Accumulation of actin in subsets of pioneer growth cone filopodia in response to neural and epithelial guidance cues in situ.

Directed outgrowth of neural processes must involve transmission of signals from the tips of filopodia to the central region of the growth cone. Here, we report on the distribution and dynamics of one possible element in this process, actin, in live growth cones which are reorienting in response to in situ guidance cues. In grasshopper embryonic limbs, pioneer growth cones respond to at least three types of guidance cues: a limb axis cue, intermediate target cells, and a circumferential band of epithelial cells. With time-lapse imaging of intracellularly injected rhodamine-phalloidin and rhodamine-actin, we monitored the distribution of actin during growth cone responses to these cues. In distal limb regions, accumulation of actin in filopodia and growth cone branches accompanies continued growth, while reduction of actin accompanies withdrawal. Where growth cones are reorienting to intermediate target cells, or along the circumferential epithelial band, actin selectively accumulates in the proximal regions of those filopodia that have contacted target cells or are extending along the band. Actin accumulations can be retrogradely transported along filopodia, and can extend into the central region of the growth cone. These results suggest that regulation and translocation of actin may be a significant element in growth cone steering.

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Microtubule behavior during guidance of pioneer neuron growth cones in situ.

The Journal of Cell Biology ◽

10.1083/jcb.115.2.381 ◽

1991 ◽

Vol 115 (2) ◽

pp. 381-395 ◽

Cited By ~ 125

Author(s):

J H Sabry ◽

T P O'Connor ◽

L Evans ◽

A Toroian-Raymond ◽

M Kirschner ◽

...

Keyword(s):

Growth Cone ◽

Growth Cones ◽

The Body ◽

Microtubule Network ◽

Guidance Cues ◽

Selective Retention ◽

Pioneer Neuron ◽

Neuron Growth

The growth of an axon toward its target results from the reorganization of the cytoskeleton in response to environmental guidance cues. Recently developed imaging technology makes it possible to address the effect of such cues on the neural cytoskeleton directly. Although high resolution studies can be carried out on neurons in vitro, these circumstances do not recreate the complexity of the natural environment. We report here on the arrangement and dynamics of microtubules in live neurons pathfinding in response to natural guidance cues in situ using the embryonic grasshopper limb fillet preparation. A rich microtubule network was present within the body of the growth cone and normally extended into the distal growth cone margin. Complex microtubule loops often formed transiently within the growth cone. Branches both with and without microtubules were regularly observed. Microtubules did not extend into filopodia. During growth cone steering events in response to identified guidance cues, microtubule behaviour could be monitored. In turns towards guidepost cells, microtubules selectively invaded branches derived from filopodia that had contacted the guidepost cell. At limb segment boundaries, microtubules displayed a variety of behaviors, including selective branch invasion, and also invasion of multiple branches followed by selective retention in branches oriented in the correct direction. Microtubule invasion of multiple branches also was seen in growth cones migrating on intrasegmental epithelium. Both selective invasion and selective retention generate asymmetrical microtubule arrangements within the growth cone, and may play a key role in growth cone steering events.

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Growth cone dynamics of embryonic grasshopper pioneer neurons in situ

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140385 ◽

1995 ◽

Vol 53 ◽

pp. 802-803

Author(s):

Tim P. O'Connor

Keyword(s):

Nervous System ◽

Growth Cone ◽

Time Lapse ◽

Pioneer Neurons ◽

The Central Nervous System ◽

Neuronal Growth Cone ◽

Time Lapse Imaging ◽

Guidance Information ◽

Extracellular Environment

During development of the nervous system, neurons extend axons over relatively long distances to contact their targets. A variety of molecules in the extracellular environment are instrumental in guiding a neuronal process. The motile tip of the process, the growth cone, senses and transduces this guidance information, resulting in a local reorganization and consolidation of the cytoskeleton. Although much work has been dedicated to isolating the molecules that guide a neuronal growth cone, relatively little is known about the dynamic processes that occur when a growth cone turns in response to guidance information. Recently, a number of biological systems have been developed that enable time lapse imaging of growth cones as they extend axons in situ. One of these systems is the embryonic grasshopper limb fillet.In the grasshopper embryo, a pair of sibling neurons, named the Til pioneers, are the first neurons to extend axons toward the central nervous system (CNS).

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The organization of myosin and actin in rapid frozen nerve growth cones.

