ULTRASTRUCTURE AND FUNCTION OF GROWTH CONES AND AXONS OF CULTURED NERVE CELLS

Dorsal root ganglion nerve cells undergoing axon elongation in vitro have been analyzed ultrastructurally. The growth cone at the axonal tip contains smooth endoplasmic reticulum, vesicles, neurofilaments, occasional microtubules, and a network of 50-A in diameter microfilaments. The filamentous network fills the periphery of the growth cone and is the only structure found in microspikes. Elements of the network are oriented parallel to the axis of microspikes, but exhibit little orientation in the growth cone. Cytochalasin B causes rounding up of growth cones, retraction of microspikes, and cessation of axon elongation. The latter biological effect correlates with an ultrastructural alteration in the filamentous network of growth cones and microspikes. No other organelle appears to be affected by the drug. Removal of cytochalasin allows reinitiation of growth cone-microspike activity, and elongation begins anew. Such recovery will occur in the presence of the protein synthesis inhibitor cycloheximide, and in the absence of exogenous nerve growth factor. The neurofilaments and microtubules of axons are regularly spaced. Fine filaments indistinguishable from those in the growth cone interconnect neurofilaments, vesicles, microtubules, and plasma membrane. This filamentous network could provide the structural basis for the initiation of lateral microspikes and perhaps of collateral axons, besides playing a role in axonal transport.     

Cell-to-substratum adhesion and guidance of axonal elongation

The behavior of axonal growth cones on surfaces with patterned variations in substratum was observed. Cells from sensory ganglia of 8-day-old chicken embryos were cultured on plastic petri dishes, plastic tissue culture dishes, and polyornithine-coated tissue culture dishes, all of which contained gridlike patterns of palladium (Pd) deposition.The results indicated that growth cones elongated on the Pd-shadowed areas vs areas lacking Pd deposits depending on the relative adhesivity of the growth cones to the substrata. In petri dishes, growth cones stay on the Pd; in tissue culture dishes, they cross from one surface to the other; and in polyornithine-coated dishes, they elongate for great distances on the Pd-free areas. Analyses of time-lapse movies showed that, on Pd-shadowed polyornithine dishes, growth cones often approach the Pd-coated areas and microspikes touch the Pd surface. Yet, the axon tip continues to elongate on the Pd-free polyornithine surface.The conclusion is offered that interactions between microspikes and the substratum adjacent to the growth cone are important determinants of the directions and pathways of axonal elongation.     

The origin,structure and occurrence of corpora amylacea in the bovine mammary gland and in milk

Summary Fibres growing from neurons of explanted dorsal root ganglia from 10 day chick embryos were transected and subsequently observed by light and electron microscopy after periods of a few to fifty minutes. Changes immediately proximal and distal to the cut together with alterations further away from the site of injury on both sides of the cut were recorded. Observations were also made on the growth cones of damaged axons and on changes in associated glial cells.Reactive and degenerative changes including the rotation, retraction and swelling of cut axons occurred rapidly. Electron microscopy revealed tracts of filamentous material close to the sealed-off ends of axons, swollen organelles such as mitochondria, and lamellar bodies of varying dimensions.Proximal to the injury and closer to the expiant, damaged and degenerating axons mingled with normal processes. Many contained only a fine granular material, others clumps of organelles, particularly mitochondria.Distal to the cut, microspikes were lost from some growth cones. The dense granular material filling microspikes and growth cones remained unchanged. Clumps of large clear vesicles, lamellar bodies and swollen degenerating mitochondria were present, not only within growth cones, but also in all parts of the axon distal to the cut.Glial cells associated with transected axons soon developed an electron dense cytoplasm containing swollen organelles. Large numbers of vesicles filled with a particulate substance were also found.The possible significance of the changes observed after transection are considered and discussed.The author wishes to thank Prof. D.W. James in whose laboratory at University College London these studies were initiated, Dr. A.R. Lieberman for his expert help and advice and the University of London Central Research Fund and Wellcome Trust for financial assistance     

Cell-substratum adhesion of neurite growth cones, and its role in neurite elongation.

