Interaction of nerve growth factor with tubulin. Studies on binding and induced polymerization

Levi, Andrea; Cimino, Mauro; Mercanti, Delio; Chen, John S.; Calissano, Pietro

doi:10.1016/0304-4165(75)90210-x

Cited by 35 publications

(10 citation statements)

References 9 publications

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“…These aggregates, at the light microscopic level, have features similar to those of the C3βT‐NIIs described in the present study. It is intriguing in this context that nerve growth factor (NGF) can induce the formation of similar structures [6,7,31]. Moreover, NGF has been localized to the nuclei of responsive cells where it appears as small aggregates which, like some of the C3βT‐NIIs, are intimately associated with nucleoli [34].…”

Section: Discussionmentioning

confidence: 99%

Tubulin immunoreactive neuronal intranuclear inclusions in the human brain

Woulfe

Muñoz

2000

Neuropathology Appl Neurobio

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Intranuclear filamentous and crystalline inclusion bodies have been described in the nuclei of a variety of cells in both normal and pathological states. The functional significance of these structures remains to be elucidated. Moreover, although the proteinaceous nature of these inclusions has been inferred in some histochemical studies, the identity of their constituent proteins remains to be determined. In the present study, immunohistochemistry was used to investigate the presence of intranuclear inclusions in neurones of the human brain which are intensely immunoreactive for the neuronal cytoskeletal protein class III beta tubulin. The ability to label these structures immunohistochemically was exploited to investigate the topographic pattern of distribution of these inclusions in the human brain. Intranuclear inclusions were rod-shaped, polygonal, or irregular in shape. They were present in neurones and ependymal cells. Intranuclear inclusion-bearing neurones were distributed in an anatomically heterogeneous pattern in the brain. Areas exhibiting relatively high densities of inclusions included the substantia inominata and anterior olfactory nucleus, dentate gyrus, substantia nigra, inferior olivary nucleus, and dentate nucleus of the cerebellum. In addition, intranuclear inclusions were prevalent in neurones in layers II, V, and VI of the cerebral cortex. They were particularly prevalent in the mesial basal temporal neocortex. The relationship of these structures to the intranuclear rods and sheets of the classical microscopists is uncertain. The demonstration that they are composed, at least in part, of tubulin, a major cytoskeletal protein, provides important clues regarding the mechanisms underlying their formation and provides a springboard for developing hypotheses regarding their functional significance. Furthermore, the ability to demonstrate these inclusions immunohistochemically provides an avenue for further studies directed at elucidating the potential involvement of these inclusions in various pathological settings.

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Section: Discussionmentioning

confidence: 99%

Tubulin immunoreactive neuronal intranuclear inclusions in the human brain

Woulfe

Muñoz

2000

Neuropathology Appl Neurobio

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“…In vitro studies of binding of NGF to tubulin and to actin in their assembled or disassembled state have found a marked effect of this ligand on their rate of assembly, association into large bundles, and other physicochemical changes (11,12,26,27). These effects have been related to the mechanism ofaction of NGF with special relevance to the process ofneurite growth and elongation.…”

Section: Discussionmentioning

confidence: 99%

Microtubules and microfilaments in fixed and permeabilized cells are selectively decorated by nerve growth factor.

Nasi¹,

Cirillo

Naldini

et al. 1982

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

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A specific antibody against nerve growth factor (NGF) and indirect immunofluorescence microscopy have been used to follow the in vitro binding of NGF to cells made permeable to large molecules. All cells tested, both target (sensory neurons and PCI2 cells) and nontarget (3T3, BKH 21, C6 glioma cells), revealed a decoration of cytoskeletal structures which on the basis of their form, reactivity with antibodies, and sensitivity to specific drugs may be identified as microtubules (MTs) and microfilaments (MFs). The decoration of either structure depends on the fixation and permeabilization conditions: MFs, in the form ofstress fibers, are stained by NGF when the plasma membrane is permeabilized with methanol/acetone; MTs become intensely stained when the plasma membrane is solubilized with a nonionic detergent in the presence of a MT-stabilizing medium. The two procedures do not affect the staining of these structures with specific antibodies.Binding of "MI-labeled NGF to PCI2 cells was not competitively inhibited by a 100-fold excess ofseveral positively charged proteins but it was markedly decreased in the presence of DNase L. 125I Labeled NGF interacted with MTs and F-actin (fixed with paraformaldehyde) in a range ofconcentrations similar to that used for their cellular localization with NGF-anti-NGF. Our studies show that the specificity and affinity of NGF binding to MTs and MFs is in the range of that of antibodies against tubulin and actin. The possible relevance of these findings to the mechanism of action of NGF in target cells is discussed.After the protein nerve growth factor (NGF) (1) binds to receptors located on the cytoplasmic membrane or at the nerve terminals of target cells (2, 3), it is internalized into the cytoplasm (4,5). In pheochromocytoma cells (clone PC12), it is localized around the nuclear outline and within the nucleus in a paranucleolar position (6). It has been suggested that such an intracellular pool ofNGF could have a role in the induction ofneurite growth (7-10). An important contribution to solving this problem would be provided by a precise localization of NGF in the cell. Some studies along this line have been attempted but the weakness of the signal detected with autoradiographic or immunofluorescence techniques (6) has permitted relatively precise localization of only a fraction of the intracellular NGF. In order to overcome the fact that only a minute amount of NGF enters living target cells, we have facilitated the intracellular diffusion of a much larger quantity of this protein into fixed and permeabilized cells and detected this ligand by indirect immunofluorescence microscopy. It has been found that NGF is not evenly distributed in the cytoplasm. It selectively binds and decorates cytoskeletal elements such as microtubules (MTs) and microfilaments (MFs) to an extent and with a specificity comparable to those obtained with specific antibodies. These findings add support to the hypothesis (11, 12) that interaction of NGF with these structures may occur in the l...