The Journal of Cell Biology ◽

10.1083/jcb.108.1.95 ◽

1989 ◽

Vol 108 (1) ◽

pp. 95-109 ◽

Cited By ~ 136

Author(s):

P C Bridgman ◽

M E Dailey

Keyword(s):

Immunoelectron Microscopy ◽

Growth Cone ◽

Central Region ◽

Growth Cones ◽

Underlying Structure ◽

Similar Distribution ◽

Intense Staining ◽

Rhodamine Phalloidin ◽

Whole Mount ◽

Ganglion Neurons

Rapid freezing and freeze substitution were used in conjunction with immunofluorescence, whole mount EM, and immunoelectron microscopy to study the organization of myosin and actin in growth cones of cultured rat superior cervical ganglion neurons. The general cytoplasmic organization was determined by whole mount EM; tight microfilament bundles formed the core of filopodia while a dense meshwork formed the underlying structure of lamellipodia. Although the central microtubule and organelle-rich region of the growth cone had fewer microfilaments, dense foci and bundles of microfilaments were usually observed. Anti-actin immunofluorescence and rhodamine phalloidin staining of f-actin both showed intense staining of filopodia and lamellipodia. In addition, staining of bundles and foci were observed in central regions suggesting that the majority of the microfilaments seen by whole mount EM are actin filaments. Anti-myosin immunofluorescence was brightest in the central region and usually had a punctate pattern. Although less intense, anti-myosin staining was also seen in peripheral regions; it was most prominent at the border with the central region, in portions of lamellipodia undergoing ruffling, and in spots along the shaft and at the base of filopodia. Immunoelectron microscopy of myosin using postembedment labeling with colloidal gold showed a similar distribution to that seen by immunofluorescence. Label was scattered throughout the growth cone, but present as distinct aggregates in the peripheral region mainly along the border with the central region. Less frequently, aggregates were also seen centrally and along the shaft and at the base of filopodia. This distribution is consistent with myosins involvement in the production of tension and movements of growth cone filopodia and lamellipodia that occur during active neurite elongation.

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Delayed Retraction of Filopodia in Gelsolin Null Mice

The Journal of Cell Biology ◽

10.1083/jcb.138.6.1279 ◽

1997 ◽

Vol 138 (6) ◽

pp. 1279-1287 ◽

Cited By ~ 105

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.

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Growth cone guidance and neuron morphology on micropatterned laminin surfaces

Journal of Cell Science ◽

10.1242/jcs.105.1.203 ◽

1993 ◽

Vol 105 (1) ◽

pp. 203-212 ◽

Cited By ~ 7

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.

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Stereotyped pathway selection by growth cones of early epiphysial neurons in the embryonic zebrafish

Development ◽

10.1242/dev.112.3.723 ◽

1991 ◽

Vol 112 (3) ◽

pp. 723-746 ◽

Cited By ~ 4

Author(s):

S.W. Wilson ◽

S.S. Easter

Keyword(s):

Growth Cone ◽

Developmental Stages ◽

Light And Electron Microscopy ◽

Local Environment ◽

Spatial Relations ◽

Growth Cones ◽

Acetylated Tubulin ◽

Guidance Cues ◽

Selective Retention ◽

Rostral Region

In this report we have examined the development of one of the earliest projections in the embryonic zebrafish brain, that from the epiphysis. Epiphysial axons and growth cones were labelled anterogradely in whole-mounted brains, using either the carbocyanine dye, diI, or horseradish peroxidase (HRP). Some embryos were also either stained with anti-acetylated tubulin or HNK-1 antibodies to reveal other axons in the brain, or were secondarily sectioned for light and electron microscopy. The epiphysial axons have a very specific projection pattern and virtually all axons grow precisely to their target regions without error. The first epiphysial growth cone extends ventrally from the epiphysis into the dorsoventral diencephalic tract at 19–20 h post-fertilisation (h PF). Several hours later, it turns rostrally to grow alongside axons in the tract of the postoptic commissure. The morphology of the leading growth cone changes in predictable ways at different locations along its pathway and these changes correlate with differences in the local environment that it encounters. In contrast to other published descriptions of other developing systems, the epiphysial growth cone is no more complex either when pioneering a pathway, or when encountering divergent axonal pathways. Indeed, it is most complex (i.e. has the greatest number of processes) when it first starts to follow the tract of the postoptic commissure. The presence and selective retention of filopodia within other axonal pathways suggests that growth cones have access to these pathways but do not select them. These observations support the notion that local guidance cues exist within the early scaffold of brain tracts. Subsequent epiphysial axons form a tight fascicle within the dorsoventral diencephalic tract, but abruptly defasciculate from each other upon turning rostrally into the tract of the postoptic commissure. Epiphysial growth cones that enter this tract at abnormal locations still turn in the appropriate direction. Therefore, guidance cues are not restricted solely to the normal intersections but may be distributed along the length of the tracts. The epiphysial growth cones and axons have very characteristic spatial relations to other axons in the tracts of the developing brain. They are restricted to the dorsal region of the tract of the postoptic commissure and the rostral region of the postoptic commissure. At early developmental stages, the epiphysial axons are the only axons within the dorsoventral diencephalic tract and they are located very superficially within the neuroepithelium. At later stages, they are displaced to deeper regions of the neuropil by non-epiphysial axons.