The adhesion of chick embryo sensory neurons to glass coverslips was examined with interference reflection optics. On untreated glass, adhesive contacts are common only beneath growth cones and are small. On polylysine-treated glass growth cones are highly spread, microspikes reach treat lengths and extensive adhesive contacts underlie growth cones, microspikes and nerve fibers. Veils, expanded from the growth cone, are adherent to the substratum either centrally or laterally, while the extending edge of the cell margin is non-adherent. Linear adhesions are frequent beneath microspikes and pass centrally beneath the growth cone margin. The distribution of linear adhesions resembles that of microfilament bundles seen within whole mounts of growth cones. Adhesive contacts stabilize extensions of the growth cone margin and may influence the organization of the microfilamentous network within the growth cone. Regulation of microfilament organization by adhesion may influence microfilament functions in growth cone mobility and the assembly of neurite structures.     

Macondo crude oil from the Deepwater Horizon oil spill disrupts specific developmental processes during zebrafish embryogenesis

Background

During nerve growth, cytoplasmic vesicles add new membrane preferentially to the growth cone located at the distal tip of extending axons. Growth cone membrane is also retrieved locally, and asymmetric retrieval facilitates membrane remodeling during growth cone repulsion by a chemorepellent gradient. Moreover, growth inhibitory factors can stimulate bulk membrane retrieval and induce growth cone collapse. Despite these functional insights, the processes mediating local membrane remodeling during axon extension remain poorly defined.

Results

To investigate the spatial and temporal dynamics of membrane retrieval in actively extending growth cones, we have used a transient labeling and optical recording method that can resolve single vesicle events. Live-cell confocal imaging revealed rapid membrane retrieval by distinct endocytic modes based on spatial distribution in Xenopus spinal neuron growth cones. These modes include endocytic "hot-spots" triggered at the base of filopodia, at the lateral margins of lamellipodia, and along dorsal ridges of the growth cone. Additionally, waves of endocytosis were induced when individual filopodia detached from the substrate and fused with the growth cone dorsal surface or with other filopodia. Vesicle formation at sites of membrane remodeling by self-contact required F-actin polymerization. Moreover, bulk membrane retrieval by macroendocytosis correlated positively with the substrate-dependent rate of axon extension and required the function of Rho-family GTPases.

Conclusions

This study provides insight into the dynamic membrane remodeling processes essential for nerve growth by identifying several distinct modes of rapid membrane retrieval in the growth cone during axon extension. We found that endocytic membrane retrieval is intensified at specific subdomains and may drive the dynamic membrane ruffling and re-absorption of filopodia and lamellipodia in actively extending growth cones. The findings offer a platform for determining the molecular mechanisms of distinct endocytic processes that may remodel the surface distribution of receptors, ion channels and other membrane-associated proteins locally to drive growth cone extension and chemotactic guidance.     

RhoA‐kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor

Growth cones are highly polarized and dynamic structures confined to the tips of axons. The polarity of growth cones is in part maintained by suppression of protrusive activity from the distal axon shaft, a process termed axon consolidation. The mechanistic basis of axon consolidation that contributes to the maintenance of growth cone polarity is not clear. We report that inhibition of RhoA‐kinase (ROCK) or myosin II resulted in unstable consolidation of the distal axon as evidenced by increased filopodial and lamellipodial extension. Furthermore, when ROCK or myosin II was inhibited lamellipodia formed at the growth cone migrated onto the axon shaft. Analysis of EYFP‐actin dynamics in the distal axon revealed that ROCK negatively regulates actin polymerization and initiation of protrusive structures from spontaneously formed axonal F‐actin patches, the latter being an effect attributable to ROCK‐mediated regulation of myosin II. Inhibition of ROCK or myosin II blocked growth cone turning toward NGF by preventing suppression of protrusive activity away from the source of NGF, resulting in aborted turning responses. These data elucidate the mechanism of growth cone polarity, provide evidence that consolidation of the distal axon is a component of guidance, and identify ROCK as a negative regulator of F‐actin polymerization underlying protrusive activity in the distal axon. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Rapid arrest of axon elongation by brefeldin A: A role for the small GTP‐binding protein ARF in neuronal growth cones

Members of the ADP‐ribosylation factor (ARF) family of small guanosine triphosphate–binding proteins play an essential role in membrane trafficking which subserves constitutive protein transport along exocytic and endocytic pathways within eukaryotic cell bodies. In growing neurons, membrane trafficking within motile growth cones distant from the cell body underlies the rapid plasmalemmal expansion which subserves axon elongation. We report here that ARF is a constituent of axonal growth cones, and that application of brefeldin A to neurons in culture produces a rapid arrest of axon extension that can be ascribed to inhibition of ARF function in growth cones. Our findings demonstrate a role for ARF in growth cones that is coupled tightly to the rapid growth of neuronal processes characteristic of developmental and regenerative axon elongation, and indicate that ARF participates not only in constitutive membrane traffic within the cell body, but also in membrane dynamics within growing axon endings. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 105–115, 1999     

