Cell cycle-dependent morphological changes in the actin cytoskeleton induced by agents which elevate cyclic AMP.

Agents which increase the intracellular concentration of cyclic adenosine-5'-monophosphate, induce a highly arborised morphology in a proportion of sub-confluent Swiss 3T3 fibroblasts. During this process the organisation of actin filaments progressively changes from a characteristic stress fibre pattern to leave a network of actin filaments within each and every arborisation. Despite this massive reorganisation of the actin cytoskeleton no changes are observed in the extent of polymerisation of actin during arborisation. The proportion of cells in asynchronous cultures undergoing arborisation at maximal concentrations of agents reaches a maximum of 30%; suggesting that the effect might be mediated only in cells during a restricted period of the cell cycle. More than 80% of serum-starved cells responded to these agents between 1 and 8 hours after readdition of serum, but not at other times, suggesting that the arborisation response can occur only in the G1 phase of the cell cycle.

Mechanisms of actin filament turnover in animal cells.

About half of the total actin in the cytoplasm of cultured animal cells is polymerised into filaments at any time. The filaments are further ordered into 3-dimensional patterns by their interaction with a number of actin-binding proteins (ABPs) to form the functional actin cytoskeleton. Three consistent patterns of organisation can be discerned both by their complement of ABPs, as determined by co-localisation, and by the characteristic arrangement of actin filaments: isotropic arrays, parallel bundles and anti-parallel bundles. These three patterns of organisation appear to have discrete functional properties which, together, give rise to the motile behavior of the cells. The proportions and locations of these actin filament organisations reflect, or give rise to, the particular properties of the cells in which they are found. In locomoting cells in particular, the extent and precise cellular location of these three classes of organisation are constantly changing during the process of locomotion. This constant adaptation of the actin cytoskeleton appears, from a number of approaches, to result from a continuous cycle of assembly and disassembly of filaments leading to continuous adaptation of the actin cytoskeleton. In some cells at least, net assembly occurs predominantly at the extreme leading edge of the lamellipodium, and it must be presumed that filaments assembled in the isotropic arrays here give rise to the other levels of architecture found in the cell. However, overlying this appears to be a continuous cycle of assembly and disassembly of filaments within all parts of the actin cytoskeleton. The underlying imperative for turnover of actin filaments derives from the ATPase associated with polymerisation. The implications of this assembly ATPase for both turnover and for the progressive evolution of actin filament architectures is discussed.

A comparison of spreading and motility behaviour of 8701-BC breast carcinoma cells on type I, I-trimer and type V collagen substrata. Evidence for a permissive effect of type I-trimer collagen on cell locomotion.

Ductal infiltrating carcinoma (d.i.c.) of human breast is a highly invasive neoplasm characterized by enhanced deposition of collagen. Paradoxically, enhanced collagen deposition is not correlated with inhibition of the migration of tumour cells into the host tissue. d.i.c. is characterized by the reappearance of 'embryonic' type I-trimer collagen and an increase in type V collagen content in the matrix. The effects of these two collagen types were compared with type I collagen as culture substrata on the spreading pattern, cytoskeletal organization and motile behaviour of 8701-BC breast carcinoma cells using rhodamine-phalloidin staining, a DNAase I-competition assay, scanning electron microscopy and time-lapse video-microscopy. Cells grown on type I collagen were stationary, showing a well-spread morphology and an extensive stress fibre pattern. Cells grown on type V collagen were also stationary, but displayed a poorly spread and elongated morphology. In contrast, cells grown on trimer collagen were motile and displayed a compact morphology and a reduced content of stress fibres. Both single-cell and group motility were detectable on trimer collagen substratum. These data are consistent with the existence of two opposite local signals, type I-trimer and type V collagens, which may confer a more or a less metastatic phenotype on breast carcinoma cells. Moreover, the synthesis of trimer collagen in d.i.c. is conceivably instrumental in providing new stromal pathways permitting tumour cells to infiltrate the host tissue.

The nature and regulation of actin filament turnover in cells.

