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PubMed2004

Length control is determined by the pattern of cytoskeleton.

In our previous experiments with linear strips of adhesive substrate, we found that elongated cultured fibroblasts preserve their length regardless of cell width and the number of cytoplasmic processes. This constancy of length was called 'length control'. In contrast to fibroblasts, single cultured epitheliocytes have nearly discoid shape on the plane substrata and have no length-controlling mechanism: their length on the narrow strips of adhesive substrate increased significantly in comparison with the diameter on the plane substrate. These results suggested that control of length is cell specific. An alternative suggestion is that length control is associated not with the cell type but with the cell cytoskeletal pattern (namely, with epithelioid circular actin bundles or straight actin bundles). Experiments described in this paper were made to choose between these two suggestions. Mouse embryo fibroblasts spreading on the planar substrate first acquire discoid epithelioid shape with a circular actin bundle. Only later did they acquire a polarized shape with straight actin bundles. Polarized, fully spread fibroblasts temporarily acquire discoid epithelioid shape when treated with the Taxol, disorganizing microtubules. However, epithelial discoid cells can be transformed into elongated fibroblast-like cells by scatter factor (HGF/SF; a cytokine) and by agents inhibiting Rho kinase. These reversible transitions from fibroblastic to epithelioid shape and vice versa were accompanied by a corresponding disappearance and appearance of length control. Fibroblasts with stress fibers destroyed by the Rho-kinase inhibitor Y27632 became considerable longer on the adhesive strips than on the plane while retaining a near-polarized shape. Thus, length control is typical not of the cell origin but of the cell phenotype (i.e. for polarized cells with microtubules and intact actin cytoskeleton).

Actin Cytoskeleton↗
PubMed1992

Attachment of steroidogenic lipid droplets to intermediate filaments in adrenal cells.

Light microscopy of living and extracted adrenal cells (Y-1 mouse adrenal tumour cells and cultured bovine fasciculata cells), using Nomarski optics and fluorescence with nile red to stain lipid, revealed in both cell types that lipid droplets remain attached to intermediate filaments when the cells are extracted to prepare these structures. Electron microscopy of thin sections shows the presence of lipid droplets in both cell types. The droplets differ in appearance but are, in both cases, surrounded by a complete capsule 5 nm wide. The droplets in Y-1 cells include those associated with lysosomes and crystalline structures in addition to typical rounded forms. Only the latter type is seen in bovine fasciculata. Intermediate filaments apparently ending in droplets can also be seen. Immunoelectron microscopy with anti-vimentin and Protein A conjugated to gold particles together with measurement of the diameter of these structures identifies them as intermediate filaments. When adrenal cells are permeabilised and extracted under mild or severe conditions using Triton X-100, thin sections showed that lipid droplets remain associated with the cytoskeleton and in particular intermediate filaments. Extraction under mild and severe conditions cleared the cell contents, revealing attachment of intermediate filaments to lipid droplets with greater clarity than in unextracted cells, i.e. homogenised cells or cells subjected to lysis. Such attachment was unequivocally demonstrated in stereo pairs. These observations support our earlier studies showing attachment of droplets to intermediate filaments, which suggests a role for these filaments in intracellular transport of cholesterol.

Adrenal Glands↗
PubMed1992

Localised application of an activating signal to a cell: experimental use of fibronectin bound to beads and the implications for mechanisms of adhesion.

Small beads derivatised with fibronectin or with bovine serum albumin are allowed to attach to BHK cells in suspension at low ratios of beads to cells. In this way populations of cells bearing predominantly one bead per cell can be prepared. We show that the attachment of one bead per cell affects the adhesion and spreading of that cell on substrata, raising adhesion and increasing spreading if the signal molecule is fibronectin, decreasing these quantities if the bead bears BSA. The experiments are conducted in the absence of other sources of exogenous fibronectin and in some cases in the additional absence of endogenous sources. The effects are especially marked if the substratum is adsorbed haemoglobin on which control cells show little attachment or spreading. We further show by interference reflection microscopy and by scanning electron microscopy that the beads are found on the non-adhering side (uppermost or outer) of the cell when fibronectin-bearing beads are used, presumably because fibronectin will not attach to haemoglobin. The increased adhesion and spreading found in such cases must be attributed to an activation produced by the bead, which spreads to other parts of the cell and which activates a fibronectin-independent mode of adhesion.

