A complex consisting of pp60c-src/pp60c-srcN and a 38 kDa protein is highly enriched in growth cones from differentiated SH-SY5Y neuroblastoma cells

1992 ◽  
Vol 103 (1) ◽  
pp. 233-243
Author(s):  
G. Meyerson ◽  
K.H. Pfenninger ◽  
S. Pahlman
Keyword(s):  
Growth Cone ◽  
Gel Electrophoresis ◽  
Neuroblastoma Cells ◽  
Growth Cones ◽  
Cell Periphery ◽  
Primary Neurons ◽  
Reducing Conditions ◽  
Oligomeric Complex ◽  
Nerve Growth Cones

Nerve growth cones of primary neurons are highly enriched in the proto-oncogene product pp60c-src. In order to investigate this molecule further in growing neuronal cells, growth cone and cell body fractions were prepared from human SH-SY5Y neuroblastoma cells differentiated neuronally in vitro under the influence of phorbol ester. The fractions were characterized ultrastructurally and by biochemical criteria. The neuronal (pp60c-srcN) and the fibroblastic (pp60c-src) forms of pp60src are slightly enriched and activated in the growth cones relative to the perikarya. Immunoprecipitates of pp60src from differentiated SH-SY5Y growth cones contain at least four phosphoproteins in addition to pp60src. One of these, pp38, migrates as a 100–140 kDa complex with pp60src under non-reducing conditions of gel electrophoresis. The pp38/pp60src complex is not easily detected in non-differentiated SH-SY5Y cells or perikarya of differentiated SH-SY5Y cells, but it is highly enriched in the growth cone preparation. These data suggest that growth-cone pp60src exists in a disulfide-linked oligomeric complex. The complex appears to be assembled only in the cell periphery and may be dependent upon neuronal differentiation.

scholarly journals Platelet-collagen adhesion: inhibition by a monoclonal antibody that binds glycoprotein IIb.

1984 ◽  
Vol 99 (6) ◽  
pp. 2056-2060 ◽  
Author(s):  
P J Shadle ◽  
M H Ginsberg ◽  
E F Plow ◽  
S H Barondes
Keyword(s):  
Monoclonal Antibody ◽  
Gel Electrophoresis ◽  
Polyclonal Antiserum ◽  
Platelet Glycoprotein ◽  
Two Dimensional Gel Electrophoresis ◽  
Vitro Assay ◽  
Platelet Surface ◽  
Reducing Conditions ◽  
Purified Antigen

To identify platelet surface structures involved in adhesion to collagen, the effect of 16 murine antiplatelet membrane hybridoma antibodies were tested in a defined, in vitro assay. Four of these antibodies inhibited platelet-collagen adhesion and reacted with a polypeptide with Mr approximately 125,000, as determined by immunoblots after gel electrophoresis under reducing conditions. Through detailed studies with one of these antibodies, the monoclonal antibody PMI-1, the relevant antigen was identified as platelet glycoprotein IIb alpha, based upon (a) co-migration with this glycoprotein in two-dimensional gel electrophoresis and (b) co-purification by immunoaffinity chromatography with a protein with apparent Mr identical to that of glycoprotein III, under conditions in which glycoproteins IIb and III form a complex. Univalent antibody fragments prepared from monoclonal antibody PMI-1 inhibited greater than 80% of platelet-collagen adhesion, and inhibition was completely blocked by the immunopurified antigen. These results indicate that glycoprotein IIb participates in some aspect of platelet-collagen adhesion. In contrast, the purified antigen only partially neutralized a polyclonal antiserum that blocked platelet-collagen adhesion, to a maximum of approximately 25%, at saturating antigen concentrations. Thus, by these immunological criteria, glycoprotein IIb is not the only molecule involved in this process.

scholarly journals BMP gradients steer nerve growth cones by a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofilin

2007 ◽  
Vol 178 (1) ◽  
pp. 107-119 ◽  
Author(s):  
Zhexing Wen ◽  
Liang Han ◽  
James R. Bamburg ◽  
Sangwoo Shim ◽  
Guo-li Ming ◽  
...  
Keyword(s):  
Growth Cone ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Growth Cones ◽  
Actin Dynamics ◽  
Lim Kinase ◽  
Nerve Growth ◽  
Nerve Growth Cones ◽  
Transient Receptor ◽  
Calcineurin Phosphatase

Bone morphogenic proteins (BMPs) are involved in axon pathfinding, but how they guide growth cones remains elusive. In this study, we report that a BMP7 gradient elicits bidirectional turning responses from nerve growth cones by acting through LIM kinase (LIMK) and Slingshot (SSH) phosphatase to regulate actin-depolymerizing factor (ADF)/cofilin-mediated actin dynamics. Xenopus laevis growth cones from 4–8-h cultured neurons are attracted to BMP7 gradients but become repelled by BMP7 after overnight culture. The attraction and repulsion are mediated by LIMK and SSH, respectively, which oppositely regulate the phosphorylation-dependent asymmetric activity of ADF/cofilin to control the actin dynamics and growth cone steering. The attraction to repulsion switching requires the expression of a transient receptor potential (TRP) channel TRPC1 and involves Ca2+ signaling through calcineurin phosphatase for SSH activation and growth cone repulsion. Together, we show that spatial regulation of ADF/cofilin activity controls the directional responses of the growth cone to BMP7, and Ca2+ influx through TRPC tilts the LIMK-SSH balance toward SSH-mediated repulsion.