Actin filaments in mammalian cells form a number of different architectures in conjunction with a number of different actin-binding proteins. In motile cells these complex architectural arrangements of actin filaments and associated proteins continuously adjust their 3-dimensional organisation to modify the shape and behaviour of cells in response to external information. Microinjection experiments with fluorescently-labelled actin monomers suggest that there is a continual exchange of monomers between the actin filaments and a soluble pool such that individual monomers exist for only a few minutes within polymers. These data suggest that remodelling of the actin filament architectures occurs by the continuous assembly of new filaments which is balanced by the disassembly of obsolete structures. The mechanisms driving and regulating the assembly and disassembly cycle are not yet clearly understood. The properties of the actin assembly ATPase in vitro suggest that the intrinsic exchange of monomers between polymers and the monomer pool is driven by the stoichiometric ATP hydrolysis which is uncoupled from monomer addition and leads to both treadmilling and to the potential for mechanisms analogous to the dynamic instability models proposed for microtubules. Because of the relatively rapid rate of ATP hydrolysis by monomers in the filament (k = 0.05-0.02/s), it is assumed that most of the F-actin in cells is in its ADP form. ADP-F-actin binds inorganic phosphate with a Kd close to that of cytoplasmic concentrations to form an ADP.Pi-F-actin form which has different kinetic, structural and behavioural properties to ADP-F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Microinjection of covalently cross-linked actin oligomers causes disruption of existing actin filament architecture in PtK2 cells.

Experiments were performed to determine the effects of interrupting the flux of actin monomers between unpolymerised and polymerised pools in PtK2 cells by (1) microinjecting exogenous polymerisation nuclei and (2) blocking endogenous assembly sites with low concentrations of cytochalasin D. Fluorescent actin oligomers were prepared by glutaraldehyde cross-linking F-actin derivatised at cysteine-374 with 5-iodoacetamido-fluorescein. These oligomers caused rapid nucleation of polymerisation of pyrene-labelled actin in vitro. Different numbers of polymerisation nuclei were injected into PtK2 cells and the cells were incubated for various times. Microinjection of between 1.2 X 10(4) and 1.8 X 10(4) nuclei per cell resulted in the complete disassembly of existing actin filament structures in nearly half of the cells within 15 min. Existing structures in such cells were replaced by foci of polymerised actin, which co-localised with concentrations of nuclei. Injection of increasing numbers of nuclei between 3 X 10(3) and 1.2 X 10(4) caused fragmentation of stress fibres in an increasing proportion of cells, whereas injection of less than 3 X 10(3) caused no obvious effects even over a 90 min incubation period. These data indicate that the degree of disruption of stress fibres was a function of the number of nuclei injected, but that it was less dependent on the incubation time. The minimum number of injected nuclei causing complete disruption of actin filament structures provides an estimate for the number of endogenous nuclei involved in filament turnover, whereas the minimum period for reorganisation (about 15min) implies a maximum time for the complete turnover of actin in the system.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ATP removal and inorganic phosphate on length redistribution of sheared actin filament populations. Evidence for a mechanism of end-to-end annealing.

The effect of inorganic phosphate (Pi) on the depolymerization of F-actin has been measured. Pi inhibits disassembly of pyrene-labelled F-actin at steady-state induced either by dilution, or by shearing, suggesting that Pi decreases the off rate constant, k-, for dissociation. This effect of Pi is maximal at 20 mM, unlike the effect of Pi in reducing the critical concentration at the pointed end (maximal at 2 mM). This difference in concentration dependence for the two effects is interpreted as different affinities of Pi for the barbed and pointed ends, presumably as ADP-Pi-actin species. The contribution of ATP/ADP phase changes at filament ends (i.e. "dynamic instability") to length redistribution in sheared polymer steady-state actin filament populations was determined by (1) converting ATP to ADP in the system to prevent phase changes, or (2) adding 20 mM-Pi to the system to inhibit depolymerization. The observed absence of effect of these treatments on length redistribution excludes all mechanisms which involve phase change-driven disassembly or monomer exchange at filament ends, and appears to constrain the mechanism to one of end-to-end annealing under these conditions.

Phorbol ester induces rapid actin assembly in neutrophil leucocytes independently of changes in [Ca2+]i and pHi.