Rapid arrest of axon elongation by brefeldin A: a role for the small GTP-binding protein ARF in neuronal growth cones

Members of the ADP-ribosylation factor (ARF) family of small guanosine triphosphate-binding proteins play an essential role in membrane trafficking which subserves constitutive protein transport along exocytic and endocytic pathways within eukaryotic cell bodies. In growing neurons, membrane trafficking within motile growth cones distant from the cell body underlies the rapid plasmalemmal expansion which subserves axon elongation. We report here that ARF is a constituent of axonal growth cones, and that application of brefeldin A to neurons in culture produces a rapid arrest of axon extension that can be ascribed to inhibition of ARF function in growth cones. Our findings demonstrate a role for ARF in growth cones that is coupled tightly to the rapid growth of neuronal processes characteristic of developmental and regenerative axon elongation, and indicate that ARF participates not only in constitutive membrane traffic within the cell body, but also in membrane dynamics within growing axon endings.     

RhoA-kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor

Growth cones are highly polarized and dynamic structures confined to the tips of axons. The polarity of growth cones is in part maintained by suppression of protrusive activity from the distal axon shaft, a process termed axon consolidation. The mechanistic basis of axon consolidation that contributes to the maintenance of growth cone polarity is not clear. We report that inhibition of RhoA-kinase (ROCK) or myosin II resulted in unstable consolidation of the distal axon as evidenced by increased filopodial and lamellipodial extension. Furthermore, when ROCK or myosin II was inhibited lamellipodia formed at the growth cone migrated onto the axon shaft. Analysis of EYFP-actin dynamics in the distal axon revealed that ROCK negatively regulates actin polymerization and initiation of protrusive structures from spontaneously formed axonal F-actin patches, the latter being an effect attributable to ROCK-mediated regulation of myosin II. Inhibition of ROCK or myosin II blocked growth cone turning toward NGF by preventing suppression of protrusive activity away from the source of NGF, resulting in aborted turning responses. These data elucidate the mechanism of growth cone polarity, provide evidence that consolidation of the distal axon is a component of guidance, and identify ROCK as a negative regulator of F-actin polymerization underlying protrusive activity in the distal axon.     

Efficient axonal localization of alphaherpesvirus structural proteins in cultured sympathetic neurons requires viral glycoprotein E

Pseudorabies virus (PRV) glycoprotein E (gE) is a type I viral membrane protein that facilitates the anterograde spread of viral infection from the peripheral nervous system to the brain. In animal models, a gE-null mutant infection spreads inefficiently from presynaptic neurons to postsynaptic neurons (anterograde spread of infection). However, the retrograde spread of infection from post- to presynaptic neurons remains unaffected. Here we show that gE is required for wild-type localization of viral structural proteins in axons of infected neurons. During a gE-null PRV infection, a subset of viral glycoproteins, capsids, and tegument proteins enter and localize to the axon inefficiently. This defect is most obvious in the distal axon and growth cones. However, axonal entry and localization of other viral membrane proteins and endogenous cellular proteins remains unaffected. Neurons infected with gE-null mutants produce wild-type levels of viral structural proteins and infectious virions in the cell body. Our results indicate that reduced axonal targeting of viral structural proteins is a compelling explanation for the lack of anterograde spread in neural circuits following infection by a gE-null mutant.     

Chondroitin sulfate proteoglycans negatively regulate the positioning of mitochondria and endoplasmic reticulum to distal axons

Chondroitin sulfate proteoglycans (CSPGs) are components of the extracellular matrix that inhibit the extension and regeneration of axons. However, the underlying mechanism of action remains poorly understood. Mitochondria and endoplasmic reticulum (ER) are functionally inter‐linked organelles important to axon development and maintenance. We report that CSPGs impair the targeting of mitochondria and ER to the growth cones of chicken embryonic sensory axons. The effect of CSPGs on the targeting of mitochondria is blocked by inhibition of the LAR receptor for CSPGs. The regulation of the targeting of mitochondria and ER to the growth cone by CSPGs is due to attenuation of PI3K signaling, which is known to be downstream of LAR receptor activation. Dynactin is a required component of the dynein motor complex that drives the normally occurring retrograde evacuation of mitochondria from growth cones. CSPGs elevate the levels of p150^Glu dynactin found in distal axons, and inhibition of the interaction of dynactin with dynein increased axon lengths on CSPGs. CSPGs decreased the membrane potential of mitochondria, and pharmacological inhibition of mitochondria respiration at the growth cone independent of manipulation of mitochondria positioning impaired axon extension. Combined inhibition of dynactin and potentiation of mitochondria respiration further increased axon lengths on CSPGs relative to inhibition of dynactin alone. These data reveal that the regulation of the localization of mitochondria and ER to growth cones is a previously unappreciated aspect of the effects of CSPGs on embryonic axons. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1351–1370, 2017     