Animals↗
PubMed1995

Localization of myosin II A and B isoforms in cultured neurons.

Tension generated by growth cones regulates both the rate and the direction of neurite growth. The most likely effectors of tension generation are actin and myosins. We are investigating the role of conventional myosin in growth cone advance. In this paper we report the localization of the two most prominent isoforms of brain myosin II in growth cones, neurites and cell bodies of rat superior cervical ganglion neurons. Affinity purified polyclonal antibodies were prepared against unique peptide sequences from human and rat A and B isoforms of myosin heavy chain. Although each of these antibodies brightly stained nonneuronal cells, antibodies to myosin heavy chain B stained neurons with greater intensity than antibodies to myosin heavy chain A. In growth cones, myosin heavy chain B was most concentrated in the margin bordering the thickened, organelle-rich central region and the thin, actin-rich peripheral region. The staining colocalized with actin bundles proximal and distal to the marginal zone, though the staining was more prominent proximally. The trailing edge of growth cones and the distal portion of the neurite often had a rimmed appearance, but more proximal regions of neurites had cytoplasmic labelling. Localizing MHC-B in growth cones previously monitored during advance (using differential interference contrast microscopy) revealed a positive correlation with edges at which retraction had just occurred and a negative correlation with lamellipodia that had recently undergone protrusion. Cell bodies were brightly labelled for myosin heavy chain B. Myosin heavy chain A staining was dimmer and its colocalization with filamentous actin bundles in growth cones was less striking than that of myosin heavy chain B. Growth cones stained for both myosin heavy chain A and B revealed that the two antigens overlapped frequently, but not exclusively, and that myosin heavy chain A lacked the elevation in the marginal zone that was characteristic of myosin heavy chain B. The pattern of staining we observed is consistent with a prominent role for myosin heavy chain B in either generating tension between widely separated areas of the growth cone, or bundling of actin filaments, which would enable other motors to effect this tension. These data support the notion that conventional myosin is important in growth cone advance and turning.

Actins↗
PubMed1995

Dynamics of fibroblast spreading.

A new technique of microinterferometry permits cellular growth and motile dynamics to be studied simultaneously in living cells. In isolated chick heart fibroblasts, we have found that the non-aqueous mass of each cell tends to increase steadily, with minor fluctuations, throughout the cell cycle. The spread area of each cell also tends to increase during interphase but fluctuates between wide limits. These limits are dependent on the cell's mass and the upper limit is particularly sharp and directly proportional to mass. From a dynamical point of view, the spread area of a cell is determined by the balance between the rates of two antagonistic processes: protrusion of cellular material into new territory and retraction of material from previously occupied territory. The spatial asymmetry of these processes determines the translocation of the cell. We have found with the chick fibroblasts that the rates of the two processes are generally closely matched to each other and appear to be dependent on the cell's area of spreading. Both continue incessantly in well spread cells, even when there is no net translocation of the cell, and the lower limit of each activity is directly proportional to spread area. The two processes show different behaviour, however, during changes in the spread area of the cell. Both increases and decreases in area appear to be brought about by changes in the rate of retraction, the rate of protrusion remaining relatively constant. A simple stochastic model based on a limited supply of adhesion molecules can simulate all our observations including the mass-limited spreading, the strong correlation between protrusion and retraction and the retraction-dominated changes in area. We conclude that the spread area of the cell is actively regulated, possibly by a simple automatic mechanism that adjusts the area of spreading in relation to the mass of the cell and controls the rate of protrusion to compensate rapidly for spontaneous fluctuations in retraction.

Animals↗
PubMed1996

Oscillating mitotic newt lung cell kinetochores are, on average, under tension and rarely push.