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.

Actin disruption alters the localization of tau in the growth cones of cerebellar granule neurons

2000 ◽  
Vol 113 (15) ◽  
pp. 2797-2809
Author(s):  
J.F. Zmuda ◽  
R.J. Rivas
Keyword(s):  
Growth Cone ◽  
Growth Cones ◽  
Cerebellar Granule Neurons ◽  
Granule Neurons ◽  
Filamentous Actin ◽  
Cerebellar Granule ◽  
Single Axon ◽  
Peripheral Domain ◽  
The Way

Cultured cerebellar granule neurons initially extend a single axon, followed by the extension of a second axon to attain a bipolar morphology. Differentiation culminates with the extension of several short dendrites from the cell body. In the present study, we determined the location of the dephosphorylated form of the microtubule-associated protein tau (dtau) within the growth cones of newly forming axons and examined whether this localization was influenced by the actin cytoskeleton. Following elongation of the initial axon at 2–3 days in vitro, dtau immunoreactivity was present along the entire length of the axon, becoming most intense just proximal to the growth cone. Dtau labeling dropped off dramatically along the microtubules of the growth cone and was undetectable along the most distal tips of these microtubules. As the initial axon continued to elongate at 3–4 days in vitro, the actin-rich growth cone peripheral domain characteristically underwent a dramatic reduction in size. Dtau immunoreactivity extended all the way to the most distal tips of the microtubules in the growth cones of these cells. Cytochalasin D and latrunculin A mimicked the effects of this characteristic reduction in growth cone size with regard to dtau localization in the growth cone. Depolymerization of filamentous actin caused the collapse of the peripheral domain and allowed dtau to bind all the way to the most distal tips of microtubules in the axon. Upon removal of the drugs, the peripheral domain of the growth cone rapidly re-formed and dtau was once again excluded from the most distal regions of growth cone microtubules. These findings suggest a novel role for actin in determining the localization of the microtubule-associated protein τ within the growth cones of neurons.

scholarly journals Microtubule behavior during guidance of pioneer neuron growth cones in situ.

1991 ◽  
Vol 115 (2) ◽  
pp. 381-395 ◽  
Author(s):  
J H Sabry ◽  
T P O'Connor ◽  
L Evans ◽  
A Toroian-Raymond ◽  
M Kirschner ◽  
...  
Keyword(s):  
Growth Cone ◽  
Growth Cones ◽  
The Body ◽  
Microtubule Network ◽  
Guidance Cues ◽  
Selective Retention ◽  
Pioneer Neuron ◽  
Neuron Growth

The growth of an axon toward its target results from the reorganization of the cytoskeleton in response to environmental guidance cues. Recently developed imaging technology makes it possible to address the effect of such cues on the neural cytoskeleton directly. Although high resolution studies can be carried out on neurons in vitro, these circumstances do not recreate the complexity of the natural environment. We report here on the arrangement and dynamics of microtubules in live neurons pathfinding in response to natural guidance cues in situ using the embryonic grasshopper limb fillet preparation. A rich microtubule network was present within the body of the growth cone and normally extended into the distal growth cone margin. Complex microtubule loops often formed transiently within the growth cone. Branches both with and without microtubules were regularly observed. Microtubules did not extend into filopodia. During growth cone steering events in response to identified guidance cues, microtubule behaviour could be monitored. In turns towards guidepost cells, microtubules selectively invaded branches derived from filopodia that had contacted the guidepost cell. At limb segment boundaries, microtubules displayed a variety of behaviors, including selective branch invasion, and also invasion of multiple branches followed by selective retention in branches oriented in the correct direction. Microtubule invasion of multiple branches also was seen in growth cones migrating on intrasegmental epithelium. Both selective invasion and selective retention generate asymmetrical microtubule arrangements within the growth cone, and may play a key role in growth cone steering events.

scholarly journals Phosphorylation-site mutagenesis of the growth-associated protein GAP-43 modulates its effects on cell spreading and morphology.

1993 ◽  
Vol 120 (2) ◽  
pp. 503-512 ◽  
Author(s):  
F Widmer ◽  
P Caroni
Keyword(s):  
Phosphorylation Site ◽  
Growth Cones ◽  
Cell Cortex ◽  
Membrane Association ◽  
Major Protein ◽  
Cell Periphery ◽  
Cortical Actin ◽  
Cortical Cytoskeleton

