scholarly journals Microtubule behavior in the growth cones of living neurons during axon elongation.

1991 ◽  
Vol 115 (2) ◽  
pp. 345-363 ◽  
Author(s):  
E M Tanaka ◽  
M W Kirschner
Keyword(s):  
Neural Tube ◽  
Growth Cone ◽  
Growth Cones ◽  
Early Step ◽  
Axon Elongation ◽  
Major Mechanism ◽  
Microtubule Growth ◽  
Future Direction ◽  
Living Neurons ◽  
Fertilized Egg

To understand how microtubules are generated in the growth cone, we have imaged fluorescently tagged microtubules in living frog embryonic neurons. The neurons were labeled by injecting rhodamine-labeled tubulin into the fertilized egg and explanting the neurons from the neural tube. Microtubules extend deep into the growth cone periphery and adopt three characteristic distributions: (a) dispersed and splayed throughout much of the growth cone; (b) looped and apparently contorted by compression; and (c) bundled into tight arrays. These distributions interconvert on a time scale of several minutes and these interconversions are correlated with the behavior of the growth cone. We observed microtubule growth and shrinkage in growth cones, but are unable to determine their contribution to net assembly. However, translocation of polymer form the axon appears to be a major mechanism of generating new polymer in the growth cone, while bundling of microtubules in the growth cone appears to be the critical step in generating new axon. Neurons that were about to turn spontaneously generated microtubules in the future direction of growth, suggesting that orientation of microtubules might be an important early step in neuronal pathfinding.

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 Microtubule and Cell Contact Dependency of ER-bound PTP1B Localization in Growth Cones

2009 ◽  
Vol 20 (6) ◽  
pp. 1878-1889 ◽  
Author(s):  
Federico Fuentes ◽  
Carlos O. Arregui
Keyword(s):  
Growth Cone ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Growth Cones ◽  
Peripheral Region ◽  
Cell Contact ◽  
Positive Role ◽  
Cellular Targets ◽  
Axon Elongation

PTP1B is an ER-bound protein tyrosine phosphatase implied in the regulation of cell adhesion. Here we investigated mechanisms involved in the positioning and dynamics of PTP1B in axonal growth cones and evaluated the role of this enzyme in axons. In growth cones, PTP1B consistently localizes in the central domain, and occasionally at the peripheral region and filopodia. Live imaging of GFP-PTP1B reveals dynamic excursions of fingerlike processes within the peripheral region and filopodia. PTP1B and GFP-PTP1B colocalize with ER markers and coalign with microtubules at the peripheral region and redistribute to the base of the growth cone after treatment with nocodazole, a condition that is reversible. Growth cone contact with cellular targets is accompanied by invasion of PTP1B and stable microtubules in the peripheral region aligned with the contact axis. Functional impairment of PTP1B causes retardation of axon elongation, as well as reduction of growth cone filopodia lifetime and Src activity. Our results highlight the role of microtubules and cell contacts in the positioning of ER-bound PTP1B to the peripheral region of growth cones, which may be required for the positive role of PTP1B in axon elongation, filopodia stabilization, and Src activity.

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 Microtubule polymer assembly and transport during axonal elongation.

1991 ◽  
Vol 115 (2) ◽  
pp. 365-379 ◽  
Author(s):  
S S Reinsch ◽  
T J Mitchison ◽  
M Kirschner
Keyword(s):  
Cell Body ◽  
Neural Tube ◽  
Growth Cone ◽  
Cell Stage ◽  
Axonal Elongation ◽  
Axon Elongation ◽  
Coherent Phase ◽  
Polymer Translocation ◽  
Characteristic Manner ◽  
Fluorescent Mark

As axons elongate, tubulin, which is synthesized in the cell body, must be transported and assembled into new structures in the axon. The mechanism of transport and the location of assembly are presently unknown. We report here on the use of tubulin tagged with a photoactivatable fluorescent group to investigate these issues. Photoactivatable tubulin, microinjected into frog embryos at the two-cell stage, is incorporated into microtubules in neurons obtained from explants of the neural tube. When activated by light, a fluorescent mark is made on the microtubules in the axon, and transport and turnover can be visualized directly. We find that microtubules are generated in or near the cell body and continually transported distally as a coherent phase of polymer during axon elongation. This vectorial polymer movement was observed at all levels on the axon, even in the absence of axonal elongation. Measurements of the rate of polymer translocation at various places in the axon suggest that new polymer is formed by intercalary assembly along the axon and assembly at the growth cone in addition to transport of polymer from the cell body. Finally, polymer movement near the growth cone appeared to respond in a characteristic manner to growth cone behavior, while polymer proximally in the axon moved more consistently. These results suggest that microtubule translocation is the principal means of tubulin transport and that translocation plays an important role in generating new axon structure at the growth cone.

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

1971 ◽  
Vol 49 (3) ◽  
pp. 614-635 ◽  
Author(s):  
Kenneth M. Yamada ◽  
Brian S. Spooner ◽  
Norman K. Wessells
Keyword(s):  
Growth Cone ◽  
Smooth Endoplasmic Reticulum ◽  
Growth Cones ◽  
Nerve Cells ◽  
Structural Basis ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Ultrastructural Alteration ◽  
Axon Elongation

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.

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.

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.

Spinal neurite reabsorption and regrowth in vitro depend on the polarity of an applied electric field

Development ◽  
1987 ◽  
Vol 100 (1) ◽  
pp. 31-41
Author(s):  
C.D. McCaig
Keyword(s):  
Electric Field ◽  
Neural Tube ◽  
Applied Field ◽  
Growth Cones ◽  
Individual Growth ◽  
Electrical Field ◽  
Field Reversal ◽  
Specific Change ◽  
Applied Electrical Field

Retraction and regrowth of frog neural tube neurites have been studied in vitro in control cultures and in the presence of a small, continuously applied electrical field. In control cultures, some degree of retraction was seen in 39% of neurites while 7% were reabsorbed completely. Reabsorption of anodal-facing neurites was at least twice as common, with 67% showing some retraction and 17% almost totally reabsorbed. Cathodal-facing neurites were spared from retraction. Following extreme reabsorption of anodal-facing neurites, reversal of the electric field promoted regeneration in 47% (9/19) of cases studied. growth cone morphology also was determined by the polarity of the applied field. Anodal-facing growth cones had fewer filopodia than cathodal-facing growth cones sharing the same cell body. Field reversal induced a polarity-specific change in filopodia number on individual growth cones: a shift from anodal to cathodal increased filopodia numbers and vice versa. Some possible mechanisms involved and the significance of these results are discussed.

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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