Experimentally introduced tension on kinetochores and their centromeres has been shown to stabilize kinetochore attachment to microtubules, modify kinetochore directional instability, and regulate cell-cycle progression into anaphase. In mitosis, kinetochore tension and the stretch of centromere chromatin are produced by the movement of sister kinetochores toward opposite poles and astral ejection forces on the chromosome arms. However, newt lung cell kinetochores oscillate between poleward and away from the pole motility states throughout mitosis, indicating kinetochores are not under constant tension. To test whether kinetochores are under net tension while they are oscillating, and how often they are under compression and pushing into the chromosome, we measured the distance between sister kinetochores in newt lung cells using both video-enhanced differential interference contrast microscopy (VE-DIC) and immunofluorescence microscopy. We found that for chromosomes in which sister kinetochores are attached to opposite spindle poles, centromeres are, on average, stretched (2.2 microns in living cells and 1.8 microns in fixed cells) with respect to the inter-kinetochore 'rest' length (1.1 microns in living and fixed cells). For chromosomes in which only one kinetochore is attached to the spindle, the centromere chromatin associated with the tethered kinetochore is, on average, stretched to approximately half of the average inter-kinetochore distance measured for chromosomes in which both kinetochores are attached. We conclude that while newt lung cell kinetochores oscillate between states of P and AP movement, they are under tension approximately 90% of the time and under compression less than 6% of the time.

Animals↗
PubMed1997

The subcellular distribution of the high molecular mass protein, HD1, is determined by the cytoplasmic domain of the integrin beta 4 subunit.

The high molecular mass protein, HD1, is a structural protein present in hemidesmosomes as well as in distinct adhesion structures termed type II hemidesmosomes. We have studied the distribution and expression of HD1 in the GD25 cells, derived from murine embryonal stem cells deficient for the beta 1 integrin subunit. We report here that these cells possess HD1 but not BP230 or BP180; two other hemidesmosomal constituents, and express only traces of the alpha 6 beta 4 integrin. By immunofluorescence and interference reflection microscopy HD1 was found together with vinculin at the end of actin filaments in focal contacts. In OVCAR-4 cells, derived from a human ovarian carcinoma which, like GD25 cells, only weakly express alpha 6 beta 4, HD1 was also localized in focal contacts. Upon transfection of both GD25 and OVCAR-4 cells with cDNA for the human beta 4 subunit the subcellular distribution of HD1 changed significantly. HD1 is then no longer present in focal contacts but in other structures at cell-substrate contacts, colocalized with alpha 6 beta 4. These junctional complexes are probably the equivalent of the type II hemidesmosomes. Transfection of GD25 cells with beta 1 cDNA did not affect the distribution of HD1, which indicates that the localization of HD1 in focal contacts was not due to the absence of beta 1. Moreover, in GD25 cells transfected with cDNA encoding a beta 4/beta 1 chimera, in which the cytoplasmic domain of beta 4 was replaced by that of beta 1, the distribution of HD1 was unaffected. Our findings indicate that the cytoplasmic domain of beta 4 determines the subcellular distribution of HD1 and emphasize the important role of alpha 6 beta 4 in the assembly of hemidesmosomes and other junctional adhesive complexes containing HD1.

Animals↗
PubMed1975

Cytoplasmic streaming in Chara: a cell model activated by ATP and inhibited by cytochalasin B.

After vacuolar perfusion of Chara internode cells, the cytoplasm remaining in situ can be reactivated by ATP to give full rates of streaming. Observations during both perfusion and reactivation indicated that the generation of the motive force was associated with fibres consisting of bundles of microfilaments. In the absence of ATP, the remaining endoplasmic organelles were immobilized along such fibres. When ATP was introduced, organelles moved along the fibres at speeds up to 50 mum S minus 1, but but were progressively released from contact to leave the fibres in a conspicuously clean state. Inorganic pyrophosphate freed the organelles from the fibres without supporting movements. Motility required millimolar Mg2nlevels, free Ca2nat 10 minus 7 M or less and was inhibited by high levels of Clminus and by pH's on either side of 7.0. The reactivated movements were rapidly and completely inhibited by 25mug ml minus 1 cytochalasin B. The results are interpreted in terms of actin filaments in the stationary cortex interacting with a myosin-like protein which is able to link to endoplasmic organelles. Movement results from an active shear type of mechanism.

