Actin filament bundles are required for microtubule reorientation during growth cone turning to avoid an inhibitory guidance cue

The extracellular matrix through which growth cones navigate contains molecules, such as chondroitin sulfate proteoglycan, that can inhibit growth cone advance and induce branching and turning. Growth cone turning is accompanied by rearrangement of the cytoskeleton. To identify changes in the organization of actin filaments and microtubules that occur as growth cones turn, we used time-lapse phase contrast videomicroscopy to observe embryonic chick dorsal root ganglion neuronal growth cones at a substratum border between fibronectin and chondroitin sulfate proteoglycan, in the presence and absence of cytochalasin B. Growth cones were fixed and immunocytochemically labeled to identify actin filaments and dynamic and stable microtubules. Our results suggest that microtubules are rearranged within growth cones to accomplish turning to avoid chondroitin sulfate proteoglycan. Compared to growth cones migrating on fibronectin, turning growth cones were more narrow, and they contained dynamic microtubules that were closer to the leading edge and were more bundled. Cytochalasin B-treated growth cones sidestepped laterally along the border instead of turning, and in sidestepping growth cones, microtubules were not bundled and aligned. We conclude that actin filament bundles are required for microtubule reorientation and growth cone turning to avoid chondroitin sulfate proteoglycan.

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Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity.

The Journal of Cell Biology ◽

10.1083/jcb.119.5.1219 ◽

1992 ◽

Vol 119 (5) ◽

pp. 1219-1243 ◽

Cited By ~ 217

Author(s):

A K Lewis ◽

P C Bridgman

Keyword(s):

Actin Filament ◽

Growth Cone ◽

Actin Filaments ◽

Membrane Surface ◽

Leading Edge ◽

Growth Cones ◽

Differential Interference Contrast Microscopy ◽

Nerve Growth ◽

Filament Bundles ◽

Two Populations

The organization and polarity of actin filaments in neuronal growth cones was studied with negative stain and freeze-etch EM using a permeabilization protocol that caused little detectable change in morphology when cultured nerve growth cones were observed by video-enhanced differential interference contrast microscopy. The lamellipodial actin cytoskeleton was composed of two distinct subpopulations: a population of 40-100-nm-wide filament bundles radiated from the leading edge, and a second population of branching short filaments filled the volume between the dorsal and ventral membrane surfaces. Together, the two populations formed the three-dimensional structural network seen within expanding lamellipodia. Interaction of the actin filaments with the ventral membrane surface occurred along the length of the filaments via membrane associated proteins. The long bundled filament population was primarily involved in these interactions. The filament tips of either population appeared to interact with the membrane only at the leading edge; this interaction was mediated by a globular Triton-insoluble material. Actin filament polarity was determined by decoration with myosin S1 or heavy meromyosin. Previous reports have suggested that the polarity of the actin filaments in motile cells is uniform, with the barbed ends toward the leading edge. We observed that the actin filament polarity within growth cone lamellipodia is not uniform; although the predominant orientation was with the barbed end toward the leading edge (47-56%), 22-25% of the filaments had the opposite orientation with their pointed ends toward the leading edge, and 19-31% ran parallel to the leading edge. The two actin filament populations display distinct polarity profiles: the longer filaments appear to be oriented predominantly with their barbed ends toward the leading edge, whereas the short filaments appear to be randomly oriented. The different length, organization and polarity of the two filament populations suggest that they differ in stability and function. The population of bundled long filaments, which appeared to be more ventrally located and in contact with membrane proteins, may be more stable than the population of short branched filaments. The location, organization, and polarity of the long bundled filaments suggest that they may be necessary for the expansion of lamellipodia and for the production of tension mediated by receptors to substrate adhesion molecules.