Non-Muscle Myosin II Regulates Neuronal Actin Dynamics by Interacting with Guanine Nucleotide Exchange Factors

Background

Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family.

Principal Findings

NM II colocalized with GEFs, such as βPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II–GEF interactions by overexpression of the DH domains of βPIX or Tiam1, or by βPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II–GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation.

Conclusion

Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF–Rho GTPase signaling pathway.     

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development,axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.     

Herpes simplex virus type 1 accumulation, envelopment, and exit in growth cones and varicosities in mid-distal regions of axons

The mechanism of anterograde transport of alphaherpesviruses in axons remains controversial. This study examined the transport, assembly, and egress of herpes simplex virus type 1 (HSV-1) in mid- and distal axons of infected explanted human fetal dorsal root ganglia using confocal microscopy and transmission electron microscopy (TEM) at 19, 24, and 48 h postinfection (p.i.). Confocal-microscopy studies showed that although capsid (VP5) and tegument (UL37) proteins were not uniformly present in axons until 24 h p.i., they colocalized with envelope (gG) proteins in axonal varicosities and in growth cones at 24 and 48 h p.i. TEM of longitudinal sections of axons in situ showed enveloped and unenveloped capsids in the axonal varicosities and growth cones, whereas in the midregion of the axons, predominantly unenveloped capsids were observed. Partially enveloped capsids, apparently budding into vesicles, were observed in axonal varicosities and growth cones, but not during viral attachment and entry into axons. Tegument proteins (VP22) were found associated with vesicles in growth cones, either alone or together with envelope (gD) proteins, by transmission immunoelectron microscopy. Extracellular virions were observed adjacent to axonal varicosities and growth cones, with some virions observed in crescent-shaped invaginations of the axonal plasma membrane, suggesting exit at these sites. These findings suggest that varicosities and growth cones are probable sites of HSV-1 envelopment of at least a proportion of virions in the mid- to distal axon. Envelopment probably occurs by budding of capsids into vesicles with associated tegument and envelope proteins. Virions appear to exit from these sites by exocytosis.     

Ultrastructural characteristics of synaptogenesis in monolayer cultures of spinal cord

Stages of formation of different types of synapse between cells of the dissociated spinal cord and spinal ganglia of 12–14 day mouse embryos, in monolayer cultures, were studied electron-microscopically. The participation of cones of growth in the formation of different junctions between structures of the monolayer was traced. It was shown that the appearance of synaptic vesicles in the growing axon precedes the onset of membrane specialization in the region of contact between axon and target cell. Ultrastructural characteristics of axo-dendritic, axo-somatic, and axo-axonal synapses formed during growth are given. On the 24th day of culture structural complexes of axonal glomerulus type, incorporating axo-axonal and axo-dendritic synapses, were discovered. It is suggested that desmosomes participate in the formation of both chemical synapses and synapses of gap junction type.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 3, pp. 336–343, May–June, 1984.     

Microtubule-mediated Src tyrosine kinase trafficking in neuronal growth cones

Src family tyrosine kinases are important signaling enzymes in the neuronal growth cone, and they have been implicated in axon guidance; however, the detailed localization, trafficking, and cellular functions of Src kinases in live growth cones are unclear. Here, we cloned two novel Aplysia Src kinases, termed Src1 and Src2, and we show their association with both the plasma membrane and the microtubule cytoskeleton in the growth cone by live cell imaging, immunocytochemistry, and cell fractionation. Activated Src2 is enriched in filopodia tips. Interestingly, Src2-enhanced green fluorescent protein–positive endocytic vesicles and tubulovesicular structures undergo microtubule-mediated movements that are bidirectional in the central domain and mainly retrograde in the peripheral domain. To further test the role of microtubules in Src trafficking in the growth cone, microtubules were depleted with either nocodazole or vinblastine treatment, resulting in an increase in Src2 plasma membrane levels in all growth cone domains. Our data suggest that microtubules regulate the steady-state level of active Src at the plasma membrane by mediating retrograde recycling of endocytosed Src. Expression of constitutively active Src2 results in longer filopodia that protrude from smaller growth cones, implicating Src2 in controlling the size of filopodia and lamellipodia.     

