scholarly journals Polymerizing Microtubules Activate Site-directed F-Actin Assembly in Nerve Growth Cones

1999 ◽  
Vol 10 (7) ◽  
pp. 2309-2327 ◽  
Author(s):  
M. William Rochlin ◽  
Michael E. Dailey ◽  
Paul C. Bridgman
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Cytochalasin B ◽  
Membrane Surface ◽  
Growth Cones ◽  
Actin Assembly ◽  
Electron Microscopic ◽  
Early Step ◽  
Microscopic Studies ◽  
Nerve Growth Cones

We identify an actin-based protrusive structure in growth cones termed “intrapodium.” Unlike filopodia, intrapodia are initiated exclusively within lamellipodia and elongate in a continuous (nonsaltatory) manner parallel to the plane of the dorsal plasma membrane causing a ridge-like protrusion. Intrapodia resemble the actin-rich structures induced by intracellular pathogens (e.g.,Listeria) or by extracellular beads. Cytochalasin B inhibits intrapodial elongation and removal of cytochalasin B produced a burst of intrapodial activity. Electron microscopic studies revealed that lamellipodial intrapodia contain both short and long actin filaments oriented with their barbed ends toward the membrane surface or advancing end. Our data suggest an interaction between microtubule endings and intrapodia formation. Disruption of microtubules by acute nocodazole treatment decreased intrapodia frequency, and washout of nocodazole or addition of the microtubule-stabilizing drug Taxol caused a burst of intrapodia formation. Furthermore, individual microtubule ends were found near intrapodia initiation sites. Thus, microtubule ends or associated structures may regulate these actin-dependent structures. We propose that intrapodia are the consequence of an early step in a cascade of events that leads to the development of F-actin-associated plasma membrane specializations.

Lipocortin 1 (annexin 1) in patches associated with the membrane of a lung adenocarcinoma cell line and in the cell cytoplasm

1998 ◽  
Vol 111 (10) ◽  
pp. 1405-1418 ◽  
Author(s):  
V. Traverso ◽  
J.F. Morris ◽  
R.J. Flower ◽  
J. Buckingham
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Lung Adenocarcinoma ◽  
Western Blotting ◽  
Signal Sequence ◽  
Membrane Surface ◽  
Integral Membrane Protein ◽  
Subcellular Fractionation ◽  
Cell Fractionation ◽  
Electron Microscopic

Lipocortin 1 (annexin I) is a calcium- and phospholipid-binding annexin protein which can be externalised from cells despite the lack of a signal sequence. To determine its cellular distribution lipocortin 1 in A549 human lung adenocarcinoma cells was localised by light- and electron-microscopic immunocytochemistry and by cell fractionation and western blotting. Lipocortin 1 immunoreactivity is concentrated in prominent patches associated with the plasma membrane. The intensity of these patches varied with the confluence and duration of the culture and was not detectably diminished by an EDTA wash before fixation. Tubulin and cytokeratin 8 were colocalized with lipocortin 1 in the patches. Within the cells lipocortin 1 was distributed throughout the cytoplasm. Electron microscopy revealed prominent immunoreactivity along the plasma membrane with occasional large clusters of gold particles in contact with the membrane surface of the cells; within the cytoplasm the membrane of some vesicle/vacuole structures and some small electron-dense bodies was immunoreactive, but no immunogold particles were associated with the multilamellar bodies. Subcellular fractionation, extraction and western blotting showed that lipocortin 1 in the membrane pellet was present as two distinct fractions; one, intimately associated with the lipid bilayer, which behaved like an integral membrane protein and one loosely attached which behaved like a peripheral membrane protein. The results show that a substantial amounts of lipocortin 1 is concentrated in focal structures associated with and immediately beneath the plasma membrane. These might form part of the mechanism by which lipocortin 1 is released from the cells.

scholarly journals Growth of neurites without filopodial or lamellipodial activity in the presence of cytochalasin B.