The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), at nanomolar concentrations, induces rapid (t1/2 approximately 30 s) protrusion of multiple petal-shaped lamellae by neutrophil leucocytes. Lamellae are richly endowed with actin filaments as determined by the localization of rhodamine-phalloidin, suggesting extensive assembly at the cell cortex. Direct measurement of the proportion of total cell actin which is polymerized, by using a deoxyribonuclease I inhibition assay, indicates that the proportion of polymerized actin approximately doubles, and that assembly initiated by 30 nM TPA occurs with no obvious lag phase and with a t1/2 of about 30 s. A half-maximal response was induced at 2 nM TPA. Since both actin assembly and protrusion of lamellae are completely inhibited by 10(-6) M cytochalasin D, protrusion of lamellae is presumably dependent on actin filament assembly. To examine whether TPA induces actin assembly via changes in [Ca2+]i or pHi, these parameters were monitored in cells loaded with the fluorescent indicators quin2 and quene1 respectively. Addition of TPA caused no change in [Ca2+]i but a biphasic change in pHi. To examine further the potential role of ionic changes in regulation of actin assembly, the morphological responses of cells to TPA were monitored in severely Ca2+-depleted cells, or cells in which pHi had been experimentally raised or lowered by simultaneous additions of a weak base (NH4Cl) or weak acid (CH3COONa) respectively. The protrusion of lamellae induced by TPA was completely unaffected by these experimental manipulations indicating that TPA can directly regulate actin assembly, and hence morphology, by mechanisms essentially independent of [Ca2+]i and pHi.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytoplasmic concentrations of inorganic phosphate affect the critical concentration for assembly of actin in the presence of cytochalasin D or ADP.

Skeletal muscle actin labelled with pyrene was used to measure the critical concentration (Cc) for assembly in conditions designed to approximate the ionic environment in the cytoplasm. Under these conditions (0.1 M-KCl, 2 mM-MgCl2, 1.1 mM-ATP, 0.1 mM-CaCl2, 0.5 mM-ethyleneglycol-bis(beta-aminoethylether)N,N'-tetraacetic acid, 0.25 mM-2-mercaptoethanol, 20 mM-imidazol X HCl, pH 7.0), the steady-state Cc value was estimated to be 0.07 microM (3.0 micrograms/ml), and, consistent with previous observations, the Cc increased to 0.20 microM (8.7 micrograms/ml) in the presence of 10(-6) M-cytochalasin D, and to 1.10 microM (47 micrograms/ml) after conversion of ATP to ADP using hexokinase and glucose. Addition of inorganic phosphate (Pi) at concentrations up to 20 mM caused only a slight decrease in the steady-state Cc, but at 2 mM-Pi (a reasonable estimate of cytoplasmic concentrations) the increase in Cc due to cytochalasin D was abolished, and at higher Pi concentrations there was even a slight decrease. Increasing Pi concentrations also progressively reduced the steady-state Cc for ADP-actin close to that for ATP-actin. These results are consistent with an increased affinity of ADP-actin for the polymer in the presence of Pi. To determine whether these effects of Pi were simply mass action effects on hydrolysis of bound ATP by polymerized actin, the stoichiometry of ATP hydrolysis during actin assembly was estimated and found to be at unity within the limits of experimental error and to be unaffected by Pi up to 20 mM. In addition, actin depolymerized by removal of ATP using glucose and hexokinase rapidly reassembled after addition of 20 mM-Pi. These results are interpreted by a mechanism involving the formation of ADP-Pi-actin species and are discussed in relation to the phenomenon of treadmilling and the theory of dynamic instability, and the potential for their occurrence in cells.

Evidence that phorbol ester interferes with stimulated Ca2+ redistribution by activating Ca2+ efflux in neutrophil leucocytes.

The free calcium ion concentration, [Ca2+]i, in the cytoplasmic matrix of quin2-loaded neutrophil leucocytes increases rapidly after addition of concanavalin A. This increase is effectively abolished by a short (3 min) preincubation with 10 nM-TPA (12-O-tetradecanoylphorbol 13-acetate). TPA also inhibits a [Ca2+]i rise of similar magnitude induced by low concentrations (10 nM) of calcium ionophore A23187, suggesting that phorbol ester does not interfere with a physiological influx mechanism. To investigate the effects of TPA further, cells were depleted of Ca2+ during quin2 loading and then re-equilibrated with normal extracellular [Ca2+]. The return to a stable [Ca2+]i value was preceded by a transient overshoot in [Ca2+]i, implying delayed activation of an efflux mechanism by rising [Ca2+]i. TPA abolished the transient, suggesting preactivation by TPA of the efflux mechanism before Ca2+ influx. TPA also stimulates net Ca2+ efflux from neutrophils and neutrophil cytoplasts. These observations are consistent with the thesis that TPA stimulates a Ca2+-efflux mechanism in these cells.

Fc receptor-directed phagocytic stimuli induce transient actin assembly at an early stage of phagocytosis in neutrophil leukocytes.