The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.

The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia, as analyzed by time-lapse phase-contrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection. This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology.     

The role of cytoskeleton in organizing growth cones: a microfilament- associated growth cone component depends upon microtubules for its localization

We are interested in the relationship between the cytoskeleton and the organization of polarized cell morphology. We show here that the growth cones of hippocampal neurons in culture are specifically stained by a monoclonal antibody called 13H9. In other systems, the antigen recognized by 13H9 is associated with marginal bands of chicken erythrocytes and shows properties of both microtubule-and microfilament- associated proteins (Birgbauer, E., and F. Solomon. 1989 J. Cell Biol. 109:1609-1620). This dual nature is manifest in hippocampal neurons as well. At early stages after plating, the antibody stains the circumferential lamellipodia that mediate initial cell spreading. As processes emerge, 13H9 staining is heavily concentrated in the distal regions of growth cones, particularly in lamellipodial fans. In these cells, the 13H9 staining is complementary to the localization of assembled microtubules. It colocalizes partially, but not entirely, with phalloidin staining of assembled actin. Incubation with nocodazole rapidly induces microtubule depolymerization, which proceeds in the distal-to-proximal direction in the processes. At the same time, a rapid and dramatic redistribution of the 13H9 staining occurs; it delocalizes along the axon shaft, becoming clearly distinct from the phalloidin staining and always remaining distal to the receding front of assembled microtubules. After longer times without assembled microtubules, no staining of 13H9 can be detected. Removal of the nocodazole allows the microtubules to reform, in an ordered proximal-to- distal fashion. The 13H9 immunoreactivity also reappears, but only in the growth cones, not in any intermediate positions along the axon, and only after the reformation of microtubules is complete. The results indicate that the antigen recognized by 13H9 is highly concentrated in growth cones, closely associated with polymerized actin, and that its proper localization depends upon intact microtubules.     

Arrival, reversal, and departure of neurofilaments at the tips of growing axons

We have investigated the movement of green fluorescent protein-tagged neurofilaments at the distal ends of growing axons by using time-lapse fluorescence imaging. The filaments moved in a rapid, infrequent, and asynchronous manner in either an anterograde or retrograde direction (60% anterograde, 40% retrograde). Most of the anterograde filaments entered the growth cone and most of the retrograde filaments originated in the growth cone. In a small number of cases we were able to observe neurofilaments reverse direction, and all of these reversals occurred in or close to the growth cone. We conclude that neurofilament polymers are delivered rapidly and infrequently to the tips of growing axons and that some of these polymers reverse direction in the growth cone and move back into the axon. We propose that 1) growth cones are a preferential site of neurofilament reversal in distal axons, 2) most retrograde neurofilaments in distal axons originate by reversal of anterograde filaments in the growth cone, 3) those anterograde filaments that do not reverse direction are recruited to form the neurofilament cytoskeleton of the newly forming axon, and 4) the net delivery of neurofilament polymers to growth cones may be controlled by regulating the reversal frequency.     

Morphology and Nanomechanics of Sensory Neurons Growth Cones following Peripheral Nerve Injury

A prior peripheral nerve injury in vivo, promotes a rapid elongated mode of sensory neurons neurite regrowth in vitro. This in vitro model of conditioned axotomy allows analysis of the cellular and molecular mechanisms leading to an improved neurite re-growth. Our differential interference contrast microscopy and immunocytochemistry results show that conditioned axotomy, induced by sciatic nerve injury, did not increase somatic size of adult lumbar sensory neurons from mice dorsal root ganglia sensory neurons but promoted the appearance of larger neurites and growth cones. Using atomic force microscopy on live neurons, we investigated whether membrane mechanical properties of growth cones of axotomized neurons were modified following sciatic nerve injury. Our data revealed that neurons having a regenerative growth were characterized by softer growth cones, compared to control neurons. The increase of the growth cone membrane elasticity suggests a modification in the ratio and the inner framework of the main structural proteins.