Actins↗
PubMed1975

Calcium regulation of the contractile state of isolated mammalian fibroblast cytoplasm.

Calcium-dependent contractions have been induced in fresh, naked cytoplasm of L-929 fibroblasts using physiological solutions (rigor, relaxing and contracting) similar to those designed to control the contractile state of vertebrate striated muscle. Free access of solutions to the cytoplasm was achieved by popping and stripping the plasma membrane from cells using 7-10 strokes of a Dounce homogenizer. Contracting solution (free Ca2+ 7 X 10(-5) M; with added MgATP) applied locally from a micropipette to cells popped in rigor (free Ca2+ less than 10(-8) M) or relaxing (free Ca2+ less than 10(-8) M; with added MgATP) solutions induced symmetrical contractions of unstretched cytoplasm and directional shortening of stretched cytoplasm. The contractions produced 12-18% shortenings and were complete in 1-3 s. The cytoplasm could be cycled repeatedly through the contracted state from the relaxed state. Exogenous MgATP was required for the Ca2+-dependent contractions. At low free Ca2+ concentrations (less than 10(-8) M), MgATP had a marked plasticizing effect on the cytoplasm. Thus cytoplasm prepared in relaxing solution was less cohesive and more easily deformed than cytoplasm prepared in rigor solution. When induced to contract, relaxed cytoplasm showed a loss of plasticity. Using this criterion, the threshold concentration of free Ca2+ for contraction was determined to lie between 7 X 10(-8) M and 5 X 10(-7) M.

Adenosine Triphosphate↗
PubMed1987

Effect of patterned surfaces of adhesive islands on the shape, cytoskeleton, adhesion and behaviour of Swiss mouse 3T3 fibroblasts.

A pattern of circular islands of adhesive substratum can be used to control cell shape and behaviour. We have shown previously that the proportion of Swiss 3T3 cells that synthesize DNA varies with the area of the island to which they are attached, within the range 500-5000 microns2. In this paper we investigate the cytoskeleton and adhesions of cells on islands using a variety of techniques including phalloidin staining and interference reflection microscopy. Islands of area 2000 microns2 or less constrain cell shape, and cause focal contacts and actin microfilament bundles to accumulate in a circle at the margin. These changes are most obvious in islands of about 1000 microns2, in which a complete ring of adhesion is sometimes formed in the periphery of the cell. This peripheral distribution is less common in cells on even smaller islands, and the focal contacts become smaller and less numerous. It is not yet clear whether any of these structural changes are associated directly with the proliferative stimulus due to contact with the substratum. However, we expect that the use of patterned substrata will contribute to the study of how cell shape and structure regulate many cell functions.

Actins↗
PubMed1979

Mechanisms of cellular adhesion. IV. Role of serum glycoproteins in fibroblast spreading on glass.

We have investigated the exogenous factors required for the transition from the round shape of suspended fibroblasts to the characteristic spread shape on serum-coated glass. Following the evidence of others that the transition is facilitated by adsorbed component(s) related to CIG/LETS (cold-insoluble globulin/large external transformation-sensitive) proteins, we have isolated 2 such preparations from chick serum. Their influence has been investigated on fibroblast adhesion, spreading and growth and they have been characterized by gel electrophoresis, immunological cross-reactivity, amino acid and carbohydrate residue analysis, sedimentation velocity behaviour and circular dichroism spectroscopy. The preparations have molecular weights of 225/215,000 and 140,000 Daltons respectively and are closely similar in composition and secondary structure. The 225/215 000 Dalton doublet is probably a product of limited proteolysis which almost certainly occurred in the avian circulation. For cells seeded on glass precoated in different ways and in different supplemented media we could detect no change in the extent of attachment but there were profound influences on cell shape following this initial adhesion. We confirm that prior adsorption of either CIG-related preparation to glass does indeed promote fibroblast spreading in the absence of other serum components and that CIG is the sole serum component with this type of activity. We now add 2 important qualifications: (i) the presence of substrate-adsorbed serum CIG is not essential for spreading when other serum components are present in the medium; and (ii) the adhesive organization shown by interference reflexion microscopy is incompletely formed unless the additional serum components are present in the medium. We therefore conclude that 16C fibroblasts have the ability when given the stimulus of soluble serum components other than CIG, but not otherwise, to synthesize all the components necessary for the highly organized contacts with glass, including endogenous CIG/LETS proteins.

