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 Evidence for the Involvement of Kif4 in the Anterograde Transport of L1-Containing Vesicles

2000 ◽  
Vol 149 (1) ◽  
pp. 141-152 ◽  
Author(s):  
Diego Peretti ◽  
Leticia Peris ◽  
Silvana Rosso ◽  
Santiago Quiroga ◽  
Alfredo Cáceres
Keyword(s):  
Growth Cone ◽  
Antisense Oligonucleotides ◽  
Cell Adhesion Molecule ◽  
Neuronal Development ◽  
Subcellular Fractionation ◽  
Complete Disappearance ◽  
Cellular Functions ◽  
Anterograde Transport ◽  
Axonal Elongation ◽  
A Cell

In this study we present evidence about the cellular functions of KIF4. Using subcellular fractionation techniques and immunoisolation, we have now identified a type of vesicle that associates with KIF4, an NH2-terminal globular motor domain kinesin-like protein. This vesicle is highly concentrated in growth cones and contains L1, a cell adhesion molecule implicated in axonal elongation. It lacks synaptic vesicle markers, receptors for neurotrophins, and membrane proteins involved in growth cone guidance. In cultured neurons, KIF4 and L1 predominantly localize to the axonal shaft and its growth cone. Suppression of KIF4 with antisense oligonucleotides results in the accumulation of L1 within the cell body and in its complete disappearance from axonal tips. In addition, KIF4 suppression prevents L1-enhanced axonal elongation. Taken collectively, our results suggest an important role for KIF4 during neuronal development, a phenomenon which may be related to the anterograde transport of L1-containing vesicles.

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 Growth cone guidance and neuron morphology on micropatterned laminin surfaces

1993 ◽  
Vol 105 (1) ◽  
pp. 203-212 ◽  
Author(s):  
P. Clark ◽  
S. Britland ◽  
P. Connolly
Keyword(s):  
Growth Cone ◽  
Morphological Differentiation ◽  
Local Environment ◽  
Growth Cones ◽  
Dorsal Root Ganglion Neurons ◽  
Neuron Morphology ◽  
Neurite Extension ◽  
Guidance Cues ◽  
Cone Morphology ◽  
Ganglion Neurons

Neurite growth cones detect and respond to guidance cues in their local environment that determine stereotyped pathways during development and regeneration. Micropatterns of laminin (which was found to adsorb preferentially to photolithographically defined hydrophobic areas of micropatterns) were here used to model adhesive pathways that might influence neurite extension. The responses of growth cones were determined by the degree of guidance of neurite extension and also by examining growth cone morphology. These parameters were found to be strongly dependent on the geometry of the patterned laminin, and on neuron type. Decreasing the spacing of multiple parallel tracks of laminin alternating with non-adhesive tracks, resulted in decreased guidance of chick embryo brain neurons. Single isolated 2 microns tracks strongly guided neurite extension whereas 2 microns tracks forming a 4 microns period multiple parallel pattern did not. Growth cones appear to be capable of bridging the narrow non-adhesive tracks, rendering them insensitive to the smaller period multiple parallel adhesive patterns. These observations suggest that growth cones would be unresponsive to the multiple adhesive cues such as would be presented by oriented extracellular matrix or certain axon fascicle structures, but could be guided by isolated adhesive tracks. Growth cone morphology became progressively simpler on progressively narrower single tracks. On narrow period multiple parallel tracks (which did not guide neurite extension) growth cones spanned a number of adhesive/non-adhesive tracks, and their morphology suggests that lamellipodial advance may be independent of the substratum by using filopodia as a scaffold. In addition to acting as guidance cues, laminin micropatterns also appeared to influence the production of primary neurites and their subsequent branching. On planar substrata, dorsal root ganglion neurons were multipolar, with highly branched neurite outgrowth whereas, on 25 microns tracks, neurite branching was reduced or absent, and neuron morphology was typically bipolar. These observations indicate the precision with which growth cone advance may be controlled by substrata and suggest a role for patterned adhesiveness in neuronal morphological differentiation, but also highlight some of the limitations of growth cone sensitivity to substratum cues.

