Transmembrane agrin regulates filopodia in rat hippocampal neurons in culture

McCroskery, Seumas; Chaudhry, Amal; Lin, Lin; Daniels, Mathew P.

doi:10.1016/j.mcn.2006.06.004

Cited by 46 publications

(79 citation statements)

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“…2 Lower). Experimentally reported lifetimes are on the order of several minutes, consistent with our results (6,(22)(23)(24)(25)(26). We found that computed filopodial lifetimes strongly depend on the amplitude of the individual filament length fluctuations (Fig.…”

supporting

confidence: 91%

Molecular noise of capping protein binding induces macroscopic instability in filopodial dynamics

Zhuravlev

Papoian

2009

Proc. Natl. Acad. Sci. U.S.A.

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Capping proteins are among the most important regulatory proteins involved in controlling complicated stochastic dynamics of filopodia, which are dynamic finger-like protrusions used by eukaryotic motile cells to probe their environment and help guide cell motility. They attach to the barbed end of a filament and prevent polymerization, leading to effective filament retraction due to retrograde flow. When we simulated filopodial growth in the presence of capping proteins, qualitatively different dynamics emerged, compared with actin-only system. We discovered that molecular noise due to capping protein binding and unbinding leads to macroscopic filopodial length fluctuations, compared with minuscule fluctuations in the actin-only system. Thus, our work shows that molecular noise of signaling proteins may induce micrometer-scale growth-retraction cycles in filopodia. When capped, some filaments eventually retract all the way down to the filopodial base and disappear. This process endows filopodium with a finite lifetime. Additionally, the filopodia transiently grow several times longer than in actin-only system, since less actin transport is required because of bundle thinning. We have also developed an accurate mean-field model that provides qualitative explanations of our numerical simulation results. Our results are broadly consistent with experiments, in terms of predicting filopodial growth retraction cycles and the average filopodial lifetimes. Eukaryotic motile cells project finger-like protrusions, called filopodia, to probe their environment and help guide cell motility (1). They play important roles in neuronal growth (2), wound healing (3), and cancer metastasis (4). The filopodial structure consists of parallel actin filaments, cross-linked into bundles by actin-binding proteins, all of which is enclosed by the cell's plasma membrane (1, 5). Despite their importance in eukaryotic biology and human health, the physical mechanisms behind filopodial regulation and dynamics are poorly understood, including what drives ubiquitous growth-retraction cycles and eventual filopodial disappearance (1-7). Using stochastic simulations of filopodial dynamics, we have discovered that molecular noise due to binding/unbinding of a capping protein results in macroscopic growth-retraction fluctuations, compared with minuscule fluctuations in the actin-only system. Because of rare fluctuations some filaments eventually retract all the way down to the filopodial base and disappear. In contrast to prior computational models that predicted stable filopodia at steady state, our simulations show that filopodial lifetimes are finite. We have also developed an accurate mean-field model that provides insights into filament disappearance kinetics.A comprehensive computational model of a filopodium should contain the following features: mechanical interactions including membrane dynamics, protrusion force, and retrograde flow; chemical interactions, including actin polymerization and depolymerization; and biological signaling int...

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supporting

confidence: 91%

Molecular noise of capping protein binding induces macroscopic instability in filopodial dynamics

Zhuravlev

Papoian

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Although some groups have examined filopodia in live cells by time lapse microscopy, structures that remained stationary (48) or went through only extension or retraction phases but not both processes (49 -51) were all scored for. In other time lapse studies, the actin content of filopodia-like protrusions was either not monitored at all or detected by F-actin staining after the end of the time lapse observations rather than simultaneously (52)(53)(54)(55). This lack of a standard definition of filopodia makes it difficult to compare results across various studies performed under different experimental conditions.…”

Section: Discussionmentioning

confidence: 99%

Rif-mDia1 Interaction Is Involved in Filopodium Formation Independent of Cdc42 and Rac Effectors

