Sally H. Zigmond

Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors.

Polymorphonuclear leukocyte (PMN) chemotaxis has been examined under conditions which allow phase microscope observations of cells responding to controlled gradients of chemotactic factors. With this visual assay, PMNs can be seen to orient rapidly and reversibly to gradients of N-formylmethionyl peptides. The level of orientation depends upon the mean concentration of peptide present as well as the concentration gradient. The response allows an estimation of the binding constant of the peptide to the cell. In optimal gradients, PMNs can detect a 1% difference in the concentration of peptide. At high cell densities, PMNs incubated with active peptides orient their locomotion away from the center of the cell population. This orientation appears to be due to inactivation of the peptides by the cells. Such inactivation in vivo could help to limit an inflammatory response.

Signal transduction and actin filament organization.

Small GTP-binding proteins of the Rho family appear to integrate extracellular signals from diverse receptor types and initiate rearrangements of F-actin. Active members of the Rho family, Rho and Rac, are now joined by Cdc42 which induces filopodia. Downstream of the Rho family proteins, actin polymerization may be induced by an increase in the availability of actin filament barbed ends. Actin organization may be affected by exposure of actin-binding sites on proteins such as vinculin and ezrin.

Formins: signaling effectors for assembly and polarization of actin filaments

Eukaryotic cells require filamentous actin to maintain their shape and for movement, growth and replication. New actin filaments are formed by the cutting of existing filaments or de novo through the action of specialized nucleators. The most highly characterized nucleator is the Arp2/3 complex, which nucleates the branched actin networks in the lamellae of migrating cells. Recently, Bni1p, which is a member of the formin family of proteins, has been shown to nucleate actin filaments in vitro. Formins are implicated in the formation of actin cables in yeast, stress fibers in tissue culture cells and cytokinesis in many cell types. Formins contain two highly conserved formin-homology domains, FH1 and FH2. The Bni1p FH2 domain is sufficient to mediate nucleation. The Bni1p FH1 domain binds profilin, an actin-monomer-binding protein that delivers actin to the growing barbed end of filaments. The Bni1p FH1-profilin interaction enhances nucleation. Formins participate in a number of signaling pathways that control the assembly of specific actin structures and bind the barbed end of actin filaments, thereby providing a cytoskeletal basis for the establishment of cell polarity.

Chemotaxis by polymorphonuclear leukocytes.

The ability of a cell or organism to direct its movement along a chemical gradient has fascinated biologists for over 100 yr. This process of chemotaxis requires transformation of directional information from the environment into a series of cellular responses resulting in directional movement. Many lower organisms including bacteria, protozoa, and slime molds exhibit chemotaxis. This ability helps them find nutrients, avoid noxious stimuli and aggregate at critical times in their development. Reports on higher organisms indicate that primordial germ cells (32, 36), neurons (123), tumor cells (124), and fibroblasts (115) exhibit chemotaxis. However, the leukocytes are the only vertebrate cells in which this ability has been shown definitively. Studies have focused on the chemotaxis exhibited by the neutrophilic polymorphonuclear leukocytes (PMNs), whose chemotaxis presumably facilitates their accumulation at sites of injury or infection. Chemical gradients are important in morphogenesis (27, 31,40, 75 ,105 ,106 ,151 ,162 ,173) . A gradient can impart at least two types of information to a given cell, positional and vectorial. Positional information is derived from the mean concentration of a given substance present around the cell. Positional information from gradients of diffusible substances has been implicated in the organization of insect epidermis (90), regeneration in hydra (174), and limb morphogenesis (135, 148, 149). Vectorial information at the cellular level depends on a cells ability to detect the direction of the chemical gradient and develop a polarity along this direction (31, 76, 90). Most ceils have a polarity which corresponds to the overall tissue architecture. Some polarities undoubtedly arise from localized stimuli to which cells respond. For example, local stimuli probably contribute to the differentiation of the luminal and basal sides of epithelial cells. We know macrophages can selectively ingest opsonized particles, leaving other unopsonized particles attached to their membrane (60). Motile cells, including slime molds (29) and fibroblasts (3), are described as extending exploratory filopodia which, upon attachment to a suitable stimuli, induce cytoplasmic flow and expand into pseudopods. Other polarizations, for example, the direction of hair growth or cell migration, may be due to gradients of fixed or diffusible agents (31, 90). If the steepness of the gradient required for cell detection in these cases is similar to that needed by leukocytes (discussed below), one would expect the size of the gradient fields to be relatively small, probably in the millimeter range. Since we do not know the chemical nature of the gradient in most developmental systems, it is difficult to study the mechanisms involved in the establishment of cell polarities. In the case of leukocyte chemotaxis, we know some of the chemical signals. Many factors have been shown to be chemotactic for polymorphonuclear leukocytes (118,163) including: (a) serum factors (85, 158,169,172), particularly a fragment of the fifth component of complement (22, 132, 137, 157); (b) bacterial metabolites (86, 152); (c) cell-derived materials from sensitized lymphocytes (159) and from PMNs (18, 30, 111, 181); and (d) denatured proteins (168). Recently, in an attempt to identify a chemotactic factor derived from bacteria, Schiffmann et al. examined the possibility that N-formylmethionyl peptides might be the chemotactic agents since bacteria initiate their protein synthesis with Nformylmethionine (127). They discovered that a number of N-formylmethionyl peptides are in fact chemotactically active, some at very low concentrations (10 -1~ M) (127, 134). It is not clear

