scholarly journals Suppression of Radixin and Moesin Alters Growth Cone Morphology, Motility, and Process Formation In Primary Cultured Neurons

1998 ◽  
Vol 143 (2) ◽  
pp. 443-455 ◽  
Author(s):  
Gabriela Paglini ◽  
Patricia Kunda ◽  
Santiago Quiroga ◽  
Kenneth Kosik ◽  
Alfredo Cáceres
Keyword(s):  
Growth Cone ◽  
Temporal Correlation ◽  
Growth Cones ◽  
Morphological Development ◽  
Cellular Functions ◽  
Erm Proteins ◽  
Process Formation ◽  
Neurite Formation ◽  
Developing Neurons ◽  
Key Aspects

In this study we have examined the cellular functions of ERM proteins in developing neurons. The results obtained indicate that there is a high degree of spatial and temporal correlation between the expression and subcellular localization of radixin and moesin with the morphological development of neuritic growth cones. More importantly, we show that double suppression of radixin and moesin, but not of ezrin–radixin or ezrin–moesin, results in reduction of growth cone size, disappearance of radial striations, retraction of the growth cone lamellipodial veil, and disorganization of actin filaments that invade the central region of growth cones where they colocalize with microtubules. Neuritic tips from radixin–moesin suppressed neurons displayed high filopodial protrusive activity; however, its rate of advance is 8–10 times slower than the one of growth cones from control neurons. Radixin–moesin suppressed neurons have short neurites and failed to develop an axon-like neurite, a phenomenon that appears to be directly linked with the alterations in growth cone structure and motility. Taken collectively, our data suggest that by regulating key aspects of growth cone development and maintenance, radixin and moesin modulate neurite formation and the development of neuronal polarity.

scholarly journals Depletion of 43-kD growth-associated protein in primary sensory neurons leads to diminished formation and spreading of growth cones.

1993 ◽  
Vol 123 (2) ◽  
pp. 417-429 ◽  
Author(s):  
L Aigner ◽  
P Caroni
Keyword(s):  
Neurite Outgrowth ◽  
Growth Cone ◽  
Growth Cones ◽  
Major Protein ◽  
Nerve Terminals ◽  
Drg Neurons ◽  
Process Formation ◽  
Neurite Formation ◽  
Cone Formation ◽  
External Signals

The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of growing axons, and of developing nerve terminals and glial cells. It is a highly hydrophilic protein associated with the cortical cytoskeleton and membranes. In neurons it is rapidly transported from the cell body to growth cones and nerve terminals, where it accumulates. To define the role of GAP-43 in neurite outgrowth, we analyzed neurite regeneration in cultured dorsal root ganglia (DRG) neurons that had been depleted of GAP-43 with any of three nonoverlapping antisense oligonucleotides. The GAP-43 depletion procedure was specific for this protein and an antisense oligonucleotide to the related PKC substrate MARCKS did not detectably affect GAP-43 immunoreactivity. We report that neurite outgrowth and morphology depended on the levels of GAP-43 in the neurons in a substrate-specific manner. When grown on a laminin substratum, GAP-43-depleted neurons extended longer, thinner and less branched neurites with strikingly smaller growth cones than their GAP-43-expressing counterparts. In contrast, suppression of GAP-43 expression prevented growth cone and neurite formation when DRG neurons were plated on poly-L-ornithine. These findings indicate that GAP-43 plays an important role in growth cone formation and neurite outgrowth. It may be involved in the potentiation of growth cone responses to external signals affecting process formation and guidance.

scholarly journals Critical Role of Collapsin Response Mediator Protein-associated Molecule CRAM for Filopodia and Growth Cone Development in Neurons

2005 ◽  
Vol 16 (1) ◽  
pp. 32-39 ◽  
Author(s):  
Azusa Hotta ◽  
Ryoko Inatome ◽  
Junichi Yuasa-Kawada ◽  
Qingyu Qin ◽  
Hirohei Yamamura ◽  
...  
Keyword(s):  
Growth Cone ◽  
Critical Role ◽  
Acquired Resistance ◽  
Immunohistochemical Analysis ◽  
Growth Cones ◽  
Axonal Guidance ◽  
Filamentous Actin ◽  
Cone Development ◽  
Developing Neurons ◽  
And Function

Collapsin response mediator proteins (CRMPs) have been implicated in signaling of axonal guidance, including semaphorins. We have previously identified a unique member of this gene family, CRMP-associated molecule CRAM (CRMP-5), which is phylogenetically divergent from the other four CRMPs. In this study, we have examined the distribution and function of CRAM in developing neurons. Immunohistochemical analysis showed accumulation of CRAM in the filopodia of growth cones. Experiments using cytochalasin D indicated that filopodial localization of CRAM was independent of filamentous actin. Overexpression of CRAM in neuronal cells significantly promoted filopodial growth and led to the formation of supernumerary growth cones, which acquired resistance to semaphorin-3A stimulation. Finally, knockdown of CRAM by using RNA interference blocked filopodial formation and revealed an aberrant morphology of growth cones. We propose that CRAM regulates filopodial dynamics and growth cone development, thereby restricting the response of growth cone to repulsive guidance cues.