The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of axonal growth cones, developing nerve terminals, regenerating axons, and adult central nervous system areas associated with plasticity. It is a cytosolic protein associated with the cortical cytoskeleton and the plasmalemma. Membrane association of GAP-43 is mediated by palmitoylation at Cys3Cys4. In vitro and in vivo, phosphorylation by PKC exclusively involves Ser41 of mammalian GAP-43 (corresponding to Ser42 in the chick protein). To identify aspects of GAP-43 function, we analyzed the actions of wild-type, membrane-association, and phosphorylation-site mutants of GAP-43 in nonneuronal cell lines. The GAP-43 constructs were introduced in L6 and COS-7 cells by transient transfection. Like the endogenous protein in neurons and their growth cones, GAP-43 in nonneuronal cells associated with the cell periphery. GAP-43 accumulated in the pseudopods of spreading cells and appeared to interact with cortical actin-containing filaments. Spreading L6 cells expressing high levels of recombinant protein displayed a characteristic F-actin labeling pattern consisting of prominent radial arrays of peripheral actin filaments. GAP-43 had dramatic effects on local surface morphology. Characteristic features of GAP-43-expressing cells were irregular cell outlines with prominent and numerous filopodia. The effects of GAP-43 on cell morphology required association with the cell membrane, since GAP-43(Ala3Ala4), a mutant that failed to associate with the cell cortex, had no morphogenetic activity. Two GAP-43 phosphorylation mutants (Ser42 to Ala42 preventing and Ser42 to Asp42 mimicking phosphorylation by PKC) modulated the effects of GAP-43 in opposite ways. Cells expressing GAP-43(Asp42) spread extensively and displayed large and irregular membranous extensions with little filopodia, whereas GAP-43(Ala42) produced small, poorly spreading cells with numerous short filopodia. Therefore, GAP-43 influences cell surface behavior and phosphorylation modulates its activity. The presence of GAP-43 in growing axons and developing nerve termini may affect the behavior of their actin-containing cortical cytoskeleton in a regulatable manner.

scholarly journals Automated profiling of growth cone heterogeneity defines relations between morphology and motility

2018 ◽  
Vol 218 (1) ◽  
pp. 350-379 ◽  
Author(s):  
Maria M. Bagonis ◽  
Ludovico Fusco ◽  
Olivier Pertz ◽  
Gaudenz Danuser
Keyword(s):  
Growth Cone ◽  
Rho Gtpase ◽  
Time Lapse ◽  
Growth Cones ◽  
Cellular Structures ◽  
Unperturbed System ◽  
Neurite Length ◽  
Manual Curation

Growth cones are complex, motile structures at the tip of an outgrowing neurite. They often exhibit a high density of filopodia (thin actin bundles), which complicates the unbiased quantification of their morphologies by software. Contemporary image processing methods require extensive tuning of segmentation parameters, require significant manual curation, and are often not sufficiently adaptable to capture morphology changes associated with switches in regulatory signals. To overcome these limitations, we developed Growth Cone Analyzer (GCA). GCA is designed to quantify growth cone morphodynamics from time-lapse sequences imaged both in vitro and in vivo, but is sufficiently generic that it may be applied to nonneuronal cellular structures. We demonstrate the adaptability of GCA through the analysis of growth cone morphological variation and its relation to motility in both an unperturbed system and in the context of modified Rho GTPase signaling. We find that perturbations inducing similar changes in neurite length exhibit underappreciated phenotypic nuance at the scale of the growth cone.

Neuronal calcium sensor-1 in nerve growth cones independently regulates both the neurite outgrowth and growth cone morphology

2007 ◽  
Vol 58 ◽  
pp. S204
Author(s):  
Masumi Iketani ◽  
Chihiro Imaizumi ◽  
Andreas Jeromin ◽  
Fumio Nakamura ◽  
Katsuhiko Mikoshiba ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Growth Cone ◽  
Growth Cones ◽  
Calcium Sensor ◽  
Nerve Growth ◽  
Neuronal Calcium Sensor ◽  
Cone Morphology ◽  
Nerve Growth Cones

A scanning electron microscope study of the development of a peripheral sensory neurite network

Development ◽  
1982 ◽  
Vol 69 (1) ◽  
pp. 237-250
Author(s):  
Alan Roberts ◽  
J. S. H. Taylor
Keyword(s):  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Basal Lamina ◽  
Growth Cone ◽  
Growth Cones ◽  
Sensory Innervation ◽  
Scanning Electron Microscope Study ◽  
Scanning Electron

The formation of the sensory neurite plexus on the basal lamina of trunk skin in Xenopus embryos has been examined using the scanning electron microscope. It is formed by Rohon-Beard and extramedullary neurons which provide the first sensory innervation of the skin. By observing the distribution of growth cones on the inside surface of the skin of embryos at different ages, the development of the plexus has been followed and related to the development of sensitivity to sensory stimulation. The general features of the plexus are illustrated using a photomontage taken at × 1100. Measurements on neurites from this, and of growth cone orientations demonstrate a general ventral growth pattern with some small regional variations. Interactions of neurites within the plexus are examined. Neurites meeting at shallow angles tend to fasciculate, whilethose meeting at close to 90° tend to cross each other. Angles of incidence and separation of neurites show few angles less than 30°, which suggests that active adjustments occur after a growth cone meets or leaves another neurite. The observations allow comparison of behaviour of growing neurites in vivo and in vitro. Our evidence suggests that adhesion between growth cones and neurites is stronger than that between growth cones and the basal lamina of the skin.

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