scholarly journals Growth Cone Collapse through Coincident Loss of Actin Bundles and Leading Edge Actin without Actin Depolymerization

2001 ◽  
Vol 153 (5) ◽  
pp. 1071-1084 ◽  
Author(s):  
Feng-quan Zhou ◽  
Christopher S. Cohan
Keyword(s):  
Growth Cone ◽  
Kinase Inhibitor ◽  
Leading Edge ◽  
Time Lapse ◽  
Growth Cones ◽  
Filamentous Actin ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Growth Cone Collapse ◽  
Substrate Adhesion

Repulsive guidance cues can either collapse the whole growth cone to arrest neurite outgrowth or cause asymmetric collapse leading to growth cone turning. How signals from repulsive cues are translated by growth cones into this morphological change through rearranging the cytoskeleton is unclear. We examined three factors that are able to induce the collapse of extending Helisoma growth cones in conditioned medium, including serotonin, myosin light chain kinase inhibitor, and phorbol ester. To study the cytoskeletal events contributing to collapse, we cultured Helisoma growth cones on polylysine in which lamellipodial collapse was prevented by substrate adhesion. We found that all three factors that induced collapse of extending growth cones also caused actin bundle loss in polylysine-attached growth cones without loss of actin meshwork. In addition, actin bundle loss correlated with specific filamentous actin redistribution away from the leading edge that is characteristic of repulsive factors. Finally, we provide direct evidence using time-lapse studies of extending growth cones that actin bundle loss paralleled collapse. Taken together, these results suggest that actin bundles could be a common cytoskeletal target of various collapsing factors, which may use different signaling pathways that converge to induce growth cone collapse.

scholarly journals Focal loss of actin bundles causes microtubule redistribution and growth cone turning

2002 ◽  
Vol 157 (5) ◽  
pp. 839-849 ◽  
Author(s):  
Feng-Quan Zhou ◽  
Clare M. Waterman-Storer ◽  
Christopher S. Cohan
Keyword(s):  
Growth Cone ◽  
Direct Evidence ◽  
Leading Edge ◽  
Actin Reorganization ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Growth Cone Collapse ◽  
Guidance Cues ◽  
First Time ◽  
Peripheral Domain

Ît is commonly believed that growth cone turning during pathfinding is initiated by reorganization of actin filaments in response to guidance cues, which then affects microtubule structure to complete the turning process. However, a major unanswered question is how changes in actin cytoskeleton are induced by guidance cues and how these changes are then translated into microtubule rearrangement. Here, we report that local and specific disruption of actin bundles from the growth cone peripheral domain induced repulsive growth cone turning. Meanwhile, dynamic microtubules within the peripheral domain were oriented into areas where actin bundles remained and were lost from areas where actin bundles disappeared. This resulted in directional microtubule extension leading to axon bending and growth cone turning. In addition, this local actin bundle loss coincided with localized growth cone collapse, as well as asymmetrical lamellipodial protrusion. Our results provide direct evidence, for the first time, that regional actin bundle reorganization can steer the growth cone by coordinating actin reorganization with microtubule dynamics. This suggests that actin bundles can be potential targets of signaling pathways downstream of guidance cues, providing a mechanism for coupling changes in leading edge actin with microtubules at the central domain during turning.

Localization of myosin II A and B isoforms in cultured neurons

1995 ◽  
Vol 108 (12) ◽  
pp. 3661-3670 ◽  
Author(s):  
M.W. Rochlin ◽  
K. Itoh ◽  
R.S. Adelstein ◽  
P.C. Bridgman
Keyword(s):  
Myosin Heavy Chain ◽  
Growth Cone ◽  
Heavy Chain ◽  
Marginal Zone ◽  
Myosin Ii ◽  
Growth Cones ◽  
Filamentous Actin ◽  
Differential Interference Contrast Microscopy ◽  
Actin Bundles ◽  
Ganglion Neurons

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

Oligodendrocytes repel axons and cause axonal growth cone collapse

1989 ◽  
Vol 92 (1) ◽  
pp. 93-100 ◽  
Author(s):  
J.W. Fawcett ◽  
J. Rokos ◽  
I. Bakst
Keyword(s):  
Growth Cone ◽  
Dorsal Root ◽  
Axonal Growth ◽  
Time Lapse ◽  
Growth Cones ◽  
Newborn Rats ◽  
Growth Cone Collapse ◽  
Axonal Growth Cone ◽  
Video Studies ◽  
Culture Surface

