scholarly journals FREEZE-FRACTURING OF NERVE GROWTH CONES AND YOUNG FIBERS

1974 ◽  
Vol 63 (1) ◽  
pp. 180-196 ◽  
Author(s):  
Karl H. Pfenninger ◽  
Richard P. Bunge
Keyword(s):  
Growth Cone ◽  
Particle Density ◽  
Growth Cones ◽  
Nerve Fibers ◽  
Nerve Tissue ◽  
Intramembranous Particle ◽  
Freeze Fracturing ◽  
Intramembranous Particles ◽  
Nerve Growth Cones

Neural and non-neural cellular processes have been studied in organotypic cultures of spinal cord and olfactory bulb by means of the freeze-fracturing technique. Identification of specific cellular elements in replicas has been achieved by comparison with thin-sectioned material in which differences in shape and contents are evident. Freeze-fracturing reveals that neural growth cones may be distinguished from glial pseudopodia by the low number of intramembranous particles within their plasma membrane; the counts of particles within the growth cone membrane average 85/µm2 (for the inner leaflet) as opposed to hundreds per square micrometer in glial pseudopodia. Whereas the intramembranous particle number in glial pseudopodia is only slightly lower than in their perikaryal plasmalemma, the number of particles in outgrowing axons increases about eightfold from the periphery towards the perikaryon. Furthermore, with prolonged time of growth in culture, the particle density in the young nerve fibers increases by about the same factor. The same phenomenon, i.e. a low intramembranous particle level at earlier stages and an increase in numbers as the nerve fiber matures, is observed in fetal nerve tissue in vivo. These findings suggest that the plasmalemma of the outgrowing nerve, and especially of the growth cone, is immature and that maturation is accompanied by the insertion of intramembranous particles. Furthermore, these data indicate that the chemistry of the growth cone membrane is distinct from that of the neuron soma which may be significant for the mechanisms of guidance and recognition in the growing nerve tip.

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 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.

scholarly journals Ultrastructure of the sodium pump: Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+, K+)-ATPase

1977 ◽  
Vol 75 (3) ◽  
pp. 619-634 ◽  
Author(s):  
N Deguchi ◽  
PL Jorgensen ◽  
AB Maunsbach
Keyword(s):  
Membrane Surface ◽  
Freeze Fracture ◽  
Negative Staining ◽  
Intramembranous Particle ◽  
Freeze Fracturing ◽  
Intramembranous Particles ◽  
Membrane Surfaces ◽  
Thin Sectioning ◽  
Surface Particle ◽  
Protein Components

Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 μm. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per μm(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per μm(2) and 390 +/- 170 per μm(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.

scholarly journals RHO-1 and the Rho GEF RHGF-1 interact with UNC-6/Netrin signaling to regulate growth cone protrusion and microtubule organization in C. elegans

2019 ◽  
Author(s):  
Mahekta R. Gujar ◽  
Aubrie M. Stricker ◽  
Erik A. Lundquist
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Neural Circuit ◽  
Growth Cones ◽  
Negative Regulator ◽  
Microtubule Organization ◽  
C Elegans ◽  
Guidance Cue ◽  
Rho Gef

AbstractUNC-6/Netrin is a conserved axon guidance cue that directs growth cone migrations in the dorsal-ventral axis of C. elegans and in the vertebrate spinal cord. UNC-6/Netrin is expressed in ventral cells, and growth cones migrate ventrally toward or dorsally away from UNC-6/Netrin. Recent studies of growth cone behavior during outgrowth in vivo in C. elegans have led to a polarity/protrusion model in directed growth cone migration away from UNC-6/Netrin. In this model, UNC-6/Netrin first polarizes the growth cone via the UNC-5 receptor, leading to dorsally biased protrusion and F-actin accumulation. UNC-6/Netrin then regulates protrusion based on this polarity. The receptor UNC-40/DCC drives protrusion dorsally, away from the UNC-6/Netrin source, and the UNC-5 receptor inhibits protrusion ventrally, near the UNC-6/Netrin source, resulting in dorsal migration. UNC-5 inhibits protrusion in part by excluding microtubules from the growth cone, which are pro-protrusive. Here we report that the RHO-1/RhoA GTPase and its activator GEF RHGF-1 inhibit growth cone protrusion and MT accumulation in growth cones, similar to UNC-5. However, growth cone polarity of protrusion and F-actin were unaffected by RHO-1 and RHGF-1. Thus, RHO-1 signaling acts specifically as a negative regulator of protrusion and MT accumulation, and not polarity. Genetic interactions suggest that RHO-1 and RHGF-1 act with UNC-5, as well as with a parallel pathway, to regulate protrusion. The cytoskeletal interacting molecule UNC-33/CRMP was required for RHO-1 activity to inhibit MT accumulation, suggesting that UNC-33/CRMP might act downstream of RHO-1. In sum, these studies describe a new role of RHO-1 and RHGF-1 in regulation of growth cone protrusion by UNC-6/Netrin.Author SummaryNeural circuits are formed by precise connections between axons. During axon formation, the growth cone leads the axon to its proper target in a process called axon guidance. Growth cone outgrowth involves asymmetric protrusion driven by extracellular cues that stimulate and inhibit protrusion. How guidance cues regulate growth cone protrusion in neural circuit formation is incompletely understood. This work shows that the signaling molecule RHO-1 acts downstream of the UNC-6/Netrin guidance cue to inhibit growth cone protrusion in part by excluding microtubules from the growth cone, which are structural elements that drive protrusion.

