Experimental modification of PC12 neurite shape with the microtubule- depolymerizing drug Nocodazole: a serial electron microscopic study of neurite shape control

The microtubule-depolymerizing drug Nocodazole has been used to experimentally manipulate the form of PC12 neurites. Both time-lapse photography and serial electron microscopy demonstrate that microtubule depolymerization leads to varicosity formation due to a clustering of membranous organelles in young neurites (nerve growth factor activated within 7 d). Neurites that have been nerve growth factor activated 7 or more d before Nocodazole application are resistant to microtubule depolymerization. These data and data from previous papers has been combined in an attempt to predict quantitatively the volume and the shape of a neurite. The relationship is described mathematically by Vn = 4.52 Vo + 0.0054 MTl, where Vn is local neurite volume, Vo is organelle volume, and MTl is MT length (the constant, 0.0054 is micron2), and 4.52 is the obligatory volume constant derived from serial electron microscopic studies. The equation predicts the total volume of neurites despite alterations of morphology due to Nocodazole and despite changes in morphology during development.     

Extracellular matrix allows PC12 neurite elongation in the absence of microtubules

Several groups have shown that PC12 will extend microtubule-containing neurites on extracellular matrix (ECM) with no lag period in the absence of nerve growth factor. This is in contrast to nerve growth factor (NGF)-induced neurite outgrowth that occurs with a lag period of several days. During this lag period, increased synthesis or activation of assembly-promoting microtubule-associated proteins (MAPs) occurs and is apparently required for neurite extension. We investigated the growth and microtubule (MT) content of PC12 neurites grown on ECM in the presence or absence of inhibitors of neurite outgrowth. On ECM, neurites of cells with or without prior exposure to NGF contain a normal density of MTs, but frequently contain unusual loops of MTs in their termini that may indicate increased MT assembly. On ECM, neurites extend from PC12 cells in the presence of 10 microM LiCl at significantly higher frequency than on polylysine. On other substrates, LiCl inhibits neurite outgrowth, apparently by inhibiting phosphorylation of particular MAPs (Burstein, D. E., P. J. Seeley, and L. A. Greene. 1985. J. Cell Biol. 101:862-870). Although 35-45% of 60 Li(+)-neurites examined were found to contain a normal array of MTs, 25-30% were found to have a MT density approximately 15% of normal. The remaining 30% of these neurites were found to be nearly devoid of MTs, containing only occasional, ambiguous, short tubular elements. We also found that neurites would extend on ECM in the presence of the microtubule depolymerizing drug, nocodazole. At 0.1 micrograms/ml nocodazole, cells on ECM produce neurites that contain a normal density of MTs. This is in contrast to the lack of neurite outgrowth and retraction of extant neurites that this dose produces in cells grown on polylysine. At 0.2 microgram/ml nocodazole, neurites again grew out in substantial number and four of five neurites examined ultrastructurally were found to be completely devoid of microtubules. We interpret these results by postulating that growth on ECM relieves the need for MTs to serve as compressive supports for neurite tension (Dennerll, T. J., H. C. Joshi, U. L. Steel, R. E. Buxbaum, and S. R. Heidemann. 1988. J. Cell Biol. 107:665). Because compression destabilizes MTs and favors disassembly, this would tend to increase MT assembly relative to other conditions, as we found. Additionally, if MTs are not needed as compressive supports, neurites could grow out in their absence, as we also observed.     

Drag Force as a Tool to Test the Active Mechanical Response of PC12 Neurites

We investigate the mechanical response of PC12 neurites subjected to a drag force imposed by a laminar flow perpendicular to the neurite axis. The curvature of the catenary shape acquired by an initially straight neurite under the action of the drag force provides information on both elongation and tension of the neurite. This method allows us to measure the rest tension and viscoelastic parameters of PC12 neurites and active behavior of neurites. Measurement of oscillations in the strain rate of neurites at constant flow rate provides insight on the response of molecular motors and additional support for the presence of a negative strain-rate sensitivity region in the global mechanical response of PC12 neurites.     

