scholarly journals Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone.

1988 ◽  
Vol 107 (4) ◽  
pp. 1505-1516 ◽  
Author(s):  
P Forscher ◽  
S J Smith
Keyword(s):  
Growth Cone ◽  
Spatial Organization ◽  
Leading Edge ◽  
Growth Cones ◽  
Actin Networks ◽  
Antibody Staining ◽  
Flow Perfusion ◽  
Rapid Flow ◽  
Lamellar Region ◽  
Neuronal Growth Cone

Actions of cytochalasin B (CB) on cytoskeletons and motility of growth cones from cultured Aplysia neurons were studied using a rapid flow perfusion chamber and digital video light microscopy. Living growth cones were observed using differential interference contrast optics and were also fixed at various time points to assay actin filament (F-actin) and microtubule distributions. Treatment with CB reversibly blocked motility and eliminated most of the phalloidin-stainable F-actin from the leading lamella. The loss of F-actin was nearly complete within 2-3 min of CB application and was largely reversed within 5-6 min of CB removal. The loss and recovery of F-actin were found to occur with a very distinctive spatial organization. Within 20-30 s of CB application, F-actin networks receded from the entire peripheral margin of the lamella forming a band devoid of F-actin. This band widened as F-actin receded at rates of 3-6 microns/min. Upon removal of CB, F-actin began to reappear within 20-30 s. The initial reappearance of F-actin took two forms: a coarse isotropic matrix of F-actin bundles throughout the lamella, and a denser matrix along the peripheral margin. The denser peripheral matrix then expanded in width, extending centrally to replace the coarse matrix at rates again between 3-6 microns/min. These results suggest that actin normally polymerizes at the leading edge and then flows rearward at a rate between 3-6 microns/min. CB treatment was also observed to alter the distribution of microtubules, assayed by antitubulin antibody staining. Normally, microtubules are restricted to the neurite shaft and a central growth cone domain. Within approximately 5 min after CB application, however, microtubules began extending into the lamellar region, often reaching the peripheral margin. Upon removal of CB, the microtubules were restored to their former central localization. The timing of these microtubule redistributions is consistent with their being secondary to effects of CB on lamellar F-actin.

scholarly journals Arp2/3 complex–dependent actin networks constrain myosin II function in driving retrograde actin flow

2012 ◽  
Vol 197 (7) ◽  
pp. 939-956 ◽  
Author(s):  
Qing Yang ◽  
Xiao-Feng Zhang ◽  
Thomas D. Pollard ◽  
Paul Forscher
Keyword(s):  
Growth Cone ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Leading Edge ◽  
Actin Dynamics ◽  
Actin Networks ◽  
Motile Cells ◽  
Nonmuscle Myosin Ii ◽  
Neuronal Growth Cone ◽  
Contractile Forces

The Arp2/3 complex nucleates actin filaments to generate networks at the leading edge of motile cells. Nonmuscle myosin II produces contractile forces involved in driving actin network translocation. We inhibited the Arp2/3 complex and/or myosin II with small molecules to investigate their respective functions in neuronal growth cone actin dynamics. Inhibition of the Arp2/3 complex with CK666 reduced barbed end actin assembly site density at the leading edge, disrupted actin veils, and resulted in veil retraction. Strikingly, retrograde actin flow rates increased with Arp2/3 complex inhibition; however, when myosin II activity was blocked, Arp2/3 complex inhibition now resulted in slowing of retrograde actin flow and veils no longer retracted. Retrograde flow rate increases induced by Arp2/3 complex inhibition were independent of Rho kinase activity. These results provide evidence that, although the Arp2/3 complex and myosin II are spatially segregated, actin networks assembled by the Arp2/3 complex can restrict myosin II–dependent contractility with consequent effects on growth cone motility.

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.

Use of calcium imaging technologies to dissect mechanisms underlying neuronal growth cone responses to environmental cues

1995 ◽  
Vol 53 ◽  
pp. 804-805
Author(s):  
C.V. Williams ◽  
S.B. Kater
Keyword(s):  
Nervous System ◽  
Growth Cone ◽  
Local Environment ◽  
Growth Cones ◽  
Neuronal Growth ◽  
Growth Inhibitory ◽  
Direct Growth ◽  
Neuronal Growth Cone ◽  
Mechanical Factors ◽  
Growth Promoting

Since calcium is a key second messenger in both the developmental formation and adult function of the nervous system, the ability to rapidly image changes in this molecule has added greatly to our understanding of how development of the nervous system is regulated. The nervous system is comprised of billions of neurons and glial cells that establish characteristic patterns of connections during development. Neurons extend processes that often must grow long distances to establish appropriate synaptic connections. Neurons perform a pathfinding behavior largely via the highly dynamic behavior of the neuronal growth cone at the distal tip of elongating processes. The motile behavior characteristic of growth cones allows the growth cone to survey the local environment, read local cues and respond to those cues with a change in behavior. A variety of cues are now known to direct growth cones (e.g. electrical activity, depolarization, growth factors, mechanical factors, neurotransmitters, substrate factors). This collection of factors includes both growth promoting and growth inhibitory influences.

