Involvement of the membrane cytoskeletal proteins and the src gene product in growth cone adhesion and movement

doi:10.1016/0921-8696(90)90035-2

Neuroscience Research Supplements

Volume 13, 1990, Pages S80-S91

https://doi.org/10.1016/0921-8696(90)90035-2 Get rights and content

Abstract

The neuronal growth cone is a highly motile and adhesive structure, leading to maintain and promote neurite outgrowth. Using immunocytochemical and biochemical techniques, we investigated the regional distribution of the membrane cytoskeletal proteins, such as α-actinin, calspectin (nonerythroid spectrin or fodrin) and actin, and the proto-oncogene product, pp60^c-src, in the growth cone. During a course of this study, the two types of α-actinin, having Ca²⁺-sensitive and -insensitive actin-binding abilities, were identified. These three membrane cytoskeletal proteins and pp60^c-src showed discrete differential distributions coinciding with the different functions of the growth cone substractures. Ca²⁺-sensitive α-actinin, calspectin and pp60^c-src were observed to localize in the growth cone body and the distal portion of neurites, which are the adhesive sites of growth cone and neurite. By contrast, Ca²⁺-sensitive α-actinin and actin were densely concentrated in the filopodia, These results suggest that Ca²-insensitive α-actinin, calspectin and pp60^c-src may be involved in adhesiveness of growth cone, and Ca²⁺-sensitive α-actinin and actin in Ca²⁺-dependent filopodial movement. Furthermore, we will discuss the functional and structural similarities between the growth cone and the motile contact which is also the adhesive site of motile, transformed and cancer cells.

