B-50/GAP43 localization on membranes of putative transport vesicle in the cell body, neurites and growth cones of cultured hippocampal neurons

1992 ◽  
Vol 137 (1) ◽  
pp. 129-132 ◽  
Author(s):  
M.Van Lookeren Campagne ◽  
C.G. Dotti ◽  
A.J. Verkleij ◽  
W.H. Gispen ◽  
A.B. Oestreicher
Keyword(s):  
Cell Body ◽  
Hippocampal Neurons ◽  
Growth Cones ◽  
Transport Vesicle ◽  
Cultured Hippocampal Neurons

scholarly journals Delayed Retraction of Filopodia in Gelsolin Null Mice

1997 ◽  
Vol 138 (6) ◽  
pp. 1279-1287 ◽  
Author(s):  
Mei Lu ◽  
Walter Witke ◽  
David J. Kwiatkowski ◽  
Kenneth S. Kosik
Keyword(s):  
Growth Cone ◽  
Hippocampal Neurons ◽  
Neurite Growth ◽  
Time Lapse ◽  
Growth Cones ◽  
Wild Type ◽  
Dynamic Studies ◽  
Shape Changes ◽  
Time Lapse Video ◽  
Cultured Hippocampal Neurons

Growth cones extend dynamic protrusions called filopodia and lamellipodia as exploratory probes that signal the direction of neurite growth. Gelsolin, as an actin filament-severing protein, may serve an important role in the rapid shape changes associated with growth cone structures. In wild-type (wt) hippocampal neurons, antibodies against gelsolin labeled the neurite shaft and growth cone. The behavior of filopodia in cultured hippocampal neurons from embryonic day 17 wt and gelsolin null (Gsn−) mice (Witke, W., A.H. Sharpe, J.H. Hartwig, T. Azuma, T.P. Stossel, and D.J. Kwiatkowski. 1995. Cell. 81:41–51.) was recorded with time-lapse video microscopy. The number of filopodia along the neurites was significantly greater in Gsn− mice and gave the neurites a studded appearance. Dynamic studies suggested that most of these filopodia were formed from the region of the growth cone and remained as protrusions from the newly consolidated shaft after the growth cone advanced. Histories of individual filopodia in Gsn− mice revealed elongation rates that did not differ from controls but an impaired retraction phase that probably accounted for the increased number of filopodia long the neutrite shaft. Gelsolin appears to function in the initiation of filopodial retraction and in its smooth progression.

scholarly journals Changes in microtubule polarity orientation during the development of hippocampal neurons in culture.

1989 ◽  
Vol 109 (6) ◽  
pp. 3085-3094 ◽  
Author(s):  
P W Baas ◽  
M M Black ◽  
G A Banker
Keyword(s):  
Cell Body ◽  
Hippocampal Neurons ◽  
Regional Differences ◽  
Morphological Characteristics ◽  
The Other ◽  
Developmental Changes ◽  
Microtubule Polarity ◽  
Temporally Correlated ◽  
Uniform Polarity ◽  
Cultured Hippocampal Neurons

Microtubules in the dendrites of cultured hippocampal neurons are of nonuniform polarity orientation. About half of the microtubules have their plus ends oriented distal to the cell body, and the other half have their minus ends distal; in contrast, microtubules in the axon are of uniform polarity orientation, all having their plus ends distal (Baas, P.W., J.S. Deitch, M. M. Black, and G. A. Banker. 1988. Proc. Natl. Acad. Sci. USA. 85:8335-8339). Here we describe the developmental changes that give rise to the distinct microtubule patterns of axons and dendrites. Cultured hippocampal neurons initially extend several short processes, any one of which can apparently become the axon (Dotti, C. G., and G. A. Banker. 1987. Nature [Lond.]. 330:477-479). A few days after the axon has begun its rapid growth, the remaining processes differentiate into dendrites (Dotti, C. G., C. A. Sullivan, and G. A. Banker. 1988. J. Neurosci. 8:1454-1468). The polarity orientation of the microtubules in all of the initial processes is uniform, with plus ends distal to the cell body, even through most of these processes will become dendrites. This uniform microtubule polarity orientation is maintained in the axon at all stages of its growth. The polarity orientation of the microtubules in the other processes remains uniform until they begin to grow and acquire the morphological characteristics of dendrites. It is during this period that microtubules with minus ends distal to the cell body first appear in these processes. The proportion of minus end-distal microtubules gradually increases until, by 7 d in culture, about equal numbers of dendritic microtubules are oriented in each direction. Thus, the establishment of regional differences in microtubule polarity orientation occurs after the initial polarization of the neuron and is temporally correlated with the differentiation of the dendrites.

scholarly journals Suppression of kinesin expression in cultured hippocampal neurons using antisense oligonucleotides.

