Assembly of actin-containing cortex occurs at distal regions of growing neurites in PC12 cells

M.C. Sanders; Y.L. Wang

doi:10.1242/jcs.100.4.771

Assembly of actin-containing cortex occurs at distal regions of growing neurites in PC12 cells

Journal of Cell Science ◽

10.1242/jcs.100.4.771 ◽

1991 ◽

Vol 100 (4) ◽

pp. 771-780 ◽

Cited By ~ 1

Author(s):

M.C. Sanders ◽

Y.L. Wang

Keyword(s):

De Novo Assembly ◽

Actin Filaments ◽

De Novo ◽

Neurite Growth ◽

Retrograde Transport ◽

Time Lapse ◽

Growth Cones ◽

Distal Region ◽

Dark Spot ◽

Neurite Elongation

Although actin filaments are known to be localized in the cortex of axons and in the growth cones of nerve cells, it is unclear how actin-containing structures are assembled during nerve growth. We have studied the formation of actin structures in growing neurites by microinjecting fluorescent phalloidin or actin into PC12 neuron-like cells to label endogenous actin filaments. Upon stimulation of neurite growth in cells microinjected with fluorescent phalloidin, little or no fluorescence was detected in nascent growth cones and adjacent neurites despite the presence of actin filaments in these regions, suggesting that actin filaments were primarily formed by de novo assembly rather than the transport and reorganization of pre-existing, phalloidin-labeled actin filaments. Time-lapse observations of the distribution of phalloidin-labeled actin filaments during neurite elongation confirmed that fluorescence associated with pre-existing neurite cortex spread out more slowly than the elongation of neurites. Furthermore, when a dark spot was photobleached with a laser microbeam along neurites of cells microinjected with either fluorescent phalloidin or actin, the spot showed no appreciable translocation during active neurite elongation. Taken together, these results suggest that de novo assembly of actin filaments plays a crucial role in the formation of growth cones and adjacent cortex in the distal region of neurites, but does not appear to require the anterograde or retrograde transport of cortical filaments, or the passive stretching of the proximal segment of the neurite cortex.

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L1-dependent neuritogenesis involves ankyrinB that mediates L1-CAM coupling with retrograde actin flow

The Journal of Cell Biology ◽

10.1083/jcb.200303060 ◽

2003 ◽

Vol 163 (5) ◽

pp. 1077-1088 ◽

Cited By ~ 70

Author(s):

Kazunari Nishimura ◽

Fumie Yoshihara ◽

Takuro Tojima ◽

Noriko Ooashi ◽

Woohyun Yoon ◽

...

Keyword(s):

Cell Adhesion Molecule ◽

Traction Force ◽

Neurite Growth ◽

Growth Cones ◽

Retrograde Flow ◽

Actin Network ◽

Filamentous Actin ◽

Neurite Elongation ◽

Neurite Initiation ◽

Cell Adhesion Molecule L1

The cell adhesion molecule L1 (L1-CAM) plays critical roles in neurite growth. Its cytoplasmic domain (L1CD) binds to ankyrins that associate with the spectrin–actin network. This paper demonstrates that L1-CAM interactions with ankyrinB (but not with ankyrinG) are involved in the initial formation of neurites. In the membranous protrusions surrounding the soma before neuritogenesis, filamentous actin (F-actin) and ankyrinB continuously move toward the soma (retrograde flow). Bead-tracking experiments show that ankyrinB mediates L1-CAM coupling with retrograde F-actin flow in these perisomatic structures. Ligation of the L1-CAM ectodomain by an immobile substrate induces L1CD–ankyrinB binding and the formation of stationary ankyrinB clusters. Neurite initiation preferentially occurs at the site of these clusters. In contrast, ankyrinB is involved neither in L1-CAM coupling with F-actin flow in growth cones nor in L1-based neurite elongation. Our results indicate that ankyrinB promotes neurite initiation by acting as a component of the clutch module that transmits traction force generated by F-actin flow to the extracellular substrate via L1-CAM.

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Growth of neurites without filopodial or lamellipodial activity in the presence of cytochalasin B.

The Journal of Cell Biology ◽

10.1083/jcb.99.6.2041 ◽

1984 ◽

Vol 99 (6) ◽

pp. 2041-2047 ◽

Cited By ~ 235

Author(s):

L Marsh ◽

P C Letourneau

Keyword(s):

Cytochalasin B ◽

Ganglion Cells ◽

Video Recording ◽

Neurite Growth ◽

Time Lapse ◽

Growth Cones ◽

Electron Microscopic ◽

Thin Sections ◽

Actin Networks ◽

Dorsal Root Ganglion Cells

To examine the role in neurite growth of actin-mediated tensions within growth cones, we cultured chick embryo dorsal root ganglion cells on various substrata in the presence of cytochalasin B. Time-lapse video recording was used to monitor behaviors of living cells, and cytoskeletal arrangements in neurites were assessed via immunofluorescence and electron microscopic observations of thin sections and whole, detergent-extracted cells decorated with the S1 fragment of myosin. On highly adhesive substrata, nerve cells were observed to extend numerous (though peculiarly oriented) neurites in the presence of cytochalasin, despite their lack of both filopodia and lamellipodia or the orderly actin networks characteristic of typical growth cones. We concluded that growth cone activity is not necessary for neurite elongation, although actin arrays seem important in mediating characteristics of substratum selectivity and neurite shape.

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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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Mechanism of the formation of contractile ring in dividing cultured animal cells. I. Recruitment of preexisting actin filaments into the cleavage furrow.

