Showing papers in "Cytoskeleton in 1989"
TL;DR: In this paper, the authors compared the short and long-term effects of latrunculins on cell shape and actin organization to those of cytochalasin D, and found that the transient effects of Latrunculin B were fully reversible for the NIL8 cells and not for the mouse N1E-115 cells.
Abstract: The latrunculins are architecturally novel marine compounds isolated from the Red Sea sponge Latrunculia magnifica. In vivo, they alter cell shape, disrupt microfilament organization, and inhibit the microfilament-mediated processes of fertilization and early development. In vitro, latrunculin A was recently found to affect the polymerization of pure actin in a manner consistent with the formation of a 1:1 molar complex with G-actin. These in vitro effects as well as previous indications that the latrunculins are more potent than the cytochalasins suggest differences in the in vivo mode of action of the two clases of drugs. To elucidate these differences we have compared the short- and long-term effects of latrunculins on cell shape and actin organization to those of cytochalasin D. Exposure of hamster fibroblast NIL8 cells for 1–3 hr to latrunculin A, latrunculin B, and cytochalasin D causes concentration-dependent changes in cell shape and actin organization. However, the latrunculin-induced changes were strikingly different from those induced by cytochalasin D. Furthermore, while initial effects were manifest with both latrunculin A and cytochalasin D already at concentrations of about 0.03 μg/ml, latrunculin A caused complete rounding up of all cells at 0.2 μg/ml, whereas with cytochalasin D maximum contraction was reached at concentrations 10–20 times higher. The short-term effects of latrunculin B were similar to those of latrunculin A although latrunculin B was slightly less potent. All three drugs inhibited cytokinesis in synchronized cells, but their long-term effects were markedly different. NIL8 cells treated with latrunculin A maintained their altered state for extended periods. In contrast, the effects of cytochalasin D progressed with time in culture, and the latrunculin B-induced changes were transient in the continued presence of the drug. These transient effects were found to be due to a gradual inactivation of latrunculin B by serum and were used to compare recovery patterns of cell shape and actin organization in two different cell lines. This comparison showed that the transient effects of latrunculin B were fully reversible for the NIL8 cells and not for the mouse neuroblastoma N1E-115 cells.
585 citations
TL;DR: Results would be consistent with axonal transport of tyrosinated alpha-tubulin followed by assembly in the growth cone and subsequent detyrosination and acetylation, and may be necessary for the provision of labile microtubules for growth cone motility and extension.
Abstract: The comparative distribution of tyrosinated, detyrosinated, and acetylated alpha-tubulins was examined in neurites of rat dorsal root ganglion neurones in culture using immunofluorescence microscopy. Phase contrast observations of single neurones revealed that the neurites were actively motile, and rhodamine phalloidin staining of actin filaments showed the extent of lamellopodia and microspike projections from the growth cones. From double-labelling experiments using antibodies against tyrosinated, detryrosinated, or acetylated alpha-tubulin, it was found that the three different isoforms were differentially localised in neurites and growth cones. Detyrosinated and acetylated forms of alpha-tubulin were in the main restricted to the neurites extending no further than the base of the growth cones. Tyrosinated alpha-tubulin was, however, distributed throughout the body of the growth cone and into the base of some microspikes. Following treatment with taxol to promote microtubule assembly, detyrosinated and acetylated alpha-tubulins were found to be colocalised with tyrosinated alpha-tubulins throughout the growth cones of all cells examined. These results would be consistent with axonal transport of tyrosinated alpha-tubulin followed by assembly in the growth cone and subsequent detyrosination and acetylation. In addition the presence of unmodified alpha-tubulin in the growth cone may be necessary for the provision of labile microtubules for growth cone motility and extension.
124 citations
TL;DR: The data indicate that although CDPK does not interact directly with actin, it may be associated with an actin-binding protein and therefore could play a role in the regulation of the plant cytoskeleton.
Abstract: We recently purified a calcium-dependent but calmodulin- and phospholipid-independent protein kinase (CDPK) from cultured plant cells (Harmon et al.: Plant Physiology 83:830–837, 1987). A monoclonal antibody (mAb 3B9) directed against CDPK was used to localize this protein in Allium root cells and Tradescantia pollen tubes using immunofluorescence techniques. The mAb 3B9 staining pattern showed that CDPK is localized within a fibrous network in the cytoplasm resembling the normal interphase network of F-actin. Treatment of tissue with 10 μM cytochalasin D (CD) prior to fixation abolished the staining pattern. Double-localization experiments in which pollen tubes were first stained with mAb 3B9 and then with rhodamine-phalloidin (RP) demonstrated that CDPK and F-actin were colocalized. Monoclonal antibody 3B9 did not react with purified actin from rabbit muscle or Dictyostelium and did not bind to proteins corresponding to the Mr of actin in crude extracts of Allium root tips and Tradescantia pollen tubes.
CDPK did not phosphorylate purified rabbit muscle or Dictyostelium actin in vitro. Binding studies showed that CDPK (1) does not cosediment with actin filaments and (2) does not form a complex with G-actin. The data indicate that although CDPK does not interact directly with actin, it may be associated with an actin-binding protein and therefore could play a role in the regulation of the plant cytoskeleton.