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.

scholarly journals Xenopus cytoplasmic linker–associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules

2013 ◽  
Vol 24 (10) ◽  
pp. 1544-1558 ◽  
Author(s):  
Astrid Marx ◽  
William J. Godinez ◽  
Vasil Tsimashchuk ◽  
Peter Bankhead ◽  
Karl Rohr ◽  
...  
Keyword(s):  
Growth Cone ◽  
Binding Protein ◽  
Leading Edge ◽  
Growth Cones ◽  
Retrograde Flow ◽  
Spinal Cord Neurons ◽  
Actin Organization ◽  
Axon Elongation ◽  
Axon Extension ◽  
Potential Link

Dynamic microtubules (MTs) are required for neuronal guidance, in which axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus cytoplasmic linker–associated protein 1 (XCLASP1) or treatment with the MT drug Taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus ends that have been fluorescently labeled by end-binding protein 3 (EB3). XCLASP1 depletion reduced MT advance rates in neuronal growth cones, very much like treatment with Taxol, demonstrating a potential link between MT dynamics in the growth cone and axon extension. Automatic tracking of EB3 comets in different compartments revealed that MTs increasingly slowed as they passed from the axon shaft into the growth cone and filopodia. We used speckle microscopy to demonstrate that MTs experience retrograde flow at the leading edge. Microtubule advance in growth cone and filopodia was strongly reduced in XCLASP1-depleted axons as compared with control axons, but actin retrograde flow remained unchanged. Instead, we found that XCLASP1-depleted growth cones lacked lamellipodial actin organization characteristic of protrusion. Lamellipodial architecture depended on XCLASP1 and its capacity to associate with MTs, highlighting the importance of XCLASP1 in actin–microtubule interactions.

scholarly journals Nanoscopic Clustering of Neuroligin-3 and Neuroligin-4X Regulates Growth Cone Organization and Size

2019 ◽  
Author(s):  
Nicholas J. F. Gatford ◽  
P. J. Michael Deans ◽  
Rodrigo R.R. Duarte ◽  
George Chennell ◽  
Pooja Raval ◽  
...  
Keyword(s):  
Growth Cone ◽  
Synapse Formation ◽  
Super Resolution ◽  
Leading Edge ◽  
Growth Cones ◽  
Autism Spectrum ◽  
Adhesion Proteins ◽  
Human Neurons ◽  
Spectrum Condition ◽  
P21 Activated Kinase

AbstractThe cell-adhesion proteins neuroligin-3 and neuroligin-4X (NLGN3/4X) have well described roles in synapse formation. NLGN3/4X are also expressed highly during neurodevelopment. However, the role these proteins play during this period is unknown. Here we show that NLGN3/4X localized to the leading edge of growth cones where itpromoted neuritogenesis in immature human neurons. Super-resolution microscopyrevealed that NLGN3/4X clustering induced growth cone enlargement and influenced actin filament organization. Critically, these morphological effects were not induced by Autism spectrum condition (ASC)-associated NLGN3/4X variants. Finally, actin regulators p21-activated kinase 1 (PAK1) and cofilin were found to be activated by NLGN3/4X and involved in mediating the effects of these adhesion proteins on actin filaments, growth cones, and neuritogenesis. These data reveal a novel role for NLGN3 and NLGN4X in the development of neuronal architecture, which may be altered in the presence of ASD-associated variants.

scholarly journals Radixin Is Involved in Lamellipodial Stability during Nerve Growth Cone Motility

1999 ◽  
Vol 10 (5) ◽  
pp. 1511-1520 ◽  
Author(s):  
Leslie Castelo ◽  
Daniel G. Jay
Keyword(s):  
Growth Cone ◽  
Critical Role ◽  
Leading Edge ◽  
Growth Cones ◽  
Forward Movement ◽  
Nerve Growth ◽  
Nerve Growth Cone

Immunocytochemistry and in vitro studies have suggested that the ERM (ezrin-radixin-moesin) protein, radixin, may have a role in nerve growth cone motility. We tested the in situ role of radixin in chick dorsal root ganglion growth cones by observing the effects of its localized and acute inactivation. Microscale chromophore-assisted laser inactivation (micro-CALI) of radixin in growth cones causes a 30% reduction of lamellipodial area within the irradiated region whereas all control treatments did not affect lamellipodia. Micro-CALI of radixin targeted to the middle of the leading edge often split growth cones to form two smaller growth cones during continued forward movement (>80%). These findings suggest a critical role for radixin in growth cone lamellipodia that is similar to ezrin function in pseudopodia of transformed fibroblasts. They are consistent with radixin linking actin filaments to each other or to the membrane during motility.

scholarly journals The organization of F-actin and microtubules in growth cones exposed to a brain-derived collapsing factor.