The phagocytic pathway for the uptake of multivalent Fc complexes by neutrophil leukocytes can be partially dissected by using a pulse-chase protocol at different chase temperatures. Utilising a deoxyribonuclease I inhibition assay, we show that an early response to recognition of phagocytic substrate by neutrophils is a rapid (less than 10 sec) polymerization of actin. Net actin assembly is transient and is reversed at higher chase temperatures which appears to allow for further progress of the pulse of phagocytic substrate along the pathway. Assembly of actin monitored in whole cells is paralleled by an increase in total actin retained in the cortical filamentous network of detergent-insoluble ghosts prepared from phagocytosing neutrophils. These data provide direct evidence for the reversible reorganisation of actin filaments in the cortical cytoplasm of phagocytic cells during phagocytosis. Furthermore, this experimental system provides an opportunity to study the characteristics of ligand-induced actin assembly, and subsequent disassembly in situ.

Immunofluorescence microscopy of the flagella and multilayered structure in two mosses, Sphagnum palustre L. and Polytrichum juniperinum Hedw.

Antibody against tubulin from porcine brain was used to examine the distribution of tubulin in developing spermatids of Polytrichum and mature spermatozoids of Sphagnum. Cells were prepared for indirect immunofluorescence microscopy after fixation in buffered paraformaldehyde and brief incubation in cellulase. Pretreatment with cold methanol resulted in considerably enhanced immunofluorescence but exposure to Triton X-100, with or without sonication, had no effect. The antibody showed similar immunological cross-reactivity with the flagella (both basal bodies and axonemes) and the spline microtubules of the multilayered structure. This is the first direct evidence that this rigid array of stable cytoskeletal microtubules consists of tubulin. Particularly intense fluorescence from the lamellar strata of the MLS in developing spermatids provides strong support for the notion that the lamellae comprise a highly structured microtubule organizing centre (MTOC), responsible for the ordered assembly of the overlying spline tubules. The demonstration of immunological cross-reactivity with antitubulin from porcine brain tubulin, within a plant structure other than fully formed microtubules, suggests that immunocytochemistry may have considerable potential for the detection of other MTOCs. By contrast, no detectable fluorescence emanated from the granular matrix cementing the flagellar basal bodies to the spline or the spindle-shaped sheath of fibres present in the spermatozoids of Sphagnum. Disruption of the mature gametes by sonication and treatment with Triton X-100 reveals the presence of particularly strong links between the spline and subjacent nuclear envelope.

Transmembrane linkage between surface glycoproteins and components of the cytoplasm in neutrophil leukocytes.

An experimental approach is described that enables the analysis of interactions between exogenous surface ligands and components of the cytoplasm in neutrophil leukocytes. Neutrophils treated with the nonionic detergent Lubrol PX, under controlled conditions, yield intact detergent-insoluble ghosts. Morphological analysis of neutrophil ghosts shows that they retain the original dimensions of the cell and consist almost entirely of a peripheral filamentous network, representing the submembranous cortical web, concentric to nuclear remnants. All intracellular membrane-bounded organelles, plasma membrane, and background cytoplasmic electron density are absent. Biochemical analysis of the ghosts shows that less than 10% of enzyme markers for the soluble and granule fractions remain, and that greater than 90% of total cell phospholipid is removed during detergent extraction. The major proteins remaining in the ghosts comigrate, on polyacrylamide gels in the presence of SDS, with chicken gizzard actin, myosin, filamin, and a 110-kdalton protein. Patches and caps induced on neutrophils with either fluorescein isothiocyanate-concanavalin A or ferritin-concanavalin A retain their original location and morphology on ghosts after lysis, as determined by both fluorescence and electron microscopy. In similar experiments, but using 125I-labeled lectins, 37% of total cell bound concanavalin A (Con A) and 25% succinylated Con A remain attached to the ghosts. A major 125I-labeled membrane glycoprotein (80 kdaltons) is associated with ghosts prepared from intact neutrophils iodinated in the presence of exogenous lactoperoxidase. Further 125I-labeled membrane glycoproteins (217, 170, and 147 kdaltons) become associated with ghosts prepared from iodinated cells treated before lysis with Con A, but not with succinylated Con A. These data taken together suggest that linkages exist in neutrophils between proteins exposed on the outer surface of the plasma membrane and the peripheral filamentous network independent of the presence of lipid bilayer. The implications of these findings for surface motile phenomena will be discussed.