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 A specific Ret receptor isoform is required for pioneer axon outgrowth and growth cone dynamics

2018 ◽  
Author(s):  
Adam M. Tuttle ◽  
Catherine M. Drerup ◽  
Molly H. Marra ◽  
Alex V. Nechiporuk
Keyword(s):  
Growth Cone ◽  
Axon Growth ◽  
Retrograde Transport ◽  
Growth Cones ◽  
Axon Outgrowth ◽  
Loss Of Function ◽  
Anterograde Transport ◽  
Interacting Protein ◽  
Pioneer Neurons

AbstractIn many cases, axon growth and guidance are driven by pioneer axons, the first axons to grow in a particular region. Despite their dynamic pathfinding capabilities and developmental importance, there are very few pioneer neuron specific markers and thus their in vivo identification and functional interrogation have been difficult. We found that a Ret receptor isoform, Ret51, is highly enriched in peripheral sensory pioneer neurons and is required for pioneer axon outgrowth. Ret null mutant pioneer neurons differentiate normally; however, they displayed defects in growth cone morphology and formation of filopodia before pioneer axon extension prematurely halts. We also demonstrate loss-of-function of a retrograde cargo adaptor, JNK-interacting protein 3 (Jip3), phenocopied many of these axonal defects. We further found that loss of Jip3 led to accumulation of activated Ret receptor in pioneer growth cones, indicating a failure in the clearance of activated Ret from growth cones. Using an axon sever approach as well as in vivo analysis of axonal transport, we showed Jip3 specifically mediates retrograde, but not anterograde, transport of activated Ret51. Finally, live imaging revealed that Jip3 and Ret51 were retrogradely co-transported in pioneer axons, suggesting Jip3 functions as an adapter for retrograde transport of Ret51. Taken together, these results identify Ret51 as a molecular marker of pioneer neurons and elucidate an important isoform-specific role for Ret51 in axon growth and growth cone dynamics during development.

scholarly journals Regulated plasmalemmal expansion in nerve growth cones.

1991 ◽  
Vol 112 (6) ◽  
pp. 1215-1227 ◽  
Author(s):  
R O Lockerbie ◽  
V E Miller ◽  
K H Pfenninger
Keyword(s):  
Growth Cone ◽  
Binding Sites ◽  
Lectin Binding ◽  
Growth Cones ◽  
Subcellular Fractionation ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Nerve Growth ◽  
High K ◽  
Nerve Growth Cones

To study the mechanisms underlying plasmalemmal expansion in the nerve growth cone, a cell-free assay was developed to quantify membrane addition, using ligand binding and sealed growth cone particles isolated by subcellular fractionation from fetal rat brain. Exposed versus total binding sites of 125I-wheat germ agglutinin were measured in the absence or presence of saponin, respectively, after incubation with various agents. Ca2(+)-ionophore A23187 in the presence of Ca2+ increases the number of binding sites (Bmax) but does not change their affinity (KD), indicating that new receptors appear on the plasma membrane. Similarly, membrane depolarization by high K+ or veratridine significantly induces, in a Ca2(+)-dependent manner, the externalization of lectin binding sites from an internal pool. Morphometric analysis of isolated growth cones indicates that A23187 and high K+ treatment cause a significant reduction in a specific cytoplasmic membrane compartment, thus confirming the lectin labeling results and identifying the plasmalemmal precursor. The isolated growth cones take up gamma-amino-butyric acid and serotonin, but show no evidence for Ca2(+)-dependent transmitter release so that transmitter exocytosis is dissociated from plasmalemmal expansion. The data demonstrate that plasmalemmal expansion in the growth cone is a regulated process and identify an internal pool of precursor membrane.

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 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.

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