123 citations
TL;DR: Neither sperm motility nor fluorescence increases were observed, which suggested that the increase of internal free Ca2+ was produced by a flux of externalCa2+ into the cell rather than by a mobilization of internal Ca2-2 stores.
Abstract: The initiation of motility of trout spermatozoa is inhibited by the presence of millimolar concentrations of external K+, but external Ca2+ might also be implicated in this control as it has been shown to antagonize the K+ inhibition of motility [S.M. Baynes et al.: J. Fish. Biol., 19:259–267, 1981]. The present work aimed to investigate internal Ca2+ levels during the motility phase of trout spermatozoa. Internal Ca2+ concentrations were monitored by the fluorescent quinoline Ca2+-indicator, “Quin-2” [R. Y. Tsien: Nature 290:527–529, 1981]. Trout spermatozoa were loaded with Quin-/ under conditions that gave efficient intracellular hydrolysis of Quin-2 and that did not impair the ability of loaded spermatozoa to initiate movement. The beat frequencies, cell velocities, and flagellar asymmetries of sperm movement were not significantly modified by the presence of the internal dye. Upon initiation of flagellar movement, an increase of the internal Quin-2 fluorescence was observed that reflected a sixfold increase of the free Ca2+ concentration. The free Ca2+ remained elevated after the cessation of movement. The variation of fluorescence was completed within 40 seconds, whereas the initiation of motility was nearly instantaneous, and the total duration of flageliar beating lasted for about 80–100 seconds (measurements at 11°C). The increase in the internal free Ca2+ concentration is completed after the initiation of flagellar beating but its occurrence correlates with that of sperm movement. Fluorescence increase was not observed in the presence of 40 mM K+, a condition in which spermatozoa did not initiate flagellar beating. In the presence of the Ca2+ channel blocker desmethoxyverapamil, neither sperm motility nor fluorescence increases were observed, which suggested that the increase of internal free Ca2+ was produced by a flux of external Ca2+ into the cell rather than by a mobilization of internal Ca2+ stores.
121 citations
TL;DR: Immunogold labelling of sections of pollen tubes prepared by rapid-freeze fixation and freeze substitution reveals that actin MF bundles are indeed present after cytochalasin treatment, and question reports in the literature, based on phalloidin staining, asserting that cy tochalasin fragments or destroys actinMFs.
Abstract: A comparison of actin localization in pollen tubes of Nicotiana has been made using a monoclonal actin antibody and rhodamine-phalloidin (RP). The monoclonal antiactin, based on Western blotting of pollen tube extract, labels a polypeptide at 45 kD that comigrates with muscle actin. A 51-kD unknown protein and three bands less than 45 kD, presumed to be proteolytic fragments of actin, are also observed. Structural observaations using this antibody reveal a network of axially oriented strands of microfilaments (MFs). The MFs are distributed throughout the length of the pollen tube except at the very tip, where diffuse staining is usually observed. A similar pattern of MFs is evident after RP staining. When pollen tubes are treated with cytochalasins (CB or CD) cytoplasmic streaming is inhibited, as is tube elongation. Microscopic analysis reveals that the microfilament (MF) pattern is markedly altered; however, the antibody and RP produce different staining patterns. The antibody reveals many MF strands that distribute throughout the tube length and extend into the very tip. In contrast, RP shows mostly a diffuse staining pattern with only a few short clumps of filamentous material. Immunogold labelling of sections of pollen tubes prepared by rapid-freeze fixation and freeze substitution reveals that actin MF bundles are indeed present after cytochalasin treatment. Our results thus question reports in the literature, based on phalloidin staining, asserting that cytochalasin fragments or destroys actin MFs.
120 citations
TL;DR: Actin and the light chains of myosin were labeled with fluorescent dyes and injected into interphase PtK2 cells in order to study the changes in distribution of actin and myOSin that occurred when the injected cells subsequently entered mitosis and divided.
Abstract: Actin and the light chains of myosin were labeled with fluorescent dyes and injected into interphase PtK2 cells in order to study the changes in distribution of actin and myosin that occurred when the injected cells subsequently entered mitosis and divided. The first changes occurred when stress fibers in prophase cells began to disassemble. During this process, which began in the center of the cell, individual fibers shortened, and in a few fibers, adjacent bands of fluorescent myosin could be seen to move closer together. In most cells, stress fiber disassembly was complete by metaphase, resulting in a diffuse distribution of the fluorescent proteins throughout the cytoplasm with the greatest concentration present in the mitotic spindle. The first evidence of actin and myosin concentration in a cleavage ring occurred at late anaphase, just before furrowing could be detected. Initially, the intensity of fluorescence and the width of the fluorescent ring increased as the ring constricted. In cells with asymmetrically positioned mitotic spindles, both protein concentration and furrowing were first evident in the cortical regions closest to the equator of the mitotic spindle. As cytokinesis progressed in such asymmetrically dividing cells, fluorescent actin and myosin appeared at the opposite side of the cell just before furrowing activity could be seen there. At the end of cytokinesis, myosin and actin were concentrated beneath the membrane of the midbody and subsequently became organized in two rings at either end of the midbody.
111 citations
TL;DR: The observation of NBBCs in the mutant bald-2, which lacks all but very rudimentary basal bodies, indicates that the assembly of the NBBC does not require fully formed basal bodies and that such assembly may not require basal bodies at all.