The Journal of Cell Biology ◽

10.1083/jcb.110.4.1089 ◽

1990 ◽

Vol 110 (4) ◽

pp. 1089-1095 ◽

Cited By ~ 107

Author(s):

L G Cao ◽

Y L Wang

Keyword(s):

De Novo Assembly ◽

Actin Filaments ◽

De Novo ◽

Rat Kidney ◽

Equatorial Region ◽

Polar Regions ◽

Cleavage Furrow ◽

Animal Cells ◽

Contractile Ring ◽

Pronounced Difference

Cytokinesis of animal cells involves the formation of the circumferential actin filament bundle (contractile ring) along the equatorial plane. To analyze the assembly mechanism of the contractile ring, we microinjected a small amount of rhodamine-labeled phalloidin (rh-pha) or rhodamine-labeled actin (rh-actin) into dividing normal rat kidney cells. rh-pha was microinjected during prometaphase or metaphase to label actin filaments that were present at that stage. As mitosis proceeded into anaphase, the labeled filaments became associated with the cortex of the cell. During cytokinesis, rh-pha was depleted from polar regions and became highly concentrated into the equatorial region. The distribution of total actin filaments, as revealed by staining the whole cell with fluorescein phalloidin, showed a much less pronounced difference between the polar and the equatorial regions. The sites of de novo assembly of actin filaments during the formation of the contractile ring were determined by microinjecting rh-actin shortly before cytokinesis, and then extracting and fixing the cell during mid-cytokinesis. Injected rhodamine actin was only slightly concentrated in the contractile ring, as compared to the distribution of total actin filaments. Our results indicate that preexisting actin filaments, probably through movement and reorganization, are used preferentially for the formation of the contractile ring. De novo assembly of filaments, on the other hand, appears to take place preferentially outside the cleavage furrow.

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Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos.

The Journal of Cell Biology ◽

10.1083/jcb.97.4.963 ◽

1983 ◽

Vol 97 (4) ◽

pp. 963-973 ◽

Cited By ~ 132

Author(s):

P C Letourneau

Keyword(s):

Growth Cone ◽

Actin Filaments ◽

Electron Microscopic Observation ◽

Neurite Growth ◽

Growth Cones ◽

Chick Embryos ◽

Heavy Meromyosin ◽

Electron Microscopic ◽

Cytochemical Localization ◽

Triton X

Sensory neurons from chick embryos were cultured on substrata that support neurite growth, and were fixed and prepared for both cytochemical localization of actin and electron microscopic observation of actin filaments in whole-mounted specimens. Samples of cells were treated with the detergent Triton X-100 before, during, or after fixation with glutaraldehyde to determine the organization of actin in simpler preparations of extracted cytoskeletons. Antibodies to actin and a fluorescent derivative of phallacidin bound strongly to the leading margins of growth cones, but in neurites the binding of these markers for actin was very weak. This was true in all cases of Triton X-100 treatment, even when cells were extracted for 4 min before fixation. In whole-mounted cytoskeletons there were bundles and networks of 6-7-nm filaments in leading edges of growth cones but very few 6-7-n filaments were present among the microtubules and neurofilaments in the cytoskeletons of neurites. These filaments, which are prominent in growth cones, were identified as actin because they were stabilized against detergent extraction by the presence of phallacidin or the heavy meromyosin and S1 fragments of myosin. In addition, heavy meromyosin and S1 decorated these filaments as expected for binding to F-actin. Microtubules extended into growth cone margins and terminated within the network of actin filaments and bundles. Interactions between microtubule ends and these actin filaments may account for the frequently observed alignment of microtubules with filopodia at the growth cone margins.

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Filopodial Initiation and a Novel Filament-organizing Center, the Focal Ring

Molecular Biology of the Cell ◽

10.1091/mbc.12.8.2378 ◽

2001 ◽

Vol 12 (8) ◽

pp. 2378-2395 ◽

Cited By ~ 34

Author(s):

Michael Steketee ◽

Kenneth Balazovich ◽

Kathryn W. Tosney

Keyword(s):

Actin Filaments ◽

Time Lapse ◽

Growth Cones ◽

Vital Role ◽

Tension Development ◽

Phase Density ◽

Adhesion Sites ◽

Organizing Center ◽

Filament Bundles ◽

Focal Ring

This study examines filopodial initiation and implicates a putative actin filament organizer, the focal ring. Filopodia were optically recorded as they emerged from veils, the active lamellar extensions of growth cones. Motile histories revealed three events that consistently preceded filopodial emergence: an influx of cytoplasm into adjacent filopodia, a focal increase in phase density at veil margins, and protrusion of nubs that transform into filopodia. The cytoplasmic influx probably supplies materials needed for initiation. In correlated time lapse-immunocytochemistry, these focal phase densities corresponded to adhesions. These adhesions persisted at filopodial bases, regardless of subsequent movements. In correlated time lapse-electron microscopy, these adhesion sites contained a focal ring (an oblate, donut-shaped structure ∼120 nm in diameter) with radiating actin filaments. Filament geometry may explain filopodial emergence at 30 degree angles relative to adjacent filopodia. A model is proposed in which focal rings play a vital role in initiating and stabilizing filopodia: 1) they anchor actin filaments at adhesions, thereby facilitating tension development and filopodial emergence; 2) “axial” filaments connect focal rings to nub tips, thereby organizing filament bundling and ensuring the bundle intersects an adhesion; and 3) “lateral” filaments interconnect focal rings and filament bundles, thereby helping stabilize lamellar margins and filopodia.

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