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

Abstract

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...

Electron microscopic radioautographic localization of iodinated nerve growth factor bound to and internalized by PC12 cells

“…Several models regarding a role for internalized NGF have been put forth. For instance, it has been postulated, on the basis of the interaction of NGF with tubulin and actin, that NGF may bind directly to cytoskeletal elements and promote their assembly and/or stabilization (Nasi et al, 1982). However, in our studies such a localization was not manifestly evident, and if such interactions were to occur, they would appear to do so at "substoichiometric" (Nasi et al, 1982) levels.…”

“…In addition to this evidence, subcellular fractionation of cells that had retrogradely transported NGF indicated that 20 to 30% of the factor was present in the nuclear fraction (Stockel et al, 1976;Johnson et al, 1978). Yet another set of studies (Nasi et al, 1982) has been interpreted to suggest that internalized NGF may directly interact with several elements of the cytoskeleton.…”

Cytoskeletal elements regulate the distribution of nerve growth factor receptors in PC12 cells

“…This may reduce the ability of tubulin to bind to the cell surface (Parness and Hor-tribution of NGFR. Previous studies described NGF associations with cytoskeletal elements following its binding to receptors on PC12 cells (Schechter and Bothwell, 1981;Nasi et al, 1982;Vale et al, 1985).…”

“…Coverslips were washed five times with HBSS and processed for TEM. TEM demonstrated on microtubules (MTs) and microfilaments (MFs) (Nasi et al, 1982), cytoskeletal altering agents were applied prior to immunolocalization of the NGFR, to determine the degree and nature of the NGFR associations to the underlying cytoskeleton.…”

Hidden receptors for nerve growth factor in PC12 cells

“…2A, and Calissano and Kinihi, unpublished). 3T3 cells show a lower affinity binding for N C F following paraformaldehyde fixation and methanol permeabilization which may be responsible for the fluorescent staining of cytoskeletal elements by N G F [12]. C6, like PC12 cells, show cryptic NGF binding sites with an equilibrium binding constant identical to that measured on intact cells (Fig.…”