1984 ◽  
Vol 99 (6) ◽  
pp. 2041-2047 ◽  
Author(s):  
L Marsh ◽  
P C Letourneau
Keyword(s):  
Cytochalasin B ◽  
Ganglion Cells ◽  
Video Recording ◽  
Neurite Growth ◽  
Time Lapse ◽  
Growth Cones ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Actin Networks ◽  
Dorsal Root Ganglion Cells

To examine the role in neurite growth of actin-mediated tensions within growth cones, we cultured chick embryo dorsal root ganglion cells on various substrata in the presence of cytochalasin B. Time-lapse video recording was used to monitor behaviors of living cells, and cytoskeletal arrangements in neurites were assessed via immunofluorescence and electron microscopic observations of thin sections and whole, detergent-extracted cells decorated with the S1 fragment of myosin. On highly adhesive substrata, nerve cells were observed to extend numerous (though peculiarly oriented) neurites in the presence of cytochalasin, despite their lack of both filopodia and lamellipodia or the orderly actin networks characteristic of typical growth cones. We concluded that growth cone activity is not necessary for neurite elongation, although actin arrays seem important in mediating characteristics of substratum selectivity and neurite shape.

scholarly journals Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity.

1992 ◽  
Vol 119 (5) ◽  
pp. 1219-1243 ◽  
Author(s):  
A K Lewis ◽  
P C Bridgman
Keyword(s):  
Actin Filament ◽  
Growth Cone ◽  
Actin Filaments ◽  
Membrane Surface ◽  
Leading Edge ◽  
Growth Cones ◽  
Differential Interference Contrast Microscopy ◽  
Nerve Growth ◽  
Filament Bundles ◽  
Two Populations

The organization and polarity of actin filaments in neuronal growth cones was studied with negative stain and freeze-etch EM using a permeabilization protocol that caused little detectable change in morphology when cultured nerve growth cones were observed by video-enhanced differential interference contrast microscopy. The lamellipodial actin cytoskeleton was composed of two distinct subpopulations: a population of 40-100-nm-wide filament bundles radiated from the leading edge, and a second population of branching short filaments filled the volume between the dorsal and ventral membrane surfaces. Together, the two populations formed the three-dimensional structural network seen within expanding lamellipodia. Interaction of the actin filaments with the ventral membrane surface occurred along the length of the filaments via membrane associated proteins. The long bundled filament population was primarily involved in these interactions. The filament tips of either population appeared to interact with the membrane only at the leading edge; this interaction was mediated by a globular Triton-insoluble material. Actin filament polarity was determined by decoration with myosin S1 or heavy meromyosin. Previous reports have suggested that the polarity of the actin filaments in motile cells is uniform, with the barbed ends toward the leading edge. We observed that the actin filament polarity within growth cone lamellipodia is not uniform; although the predominant orientation was with the barbed end toward the leading edge (47-56%), 22-25% of the filaments had the opposite orientation with their pointed ends toward the leading edge, and 19-31% ran parallel to the leading edge. The two actin filament populations display distinct polarity profiles: the longer filaments appear to be oriented predominantly with their barbed ends toward the leading edge, whereas the short filaments appear to be randomly oriented. The different length, organization and polarity of the two filament populations suggest that they differ in stability and function. The population of bundled long filaments, which appeared to be more ventrally located and in contact with membrane proteins, may be more stable than the population of short branched filaments. The location, organization, and polarity of the long bundled filaments suggest that they may be necessary for the expansion of lamellipodia and for the production of tension mediated by receptors to substrate adhesion molecules.