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.

Growth cones stall and collapse during axon outgrowth in Caenorhabditis elegans

Development ◽  
1999 ◽  
Vol 126 (20) ◽  
pp. 4489-4498 ◽  
Author(s):  
K.M. Knobel ◽  
E.M. Jorgensen ◽  
M.J. Bastiani
Keyword(s):  
Caenorhabditis Elegans ◽  
Growth Cone ◽  
Nerve Cord ◽  
Body Wall ◽  
Growth Cones ◽  
The Body ◽  
Axon Outgrowth ◽  
Attachment Structures ◽  
Dorsal Nerve

During nervous system development, neurons form synaptic contacts with distant target cells. These connections are formed by the extension of axonal processes along predetermined pathways. Axon outgrowth is directed by growth cones located at the tips of these neuronal processes. Although the behavior of growth cones has been well-characterized in vitro, it is difficult to observe growth cones in vivo. We have observed motor neuron growth cones migrating in living Caenorhabditis elegans larvae using time-lapse confocal microscopy. Specifically, we observed the VD motor neurons extend axons from the ventral to dorsal nerve cord during the L2 stage. The growth cones of these neurons are round and migrate rapidly across the epidermis if they are unobstructed. When they contact axons of the lateral nerve fascicles, growth cones stall and spread out along the fascicle to form anvil-shaped structures. After pausing for a few minutes, they extend lamellipodia beyond the fascicle and resume migration toward the dorsal nerve cord. Growth cones stall again when they contact the body wall muscles. These muscles are tightly attached to the epidermis by narrowly spaced circumferential attachment structures. Stalled growth cones extend fingers dorsally between these hypodermal attachment structures. When a single finger has projected through the body wall muscle quadrant, the growth cone located on the ventral side of the muscle collapses and a new growth cone forms at the dorsal tip of the predominating finger. Thus, we observe that complete growth cone collapse occurs in vivo and not just in culture assays. In contrast to studies indicating that collapse occurs upon contact with repulsive substrata, collapse of the VD growth cones may result from an intrinsic signal that serves to maintain growth cone primacy and conserve cellular material.

scholarly journals Gradients and Growth Cone Guidance of Grasshopper Neurons

2003 ◽  
Vol 51 (4) ◽  
pp. 445-454 ◽  
Author(s):  
Arthur T. Legg ◽  
Timothy P. O'Connor
Keyword(s):  
Nervous System ◽  
Long Range ◽  
Growth Cone ◽  
Growth Cones ◽  
Path Finding ◽  
Guidance Cues ◽  
Cone Function ◽  
The Absolute ◽  
Growth Cone Guidance

The generation of a functional nervous system is dependent on precise path-finding of axons during development. This pathfinding is directed by the distribution of local and long-range guidance cues, the latter of which are believed to be distributed in gradients. Gradients of guidance cues have been associated with growth cone function for over a hundred years. However, little is known about the mechanisms used by growth cones to respond to these gradients, in part owing to the lack of identifiable gradients in vivo. In the developing grasshopper limb, two gradients of the semaphorin Sema-2a are necessary for correct neuronal pathfinding in vivo. The gradients are found in regions where growth cones make critical steering decisions. Observations of different growth cone behaviors associated with these gradients have provided some insights into how growth cones respond to them. Growth cones appear to respond more faithfully to changes in concentration, rather than absolute levels, of Sema-2a expression, whereas the absolute levels may regulate growth cone size.

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.

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