The Platelet Cytoskeleton Regulates the Affinity of the Integrin αIIbβ3 for Fibrinogen

Agonist-generated inside-out signals enable the platelet integrin αIIbβ3 to bind soluble ligands such as fibrinogen. We found that inhibiting actin polymerization in unstimulated platelets with cytochalasin D or latrunculin A mimics the effects of platelet agonists by inducing fibrinogen binding to αIIbβ3. By contrast, stabilizing actin filaments with jasplakinolide prevented cytochalasin D-, latrunculin A-, and ADP-induced fibrinogen binding. Cytochalasin D- and latrunculin A-induced fibrinogen was inhibited by ADP scavengers, suggesting that subthreshold concentrations of ADP provided the stimulus for the actin filament turnover required to see cytochalasin D and latrunculin A effects. Gelsolin, which severs actin filaments, is activated by calcium, whereas the actin disassembly factor cofilin is inhibited by serine phosphorylation. Consistent with a role for these factors in regulating αIIbβ3 function, cytochalasin D- and latrunculin A-induced fibrinogen binding was inhibited by the intracellular calcium chelators 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester and EGTA acetoxymethyl ester and the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A. Our results suggest that the actin cytoskeleton in unstimulated platelets constrains αIIbβ3 in a low affinity state. We propose that agonist-stimulated increases in platelet cytosolic calcium initiate actin filament turnover. Increased actin filament turnover then relieves cytoskeletal constraints on αIIbβ3, allowing it to assume the high affinity conformation required for soluble ligand binding.

Chemotactic factor concentration gradients in chemotaxis assay systems

In most assays of chemotaxis the gradient of the chemotactic factor is established and later destroyed by its diffusion through some matrix. The characteristics of the gradient depend upon the geometry of the assay system, the diffusion coefficient of the chemotactic factor and the concentration of the chemotactic factor added. We have solved the diffusion equations to characterize the gradients present in 3 assays of chemotaxis in current use: the millipore, under-agarose and visual assay systems. In each case of the solutions are presented for various assay times and for chemotactic factors with various diffusion coefficients.