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Gradients and Growth Cone Guidance of Grasshopper Neurons

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540305100406 ◽

2003 ◽

Vol 51 (4) ◽

pp. 445-454 ◽

Cited By ~ 10

Author(s):

Arthur T. Legg ◽

Timothy P. O'Connor

Keyword(s):

Nervous System ◽

Long Range ◽

Growth Cone ◽

Growth Cones ◽

Path Finding ◽

Guidance Cues ◽

Cone Function ◽

The Absolute ◽

Growth Cone Guidance

The generation of a functional nervous system is dependent on precise path-finding of axons during development. This pathfinding is directed by the distribution of local and long-range guidance cues, the latter of which are believed to be distributed in gradients. Gradients of guidance cues have been associated with growth cone function for over a hundred years. However, little is known about the mechanisms used by growth cones to respond to these gradients, in part owing to the lack of identifiable gradients in vivo. In the developing grasshopper limb, two gradients of the semaphorin Sema-2a are necessary for correct neuronal pathfinding in vivo. The gradients are found in regions where growth cones make critical steering decisions. Observations of different growth cone behaviors associated with these gradients have provided some insights into how growth cones respond to them. Growth cones appear to respond more faithfully to changes in concentration, rather than absolute levels, of Sema-2a expression, whereas the absolute levels may regulate growth cone size.

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Automated profiling of growth cone heterogeneity defines relations between morphology and motility

The Journal of Cell Biology ◽

10.1083/jcb.201711023 ◽

2018 ◽

Vol 218 (1) ◽

pp. 350-379 ◽

Cited By ~ 2

Author(s):

Maria M. Bagonis ◽

Ludovico Fusco ◽

Olivier Pertz ◽

Gaudenz Danuser

Keyword(s):

Growth Cone ◽

Rho Gtpase ◽

Time Lapse ◽

Growth Cones ◽

Cellular Structures ◽

Unperturbed System ◽

Neurite Length ◽

Manual Curation

Growth cones are complex, motile structures at the tip of an outgrowing neurite. They often exhibit a high density of filopodia (thin actin bundles), which complicates the unbiased quantification of their morphologies by software. Contemporary image processing methods require extensive tuning of segmentation parameters, require significant manual curation, and are often not sufficiently adaptable to capture morphology changes associated with switches in regulatory signals. To overcome these limitations, we developed Growth Cone Analyzer (GCA). GCA is designed to quantify growth cone morphodynamics from time-lapse sequences imaged both in vitro and in vivo, but is sufficiently generic that it may be applied to nonneuronal cellular structures. We demonstrate the adaptability of GCA through the analysis of growth cone morphological variation and its relation to motility in both an unperturbed system and in the context of modified Rho GTPase signaling. We find that perturbations inducing similar changes in neurite length exhibit underappreciated phenotypic nuance at the scale of the growth cone.

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Early cytoplasmic uncoating is associated with infectivity of HIV-1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1706245114 ◽

2017 ◽

Vol 114 (34) ◽

pp. E7169-E7178 ◽

Cited By ~ 55

Author(s):

João I. Mamede ◽

Gianguido C. Cianci ◽

Meegan R. Anderson ◽

Thomas J. Hope

Keyword(s):

Fluid Phase ◽

Time Lapse ◽

Fluorescent Imaging ◽

Individual Particle ◽

Target Cells ◽

Cytoplasmic Process ◽

Particle Level ◽

The Individual ◽

Time Lapse Imaging ◽

Hiv 1

After fusion, HIV delivers its conical capsid into the cytoplasm. To release the contained reverse-transcribing viral genome, the capsid must disassemble in a process termed uncoating. Defining the kinetics, dynamics, and cellular location of uncoating of virions leading to infection has been confounded by defective, noninfectious particles and the stochastic minefield blocking access to host DNA. We used live-cell fluorescent imaging of intravirion fluid phase markers to monitor HIV-1 uncoating at the individual particle level. We find that HIV-1 uncoating of particles leading to infection is a cytoplasmic process that occurs ∼30 min postfusion. Most, but not all, of the capsid protein is rapidly shed in tissue culture and primary target cells, independent of entry pathway. Extended time-lapse imaging with less than one virion per cell allows identification of infected cells by Gag-GFP expression and directly links individual particle behavior to infectivity, providing unprecedented insights into the biology of HIV infection.

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