1993 ◽  
Vol 121 (4) ◽  
pp. 867-878 ◽  
Author(s):  
J Fan ◽  
S G Mansfield ◽  
T Redmond ◽  
P R Gordon-Weeks ◽  
J A Raper
Keyword(s):  
Growth Cone ◽  
Relative Concentration ◽  
Leading Edge ◽  
Growth Cones ◽  
Rhodamine Phalloidin ◽  
Alpha Tubulin ◽  
Microtubule Polymerization ◽  
Before And After ◽  
General Protein ◽  
The Brain

In previous work we characterized a brain derived collapsing factor that induces the collapse of dorsal root ganglion growth cones in culture (Raper and Kapfhammer, 1990). To determine how the growth cone cytoskeleton is rearranged during collapse, we have compared the distributions of F-actin and microtubules in normal and partially collapsed growth cones. The relative concentration of F-actin as compared to all proteins can be measured in growth cones by rationing the intensity of rhodamine-phalloidin staining of F-actin to the intensity of a general protein stain. The relative concentration of F-actin is decreased by about one half in growth cones exposed to collapsing factor for five minutes, a time at which they are just beginning to collapse. During this period the relative concentration of F-actin in the leading edges of growth cones decreases dramatically while the concentration of F-actin in the centers decreases little. These results suggest that collapse is associated with a net loss of F-actin at the leading edge. The distributions of microtubules in normal and collapsing factor treated growth cones were examined with antibodies to tyrosinated and detyrosinated isoforms of alpha-tubulin. The tyrosinated form is found in newly polymerized microtubules while the detyrosinated form is not. The relative proximal-distal distributions of these isoforms are not altered during collapse, suggesting that rates of microtubule polymerization and depolymerization are not greatly affected by the presence of collapsing factor. An analysis of the distributions of microtubules before and after collapse suggests that microtubules are rearranged, but their polymerization state is unaffected during collapse. These results are consistent with the hypothesis that the brain derived collapsing factor has little effect on microtubule polymerization or depolymerization. Instead it appears to induce a net loss of F-actin at the leading edge of the growth cone.

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 Myosin 1c and myosin IIB serve opposing roles in lamellipodial dynamics of the neuronal growth cone

2002 ◽  
Vol 158 (7) ◽  
pp. 1207-1217 ◽  
Author(s):  
Thomas J. Diefenbach ◽  
Vaughan M. Latham ◽  
Dean Yimlamai ◽  
Canwen A. Liu ◽  
Ira M. Herman ◽  
...  
Keyword(s):  
Dorsal Root Ganglion ◽  
Neurite Outgrowth ◽  
Growth Cone ◽  
Dorsal Root ◽  
Myosin Ii ◽  
Growth Cones ◽  
Neuronal Growth ◽  
Rapid Reduction ◽  
Neuronal Growth Cone

The myosin family of motor proteins is implicated in mediating actin-based growth cone motility, but the roles of many myosins remain unclear. We previously implicated myosin 1c (M1c; formerly myosin Iβ) in the retention of lamellipodia (Wang et al., 1996). Here we address the role of myosin II (MII) in chick dorsal root ganglion neuronal growth cone motility and the contribution of M1c and MII to retrograde F-actin flow using chromophore-assisted laser inactivation (CALI). CALI of MII reduced neurite outgrowth and growth cone area by 25%, suggesting a role for MII in lamellipodial expansion. Micro-CALI of MII caused a rapid reduction in local lamellipodial protrusion in growth cones with no effects on filopodial dynamics. This is opposite to micro-CALI of M1c, which caused an increase in lamellipodial protrusion. We used fiduciary beads (Forscher et al., 1992) to observe retrograde F-actin flow during the acute loss of M1c or MII. Micro-CALI of M1c reduced retrograde bead flow by 76%, whereas micro-CALI of MII or the MIIB isoform did not. Thus, M1c and MIIB serve opposite and nonredundant roles in regulating lamellipodial dynamics, and M1c activity is specifically required for retrograde F-actin flow.

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