Abstract: In the unicellular biflagellate green alga Chlamydomonas reinhardtii each basal body is linked to the nucleus by a fibrous nucleus-basal body connector (NBBC) that contains the calcium-binding protein centrin. (Wright et al.: Journal of Cell Biology 101:1903–1912.; Salisbury et al.: Journal of Cell Biology 107:635–642; Huang et al.: Journal of Cell Biology 107:121–131). In order to explore the cellular function of the NBBC we used antiserum directed against centrin to examine a number of mutants known to be defective for basal body assembly and/or localization. Of three variable flagella-number mutants examined, one, vfl-2, is dramatically defective with respect to the NBBC in that (1) the union between basal bodies and nucleus is very labile, (2) there is no detectible centrin in the NBBC region, and (3) total cellular centrin levels are reduced 75–80% relative to wild type. The existence of these defects in a mutant incapable of maintaining normal flagellar number supports the view that the NBBC plays an important role in determining proper basal body localization and/or segregation. In contrast to vfl-2, the mutants vfl-1, vfl-3, uni-1, and bald-2 contain approximately normal levels of centrin and possess stable NBBCs. The observation of NBBCs in the mutant bald-2, which lacks all but very rudimentary basal bodies, indicates that the assembly of the NBBC does not require fully formed basal bodies and that such assembly may not require basal bodies at all. Finally, the possibility that the NBBC is required for induction of gene expression following deflagellation was tested by examining vfl-2 for such induction. Results indicate that the connector does not play a necessary role in the induction process.
95 citations
TL;DR: An experimental system allowed the capacity of the centrosomal microtubule organizing center to double regularly in the absence of centrioles to be characterized, and it is shown that a centrosome without centriole does not reproduce between mitoses.
Abstract: For animal cells, the relative roles of the centrioles and the pericentriolar material (the centrosomal microtubule organizing center) in controlling the precise doubling of the centrosome before mitosis have not been well defined. To this end we devised an experimental system that allowed us to characterize the capacity of the centrosomal microtubule organizing center to double regularly in the absence of centrioles. Sea urchin eggs were fertilized, stripped of their fertilization envelopes, and fragmented before syngamy. Those activated egg fragments containing just the female pronucleus assembled a monaster at first mitosis. A serial section ultrastructural analysis of such monasters revealed that the radially arrayed microtubules were organized by a hollow fenestrated sphere of electron-dense material, of the same appearance as pericentriolar material, that was devoid of centrioles. We followed individual fragments with only a female pronucleus through at least three cell cycles and found that the monasters did not double between mitoses. The observation that fragments with only a male pronucleus repeatedly divided in a normal fashion indicates that the assembly and behavior of monasters were not artifacts of egg fragmentation. Our results demonstrate that the activity that controls the precise doubling of the centrosome before mitosis is distinct and experimentally separable from the centrosomal microtubule organizing center. Our observations also extend the correlation between the reproductive capacity of a centrosome and the number of centrioles it contains (G Sluder and CL Rieder, 1985a: J. Cell Biol. 100:887-896). For a cell that normally has centrioles, we show that a centrosome without centrioles does not reproduce between mitoses.
80 citations
TL;DR: This study showed that the actin- and myosin-containing cytoskeleton was reversibly redistributed between the cortical ectoplasm and the endoplasm during prophase and telophase, demonstrating that actin andMyosin are reorganized by a temporally coordinated but spatially different mechanism during cytokinesis of Dictyostelium.
Abstract: Synchronized cultures of Dictyostelium discoideum were used to study organizational changes of the cytoskeleton during mitotic cell division. The agar-overlay technique (Yumura et al.: J. Cell Biol. 99:894-899, 1984) was employed for immunofluorescence localization and video microscopic observation of living mitotic cells. The mitotic phase was defined by changes in chromosome configuration by using a double stain with the fluorescent dye DAPI. This study showed that the actin- and myosin-containing cytoskeleton was reversibly redistributed between the cortical ectoplasm and the endoplasm during prophase and telophase. Both actin and myosin filaments were dissociated from the cell cortex in prophase. Most of the actin and myosin was filamentous and remained in the endoplasm until telophase. Saltatory movements of organelles stopped suddenly, coincident with the breakdown of the cytoplasmic microtubule network. This change in the microtubule system was temporally coupled with the disappearance of actomyosin from the cortex. At the same time, the local vibrating movement of particles almost stopped, suggesting that the viscoelastic nature of the endoplasm was altered. In the late anaphase, actin and myosin relocalized to the cortical ectoplasm. Early in this phase, myosin filaments were localized specifically at the anticipated cleavage furrow region of the cleavage furrow, whereas actin filaments were redistributed more uniformly in the cell cortex, with an extremely large accumulation in the polar pseudopods. Subsequently the actin formed an orderly parallel array of cables along with myosin filaments in the contractile ring. The spatial segregation of actin and myosin in late anaphase was clearly demonstrated by multipolar cell division of artificially induced giant cells. Actin was relocalized in both the polar and the proximal constricting regions whereas myosin was only localized in the center of each pair of daughter microtubule networks where the cleavage furrow was formed. This study demonstrates that actin and myosin are reorganized by a temporally coordinated but spatially different mechanism during cytokinesis of Dictyostelium.
