Günther Gerisch

Discrete interactions in cell adhesion measured by single-molecule force spectroscopy

Cell–cell adhesion mediated by specific cell-surface molecules is essential for multicellular development. Here we quantify de-adhesion forces at the resolution of individual cell-adhesion molecules, by controlling the interactions between single cells and combining single-molecule force spectroscopy with genetic manipulation. Our measurements are focused on a glycoprotein, contact site A (csA), as a prototype of cell-adhesion proteins. csA is expressed in aggregating cells of Dictyostelium discoideum, which are engaged in development of a multicellular organism. Adhesion between two adjacent cell surfaces involves discrete interactions characterized by an unbinding force of 23 ± 8 pN, measured at a rupture rate of 2.5 ± 0.5 µm s–1.

Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag.

Coronin is a protein involved in cell locomotion and cytokinesis of Dictyostelium discoideum. Here we show that coronin is strongly enriched in phagocytic cups formed in response to particle attachment. A fusion of coronin with green fluorescent protein (GFP) accumulates in the cups within less than 1 min upon attachment of a particle and is gradually released from the phagosome within 1 min after engulfment is completed. Phagocytic cup formation competes with leading edge formation and can be interrupted at any stage. When the cup regresses, coronin dissociates from the site of accumulation. TRITC-labeled yeast cells have been used to assay phagocytosis quantitatively in wild-type and coronin-null cells. In the mutant, the rate of uptake is reduced to about one third, which shows that coronin contributes to the efficiency of phagocytosis to about the same extent as it improves the speed of cell locomotion.

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.

Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis

Calreticulin and calnexin are Ca2+‐binding proteins with chaperone activity in the endoplasmic reticulum. These proteins have been eliminated by gene replacement in Dictyostelium, the only microorganism known to harbor both proteins; family members in Dictyostelium are located at the base of phylogenetic trees. A dramatic decline in the rate of phagocytosis was observed in double mutants lacking calreticulin and calnexin, whereas only mild changes occurred in single mutants. Dictyostelium cells are professional phagocytes, capable of internalizing particles by a sequence of activities: adhesion of the particle to the cell surface, actin‐dependent outgrowth of a phagocytic cup, and separation of the phagosome from the plasma membrane. In the double‐null mutants, particles still adhered to the cell surface, but the outgrowth of phagocytic cups was compromised. Green fluorescent protein‐tagged calreticulin and calnexin, expressed in wild‐type cells, revealed a direct link of the endoplasmic reticulum to the phagocytic cup enclosing a particle, such that the Ca2+ storage capacity of calreticulin and calnexin might directly modulate activities of the actin system during particle uptake.

Sequence and analysis of chromosome 2 of Dictyostelium discoideum

The genome of the lower eukaryote Dictyostelium discoideum comprises six chromosomes. Here we report the sequence of the largest, chromosome 2, which at 8 megabases (Mb) represents about 25% of the genome. Despite an A + T content of nearly 80%, the chromosome codes for 2,799 predicted protein coding genes and 73 transfer RNA genes. This gene density, about 1 gene per 2.6 kilobases (kb), is surpassed only by Saccharomyces cerevisiae (one per 2 kb) and is similar to that of Schizosaccharomyces pombe (one per 2.5 kb). If we assume that the other chromosomes have a similar gene density, we can expect around 11,000 genes in the D. discoideum genome. A significant number of the genes show higher similarities to genes of vertebrates than to those of other fully sequenced eukaryotes. This analysis strengthens the view that the evolutionary position of D. discoideum is located before the branching of metazoa and fungi but after the divergence of the plant kingdom, placing it close to the base of metazoan evolution.

Cyclic GMP in Dictyostelium discoideum Oscillations and pulses in response to folic acid and cyclic AMP signals

respond chemo- tactically to.cyclic AMP [l] and to folic acid [3]. Cyclic AMP is a most efficient attractant for cells of the aggregation phase [2], folic acid is more effective with preaggregation cells [4]. Both cyclic AMP and folic acid stimulate also cell development from the preaggregation phase to the aggregation competent state [S-8]. During this process the cells acquire the capacity to synthesize cyclic AMP periodically, and to release it into the extracellular space in form of pulses [9] . The administration of cyclic AMP or folic acid pulses accelerates the onset of sustained oscillations [5,8] . The cyclic-AMP induced responses are known to be mediated by cell-surface receptors [lo-121. The intermediate steps in signal processing from the receptors to the various intracellular targets are unknown. In the present communication we show that upon stimulation of early preaggregation cells with folic acid a rapid increase of the cyclic GMP concentration is induced. A biphasic increase of cyclic GMP is observed in late preaggregation cells stimulated by cyclic AMP. The second cyclic GMP peak is suc- ceeded by a cyclic AMP peak known to be based on the activation of adenylate cyclase [ 131. Free-running oscillations of cyclic GMP were observed together with the periodic formation of cyclic AMP pulses, and the cyclic GMP peaks seemed to occur slightly in advance of the cyclic AMP peaks. 2.

