolism , Cytokinesis , Dictyostelium/genetics , Dictyostelium/metabolism , Microtubules/metabolism , Myosin Type II/metabolismABSTRACT
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.
Subject(s)
Actins , Dictyostelium , Actins/metabolism , Formins/metabolism , Dictyostelium/metabolism , Membranes/metabolism , Cell Membrane/metabolism , Actin Cytoskeleton/metabolismABSTRACT
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.
Subject(s)
Dictyostelium , Actins/metabolism , Cell Movement/physiology , Chemotactic Factors/pharmacology , Chemotaxis , Myosin Type II/metabolism , Pseudopodia/metabolismABSTRACT
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.
Subject(s)
Dictyostelium/cytology , Phagocytosis , Bacteria/cytology , Phagocytes/cytology , Phagosomes/metabolismABSTRACT
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.
Subject(s)
Cell Polarity , Dictyostelium/cytology , Cytoskeleton/metabolism , Glass , Single-Cell Analysis , Surface PropertiesABSTRACT
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.
Subject(s)
Actins/metabolism , Cell Size , Chemotaxis , Dictyostelium/cytology , Dictyostelium/metabolism , PTEN Phosphohydrolase/metabolism , Rheology , ras Proteins/metabolism , Cell Size/drug effects , Cell Surface Extensions/metabolism , Chemotactic Factors/pharmacology , Chemotaxis/drug effects , Cyclic AMP/metabolism , Dictyostelium/drug effects , Diffusion , Pinocytosis/drug effectsABSTRACT
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.
Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Cell Membrane/metabolism , Dictyostelium/physiology , Giant Cells/physiology , Cell Fusion/methods , Cell Movement , Cell Polarity , Cell Size , Dictyostelium/metabolism , Electromagnetic Radiation , Phosphatidylinositol Phosphates/metabolismABSTRACT
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.
Subject(s)
Actins/metabolism , Cell Membrane/metabolism , Dictyostelium/metabolism , Giant Cells/metabolism , Protozoan Proteins/metabolism , Actins/genetics , Cell Fusion , Dictyostelium/cytology , Giant Cells/cytology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Protozoan Proteins/geneticsABSTRACT
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.
Subject(s)
Actins/metabolism , Cell Membrane/metabolism , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/ultrastructure , Actins/chemistry , Biomechanical Phenomena , Cell Membrane/ultrastructure , Cell Survival , Cryoelectron Microscopy , Dictyostelium/cytology , Glass/chemistry , Microtubules/metabolism , Myosin Type II/metabolism , Protein Multimerization , Protein Structure, Quaternary , Surface PropertiesABSTRACT
Normal-sized cells of Dictyostelium build up a front-tail polarity when they respond to a gradient of chemoattractant. To challenge the polarity-generating system, cells are fused to study the chemotactic response of oversized cells that extend multiple fronts toward the source of attractant. An aspect that can be explored in these cells is the relationship of spontaneously generated actin waves to actin reorganization in response to chemoattractant.
Subject(s)
Chemotaxis , Dictyostelium , Dictyostelium/physiology , Dictyostelium/cytology , Chemotactic Factors/pharmacology , Chemotactic Factors/metabolism , Actins/metabolism , Cell Fusion/methods , Giant Cells/cytology , Giant Cells/metabolism , Cell PolarityABSTRACT
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.
Subject(s)
Dictyostelium , Microfilament Proteins , Microtubules , Mitosis , Microtubules/metabolism , Dictyostelium/metabolism , Microfilament Proteins/metabolism , Microfilament Proteins/genetics , Actin-Related Protein 2-3 Complex/metabolism , Protozoan Proteins/metabolism , Protozoan Proteins/genetics , Protein Transport , Cytokinesis , Actins/metabolismABSTRACT
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.
Subject(s)
Dictyostelium , Dictyostelium/metabolism , Mitosis , Microtubules/metabolism , Myosin Type II/metabolism , Cell Membrane , Cytoskeletal Proteins/metabolismABSTRACT
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.
Subject(s)
Cell Polarity , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol Phosphates/metabolism , Actins/chemistry , Dictyostelium/cytology , Dictyostelium/enzymology , Kinetics , Microscopy, Fluorescence , Protein Multimerization , Protein Structure, Quaternary , Stochastic ProcessesABSTRACT
BACKGROUND: In a motile polarized cell the actin system is differentiated to allow protrusion at the front and retraction at the tail. This differentiation is linked to the phosphoinositide pattern in the plasma membrane. In the highly motile Dictyostelium cells studied here, the front is dominated by PI3-kinases producing PI(3,4,5)tris-phosphate (PIP3), the tail by the PI3-phosphatase PTEN that hydrolyses PIP3 to PI(4,5)bis-phosphate. To study de-novo cell polarization, we first depolymerized actin and subsequently recorded the spontaneous reorganization of actin patterns in relation to PTEN. RESULTS: In a transient stage of recovery from depolymerization, symmetric actin patterns alternate periodically with asymmetric ones. The switches to asymmetry coincide with the unilateral membrane-binding of PTEN. The modes of state transitions in the actin and PTEN systems differ. Transitions in the actin system propagate as waves that are initiated at single sites by the amplification of spontaneous fluctuations. In PTEN-null cells, these waves still propagate with normal speed but loose their regular periodicity. Membrane-binding of PTEN is induced at the border of a coherent PTEN-rich area in the form of expanding and regressing gradients. CONCLUSIONS: The state transitions in actin organization and the reversible transition from cytoplasmic to membrane-bound PTEN are synchronized but their patterns differ. The transitions in actin organization are independent of PTEN, but when PTEN is present, they are coupled to periodic changes in the membrane-binding of this PIP3-degrading phosphatase. The PTEN oscillations are related to motility patterns of chemotaxing cells.