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Schwann cell chondroitin sulfate proteoglycan inhibits dorsal root ganglion neuron neurite outgrowth and substrate specificity via a soma and not a growth cone mechanism

Journal of Neuroscience Research ◽

10.1002/jnr.22132 ◽

2009 ◽

Vol 87 (13) ◽

pp. 2863-2871 ◽

Cited By ~ 10

Author(s):

Damien P. Kuffler ◽

Ivan J. Sosa ◽

Onix Reyes

Keyword(s):

Dorsal Root Ganglion ◽

Schwann Cell ◽

Substrate Specificity ◽

Chondroitin Sulfate ◽

Neurite Outgrowth ◽

Dorsal Root Ganglion Neuron ◽

Growth Cone ◽

Dorsal Root ◽

Chondroitin Sulfate Proteoglycan ◽

Sulfate Proteoglycan

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GROWTH CONE BEHAVIOR IN THE PRESENCE OF SOLUBLE CHONDROITIN SULFATE PROTEOGLYCAN (CSPG), COMPARED TO BEHAVIOR ON CSPG BOUND TO LAMININ OR FIBRONECTIN

International Journal of Developmental Neuroscience ◽

10.1016/0736-5748(96)00017-2 ◽

1996 ◽

Vol 14 (3) ◽

pp. 331-349 ◽

Cited By ~ 70

Author(s):

Diane M. Snow ◽

Eric M. Brown ◽

Paul C. Letourneau

Keyword(s):

Chondroitin Sulfate ◽

Growth Cone ◽

Chondroitin Sulfate Proteoglycan ◽

Sulfate Proteoglycan

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A chondroitin sulfate proteoglycan may influence the direction of retinal ganglion cell outgrowth

Development ◽

10.1242/dev.113.4.1473 ◽

1991 ◽

Vol 113 (4) ◽

pp. 1473-1485 ◽

Cited By ~ 3

Author(s):

D.M. Snow ◽

M. Watanabe ◽

P.C. Letourneau ◽

J. Silver

Keyword(s):

Ganglion Cell ◽

Chondroitin Sulfate ◽

Retinal Ganglion Cell ◽

Growth Cones ◽

Chondroitin Sulfate Proteoglycan ◽

Retinal Ganglion ◽

Sulfate Proteoglycan ◽

Cell Outgrowth ◽

Axon Elongation

In the developing retina, retinal ganglion cell (RGC) axons elongate toward the optic fissure, even though no obvious directional restrictions exist. Previous studies indicate that axon-matrix interactions are important for retinal ganglion cell axon elongation, but the factors that direct elongation are unknown. Chondroitin sulfate proteoglycan (CS-PG), a component of the extracellular matrix, repels elongating dorsal root ganglion (DRG) axons in vitro and is present in vivo in the roof plate of the spinal cord, a structure that acts as a barrier to DRG axons during development. In this study, we examined whether CS-PG may regulate the pattern of retinal ganglion cell outgrowth in the developing retina. Immunocytochemical analysis showed that CS-PG was present in the innermost layers of the developing rat retina. The expression of CS-PG moved peripherally with retinal development, always remaining at the outer edge of the front of the developing axons. CS-PG was no longer detectable with immunocytochemical techniques when RGC axon elongation in the retina is complete. Results of studies in vitro showed that CS-PG, isolated from bovine nasal cartilage and chick limb, was inhibitory to elongating RGC axons and that RGC growth cones were more sensitive to CS-PG than were DRG neurites tested at the same concentrations of CS-PG. The behavior of retinal growth cones as they encounter CS-PG was characterized using time-lapse video microscopy. Filopodia of the RGC growth cones extended to and sampled the CS-PG repeatedly. With time, the growth cones turned to avoid outgrowth on the CS-PG and grew only on laminin. While numerous studies have shown the presence of positive factors within the retina that may guide developing RGC axons, this is the first demonstration of an inhibitory or repelling molecule in the retina that may regulate axon elongation. Taken together, these data suggest that the direction of RGC outgrowth in the retina may be regulated by the proper ratio of growth-promoting molecules, such as laminin, to growth-inhibiting molecules, like CS-PG, present in the correct pattern and concentrations along the retinal ganglion cell pathway.

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