Amplification of cyclic-AMP signals in aggregating cells of Dictyostelium discoideum

Aggregating cells of the slime mold, Dictyostelium discoideum, respond to cyclic AMP by chemotactic orientation [ 1,2] and by propagating chemotactic signals in the form of waves from one cell to the next [3,6]. It has been proposed that each cell acts as a biochemical signal amplifier [3-81 so that cyclic AMP would play the role of a transmitter in a relay system. In order to investigate the amplifier system quantitatively, methods have been developed to measure signal generation, cyclic-AMP binding to receptors as well as cellular responses to cyclic AMP in stirred cell suspensions [9-l 11. Under these conditions evidence for signal amplification has been obtained [ 1 1 13 ] using the protein-binding assay for cyclic-AMP [ 141. In the present paper we report results obtained by an independent method for measuring cyclic-AMP stimulated cyclic-AMP release: prelabelling of cells with [3H]adenine and chromatographic separation of the labelled compounds from the extracellular fluid after stimulation of the cells by unlabelled cyclic AMP.

Membrane Bending Modulus and Adhesion Energy of Wild-Type and Mutant Cells of Dictyostelium Lacking Talin or Cortexillins

We have employed an interferometric technique for the local measurement of bending modulus, membrane tension, and adhesion energy of motile cells adhering to a substrate. Wild-type and mutant cells of Dictyostelium discoideum were incubated in a flow chamber. The flow-induced deformation of a cell near its adhesion area was determined by quantitative reflection interference contrast microscopy (RICM) and analyzed in terms of the elastic boundary conditions: equilibrium of tensions and bending moments at the contact line. This technique was employed to quantify changes caused by the lack of talin, a protein that couples the actin network to the plasma membrane, or by the lack of cortexillin I or II, two isoforms of the actin-bundling protein cortexillin. Cells lacking either cortexillin I or II exhibited reduced bending moduli of 95 and 160 k(B)T, respectively, as compared to 390 k(B)T, obtained for wild-type cells. No significant difference was found for the adhesion energies of wild-type and cortexillin mutant cells. In cells lacking talin, not only a strongly reduced bending modulus of 70 k(B)T, but also a low adhesion energy one-fourth of that in wild-type cells was measured.

Cortexillins, major determinants of cell shape and size, are actin-bundling proteins with a parallel coiled-coil tail.

Cortexillins I and II of D. discoideum constitute a novel subfamily of proteins with actin-binding sites of the alpha-actinin/spectrin type. The C-terminal halves of these dimeric proteins contain a heptad repeat domain by which the two subunits are joined to form a two-stranded, parallel coiled coil, giving rise to a 19 nm tail. The N-terminal domains that encompass a consensus actin-binding sequence are folded into globular heads. Cortexillin-linked actin filaments form preferentially anti-parallel bundles that associate into meshworks. Both cortexillins are enriched in the cortex of locomoting cells, primarily at the anterior and posterior ends. Elimination of the two isoforms by gene disruption gives rise to large, flattened cells with rugged boundaries, portions of which are often connected by thin cytoplasmic bridges. The double-mutant cells are multinucleate owing to a severe impairment of cytokinesis.

A role for myosin VII in dynamic cell adhesion

BACKGROUND The initial stages of phagocytosis and cell motility resemble each other. The extension of a pseudopod at the leading edge of a migratory cell and the formation of a phagocytic cup are actin dependent, and each rely on the plasma membrane adhering to a surface during dynamic extension. RESULTS A myosin VII null mutant exhibited a drastic loss of adhesion to particles, consistent with the extent of an observed decrease in particle uptake. Additionally, cell-cell adhesion and the adhesion of the leading edge to the substratum during cell migration were defective in the myosin VII null cells. GFP-myosin VII rescued the phagocytosis defect of the null mutant and was distributed in the cytosol and recruited to the cortical cytoskeleton, where it appeared to be enriched at the tips of filopods. It was also localized to phagocytic cups, but only during the initial stages of particle engulfment. During migration, GFP-myosin VII is found at the leading edge of the cell. CONCLUSIONS Myosin VII plays an important role in mediating the initial binding of cells to substrata, a novel role for an unconventional myosin.