Subcellular localization of myosin V in nerve growth cones and outgrowth from dilute-lethal neurons

1997 ◽  
Vol 110 (4) ◽  
pp. 439-449 ◽  
Author(s):  
L.L. Evans ◽  
J. Hammer ◽  
P.C. Bridgman
Keyword(s):  
Growth Cone ◽  
Actin Filaments ◽  
Traction Force ◽  
Growth Cones ◽  
Neuronal Function ◽  
Neuronal Structure ◽  
Myosin V ◽  
Cytoskeletal Organization ◽  
Cervical Ganglion ◽  
Nerve Growth Cones

Myosin V-null mice (dilute-lethal mutants) exhibit apparent neurological defects that worsen from birth until death in the third postnatal week. Although myosin V is enriched in brain, the neuronal function of myosin V is unclear and the underlying cause of the neurological defects in these mice is unknown. To aide in understanding myosin V function, we examined the distribution of myosin V in the rodent superior cervical ganglion (SCG) growth cone, a well characterized neuronal structure in which myosin V is concentrated. Using affinity purified, myosin V-specific antibodies in immunofluorescence and immunoelectron microscopy, we observed that myosin V is concentrated in organelle-rich regions of the growth cone. Myosin V is present on a distinct population of small (50–100 nm) organelles, and on actin filaments and the plasma membrane. Myosin V-associated organelles are present on both microtubules and actin filaments. These results indicate that myosin V may be carried as a passenger on organelles that are transported along microtubules, and that these organelles may also be capable of movement along actin filaments. In addition, we found no abnormalities in outgrowth, morphology, or cytoskeletal organization of SCG growth cones from dilute-lethal mice. These results indicate that myosin V is not necessary for the traction force needed for growth cone locomotion, for organization of the actin cytoskeleton, or for filopodial dynamics.

scholarly journals PHAGOCYTOSIS OF LATEX BEADS BY ACANTHAMOEBA

1967 ◽  
Vol 34 (1) ◽  
pp. 219-227 ◽  
Author(s):  
Edward D. Korn ◽  
Robert A. Weisman
Keyword(s):  
Plasma Membrane ◽  
Biochemical Study ◽  
Electron Microscopic ◽  
Latex Beads ◽  
Microscopic Studies

Electron microscopic studies confirm and extend the conclusions derived previously from a quantitative biochemical study of the phagocytosis of polystyrene and polyvinyltoluene latex beads by Acanthamoeba (1). Latex beads 1.305, 1.90, and 2.68 µ in diameter are ingested individually, with each bead tightly surrounded by a membrane derived from the plasma membrane. Latex beads 0.557, 0.264, 0.126, and 0.088 µ in diameter are accumulated at the surface of the ameba and then phagocytosed, with many beads tightly packed within one membrane-bounded vesicle.

Evidence that cell surface motility in Allogromia is mediated by cytoplasmic microtubules

1985 ◽  
Vol 63 (6) ◽  
pp. 608-620 ◽  
Author(s):  
Samuel S. Bowser ◽  
Conly L. Rieder
Keyword(s):  
Plasma Membrane ◽  
Membrane Surface ◽  
Organelle Transport ◽  
Polystyrene Microspheres ◽  
Electron Microscopic ◽  
Surface Transport ◽  
Cytoplasmic Microtubules ◽  
Surface Motility ◽  
Plasma Membrane Surface ◽  
Nonionic Detergents

We have previously shown that reticulopods of Allogromia sp. (strain NF) and Allogromia laticollaris display rapid, bidirectional saltatory transport of plasma membrane surface markers (i.e., polystyrene microspheres). Correlative video microscopic and electron microscopic methods were used to determine whether cytoskeletal components are involved in this surface transport. Such transport was observed only where the plasma membrane overlay cytoplasmic fibrils, which have been shown to be involved in organelle transport. Ultrastuctural analysis indicated that these fibrils contain microtubules and an associated flocculent fibrillar material. In studies with nonionic detergents the surface marker particles remained bound to these microtubule-containing fibrils, even after the plasma membrane had been removed. Surface transport was inhibited when reticulopods were treated with agents that induce microtubule disassembly. Together these observations provide strong evidence that surface motility in Allogromia is mediated by labile cytoplasmic microtubules.