Goh

Sudhaharan

Lim³

et al. 2011

Journal of Biological Chemistry

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Filopodia are cellular protrusions important for axon guidance, embryonic development, and wound healing. The Rho GTPase Cdc42 is the best studied inducer of filopodium formation, and several of its effectors and their interacting partners have been linked to the process. These include IRSp53, N-WASP, Mena, and Eps8. The Rho GTPase, Rif, also drives filopodium formation. The signaling pathway by which Rif induces filopodia is poorly understood, with mDia2 being the only protein implicated to date. It is thus not clear how distinct the Rif-driven pathway for filopodium formation is from the one mediated by Cdc42. In this study, we characterize the dynamics of Rif-induced filopodia by time lapse imaging of live neuronal cells and show that Rif drives filopodium formation via an independent pathway that does not involve the Cdc42 effectors N-WASP and IRSp53, the IRSp53 binding partner Mena, or the Rac effectors WAVE1 and WAVE2. Rif formed filopodia in the absence of N-WASP or Mena and when IRSp53, WAVE1, or WAVE2 was knocked down by RNAi. Rif-mediated filopodial protrusion was instead reduced by silencing mDia1 expression or overexpressing a dominant negative mutant of mDia1. mDia1 on its own was able to form filopodia. Data from acceptor photobleaching FRET studies of protein-protein interaction demonstrate that Rif interacts directly with mDia1 in filopodia but not with mDia2. Taken together, these results suggest a novel pathway for filopodia formation via Rif and mDia1.Filopodia are dynamic, actin-rich cellular protrusions that are important for processes such as cell migration, neuritogenesis, axon guidance, wound healing, angiogenesis, embryonic development, and phagocytosis (1, 2). Elucidating the exact mechanism(s) by which filopodia form will give a greater understanding of these cellular processes and how such structures play a role in pathological conditions such as metastasis (3) and pathogen invasion (4, 5). The Rho GTPase Cdc42 is a key regulator of cell signaling events that lead to filopodium formation in mammalian cells. It binds to and activates IRSp53 (insulin receptor substrate protein 53 kDa) (6 -8). The Cdc42-IRSp53 complex induces filopodia by coupling membrane protrusion with actin dynamics (8). Interacting partners of IRSp53 include N-WASP (neural Wiskott-Aldrich syndrome protein) (8), Mena (mammalian enabled) (7, 9), Eps8 (EGF receptor kinase substrate 8) (10), and the Rac effectors WAVE (WASP family verprolin homology) isoforms WAVE1 (11-14) and WAVE2 (15). Yeast two-hybrid experiments have shown that IRSp53 binds mDia1 (mammalian Diaphanous 1) (16), but little else is known about this interaction. mDia1 and mDia2 (mammalian Diaphanous 2) belong to the formin family of multidomain eukaryotic proteins that are involved in a wide range of cellular processes that require actin polymerization (17). Formins contain an actin-nucleating formin homology 2 domain, and a proline-rich, formin homology 1 domain that binds Src homology 3 and WW domaincontaining proteins and profilin (18). Diaphanous-r...

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“…Functional studies provided additional evidence for a regulatory role of agrin on the rate of axonal and dendritic elongation in central neurons (Chang et al, 1997;Gautman et al, 1996;Ferreira, 1999;Mantych and Ferreira, 2001). This agrin effect was accompanied by clear changes in neuronal morphology including an enhanced branching pattern and the formation of filopodia-like processes protruding from axonal and dendritic shafts (Ferreira, 1999;Mantych and Ferreira, 2001;Annies et al, 2006;McCroskery et al, 2006). In this study, we showed that agrin also induced morphological changes in the growth cones of hippocampal neurons.…”

Section: Discussionmentioning

confidence: 54%

Agrin induced morphological and structural changes in growth cones of cultured hippocampal neurons

2007

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The role of agrin in synaptogenesis has been extensively studied. On the other hand, little is known about the function of this extracellular matrix protein during developmental processes that precede the formation of synapses. Recently, it has been shown that agrin regulates the rate of neurite elongation and the behavior of growth cones in hippocampal and spinal neurons, respectively. However, the molecular mechanisms underlying these effects have not been completely elucidated.In the present study, we analyzed the morphological and molecular changes induced by agrin in growth cones of hippocampal neurons that developed in culture. Morphometric analysis showed a significant enlargement of growth cones of hippocampal neurons cultured in the presence of agrin. These agrin-induced growth cone changes were accompanied by the formation of loops of microtubules highly enriched in acetylated tubulin and an increase in the content of the microtubuleassociated protein MAP1B. Together, these data provide further insights into the potential molecular mechanisms underlying the effects of agrin on neurite outgrowth in central neurons.

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Transmembrane agrin regulates filopodia in rat hippocampal neurons in culture

Cited by 46 publications

References 47 publications

Molecular noise of capping protein binding induces macroscopic instability in filopodial dynamics

Molecular noise of capping protein binding induces macroscopic instability in filopodial dynamics

Rif-mDia1 Interaction Is Involved in Filopodium Formation Independent of Cdc42 and Rac Effectors

Agrin induced morphological and structural changes in growth cones of cultured hippocampal neurons

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