scholarly journals LIMK1 Regulates Golgi Dynamics, Traffic of Golgi-derived Vesicles, and Process Extension in Primary Cultured Neurons

2004 ◽  
Vol 15 (7) ◽  
pp. 3433-3449 ◽  
Author(s):  
Silvana Rosso ◽  
Flavia Bollati ◽  
Mariano Bisbal ◽  
Diego Peretti ◽  
Tomoyuki Sumi ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Growth Cone ◽  
Pdz Domain ◽  
Growth Cones ◽  
Subcellular Fractionation ◽  
Wild Type ◽  
Growth Cone Collapse ◽  
Golgi Localization ◽  
Axon Retraction ◽  
Developing Neurons

In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.

scholarly journals Suppression of KIF2 in PC12 Cells Alters the Distribution of a Growth Cone Nonsynaptic Membrane Receptor and Inhibits Neurite Extension

1997 ◽  
Vol 138 (3) ◽  
pp. 657-669 ◽  
Author(s):  
Gerardo Morfini ◽  
Santiago Quiroga ◽  
Alberto Rosa ◽  
Kenneth Kosik ◽  
Alfredo Cáceres
Keyword(s):  
Pc12 Cells ◽  
Nerve Cell ◽  
Growth Cone ◽  
Growth Cones ◽  
Subcellular Fractionation ◽  
Membrane Receptor ◽  
Complete Disappearance ◽  
Anterograde Transport ◽  
Neurite Extension ◽  
Developing Neurons

In the present study, we present evidence about the cellular functions of KIF2, a kinesin-like superfamily member having a unique structure in that its motor domain is localized at the center of the molecule (Noda Y., Y. Sato-Yoshitake, S. Kondo, M. Nangaku, and N. Hirokawa. 1995. J. Cell Biol. 129:157–167.). Using subcellular fractionation techniques, isopicnic sucrose density centrifugation of microsomal fractions from developing rat cerebral cortex, and immunoisolation with KIF2 antibodies, we have now identified a type of nonsynaptic vesicle that associates with KIF2. This type of organelle lacks synaptic vesicle markers (synapsin, synaptophysin), amyloid precursor protein, GAP-43, or N-cadherin. On the other hand, it contains βgc, which is a novel variant of the β subunit of the IGF-1 receptor, which is highly enriched in growth cone membranes. Both βgc and KIF2 are upregulated by NGF in PC12 cells and highly concentrated in growth cones of developing neurons. We have also analyzed the consequences of KIF2 suppression by antisense oligonucleotide treatment on nerve cell morphogenesis and the distribution of synaptic and nonsynaptic vesicle markers. KIF2 suppression results in a dramatic accumulation of βgc within the cell body and in its complete disappearance from growth cones; no alterations in the distribution of synapsin, synaptophysin, GAP-43, or amyloid percursor protein are detected in KIF2-suppressed neurons. Instead, all of them remained highly enriched at nerve terminals. KIF2 suppression also produces a dramatic inhibition of neurite outgrowth; this phenomenon occurs after βgc has disappeared from growth cones. Taken collectively, our results suggest an important role for KIF2 in neurite extension, a phenomenon that may be related with the anterograde transport of a type of nonsynaptic vesicle that contains as one of its components a growth cone membrane receptor for IGF-1, a growth factor implicated in nerve cell development.

scholarly journals Regulated Actin Cytoskeleton Assembly at Filopodium Tips Controls Their Extension and Retraction

1999 ◽  
Vol 146 (5) ◽  
pp. 1097-1106 ◽  
Author(s):  
Aneil Mallavarapu ◽  
Tim Mitchison
Keyword(s):  
Actin Cytoskeleton ◽  
Growth Cone ◽  
Actin Filaments ◽  
Neuroblastoma Cell ◽  
Initial Step ◽  
Growth Cones ◽  
Neuroblastoma Cell Line ◽  
Dominant Factor ◽  
Retrograde Flow ◽  
Assembly Rate

The extension and retraction of filopodia in response to extracellular cues is thought to be an important initial step that determines the direction of growth cone advance. We sought to understand how the dynamic behavior of the actin cytoskeleton is regulated to produce extension or retraction. By observing the movement of fiduciary marks on actin filaments in growth cones of a neuroblastoma cell line, we found that filopodium extension and retraction are governed by a balance between the rate of actin cytoskeleton assembly at the tip and retrograde flow. Both assembly and flow rate can vary with time in a single filopodium and between filopodia in a single growth cone. Regulation of assembly rate is the dominant factor in controlling filopodia behavior in our system.

scholarly journals ERM proteins regulate growth cone responses to Sema3A

2008 ◽  
Vol 510 (4) ◽  
pp. 351-366 ◽  
Author(s):  
C. David Mintz ◽  
Ioana Carcea ◽  
Daniel G. McNickle ◽  
Tracey C. Dickson ◽  
Yongchao Ge ◽  
...  
Keyword(s):  
Growth Cone ◽  
Erm Proteins

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.