We have examined the interactions between axons regenerating from dorsal root ganglia (DRGs) derived from newborn rats and oligodendrocytes cultured by three different techniques. Cultures examined after 2 days have a profuse outgrowth of axons from the DRGs, forming a dense mat on the culture surface. However, the axons avoid growing on oligodendrocytes; axons are seen all around these cells, but do not grow over them. We have also performed time-lapse video studies of the interactions between axonal growth cones and oligodendrocytes. Axons grow normally until their growth cone comes into direct contact with an oligodendrocyte, following which the growth cone remains motile for 30–60 min, but without making any progress over the cell. The growth cone then suddenly collapses, and the axon retracts, leaving a thin strand in contact with the cell. After this a new growth cone is usually elaborated and the process repeated. Oligodendrocytes are therefore inhibitory to axonal growth, and this may partially explain the failure of axons to regenerate in the mammalian central nervous system.

scholarly journals Phosphorylation by Rho Kinase Regulates CRMP-2 Activity in Growth Cones

2005 ◽  
Vol 25 (22) ◽  
pp. 9973-9984 ◽  
Author(s):  
Nariko Arimura ◽  
Céline Ménager ◽  
Yoji Kawano ◽  
Takeshi Yoshimura ◽  
Saeko Kawabata ◽  
...  
Keyword(s):  
Growth Cone ◽  
Actin Filaments ◽  
Rho Kinase ◽  
Growth Cones ◽  
Binding Activity ◽  
Microtubule Assembly ◽  
Coated Pits ◽  
Growth Cone Collapse ◽  
Mediator Protein ◽  
Neuron Growth

ABSTRACT Collapsin response mediator protein 2 (CRMP-2) enhances the advance of growth cones by regulating microtubule assembly and Numb-mediated endocytosis. We previously showed that Rho kinase phosphorylates CRMP-2 during growth cone collapse; however, the roles of phosphorylated CRMP-2 in growth cone collapse remain to be clarified. Here, we report that CRMP-2 phosphorylation by Rho kinase cancels the binding activity to the tubulin dimer, microtubules, or Numb. CRMP-2 binds to actin, but its binding is not affected by phosphorylation. Electron microscopy revealed that CRMP-2 localizes on microtubules, clathrin-coated pits, and actin filaments in dorsal root ganglion neuron growth cones, while phosphorylated CRMP-2 localizes only on actin filaments. The phosphomimic mutant of CRMP-2 has a weakened ability to enhance neurite elongation. Furthermore, ephrin-A5 induces phosphorylation of CRMP-2 via Rho kinase during growth cone collapse. Taken together, these results suggest that Rho kinase phosphorylates CRMP-2, and inactivates the ability of CRMP-2 to promote microtubule assembly and Numb-mediated endocytosis, during growth cone collapse.

scholarly journals The role of microtubules in growth cone turning at substrate boundaries.

1995 ◽  
Vol 128 (1) ◽  
pp. 127-137 ◽  
Author(s):  
E Tanaka ◽  
M W Kirschner
Keyword(s):  
Growth Cone ◽  
Collagen Type ◽  
Axon Growth ◽  
Growth Cones ◽  
Collagen Type Iv ◽  
Early Step ◽  
Growth Cone Collapse ◽  
Type Iv ◽  
And Behaviors

To understand the role of microtubules in growth cone turning, we observed fluorescently labeled microtubules in neurons as they encountered a substrate boundary. Neurons growing on a laminin-rich substrate avoided growing onto collagen type IV. Turning growth cones assumed heterogeneous morphologies and behaviors that depended primarily in their extent of adhesion to the substrate. We grouped these behaviors into three categories-sidestepping, motility, and growth-mediated reorientation. In sidestepping and motility-mediated reorientation, the growth cone and parts of the axon were not well attached to the substrate so the acquisition of an adherent lamella caused the entire growth cone to move away from the border and consequently reoriented the axon. In these cases, since the motility of the growth cone dominates its reorientation, the microtubules were passive, and reorientation occurred without significant axon growth. In growth-mediated reorientation, the growth cone and axon were attached to the substrate. In this case, microtubules reoriented within the growth cone to stabilize a lamella. Bundling of the reoriented microtubules was followed by growth cone collapse to form new axon, and further, polarized lamellipodial extension. These observations indicate that when the growth cone remains adherent to the substrate during turning, the reorientation and bundling of microtubules is an important, early step in growth cone turning.