69 citations
TL;DR: The utility of a commercially available motion analysis system for the analysis of the Chlamydomonas stop response is presented and a kinetic model for the description of adaptation is presented.
Abstract: In darkness Chlamydomonas cells swim forward with a helical motion of low amplitude. When cells are exposed to light conditions they are not adapted to, they perform direction changes or stop responses depending on how far the stimulant irradiance is shifted from the former adaptation level. Here we present the utility of a commercially available motion analysis system for the analysis of the Chlamydomonas stop response.
Chlamydomonas cells stop in darkness only occasionally with a random temporal distribution but with a highly increased frequency after a flash or a step-up light stimulation. The delay time, tD, defined as the minimal time difference between flash and a light-induced stop, was below 50 ms. The reaction time, tR, defined as the time difference between flash and the maximal probability for a cell to stop was found to be 140 ms. During a stop the cells swim revers for some 300 ms with 20% of the forward swimming speed.
To a given stimulation program cells adapt with a first-order kinetic. In the case of a single step-up or step-down stimulation this adaptation consists of a single stop response followed by direction changes which decrease in frequency to a certain steady-state level. To repetitive light pulses the cells respond with a gradual disappearance of step-up stop responses and a concurrent appearance of step-down responses.
External calcium influences the stop response in a multifunctional way. For stop responses to occur 300 nM calcium are required. At increasing calcium concentrations the duration of a stop response is extended. Besides light, calcium regulates the time course of light-adaption and the absolute adaptation level.
A kinetic model for the description of adaptation is presented.
62 citations
TL;DR: Results indicate that ABP-120 is involved in cross-linking newly assembled actin filaments into the cytoskeleton during chemoattractant-stimulated pseudopod extension.
Abstract: Triton-insoluble cytoskeletons were isolated from Dictyostelium discoideum AX3 cells prior to and following stimulation with 2'deoxy cyclic adenosine monophosphate (cAMP). Temporal changes in the content of actin and a 120,000 dalton actin-binding protein (ABP-120) in cytoskeletons following stimulation were monitored. Both actin and ABP-120 were incorporated into the cytoskeleton at 30-40 seconds following stimulation, which is cotemporal with the onset of pseudopod extension during stimulation of amoebae with chemoattractants. Changes in the content of total cytoskeletal protein and cytoskeletal myosin were determined under the same experimental conditions as controls. These proteins exhibited different kinetics from those of cytoskeletal ABP-120 and actin following the addition of 2'deoxy cAMP. The authors concluded that the association of ABP-120 with the cytoskeleton is regulated during cAMP signalling. Furthermore, these results indicate that ABP-120 is involved in cross-linking newly assembled actin filaments into the cytoskeleton during chemoattractant-stimulated pseudopod extension.
TL;DR: Video-enhanced contrast microscopy was used to monitor the motile behavior of cells to classify protrusions into the temporal stages of initial and establishedprotrusions, and multiple parameter immunofluorescence imaging methods were used to determine and to map accurately the distributions of actin, myosin and microtubules in specific types of protrusion.
Abstract: The formation of lamellipodia in migrating cells involves dynamic processes that occur in a cyclic manner as the leading edge of a cell slowly advances. We used video-enhanced contrast microscopy (VEC) to monitor the motile behavior of cells to classify protrusions into the temporal stages of initial and established protrusions (Fisher et al.: Cell Motility and the Cytoskeleton 11:235–247, 1988), and to monitor the fixation of cells. Multiple parameter fluorescence imaging methods (DeBiasio et al.: Journal of Cell Biology 105:1613–1622, 1987; Waggoner et al.: Methods in Cell Biology, Vol. 30, Part B, pp. 449–478, 1989) were then used to determine and to map accurately the distributions of actin, myosin and microtubules in specific types of protrusions. Initial protrustions exhibited no substructure as evidenced by VEC and actin was diffusely arranged, while myosin and microtubules were absent. Newly established protrusions contained diffuse actin as well as actin in microspikes. There was a delay in the appearance of myosin into established protrusions relative to the presence of actin. Microtubules were found in established protrusions after myosin was detected, and they were oriented parallel to the direction of migration. Actin and myosin were also localized in fibers transverse to the direction of migration at the base of initial and established protrusions. Image analysis was used to quantify the orientation of actin fibers relative to the leading edge of motile cells. The combined use of VEC, multiple parameter immunofluorescence, and image analysis should have a major impact on defining complex relationships within cells.
TL;DR: Exogenous ATP could abolish thiophosphate-staining more effectively when added with phosphatase inhibitors, suggesting a dynamic state in which centrosomal proteins are being phosphorylated and dephosphorylated in rapid succession by the action of protein kinase(s) and phosphat enzyme(s).