The distribution and movement of organelles in maturing growth cones: correlated video-enhanced and electron microscopic studies

1988 ◽  
Vol 17 (6) ◽  
pp. 783-795 ◽  
Author(s):  
Donald W. Burmeister ◽  
Mary Chen ◽  
Craig H. Bailey ◽  
Daniel J. Goldberg
Keyword(s):  
Growth Cones ◽  
Electron Microscopic ◽  
Microscopic Studies

Cell-cell channel formation and lectins

1991 ◽  
Vol 261 (6) ◽  
pp. C1025-C1032 ◽  
Author(s):  
E. Levine ◽  
R. Werner ◽  
G. Dahl
Keyword(s):  
Membrane Surface ◽  
Significant Inhibition ◽  
Carbohydrate Binding ◽  
Binding Affinities ◽  
Channel Formation ◽  
Electron Microscopic ◽  
Lectin Receptors ◽  
Microscopic Studies ◽  
Free Membrane ◽  
Cell Cell

The oocyte cell-cell channel assay was used to investigate determinants of the rate of channel formation. After injection of connexin-specific mRNA, oocytes accumulate a pool of precursors from which cell-cell channels can form after oocytes are paired. Channel formation was found to be increased if oocytes are pretreated with lectins before pairing. Several lectins differing in their carbohydrate binding affinities can exert this effect. Lectin-specific sugars suppress the effect on cell-cell channel formation only if the sugar is mixed with the lectin before application to the oocyte. If the lectin is first bound to the oocyte and then the sugar is added, no significant inhibition is seen. The promotion of channel formation by lectins is enhanced by adding an incubation period in regular medium after lectin treatment, before pairing of the oocytes. Electron microscopic studies with gold-conjugated lectins show that the lectin receptors are clustered on the free membrane surface and are taken up in endocytotic vesicles. These data suggest that the observed acceleration of cell-cell channel formation by lectins can be attributed to the removal of steric hindrance, which is a consequence of clustering of the bulky glycoprotein lectin receptors as well as of the removal from the surface by endocytosis.

The normal human hippocampus and that of Alzheimer’s patients contain filopodia like those of octopus neuropils and nerve growth cones

1992 ◽  
Vol 50 (1) ◽  
pp. 478-479
Author(s):  
J. David Robertson ◽  
Psyche Lee
Keyword(s):  
Actin Polymerization ◽  
Calcium Influx ◽  
Growth Cones ◽  
Good Evidence ◽  
Octopus Vulgaris ◽  
Short Term ◽  
Electron Microscopic ◽  
Nerve Growth ◽  
Normal Human ◽  
Nerve Growth Cones

New synapses are generally formed in long-term learning [See (3) for references], but it is believed that short-term learning involves only alterations in synaptic efficacy with activity (1). We have studied this problem extensively in Octopus vulgaris (2-5). Electron Microscopic (EM) studies of serial sections reveal that filopodia like those in nerve growth cones occur in its touch learning neuropils (2). They increase in numbers transiently during learning and some form new synapses, while others retract. Calcium influx into neurites during activity triggers actin polymerization and related filopodial extension. This, by interpolation of new membranes, results in separation of interactive membranes, formerly in contact. An alteration in the total electrochemical interactions of the membranes results that alters the summation of interactions between all the membranes and changes the overall integrated output. This can be defined as learning and one of us has advanced a filopodial theory of learning based on these ideas (5). Accordingly, there is a localized reactivation of growth processes normally only operating during development to bring about structural alterations beginning with short-term learning. If correct, leaning should be blocked by cytochalasins B (CB) and D (CD) that interfere with actin polymerization and it should be stimulated by nerve growth factor (NGF). We have good evidence that CB and CD block touch learning reversibly in Octopus (2-5) and recently that NGF stimulates touch learning.

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