scholarly journals Delayed Retraction of Filopodia in Gelsolin Null Mice

1997 ◽  
Vol 138 (6) ◽  
pp. 1279-1287 ◽  
Author(s):  
Mei Lu ◽  
Walter Witke ◽  
David J. Kwiatkowski ◽  
Kenneth S. Kosik
Keyword(s):  
Growth Cone ◽  
Hippocampal Neurons ◽  
Neurite Growth ◽  
Time Lapse ◽  
Growth Cones ◽  
Wild Type ◽  
Dynamic Studies ◽  
Shape Changes ◽  
Time Lapse Video ◽  
Cultured Hippocampal Neurons

Growth cones extend dynamic protrusions called filopodia and lamellipodia as exploratory probes that signal the direction of neurite growth. Gelsolin, as an actin filament-severing protein, may serve an important role in the rapid shape changes associated with growth cone structures. In wild-type (wt) hippocampal neurons, antibodies against gelsolin labeled the neurite shaft and growth cone. The behavior of filopodia in cultured hippocampal neurons from embryonic day 17 wt and gelsolin null (Gsn−) mice (Witke, W., A.H. Sharpe, J.H. Hartwig, T. Azuma, T.P. Stossel, and D.J. Kwiatkowski. 1995. Cell. 81:41–51.) was recorded with time-lapse video microscopy. The number of filopodia along the neurites was significantly greater in Gsn− mice and gave the neurites a studded appearance. Dynamic studies suggested that most of these filopodia were formed from the region of the growth cone and remained as protrusions from the newly consolidated shaft after the growth cone advanced. Histories of individual filopodia in Gsn− mice revealed elongation rates that did not differ from controls but an impaired retraction phase that probably accounted for the increased number of filopodia long the neutrite shaft. Gelsolin appears to function in the initiation of filopodial retraction and in its smooth progression.

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.

NrCAM, cerebellar granule cell receptor for the neuronal adhesion molecule F3, displays an actin-dependent mobility in growth cones

1999 ◽  
Vol 112 (18) ◽  
pp. 3015-3027 ◽  
Author(s):  
C. Faivre-Sarrailh ◽  
J. Falk ◽  
E. Pollerberg ◽  
M. Schachner ◽  
G. Rougon
Keyword(s):  
Growth Cone ◽  
Actin Filaments ◽  
Cerebellar Granule Cells ◽  
Leading Edge ◽  
Growth Cones ◽  
Inhibitory Effect ◽  
Immunoglobulin Superfamily ◽  
Cerebellar Granule ◽  
Axonal Elongation ◽  
Neuronal Adhesion

The neuronal adhesion glycoprotein F3 is a multifunctional molecule of the immunoglobulin superfamily that displays heterophilic binding activities. In the present study, NrCAM was identified as the functional receptor mediating the inhibitory effect of F3 on axonal elongation from cerebellar granule cells. F3Fc-conjugated microspheres binding to neuronal growth cones resulted from heterophilic interaction with NrCAM but not with L1. Time-lapse video-microscopy indicated that F3Fc beads bind at the leading edge and move retrogradely to reach the base of the growth cone within a lapse of 30–60 seconds. Such velocity (5.7 microm/minute) is consistent with a coupling between F3 receptors and the retrograde flow of actin filaments. When actin filaments were disrupted by cytochalasin B, the F3Fc beads remained immobile at the leading edge. The retrograde mobility appeared to be dependent on NrCAM clustering since it was induced upon binding with cross-linked but not dimeric F3Fc chimera. These data indicate that F3 may control growth cone motility by modulating the linkage of its receptor, NrCAM, to the cytoskeleton. They provide further insights into the mechanisms by which GPI-anchored adhesion molecules may exert an inhibitory effect on axonal elongation.

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 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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