Abstract: Protein phosphorylation during development of sea urchin eggs from fertilization to first cleavage was examined by labeling cells with specific antiphosphoprotein antibodies. Indirect immunofluorescence staining with monoclonal antithiophosphoprotein antibody (Gerhart et al.: Cytobios 43:335-347, 1985) has revealed that nuclei as well as centrosomes, kinetochores, and midbodies were specifically thiophosphorylated in developing eggs incubated with adenosine 5'-O (3-thiotriphosphate) (ATP-gamma-S). The phosphorylation reaction required Mg2+ but was not dependent on cAMP or calmodulin in detergent-extracted models. Centrosomes were purified by fractionation of isolated mitotic spindles with 0.5 M KCl extraction. The thiophosphoproteins were retained in the purified centrosomes and the antibody recognized a major 225-Kd polypeptide on immunoblots. In an independent preparation, a monoclonal antiphosphoprotein antibody (CHO3) was found also to react with mitotic poles and stained a 225-Kd polypeptide, confirming the centrosome specificity of this protein. Immunoelectron microscopy showed that the 225-Kd thiophosphoprotein was found at mitotic poles associated with granules to which mitotic microtubules were directly attached. Unlike centrosomes in permeabilized eggs, those in isolated spindles could not be thiophosphorylated, possibly due to inactivation or loss of either phosphorylation enzymes or cofactors, or both, during isolation. The immunofluorescence labeling of thiophosphate could be inhibited by ATP and AMP.PNP in a concentration-dependent manner. Exogenous ATP could abolish thiophosphate-staining more effectively when added with phosphatase inhibitors, suggesting a dynamic state in which centrosomal proteins are being phosphorylated and dephosphorylated in rapid succession by the action of protein kinase(s) and phosphatase(s).
TL;DR: Based upon their size and density, RNA and protein composition, and sedimentation in sucrose gradients, the microtubule-associated particles are identified as ribosomes.
Abstract: Microtubules were purified from unfertilized eggs of the sea urchins Arbacia punctulata, Lytechinus pictus, Lytechinus variegatus, and Strongylocentrotus purpuratus. Numerous densely stained particles (24 × 26 nm) are associated with microtubules isolated from each of these sea urchins. The most striking aspect of this structure is an extended, slightly curved arm that appears to attach the particles to the microtubule. Morphologically similar particles are associated with microtubules of the isolated first cleavage mitotic apparatus. The particles are attached to the microtubules by ionic interactions and contain large amounts of extractable RNA. Based upon their size and density, RNA and protein composition, and sedimentation in sucrose gradients, the microtubule-associated particles are identified as ribosomes.
TL;DR: The dynamic changes of the endoplasmic reticulum (ER) in interphase and mitotic cells was detected by the vital fluorescent dye 3,3'-dihexyloxacarbocyanine iodide and the ER and SR arrays seen in living cells were also detected in fixed cells stained with antibodies directed against proteins of the ENDO and SR.
Abstract: The dynamic changes of the endoplasmic reticulum (ER) in interphase and mitotic cells was detected by the vital fluorescent dye 3,3′-dihexyloxacarbocyanine iodide. Two types of arrays characterize the continuous ER system in the non-muscle PtK2 cell: (1) a lacy network of irregular polygons and (2) long strands of ER that are found aligned along stress fibers. In cross-striated myotubes there was a periodic localization of fluorescence over each I-band corresponding to the positions of the terminal cisternae of the sarcoplasmic reticulum (SR). In contrast to the arrangement in muscle cells, the aligment of the long strands of ER along stress fibers showed no strict periodicity that could be correlated with the sarcomeric units of the stress fibers. The ER and SR arrays seen in living cells were also detected in fixed cells stained with antibodies directed against proteins of the endoplasmic reticulum and sarcoplasmic reticulum, respectively. Observations of vitally stained PtK2 cells at 1 to 2 minute intervals using low light level video cameras and image processing techniques enabled us to see the polygonal ER units form and undergo changes in their shapes. During cell division, the ER, rhodamine 123-stained mitochondria, and phagocytosed fluorescent beads were excluded from the mitotic spindle while soluble proteins were not. No obvious concentration or alignment of membranes could be found associated with the contractile proteins in the cleavage furrow. After completion of cell division there was a redeployment of the ER network in each daughter cell.
TL;DR: Changes in cell shape, in the organization of microfilaments, and in cell-cell and cell-substratum interactions during this time were examined by epifluorescence and transmission electron microscopy.
Abstract: Retinal pigment epithelial (RPE) cells maintained in organ culture on Bruch's membrane and the associated choroid spread and migrate into a linear wound along the exposed basal lamina. Changes in cell shape, in the organization of microfilaments, and in cell-cell and cell-substratum interactions during this time were examined by epifluorescence and transmission electron microscopy. In contrast to cuboidal stationary cells distant from the wound edge, which display well-developed apical circumferential microfilament bundles (CMBs) associated with zonulae adhaerentes junctions, the migrating RPE cells near the wound edge instead are flat, and, in addition to microfilament bundles near junctions between adjacent cells, display prominent stress fibers. Furthermore, monoclonal antibodies to vinculin labeled regions at the terminal ends of these stress fibers indicating that the RPE cells form focal contacts with the basal lamina at these sites. Electron microscopy of these regions of cell-substratum interaction confirmed the presence of microfilament bundles that terminate on the cell membrane. Folds present in the basal lamina near these sites suggest that tension is being generated by the microfilaments in the stress fibers as the migrating cells pull on the underlying basal lamina through these adhesion points.