Commissure formation in the embryonic CNS of Drosophila

Development ◽  
1999 ◽  
Vol 126 (4) ◽  
pp. 771-779 ◽  
Author(s):  
T. Hummel ◽  
K. Schimmelpfeng ◽  
C. Klambt
Keyword(s):  
Nervous System ◽  
Glial Cells ◽  
Ventral Nerve Cord ◽  
Growth Cones ◽  
Cellular Functions ◽  
Detailed Model ◽  
Present Evidence ◽  
Embryonic Nervous System ◽  
Dependent Signal ◽  
Embryonic Cns

Most of the neurons of the ventral nerve cord send out long projecting axons which cross the midline. In the Drosophila central nervous system (CNS) cells of the midline give rise to neuronal and glial lineages with different functions during the establishment of the commissural pattern. Here we present evidence that beside the previously known NETRIN/FRAZZLED (DCC) signalling system an additional attractive system(s) is operating in the developing embryonic nervous system of Drosophila. Attractive cues appear to be provided by the midline neurons. We show that the glial cells present repulsive signals to the previously described ROUNDABOUT receptor in addition to a permissive contact-dependent signal helping commissural growth cones across the midline. A novel repulsive component is encoded by the karussell gene. Furthermore the midline glial cells separate anterior and posterior commissures. By genetic criteria we demonstrate that some of the genes we have identified are acting in the midline glia whereas other genes are required in the midline neurons. The results lead to a detailed model relating different cellular functions to axonal patterning at the midline.

Pioneer neurones use basal lamina as a substratum for outgrowth in the embryonic grasshopper limb

Development ◽  
1988 ◽  
Vol 104 (4) ◽  
pp. 601-608 ◽  
Author(s):  
H. Anderson ◽  
R.P. Tucker
Keyword(s):  
Electron Microscopy ◽  
Horseradish Peroxidase ◽  
Basal Lamina ◽  
Growth Cone ◽  
Growth Cones ◽  
Axon Outgrowth ◽  
Stages Of Development

During axonogenesis, contacts made by the growth cone with its substratum are important in guiding the direction of neurone outgrowth. This study examines the contacts made by the growth cones of pioneer neurones in the embryonic grasshopper limb. Individual pioneer neurones at different stages of development were injected with horseradish peroxidase and the contacts made by the filopodia at the tip of their growth cones were examined by electron microscopy. Filopodia made few contacts with mesodermal cells, some contacts with ectodermal cells and very frequent contacts with basal lamina underlying the ectoderm. Components of the basal lamina may therefore play a role in guiding pioneer axon outgrowth.

scholarly journals Axon growth regulation by a bistable molecular switch

2018 ◽  
Vol 285 (1877) ◽  
pp. 20172618 ◽  
Author(s):  
Pranesh Padmanabhan ◽  
Geoffrey J. Goodhill
Keyword(s):  
Growth Cone ◽  
Neural Development ◽  
Growth Regulation ◽  
Axon Growth ◽  
Interaction Network ◽  
Growth Cones ◽  
Extrinsic Factors ◽  
Environmental Signals ◽  
Switching Behaviour ◽  
The Right

For the brain to function properly, its neurons must make the right connections during neural development. A key aspect of this process is the tight regulation of axon growth as axons navigate towards their targets. Neuronal growth cones at the tips of developing axons switch between growth and paused states during axonal pathfinding, and this switching behaviour determines the heterogeneous axon growth rates observed during brain development. The mechanisms controlling this switching behaviour, however, remain largely unknown. Here, using mathematical modelling, we predict that the molecular interaction network involved in axon growth can exhibit bistability, with one state representing a fast-growing growth cone state and the other a paused growth cone state. Owing to stochastic effects, even in an unchanging environment, model growth cones reversibly switch between growth and paused states. Our model further predicts that environmental signals could regulate axon growth rate by controlling the rates of switching between the two states. Our study presents a new conceptual understanding of growth cone switching behaviour, and suggests that axon guidance may be controlled by both cell-extrinsic factors and cell-intrinsic growth regulatory mechanisms.

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