Jana Köhler

Dynamic Actin Patterns and Arp2/3 Assembly at the Substrate-Attached Surface of Motile Cells

BACKGROUND In the cortical region of motile cells, the actin network rapidly reorganizes as required for movement in various directions and for cell-to-substrate adhesion. The analysis of actin network dynamics requires the combination of high-resolution imaging with a specific fluorescent probe that highlights the filamentous actin structures in live cells. RESULTS Combining total internal reflection fluorescence (TIRF) microscopy with a method for labeling actin filaments, we analyze the dynamics of actin patterns in the highly motile cells of Dictyostelium. A rapidly restructured network of single or bundled actin filaments provides a scaffold for the assembly of differentiated actin complexes. Recruitment of the Arp2/3 complex characterizes stationary foci with a lifetime of 7-10 s and traveling waves. These structures are also formed in the absence of myosin-II. Arp2/3-actin assemblies similar to those driving the protrusion of a leading edge form freely at the inner face of the plasma membrane. CONCLUSIONS The actin system of highly motile cells runs far from equilibrium and generates a multitude of patterns within a dynamic filamentous network. Traveling waves are the most complicated patterns based on recruitment of the Arp2/3 complex. They are governed by the propagated induction of actin polymerization. We hypothesize that the actin system autonomously generates primordia of specialized structures such as phagocytic cups or lamellipodia. These primordia would represent an activated state of the actin system and enable cells to respond within seconds to local stimuli by chemotaxis or phagocytic-cup formation.

Dynein motor regulation stabilizes interphase microtubule arrays and determines centrosome position.

Cytoplasmic dynein is a microtubule‐based motor protein responsible for vesicle movement and spindle orientation in eukaryotic cells. We show here that dynein also supports microtubule architecture and determines centrosome position in interphase cells. Overexpression of the motor domain in Dictyostelium leads to a collapse of the interphase microtubule array, forming loose bundles that often enwrap the nucleus. Using green fluorescent protein (GFP)–α‐tubulin to visualize microtubules in live cells, we show that the collapsed arrays remain associated with centrosomes and are highly motile, often circulating along the inner surface of the cell cortex. This is strikingly different from wild‐type cells where centrosome movement is constrained by a balance of tension on the microtubule array. Centrosome motility involves force‐generating microtubule interactions at the cortex, with the rate and direction consistent with a dynein‐mediated mechanism. Mapping the overexpression effect to a C‐terminal region of the heavy chain highlights a functional domain within the massive sequence important for regulating motor activity.

Recruitment of cortexillin into the cleavage furrow is controlled by Rac1 and IQGAP‐related proteins

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 pro teins 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.

Dynamic organization of the actin system in the motile cells of Dictyostelium

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.

Golvesin-GFP fusions as distinct markers for Golgi and post- Golgi vesicles in Dictyostelium cells

Golvesin is a new protein associated with membranes of the Golgi apparatus and post‐Golgi vesicles in Dictyostelium cells. An internal hydrophobic sequence of 24 amino‐acid residues is responsible for anchoring golvesin to the membranes of these organelles. In an attempt to visualize organelle dynamics in vivo, we have used specific antibody and other labels to localize golvesin—green fluorescent protein (GFP) constructs to different cellular compartments. With a GFP tag at its N‐terminus, golvesin shows the same localization as the untagged protein. It is transferred to two post‐Golgi compartments, the endosomal and contractile vacuole systems. Endosomes are decorated with GFP—golvesin within less than 10 min of their internalisation, and keep the label during the acidic phase of the pathway.

Endosome fusion and microtubule-based dynamics in the early endocytic pathway of Dictyostelium

Dictyostelium amoebae, like mammalian macrophages, take up fluid by macropinocytosis. The present study used fluorescent fluid‐phase markers and GFP‐labeled microtubules to visualize the uptake, dynamics, and fusion of early endosomes in Dictyostelium. Consecutive labeling with two fluorescent fluid‐phase markers demonstrated that within the first few minutes after uptake, new macropinosomes underwent fusion with pre‐existing endosomes. The fusing endosomes, which represent the mixing compartment, displayed extreme shape changes and rapid transport about the cell in association with microtubules. The great plasticity of endosomes at this stage of maturation was also evident by electron microscopy. The constant undulatory motion of microtubules was implemental in establishing contact with endosomes. Treatment of cells with agents that selectively disrupted either actin filaments or microtubules confirmed that endosome dynamics were microtubule based. Further maturation of endosomes led to loss of pleiomorphy in favor of a spherical shape, inability to fuse with new macropinosomes, and diminished motility.

Two-step positioning of a cleavage furrow by cortexillin and myosin II

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.

GFP-golvesin constructs to study Golgi tubulation and post-Golgi vesicle dynamics in phagocytosis.

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.