Amino Acids↗
PubMed1981

Structure of the chloroplast and its DNA in chloromonadophycean algae.

The arrangement and ultrastructure of chloroplasts is described for the Chloromonadophycean algae gonyostomum semen Diesing and Vacuolaria virescens Cienkowsky. The chloroplasts are present in large numbers and are discoid structures approximately 3-4 micrometer in length by 2-3 micrometer in width. In Gonyostomum semen the chloroplasts form a single layer immediately interior to the cell membrane; frequently their longitudinal axis parallels the longitudinal axis of the cell. The chloroplasts in Vacuolaria virescens are more than I layer deep and do not appear to be preferentially oriented. In both organisms, chloroplast bands usually consist of 3 apposed thylakoids, although fusion and interconnections between adjacent bands frequently occur. External to the girdle band (the outermost thylakoids) is the chloroplast envelope. This is bounded by endoplasmic reticulum but there is no immediately apparent continuity between this endoplasmic reticulum and the nuclear envelope. Electron-dense spheres in the chloroplast stroma are thought to be lipid food reserve. Ring-shaped electron-translucent regions in the chloroplast contain chloroplast DNA. The DNA is distributed along this ring in an uneven fashion and, when stained, resembles a string of beads. Each plastid has I ring, and the ring is unbroken in the intact plastid.

Chloroplasts↗
PubMed1981

Calcium-dependent shortening of fibroblasts induced by the ionophore, A23187.

Attached, spread, chick heart fibroblasts were induced to shorten by treatment with the ionophore, A23187. The shortening resulted from the retraction of the leading lamellae and other major cell processes. The response was dependent on external calcium with a threshold close to, and a maximal effect at, physiological concentrations. The shortening was also induced in Colcemid-treated cells and therefore did not involve a depolymerization of microtubules. Indirect evidence indicates that the shortening was preceded by an increase in tension in the spread cell. The response is consistent with the effect of an increase in the cytoplasmic concentration of free calcium on a calcium-sensitive actomyosin system in the spread fibroblast. Although the retraction of non-spreading processes mimicked the intermittent retraction of similar trailing processes during normal movement of fibroblasts, the response to the ionophore differed in that the leading lamellae were also induced to retract. This difference implies that a general increase in the cytoplasmic concentration of free calcium alone cannot account for the intermittent shortening that occurs in normal movement.

Animals↗
PubMed1982

Rapid cellular translocation is related to close contacts formed between various cultured cells and their substrata.

Interference-reflection microscopy combined with time-lapse cinemicrography was used to examine the relationship between cell-to-substratum contact patterns and the speeds of translocation for a variety of cell types. Rapid translocation of amphibian leukocytes (average speed = 9.0 micron/min), amphibian epidermal cells (7 micron/min) and teleost epidermal cells (7 micron/min) was found to correlate with patterns of broad grey close contacts. Similar contact patterns were found under freshly seeded (2 h) chick heart fibroblasts (moving 1-3 micron/min), the rapidly advancing (1-5 micron/min) margin of spreading human WI-38 fibroblasts, and isolated MDCK canine epithelial cells (0.5-1.0 micron/min). Conversely, numerous dark streaks of focal contact were found associated with the slow rate of translocation displayed by older cultures (72 h) of chick fibroblasts (less than 0.1 micron/min), well-spread WI-38 cells (less than or equal to 0.3 micron/min) and confluent MDCK cells (less than 0.01 micron/min). It is concluded that close contacts, but not focal contacts, are associated with rapid cellular translocation, and that the build-up of focal contacts is associated with reduced cellular translocation and maintenance of the spread cell shape.

Animals↗