TL;DR: Bipolar filaments of myosin in actin-free cytoskeletons were often organized in chains and lattices formed by end-to-end contacts of individual filaments at their head-containing regions, and it was possible for the first time to display bipolar myOSin filaments in the stress fibers of cultured cells.
Abstract: The authors examined the molecular organization of myosin in stress fibers (microfilament bundles) of cultured mouse embryo fibroblasts. To visualize the organization of myosin filaments in these cells, fibroblast cytoskeletons were treated with gelsolin-like protein from bovine brain (hereafter called brain gelsolin), which selectively disrupts actin filaments. As shown earlier [Verkhovsky et al., 1987], this treatment did not remove myosin from the stress fibers. The actin-free cytoskeletons then were lightly sonicated to loosen the packing of the remaining stress fiber components and fixed with glutaraldehyde. Electron microscopy of platinum replicas of these preparations revealed dumbbell-shaped structures of approximately 0.28 micron in length, which were identified as bipolar myosin filaments by using antibodies to fragments of myosin molecule (subfragment 1 and light meromyosin) and colloidal gold label. Bipolar filaments of myosin in actin-free cytoskeletons were often organized in chains and lattices formed by end-to-end contacts of individual filaments at their head-containing regions. Therefore, after extraction of actin, it was possible for the first time to display bipolar myosin filaments in the stress fibers of cultured cells.
TL;DR: This observation supports the hypothesis that tubulin subunits add at the kinetochore in metaphase and that acetylation occurs subsequent to addition and may be useful as a marker in anaphase to distinguish between different hypotheses of chromosome motion.
Abstract: We studied the distribution of acetylated α-tubulin in the microtubules of spermatogenic cells from the crane fly Nephrotoma suturalis (Loew) using a mono-clonal antibody specific for acetylated α-tubulin (6-11B-1). We found that cells in all stages of spermatogenesis contained acetylated microtubules including primary spermatocytes, meiotic cells, spermatids, and sperm. A subset of the acety-lated microtubules (those in midbodies and flagella) were resistant to cold depolymerization. Newly polymerized microtubules in nondividing cells were not acetylated for up to 15 min. indicating that acetylation lagged behind polymerization. In spindles, newly polymerized microtubules were acetylated after 5 min. Antibodies to acetylated α-tubulin selectively stained chromosome-to-pole fibers in dividing cells, but the staining appeared to decrease and taper of at the kinetochores. This observation supports the hypothesis that tubulin subunits add at the kinetochore in metaphase and that acetylation occurs subsequent to addition. Further, this taper may be useful as a marker in anaphase, to distinguish between different hypotheses of chromosome motion.
TL;DR: It is suggested that the microfilament bundles that frequently line the lateral contacts between epithelial cells in vivo may be related to the actin edge-bundle, a specialized cytoskeletal structure that supports the webbed edges of interphase 3T3 fibroblasts.
Abstract: The outline of cells in sparse cultures consists predominantly of concave and convex segments; straight segments are rare and ephemeral. The convex segments are areas of active cell expansion. The concave segments are stationary and web-shaped, similar in profile to the cables of a suspension bridge. In 3T3 fibroblasts, we have found a single microfilament bundle following the outline of every webbed edge and have called it the actin edge-bundle (AEB). While the AEB is composed predominantly of actin, alpha-actinin and myosin are also present. In contrast to normal stress fibers, AEBs are more resistant to several treatments that depolymerize F-actin. Once an AEB disassembles, however, the webbed edge collapses and retracts, suggesting that the actin edge-bundle is a specialized cytoskeletal structure that supports the webbed edges of interphase 3T3 fibroblasts. The stability of AEBs is independent of microtubules. We suggest that the microfilament bundles that frequently line the lateral contacts between epithelial cells in vivo may be related to the actin edge-bundle.
TL;DR: The relationship of nascent myofibrils with the accumulation of adhesion plaque proteins and the formation of focal cell contacts was studied in embryonic chick cardiac myocytes in vitro and polarized lines appeared to be generated within the cells in response to their physical and biochemical environment.
Abstract: The relationship of nascent myofibrils with the accumulation of adhesion plaque proteins and the formation of focal cell contacts was studied in embryonic chick cardiac myocytes in vitro. The cultures were double-stained with various combinations of the specific antiactin drug phalloidin and antibodies against vinculin, α-actinin, connectin (titin), myosin heavy chain, fibronectin, and desmin and examined under fluorescence and interference reflection microscopy.
In the areas of myofibril assembly, vinculin and α-actinin plaques were formed at the ventral sarcolemmae. These areas overlapped with the sites of cell-to-substrate focal contacts and extracellular fibronectin. Because the myofibrils always ran in a straight line between these sites, polarized lines appeared to be generated within the cells in response to their physical (e.g., stress) and/or biochemical environment (e.g., adhesion plaque proteins). The possible presence of other factors cannot be ruled out for the proper alignment of myofibrils. As soon as myofibrils came to span between these adhesion sites, they exhibited typically mature cross-striated characteristics. Thus, the formation of these inferred lines has some relation to or is in fact necessary for the maturation of myofibrils, in addition to the directional arrangement of sarcomeric proteins.
Additionally, synthesis and distribution of myosin and connectin were tightly linked during early developmental (premyofibril and myofibril) stages. The spatial deployment of desmin was not coupled with vinculin. Thus, connectin and desmin do not appear to form the initial scaffold of sarcomeres.
