Comparison of the Force Exerted by Hippocampal and Drg Growth Cones
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Filopodia
Lamellipodium
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A role of Arp2/3 complex in lamellipodia is well established, whereas its roles in filopodia formation remain obscure. We addressed this question in neuronal cells, in which motility is heavily based on filopodia, and we found that Arp2/3 complex is involved in generation of both lamellipodia and filopodia in growth cones, and in neuritogenesis, the processes thought to occur largely in Arp2/3 complex-independent manner. Depletion of Arp2/3 complex in primary neurons and neuroblastoma cells by small interfering RNA significantly decreased the F-actin contents and inhibited lamellipodial protrusion and retrograde flow in growth cones, but also initiation and dynamics of filopodia. Using electron microscopy, immunochemistry, and gene expression, we demonstrated the presence of the Arp2/3 complex-dependent dendritic network of actin filaments in growth cones, and we showed that individual actin filaments in filopodia originated at Arp2/3 complex-dependent branch points in lamellipodia, thus providing a mechanistic explanation of Arp2/3 complex functions during filopodia formation. Additionally, Arp2/3 complex depletion led to formation of multiple neurites, erratic pattern of neurite extension, and excessive formation of stress fibers and focal adhesions. Consistent with this phenotype, RhoA activity was increased in Arp2/3 complex-depleted cells, indicating that besides nucleating actin filaments, Arp2/3 complex may influence cell motility by altering Rho GTPase signaling.
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Capping protein (CP) binds to barbed ends of growing actin filaments and inhibits elongation. CP is essential for actin-based motility in cell-free systems and in Dictyostelium. Even though CP is believed to be critical for creating the lamellipodial actin structure necessary for protrusion and migration, CP's role in mammalian cell migration has not been directly tested. Moreover, recent studies have suggested that structures besides lamellipodia, including lamella and filopodia, may have unappreciated roles in cell migration. CP has been postulated to be absent from filopodia, and thus its role in filopodial activity has remained unexplored. We report that silencing CP in both cultured mammalian B16F10 cells and in neurons of developing neocortex impaired cell migration. Moreover, we unexpectedly observed that low levels of CP were detectable in the majority of filopodia. CP depletion decreased filopodial length, altered filopodial shape, and reduced filopodial dynamics. Our results support an expansion of the potential roles that CP plays in cell motility by implicating CP in filopodia as well as in lamellipodia, both of which are important for locomotion in many types of migrating cells.
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Src tyrosine kinases have been implicated in axonal growth and guidance; however, the underlying cellular mechanisms are not well understood. Specifically, it is unclear which aspects of actin organization and dynamics are regulated by Src in neuronal growth cones. Here, we investigated the function of Src2 and one of its substrates, cortactin, in lamellipodia and filopodia of Aplysia growth cones. We found that up-regulation of Src2 activation state or cortactin increased lamellipodial length, protrusion time, and actin network density, whereas down-regulation had opposite effects. Furthermore, Src2 or cortactin up-regulation increased filopodial density, length, and protrusion time, whereas down-regulation promoted lateral movements of filopodia. Fluorescent speckle microscopy revealed that rates of actin assembly and retrograde flow were not affected in either case. In summary, our results support a model in which Src and cortactin regulate growth cone motility by increasing actin network density and protrusion persistence of lamellipodia by controlling the state of actin-driven protrusion versus retraction. In addition, both proteins promote the formation and stability of actin bundles in filopodia.
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We have analyzed the dynamics of growth cone configuration using video-enhanced contrast differential interference contrast microscopy. Regenerating neurites from NGF-treated PC12 cells and sympathetic neurons were observed in real time during their elongation and reviewed by time-lapse video recordings. This technique provided a high-resolution view of motile growth cone elements including filopodia, microspikes, lamellipodia and ruffles. On the basis of our observations, a multistage model for growth cone advance is proposed. Elongation commences with lamellipodial spreading. If the newly extended lamellipodium does not retract or lift off the substrate in the form of a ruffle, a second phase--consolidation--occurs, in which the lamellipodium thickens as it fills with cytoplasm and organelles. The consolidated area then undergoes further transformation into an area of neuritic shaft as new lamellipodia form at the leading and peripheral zones of the distal process. We never observed filopodia or microspikes contracting to propel the growth cone forward. We also noted that elongating tips generally had large varicosities within 20 micron of their leading edges. These may play a role in neurite outgrowth and in the formation of smaller, synaptic vesicle-containing varicosities. The dynamic behavior of the growth cone was under the control of NGF. Withdrawal of NGF resulted in the disappearance of motile structures and cessation of growth, while readdition of NGF triggered the rapid reappearance of these structures and the resumption of growth. The high-resolution video microscopy of living growth cones provides necessary baseline information, as well as a bioassay paradigm, for future studies on the molecular mechanisms of nerve growth.