Subject(s)
Actin Cytoskeleton/ultrastructure , Cell Surface Extensions/metabolism , Dictyostelium/physiology , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Biological Clocks/drug effects , Biological Clocks/physiology , Cell Movement/physiology , Cell Polarity , Cells, Cultured , Gene Knockout Techniques , Protein Binding/drug effects , Protein TransportABSTRACT
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.
Subject(s)
Organelle Shape/physiology , Phagocytes/physiology , Phagocytosis/physiology , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/metabolism , Actins/metabolism , Dictyostelium/cytology , Dictyostelium/genetics , Dictyostelium/metabolism , Dictyostelium/physiology , Fluorescence Recovery After Photobleaching , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Models, Biological , Myosins/metabolism , Organisms, Genetically Modified , Phagocytes/metabolism , Phagocytosis/genetics , Phosphatidylinositols/metabolism , Phosphatidylinositols/physiology , Physical Phenomena , Protein Multimerization/physiology , ras Proteins/metabolism , ras Proteins/physiologyABSTRACT
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.
Subject(s)
Chromosome Segregation , Dictyostelium/genetics , Genomic Instability , Mitosis , Mutation , Spindle Apparatus/genetics , Dictyostelium/growth & development , Dictyostelium/metabolism , Microtubules/genetics , Microtubules/metabolism , Spindle Apparatus/metabolism , Spindle Apparatus/pathology , rac GTP-Binding Proteins/genetics , rac GTP-Binding Proteins/metabolismABSTRACT
Filopodia are finger-like extensions of the cell surface that are involved in sensing the environment, in attachment of particles for phagocytosis, in anchorage of cells on a substratum, and in the response to chemoattractants or other guidance cues. Filopodia present an excellent model for actin-driven membrane protrusion. They grow at their tips by the assembly of actin and are stabilized along their length by a core of bundled actin filaments. To visualize actin networks in their native membrane-anchored state, filopodia of Dictyostelium cells were subjected to cryo-electron tomography. At the site of actin polymerization, a peculiar structure, the "terminal cone," is built of short filaments fixed with their distal end to the filopod's tip and with their proximal end to the flank of the filopod. The backbone of the filopodia consists of actin filaments that are shorter than the entire filopod and aligned in parallel or obliquely to the filopod's axis. We hypothesize that growth of the highly dynamic filopodia of Dictyostelium is accompanied by repetitive nucleation of actin polymerization at the filopod tip, followed by the rearrangement of filaments within the shaft.
Subject(s)
Actins/ultrastructure , Dictyostelium/ultrastructure , Pseudopodia/ultrastructure , Actins/metabolism , Animals , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cryoelectron Microscopy/methods , Dictyostelium/metabolism , Dictyostelium/physiology , Movement/physiology , Pseudopodia/metabolism , Pseudopodia/physiology , Tomography/methodsABSTRACT
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.
Subject(s)
Cytokinesis/physiology , Dictyostelium/ultrastructure , Protein Serine-Threonine Kinases/metabolism , Actins/metabolism , Actins/ultrastructure , Animals , Cell Cycle Proteins/genetics , Cell Surface Extensions/ultrastructure , Centrosome/metabolism , Centrosome/ultrastructure , Dictyostelium/chemistry , Microscopy, Confocal , Protein Serine-Threonine Kinases/genetics , Schizosaccharomyces pombe Proteins/genetics , Spindle Apparatus/physiology , Spindle Apparatus/ultrastructureABSTRACT
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.
Subject(s)
Cytokinesis/physiology , Dictyostelium/cytology , Dictyostelium/physiology , Cell Division/genetics , Cell Division/physiology , Cell Fusion/methods , Cell Membrane/physiology , Cytokinesis/genetics , Dictyostelium/genetics , Gene Knockout Techniques , Genes, Protozoan , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Microfilament Proteins/genetics , Microfilament Proteins/physiology , Myosin Type II/deficiency , Myosin Type II/genetics , Myosin Type II/physiology , Protozoan Proteins/genetics , Protozoan Proteins/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
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.