TL;DR: In PtK2 cells, an epithelial cell line, injected desmin formed a filamentous network, which colocalized with the native vimentin intermediate filaments but not with the cytokeratin networks and microtubular arrays.
Abstract: Fluorescently labeled desmin was incorporated into intermediate filaments when microinjected into living tissue culture cells. The desmin, purified from chicken gizzard smooth muscle and labeled with the fluorescent dye iodoacetamido rhodamine, was capable of forming a network of 10-nm filaments in solution. The labeled protein associated specifically with the native vimentin filaments in permeabilized, unfixed interphase and mitotic PtK2 cells. The labeled desmin was microinjected into living, cultured embryonic skeletal myotubes, where it became incorporated in straight fibers aligned along the long axis of the myotubes. Upon exposure to nocodazole, microinjected myotubes exhibited wavy, fluorescent filament bundles around the muscle nuclei. In PtK2 cells, an epithelial cell line, injected desmin formed a filamentous network, which colocalized with the native vimentin intermediate filaments but not with the cytokeratin networks and microtubular arrays. Exposure of the injected cells to nocadazole or acrylamide caused the desmin network to collapse and form a perinuclear cap that was indistinguishable from vimentin caps in the same cells. During mitosis, labeled desmin filaments were excluded from the spindle area, forming a cage around it. The filaments were partitioned into two groups either during anaphase or at the completion of cytokinesis. In the former case, the perispindle desmin filaments appeared to be stretched into two parts by the elongating spindle. In the latter case, a continuous bundle of filaments extended along the length of the spindle and appeared to be pinched in two by the contracting cleavage furrow. In these cells, desmin filaments were present in the midbody where they gradually were removed as the desmin filament network became redistributed throughout the cytoplasm of the spreading daughter cells.
TL;DR: The inability of antibodies to myosin-II to inhibit completely any of the movements studied suggests that although myos in-II probably plays a role in these motilities, the cell either routinely uses or can draw upon another cytoplasmic motor to maintain locomotion, organelle movement, contractile vacuole activity, and endocytosis.
Abstract: To study the in vivo role of myosin-II in Acanthamoeba castellanii, motile cells were microinjected with monoclonal antibodies raised against the myosin-II heavy chain. All injected cells underwent a transient shock response. It was found that although injection of buffer alone or of an endogenous Acanthamoeba protein decreased the motility of injected cells from 7 microns/min to approximately 3 microns/min, injection of monoclonal antibodies specific for myosin-II decreased motility further to approximately 0.8 micron/min. This effect was seen whether or not the monoclonal antibody to myosin-II inhibited the actomyosin-II MgATPase activity in vitro. Levels of antibody far in excess of endogenous myosin-II concentrations could not completely block amoeboid movement. The morphology of moving antimyosin-II-injected cells was unusual, suggesting a greater defect in the ability to retract the trailing edge of the cell rather than to extend the leading edge. Endosomes frequently disappeared from injected cells, and although buffer-injected cells rapidly recovered visible endosomes (50% recovery at 5 min), endosomes were not seen in antimyosin-II-injected cells until, on the average, approximately 50 min after injection. Injection of a nonspecific antibody or of a nonspecific exogenous protein (ovalbumin) also decreased the mobility of the injected cells beyond that of buffer-injected cells (to approximately 1 micron/min). These cells tended to recover endosomes more rapidly (approximately 25 min) than cells injected with antimyosin-II monoclonal antibodies. The inability of antibodies to myosin-II to inhibit completely any of the movements studied suggests that although myosin-II probably plays a role in these motilities, the cell either routinely uses or can draw upon another cytoplasmic motor to maintain locomotion, organelle movement, contractile vacuole activity, and endocytosis.
TL;DR: Age-related differences in neurite extension thus reflected the proportion of lamellipodial growth cones present rather than disparities in basic structure or in the rates at which growth cones of a given type moved at different ages.
Abstract: To determine the relationship between growth cone structure and motility, we compared the neurite extension rate, the form of individual growth cones, and the organization of f-actin in embryonic (E21) and postnatal (P30) sympathetic neurons in culture. Neurites extended faster on laminin than on collagen, but the P30 nerites were less than half as long as E21 neurites on both substrata. Growth cone shape was classified into one of five categories, ranging from fully lamellipodial to blunt endings. The leading margins of lamellipodia advanced smoothly across the substratum ahead of any filopodial activity and contained meshworks of actin filaments with no linear f-actin bundles, indicating that filopodia need not underlie lamellipodia. Rapid translocation (averaging 0.9-1.4 microns/min) was correlated with the presence of lamellipodia; translocation associated with filopodia averaged only 0.3-0.5 microns/min. This relationship extended to growth cones on a branched neurite where the translocation of each growth cone was dependent on its shape. Growth cones with both filopodial and lamellipodial components moved at intermediate rates. The prevalence of lamellipodial growth cones depended on age of the neurites; early in culture, 70% of E21 growth cones were primarily lamellipodial compared to 38% of P30 growth cones. A high percentage of E21 lamellipodial growth cones were associated with rapid neurite elongation (1.2 mm/day), whereas a week later, only 16% were lamellipodial, and neurites extended at 0.5 mm/day. Age-related differences in neurite extension thus reflected the proportion of lamellipodial growth cones present rather than disparities in basic structure or in the rates at which growth cones of a given type moved at different ages. Filopodia and lamellipodia are each sufficient to advance the neurite margin; however, rapid extension of superior cervical ganglion neurites was supported by lamellipodia independent of filopodial activity.