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“…It has been shown that NGF interacts in vitro with tubulin (4) and actin (5) precursor elements of microtubules and microfilaments (MFs) and changes some of their most important physicochemical properties (6)(7)(8)(9). These findings led us to hypothesize a relationship between these in vitro effects and in vivo NGF induction of fiber growth, postulating a direct action of this growth factor on some functional properties of filamentous proteins (5,8).…”

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confidence: 99%

“…Binding to G actin was performed with the same procedure adopted for studies with tubulin (5,6 tating with actin was measured after subtracting the contribution of the same amount of NGF incubated and centrifuged in the absence of actin. Stoichiometry of NGF bound to F-actin was calculated by measuring the amount of protein in the pellet induced to precipitate by NGF and subtracting its contribution (generally between 0.1 and 5%) to the whole amount of protein measured.…”

mentioning

confidence: 99%

Nerve growth factor potentiates actomyosin adenosinetriphosphatase.

Calissano¹,

Monaco²,

Castellani³

et al. 1978

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

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The nerve growth factor protein (NGF) favors polymerization of brain actin and induces its organization to form paracrystalline structures that activate myosin ATPase (ATP phosphohydrolase, EC 3.6.1.3) to an extent greater than actin alone. Binding studies show that the initial 1:1 stoichiometry of NGF-G-actin complexes decreases to 1:7-10 when polymerization is ended and paracrystalline structures are formed. The ratio becomes even lower when heavy meromyosin is added in the absence of ATP, suggesting that heavy meromyosin displaces NGF bound to actin microfilaments. This conclusion is supported by the finding that when heavy meromyosin is added to NGF-microfilament complexes, under conditions for "decorating" microfilaments, the usual paracrystalline structure of the complexes disappears. The NGFmediated organization of actin and activation of myosin ATPase is visualized as a self-regulatory and self-propagating mechanism, because progressive displacement of the growth factor induced by heavy meromyosin binding to F actin as ATP consumption proceeds renders an increasingly higher amount of NGF free for new interactions. These findings are discussed in the light of the mechanism of action of NGF in the target cells. The earliest ultrastructural change accompanying neurite growth induced by nerve growth factor (NGF) (1) in the target cells is the appearance and organization of microtubules and other filamentous proteins (2). These contractile elements play an essential role in fiber growth and elongation, as shown by the finding that inhibitors of their assembly and/or function, such as colchicine or cytochalasin B, block neurite growth or induce fiber retraction when added to preformed neurites (3).It has been shown that NGF interacts in vitro with tubulin (4) and actin (5) precursor elements of microtubules and microfilaments (MFs) and changes some of their most important physicochemical properties (6-9). These findings led us to hypothesize a relationship between these in vitro effects and in vivo NGF induction of fiber growth, postulating a direct action of this growth factor on some functional properties of filamentous proteins (5,8).This paper describes the properties of the NGF-actin complexes, shows how they activate myosin ATPase (ATP phosphohydrolase, EC 3.6.1.3), and discusses how these findings could fit with the hypothesis that NGF-mediated neutrite growth may involve a direct interaction with the contractile network formed by microtubules, MFs, and their precursor elements.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.METHODS AND MATERIALS Purification of NGF, Brain Actin, Muscle Myosin, and Heavy Meromyosin. The 2.5S NGF was purified from mouse salivary gland according to the procedure described by Bocchini and Angeletti (10). The slight ATPase activity still contaminating the growth factor after the first CM-cellulo...

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Interaction of nerve growth factor with tubulin. Studies on binding and induced polymerization

Cited by 35 publications

References 9 publications

Tubulin immunoreactive neuronal intranuclear inclusions in the human brain

Tubulin immunoreactive neuronal intranuclear inclusions in the human brain

Microtubules and microfilaments in fixed and permeabilized cells are selectively decorated by nerve growth factor.

Nerve growth factor potentiates actomyosin adenosinetriphosphatase.

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