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Large numbers of growth cones were present in 6-day-old primary cultures of cerebral hemispheres from fetal rats. The average size of the growth cones was 24 by 28 microns. Many of these growth cones had both veil-like lamellipodia and filopodia. A few cones remained in 21-day-old cultures. These also had lamellipodia and filopodia. Ganglioside GM1 was present in both 6-day-old and 21-day-old cultured growth cones.
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Actin filaments forming at the anterior margin of a migrating cell are essential for the formation of filopodia, lamellipodia, and pseudopodia, the "feet" that the cell extends before it. These structures in turn are required for cell locomotion. Yet the molecular nature of the "nucleator" that seeds the polymerization of actin at the leading edge is unknown. Recent advances, including video microscopy of actin dynamics, discovery of proteins unique to the leading edge such as ponticulin, the Mab 2E4 antigen, and ABP 120, and novel experimental models of actin polymerization such as the actin-based movements of intracellular parasites, promise to shed light on this problem in the near future.
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Electric fields (EFs) resembling those in the developing and regenerating nervous systems steer growth cones towards the cathode. Requirements for actin microfilaments, microtubules and their interactions during EF growth cone steering have been presumed, but remain unproven. Here, we demonstrate essential roles for dynamic microfilaments and microtubules in cathode-directed migration. Cathodal turning of growth cones on cultured Xenopus embryonic spinal neurons was attenuated significantly by nanomolar concentrations of the microfilament inhibitor latrunculin, the microtubule-stabilising drug taxol, or the microtubule-destabilising drugs vinblastine or nocodazole. Dynamically, the cathodal bias of filopodia preceded cathodal turning of the growth cone, suggesting an instructive role in EF-induced steering. Lamellipodial asymmetry accompanied turning. Filopodia and lamellipodia are regulated by the GTPases Cdc42 and Rac, respectively, and, as shown in the companion paper in this issue, peptides that selectively prevented effector binding to the CRIB domains of Cdc42 or Rac abolished cathodal growth cone turning during 3 hours of EF exposure. Here, the Rac peptide suppressed lamellipodium formation, increased the number of filopodia, abolished cathodal filopodial orientation, and prevented cathodal steering. The Cdc42 peptide suppressed filopodium formation, increased lamellipodial area and prevented cathodal steering. The cathodal bias of lamellipodia was independent of Cdc42 CRIB activity and was not sufficient for cathodal steering in the absence of filopodia, but the cathodal bias of filopodia through Rac CRIB activity was necessary for cathodal turning. Understanding the mechanism for cathodal growth cone guidance will enhance the emerging clinical effort to stimulate human spinal cord regeneration through EF application.
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The current dominant model of cell locomotion proposes that actin polymerization pushes against the membrane at the leading edge producing filopodia and lamellipodia that move the cell forward. Despite its success, this model does not fully explain the complex process of amoeboid motility, such as that occurring during embryogenesis and metastasis. Here, we show that Dictyostelium cells moving in a physiological milieu continuously produce `blebs' at their leading edges, and demonstrate that focal blebbing contributes greatly to their locomotion. Blebs are well-characterized spherical hyaline protrusions that occur when a patch of cell membrane detaches from its supporting cortex. Their formation requires the activity of myosin II, and their physiological contribution to cell motility has not been fully appreciated. We find that pseudopodia extension, cell body retraction and overall cell displacement are reduced under conditions that prevent blebbing, including high osmolarity and blebbistatin, and in myosin-II-null cells. We conclude that amoeboid motility comprises two mechanically different processes characterized by the production of two distinct cell-surface protrusions, blebs and filopodia-lamellipodia.
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