TL;DR: Indirect immunofluorescence microscopy and immunoblot analysis demonstrate that human polymorphonuclear leukocytes also contain a 17 kD protein that specifically cross-reacts with antibodies affinity-purified against D. discoideum ponticulin, and this protein appears to be localized in plasma membrane and is evident in actin-rich cell extensions.
Abstract: Ponticulin is the major actin-binding integral glycoprotein in plasma membranes isolated from log-phase Dictyostelium discoideum amebae. As such, this protein appears to be an important link between the plasma membrane and actin filaments (Wuestehube and Luna: Journal of Cell Biology 105:1741–1751, 1987). In this study, indirect immunofluorescence microcopy was used to examine the distribution of ponticulin in randomly moving D. discoideum amebae and in amebae engaged in cell migration and phagocytosis. Ponticulin is distributed throughout the plasma membrane and also is present in intracellular vesicles associated with the microtubule-organizing center-Golgi complex adjacent to the nucleus. In aggreating amebae, ponticulin is concentrated in regions of lateral cell-cell contact and in arched regions of the plasma membrane. Ponticulin also is present, but not obviously enriched, in filopodia, in the actin-rich anterior end of polarized cells, and in detergent-insoluble cytoskeletons. In amebae engaged in phagocytosis of yeast, ponticulin is present but not enriched in phagocytic cups and is associated with intracellular vesicles around engulfed yeast. These results suggest that ponticulin is stably associated with actin filaments in certain regions of the plasma membrace and that the actin-binding activity of ponticulin may be tightly controlled.
Indirect immupofluorescence microscopy and immunoblot analysis demonstrate that human polymorphonuclear leukocytes also contain a 17 kD protein that specifically cross-reacts with antibodies affinity-purified aganst D. discoideum ponticulin. As in D. discoideum, the mammalian 17 kD ponticulin-analog appears to be localized in plasma membrane and is evident in actin-rich cell extensions. These results indicate that ponticulin-mediated linkages between the plasma membrane and actin may be present in higher eukaryotic cells.
TL;DR: The demembranated spermatozoa of the rainbow trout showed vigorous motility in the reactivation solution containing Ca2+ at the concentrations below 10−8.5M in the presence of cAMP, and it is likely that the Ca2-dependent regulatory system of flagellar movement is independent of the cAMP-induced initiation mechanism.
Abstract: Spermatozoa of the rainbow trout, Salmo gairdneri, were demembranated with Triton X-100. The demembranated spermatozoa showed vigorous motility in the reactivation solution containing Ca2+ at the concentrations below 10−8.5M in the presence of cAMP. The motility was lost at 10−8M Ca2+ or more. The shape of the immotile flagella in the presence of high concentration of Ca2+ was not uniform: Some showed the cane shape and some were almost straight. The change in Ca2+ concentration of the extraction solution did not alter the motility of the reactivated spermatozoa. These results were different from those obtained from the sea urchin spermatozoa. When the concentration of cAMP was changed from 0.5 to 100 μM, the concentration of Ca2+ for converting the motile to immotile state was not altered. Thus, it is likely that the Ca2+-dependent regulatory system of flagellar movement is independent of the cAMP-induced initiation mechanism, which is assumed to require the transient influx of Ca2+ in rainbow trout spermatozoa.
TL;DR: It is confirmed that cells that move through collagen lattices lack the well-spread morphology of their counterparts moving on glass, and the flattened shape of lamellipodia may be part of the mechanism by which cells sense the orientation of fibrillar extracellular matrices during embryonic morphogenesis.
Abstract: We have used 400 kilovolt intermediate voltage electron microscopy (IVEM) to examine thick sections of fibroblasts cultured in collagen gels. In these 3D collagen lattices, the long, narrow pseudopodial extensions that extend out and make contact with the collagen matrix exhibit a complex topography not seen in the processes put out by cells moving on planar substrata. For this reason, sections 1 to 2 microns thick that enclose a whole cell process are more informative of the overall morphology of the interaction between cells and the collagen than are thin sections. To aid the discrimination of topography of cell processes in stereo views of micrographs, some cells were labeled with antibodies and protein A-colloidal gold conjugates. The gold particles provided clear 3D reference points for computer-aided reconstructions of membrane topography from tilt series of IVEM images. Our results confirm that cells that move through collagen lattices lack the well-spread morphology of their counterparts moving on glass. They are generally rather spindly with several long branching anterior pseudopodia. We found that the cell bodies and major pseudopodial processes were cylindrical, as one might expect of cells in a 3D environment, but at the leading edge of advancing pseudopodia there are small flat extensions similar to those seen in cells on glass. This similarity suggests that the lamellipodium is a basic type of protrusive structure used by fibroblasts during locomotion on all types of substratum. The flattened shape of lamellipodia may be part of the mechanism by which cells sense the orientation of fibrillar extracellular matrices during embryonic morphogenesis.