1992 ◽  
Vol 117 (3) ◽  
pp. 595-606 ◽  
Author(s):  
A Ferreira ◽  
J Niclas ◽  
R D Vale ◽  
G Banker ◽  
K S Kosik
Keyword(s):  
Neurite Outgrowth ◽  
Cell Body ◽  
Heavy Chain ◽  
Antisense Oligonucleotides ◽  
Hippocampal Neurons ◽  
Full Recovery ◽  
Synapsin I ◽  
Neurite Length ◽  
Protein Levels ◽  
Cultured Hippocampal Neurons

Kinesin, a microtubule-based force-generating molecule, is thought to translocate organelles along microtubules. To examine the function of kinesin in neurons, we sought to suppress kinesin heavy chain (KHC) expression in cultured hippocampal neurons using antisense oligonucleotides and study the phenotype of these KHC "null" cells. Two different antisense oligonucleotides complementary to the KHC sequence reduced the protein levels of the heavy chain by greater than 95% within 24 h after application and produced identical phenotypes. After inhibition of KHC expression for 24 or 48 h, neurons extended an array of neurites often with one neurite longer than the others; however, the length of all these neurites was significantly reduced. Inhibition of KHC expression also altered the distribution of GAP-43 and synapsin I, two proteins thought to be transported in association with membranous organelles. These proteins, which are normally localized at the tips of growing neurites, were confined to the cell body in antisense-treated cells. Treatment of the cells with the corresponding sense oligonucleotides affected neither the distribution of GAP-43 and synapsin I, nor the length of neurites. A full recovery of neurite length occurred after removal of the antisense oligonucleotides from the medium. These data indicate that KHC plays a role in the anterograde translocation of vesicles containing GAP-43 and synapsin I. A deficiency in vesicle delivery may also explain the inhibition of neurite outgrowth. Despite the inhibition of KHC and the failure of GAP-43 and synapsin I to move out of the cell body, hippocampal neurons can extend processes and acquire as asymmetric morphology.

scholarly journals Transport of dendritic microtubules establishes their nonuniform polarity orientation.

1995 ◽  
Vol 130 (1) ◽  
pp. 93-103 ◽  
Author(s):  
D J Sharp ◽  
W Yu ◽  
P W Baas
Keyword(s):  
Chemical Composition ◽  
Cell Body ◽  
Hippocampal Neurons ◽  
Essential Feature ◽  
Proximal Region ◽  
The Other ◽  
Stage Of Development ◽  
Timely Fashion ◽  
Dendritic Development ◽  
Cultured Hippocampal Neurons

The immature processes that give rise to both axons and dendrites contain microtubules (MTs) that are uniformly oriented with their plus-ends distal to the cell body, and this pattern is preserved in the developing axon. In contrast, developing dendrites gradually acquire nonuniform MT polarity orientation due to the addition of a subpopulation of oppositely oriented MTs (Baas, P. W., M. M. Black, and G. A. Banker. 1989. J. Cell Biol. 109:3085-3094). In theory, these minus-end-distal MTs could be locally nucleated and assembled within the dendrite itself, or could be transported into the dendrite after their nucleation within the cell body. To distinguish between these possibilities, we exposed cultured hippocampal neurons to nanomolar levels of vinblastine after one of the immature processes had developed into the axon but before the others had become dendrites. At these levels, vinblastine acts as a kinetic stabilizer of MTs, inhibiting further assembly while not substantially depolymerizing existing MTs. This treatment did not abolish dendritic differentiation, which occurred in timely fashion over the next two to three days. The resulting dendrites were flatter and shorter than controls, but were identifiable by their ultrastructure, chemical composition, and thickened tapering morphology. The growth of these dendrites was accompanied by a diminution of MTs from the cell body, indicating a net transfer of MTs from one compartment into the other. During this time, minus-end-distal microtubules arose in the experimental dendrites, indicating that new MT assembly is not required for the acquisition of nonuniform MT polarity orientation in the dendrite. Minus-end-distal microtubules predominated in the more proximal region of experimental dendrites, indicating that most of the MTs at this stage of development are transported into the dendrite with their minus-ends leading. These observations indicate that transport of MTs from the cell body is an essential feature of dendritic development, and that this transport establishes the nonuniform polarity orientation of MTs in the dendrite.

scholarly journals Kinesin-mediated organelle translocation revealed by specific cellular manipulations.

1994 ◽  
Vol 127 (4) ◽  
pp. 1021-1039 ◽  
Author(s):  
F Feiguin ◽  
A Ferreira ◽  
K S Kosik ◽  
A Caceres
Keyword(s):  
Hippocampal Neurons ◽  
Brefeldin A ◽  
Growth Cones ◽  
Cell Periphery ◽  
Fluorescent Staining ◽  
Anterograde Transport ◽  
Membrane Bound ◽  
Living Neurons ◽  
Vacuolar System ◽  
Cultured Hippocampal Neurons

The distribution of membrane-bound organelles was studied in cultured hippocampal neurons after antisense oligonucleotide suppression of the kinesin-heavy chain (KHC). We observed reduced 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) fluorescent staining in neurites and growth cones. In astrocytes, KHC suppression results in the disappearance of the DiOC6(3)-positive reticular network from the cell periphery, and a parallel accumulation of label within the cell center. On the other hand, mitochondria microtubules and microfilaments display a distribution that closely resembles that observed in control cells. KHC suppression of neurons and astrocytes completely inhibited the Brefeldin A-induced spreading and tubulation of the Golgi-associated structure enriched in mannose-6-phosphate receptors. In addition, KHC suppression prevents the low pH-induced anterograde redistribution of late endocytic structures. Taken collectively, these observations suggest that in living neurons, kinesin mediates the anterograde transport of tubulovesicular structures originated in the central vacuolar system (e.g., the endoplasmic reticulum) and that the regulation of kinesin-membrane interactions may be of key importance for determining the intracellular distribution of selected organelles.

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