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The podosome and invadopodium are dynamic cell-adhesion structures that degrade the extracellular matrix (ECM) and promote cell invasion. We recently reported that the actin-binding protein caldesmon is a pivotal regulator of podosome formation. Here, we analyzed the caldesmon’s involvement in podosome/invadopodium-mediated invasion by transformed and cancer cells. The ectopic expression of caldesmon reduced the number of podosomes/invadopodia and decreased the ECM degradation activity, resulting in the suppression of cell invasion. Conversely, the depletion of caldesmon facilitated the formation of podosomes/invadopodia and cell invasion. Taken together, our results indicate that caldesmon acts as a potent repressor of cancer cell invasion.
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Podosomes are dynamic cell adhesion structures that degrade the extracellular matrix, permitting extracellular matrix remodeling. Accumulating evidence suggests that actin and its associated proteins play a crucial role in podosome dynamics. Caldesmon is localized to the podosomes, and its expression is down-regulated in transformed and cancer cells. Here we studied the regulatory mode of caldesmon in podosome formation in Rous sarcoma virus-transformed fibroblasts. Exogenous expression analyses revealed that caldesmon represses podosome formation triggered by the N-WASP-Arp2/3 pathway. Conversely, depletion of caldesmon by RNA interference induces numerous small-sized podosomes with high dynamics. Caldesmon competes with the Arp2/3 complex for actin binding and thereby inhibits podosome formation. p21-activated kinases (PAK)1 and 2 are also repressors of podosome formation via phosphorylation of caldesmon. Consequently, phosphorylation of caldesmon by PAK1/2 enhances this regulatory mode of caldesmon. Taken together, we conclude that in Rous sarcoma virus-transformed cells, changes in the balance between PAK1/2-regulated caldesmon and the Arp2/3 complex govern the formation of podosomes.
C-src activation by ErbB2 leads to attachment-independent growth of human breast epithelial cells
1998, Biochemical and Biophysical Research Communications
Nontumorigenic human mammary epithelial cells (184.A1 line) were stably transfected with ErbB2 or with Ha-Ras. Transformation with ErbB2, but not ras, resulted in a 5-6 fold increase in c-src activity without affecting c-src content of cells. Similar activation of c-src by ErbB2 was also observed in other non-tumorigenic mammary epithelial cells, including the human line MCF10A and the mouse line NMuMG. Activation of c-src appeared to be dependent on active ErbB2 tyrosine kinase, as the ErbB2 inhibitor tyrphostin AG 825 blocked the induction of c-src kinase activity, as well as the ability of transformed cells to grow on soft agar, but not plastic. The src-selective inhibitor PP1 effectively reduced c-src activity, as well as growth of ErbB2-transformed cells on soft agar, but not on plastic. These results indicate that activation of c-src is a consequence of ErbB2 kinase activity in human breast cancer cells overexpressing ErbB2, and that increased activity of c-src may be responsible for attachment-independent growth of the cells.
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To investigate whether overexpression of human c-Src leads to cell rounding in anchorage-dependent NIH 3T3 fibroblasts, we have established c-Src-inducible cell lines using a lac repressor–operator system. RN1 cells, which expressed c-Src at a high level after induction, exhibited a spherical morphology and ceased to grow in monolayer culture. RN1 cells, however, exhibited neither focus-forming ability nor anchorage-independent growth potential with or without induction. Induced RN1 c-Src was phosphorylated at Ser⁷⁵, a previously reported spherical cell-associated site, and at Tyr⁴¹⁹. These data demonstrated for the first time that highly elevated human c-Src tyrosine kinase activity can cause NIH 3T3 cells to have a spherical morphology without loss of anchorage-dependent growth. The inducible cell line should be useful to study the mechanism for cell rounding by c-Src.
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Effects of exposure to ethylnitrosourea (ENU) on expression of proteins that play a role in neuronal differentiation were examined in central nervous system (CNS) micromass embryo cell cultures. ENU is a known developmental toxicant which affects neuronal development. The proteins selected were the protein product of the src proto-oncogene (pp60^c-src) and high-molecular-weight neurofilament protein (NF). pp60^c-src has marked increases in amount and kinase activity in neurons at the time of differentiation and NF is found in differentiated neurons. CNS micromass cultures are primary cells from Day 12 rat embryo midbrains which are plated at high density and differentiate into neurons during 5 days in culture. Proteins were quantitated by polyacrylamide gel electrophoresis separation of equal amounts of total cell protein followed by transfer to membranes, immunoblotting, and densitometric scanning of blots. Dose-dependent decreases in cell growth and differentiation were confirmed using endpoints of cell number, protein content of cultures, neutral red uptake, hematoxylin staining of differentiated cells, and levels of binding of the neurotransmitter [³H]γ-aminobutyric acid. Concentrations,which inhibited response by 50% compared to controls ranged from 232 to 455 μM ENU. Dose-related decreases in amounts of pp60^c-src and NF proteins relative to total protein were seen in CNS cultures treated with ENU. Results confirm the usefulness of the micromass cultures in following chemical effects on neuronal differentiation. The effects of ENU on specific proteins associated with neuronal differentiation were shown.
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This chapter describes the early development of the retinotectal system of the African clawed frog Xenopus laevis. Early work on the Xenopus retinotectal projection was done by manipulating the embryo and assaying the effects after metamorphosis, but, over the past decade, attention has shifted to embryonic assays. Embryonic experiments have the practical advantage of taking days instead of months, and they also have a great conceptual advantage. Postmetamorphic studies show the distant shadows of initial development. Embryonic experiments, on the other hand, allow direct observation of the processes of initial development. Amphibians have long been favored for embryological studies because their external fertilization and development make them easy to observe and manipulate. Xenopus are especially useful because they are easily raised in captivity, can be induced to breed by hormonal injection, produce hundreds of embryos per mating, and develop very quickly during embryonic stages.

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Mechanism of neurite growthInvolvement of the membrane cytoskeletal proteins and the src gene product in growth cone adhesion and movement

Abstract

Dev. Biol.

Biochem. Biophys. Res. Commun.

Exp. Cell Res.

Neuron

Dev. Biol.

Cellular oncogenes and retroviruses

Ann. Rev. Biochem.

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

J. Cell Biol.

Changes in the pattern of expression of pp60c-src in cerebellar mutants of mice

J. Neurosci. Res.

Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum

Mol. Cell. Biol.

Digital imaging of free calcium changes and of spatial gradients in growing processes in single, mammalian central nervous system cells

Neural tissues express high levels of the cellular src gene product pp60c-src

Mol. Cell. Biol.

PC12 pheochromocytoma cultures in neurobiological research

Adv. Cell. Neurobiol.

Mechanism of neurite growth
Involvement of the membrane cytoskeletal proteins and the src gene product in growth cone adhesion and movement

Changes in the pattern of expression of pp60^c-src in cerebellar mutants of mice

Alterations in pp60^c-src accompany differentiation of neurons from rat embryo striatum

Neural tissues express high levels of the cellular src gene product pp60^c-src