Phospholipase Cβ Is Critical for T Cell Chemotaxis

Chemokines acting through G protein-coupled receptors play an essential role in the immune response. PI3K and phospholipase C (PLC) are distinct signaling molecules that have been proposed in the regulation of chemokine-mediated cell migration. Studies with knockout mice have demonstrated a critical role for PI3K in Gαi protein-coupled receptor-mediated neutrophil and lymphocyte chemotaxis. Although PLCβ is not essential for the chemotactic response of neutrophils, its role in lymphocyte migration has not been clearly defined. We compared the chemotactic response of peripheral T cells derived from wild-type mice with mice containing loss-of-function mutations in both of the two predominant lymphocyte PLCβ isoforms (PLCβ2 and PLCβ3), and demonstrate that loss of PLCβ2 and PLCβ3 significantly impaired T cell migration. Because second messengers generated by PLCβ lead to a rise in intracellular calcium and activation of PKC, we analyzed which of these responses was critical for the PLCβ-mediated chemotaxis. Intracellular calcium chelation decreased the chemotactic response of wild-type lymphocytes, but pharmacologic inhibition of several PKC isoforms had no effect. Furthermore, calcium efflux induced by stromal cell-derived factor-1α was undetectable in PLCβ2β3-null lymphocytes, suggesting that the migration defect is due to the impaired ability to increase intracellular calcium. This study demonstrates that, in contrast to neutrophils, phospholipid second messengers generated by PLCβ play a critical role in T lymphocyte chemotaxis.

ACTIN POLYMERIZATION : WHERE THE WASP STINGS

How do extracellular signals induce actin polymerization, as required for many cellular responses? Key signal transducers, such as the small GTPases Cdc42 and Rac, have now been shown to link via proteins of the WASP family to the Arp2/3 complex, which nucleates actin polymerization.

Beginning and ending an actin filament: control at the barbed end.

Dynamic actin filaments contribute to cell migration, organelle movements, memory, and gene regulation. These dynamic processes are often regulated by extracellular and?or cell cycle signals. Regulation targets, not actin itself, but the factors that determine its dynamic properties. Thus, filament nucleation, rate and duration of elongation, and depolymerization are each controlled with regard to time and?or space. Two mechanisms exist for nucleating filaments de novo, the Arp23 complex and the formins; multiple pathways regulate each. A new filament elongates rapidly but transiently before its barbed end is capped. Rapid capping allows the cell to maintain fine temporal and spatial control over F-actin distribution. Modulation of capping protein activity and its access to barbed ends is emerging as a site of local regulation. Finally, to maintain a steady state filaments must depolymerize. Depolymerization can limit the rate of new filament nucleation and elongation. The activity of ADF?cofilin, which facilitates depolymerization, is also regulated by multiple inputs. This chapter describes (1) mechanism and regulation of new filament formation, (2) mechanism of enhancing elongation at barbed ends, (3) capping proteins and their regulators, and (4) recycling of actin monomers from filamentous actin (F-actin) back to globular actin (G-actin).

Organization of myosin in a submembranous sheath in well-spread human fibroblasts☆

Abstract Using an indirect immunofluorescence technique, antibodies against human platelet myosin stain a highly organized layer of sheath of fibers in well-spread human fibroblasts. By adjusting the focus, the sheath is seen to be continuous over the top of the cell. Myosin staining along a fiber is periodic with a mean spacing of 0.71 ± 0.1 μm. Although the sheath becomes progressively disorganized and eventually disappears upon cell rounding after trypsinization, the mean spacing in the fibers remaining at intermediate stages does not appear to change. The organization is sensitive to cytochalasin B (CB), but insensitive to colchicine. Anti-myosin staining of the sheath persists in cells lysed in 0.1–0.5% Triton X-100 at low ionic strength despite some myosin extraction. Preparation of the cytoskeleton at increasingly higher ionic strength or extraction with ATP or pyrophosphate diminishes the sheath staining with anti-myosin. A detailed analysis of the mean myosin spacing in the cytoskeleton after various treatments designed to induce contraction suggests (1) that reagents which extract myosin, i.e., ATP, high salt and pyrophosphate, all cause contraction and (2) that the mean spacing does not detectably change. Anti-alpha-actinin and anti-tropomyosin also localize and demonstrate a periodicity for their respective antigens in the sheath fibers. Double staining with anti-myosin and anti-tropomyosin gives an enhancement of the basic myosin perodicity indicating co-localization. Double staining with anti-alpha-actinin and anti-myosin gives a nearly continuous fluorescence along the sheath fiber suggesting a complementary periodicity. This finding was confirmed with a direct anti-myosin-indirect anti-alpha-actinin staining experiment. A possible chemical mechanism, based on a competition of these proteins for actin, for the generation of the observed periodicity and formation of the sheath is considered.