Tension and compression in the cytoskeleton of PC 12 neurites

We report in this article that the retraction of PC 12 neurites, unlike that of other cultured neurons, is due to tension within the neurite. Retraction is rapid and independent of metabolic energy. Transection of one arm of a branched neurite immediately causes the remaining arm to take up a new equilibrium position between attachment points. Similarly, detachment of one growth cone of a cell causes the cell body to move to a new equilibrium position between the remaining neurites. These observations provide direct evidence for the suspension of the cell soma among a network of tensioned neurites. We used retraction as an assay for neurite tension to examine the role of actin filaments and microtubules in neurite support and elongation. Our data suggest that microtubules (MTs) within PC 12 neurites are under compression, supporting tension within the actin network. Treatment of cells with drugs that disrupt actin networks, cytochalasin D or erythro-9-[3-(2-hydroxynonyl)]adenosine eliminates retraction regardless of the absence of MTs, lack of adhesion to the substratum, or integrity of the neurite. Conversely, stimulation of actin polymerization by injection of phalloidin causes retraction of neurites. Treatments that depolymerize MTs, nocodazole or cold, cause retraction of neurites, which suggests that microtubules support this tension, i.e., are under compression. Stabilization of MTs with taxol stabilizes neurites to retraction and under appropriate circumstances can drive neurite extension. Taxol-stimulated neurite extension is augmented by combined treatment with anti-actin drugs. This is consistent with the actin network's normally exerting a force opposite that of MT assembly. Cytochalasin and erythro-9-[3-(2-hydroxynonyl)] adenosine were found to increase slightly the dose of nocodazole required for MT depolymerization. This is consistent with the postulated balance of forces and also suggests that alteration of the compression borne by the microtubules could serve as a local regulator for MT polymerization during neurite outgrowth.     

Effects of soluble laminin on organelle transport and neurite growth in cultured mouse dorsal root ganglion neurons: difference between primary neurites and branches

Laminin, an extracellular matrix molecule, is known to promote neurite growth. In the present study, the effects of soluble laminin on organelle transport and their relation to neurite growth were investigated in cultured dissociated mouse dorsal root ganglion (DRG) neurons. Laminin added into the extracellular medium was deposited on the surface of DRG neurons. DRG neurons incubated with soluble laminin exhibited branched, long, and thin neurites. Time-lapse study demonstrated that many small-diameter branches were newly formed after the addition of laminin. Thus, the growths of large-diameter primary neuritis, arising from cell bodies and branches extended from growth cones of primary neuritis, were analyzed separately. Laminin decreased the growth rate of primary neurites but increased that of branches. In primary neurites, acute addition of laminin rapidly decreased organelle movement in the neurite shaft and growth cone, accompanied by slowing of the growth cone advance. Branching of primary neurites occurred in response to laminin in some growth cones. In these growth cones, organelles protruded into nascent branches. In branches, soluble laminin increased organelle movement in the growth cone and the distal portion of the shaft. These results suggest that laminin inhibits the elongation of primary neurites but promotes branching and elongation of branches, all of which seem to be closely related to organelle transport.     

The cytoskeleton of neurites after microtubule depolymerization

We previously reported a positive correlation between the number of cold-stable microtubules (MTs) remaining after cold treatment of cat sympathetic nerve and the extent to which the original uniform polarity orientation of axonal MTs was recapitulated after rewarming (J cell biol 99 (1984) 1289). We interpreted these data to indicate that cold-stable fragments, part of larger, generally labile MTs, could act as seeds to organize MT assembly in axons. We report here a direct investigation of the form of cold-stable MTs in neurites of PC-12 cells using two-dimensional reconstruction of serial thin sections. Our data provides strong support for our previous interpretation. The number of MTs in cold-treated neurites was 2-3 times as great while the total length of polymer was approximately half that in control neurites. The average length of MTs in cold-treated neurites was 7-10 times lower than in control neurites. We observed that treatments that depolymerize axonal microtubules cause a marked increase in the number of membranous elements within the axoplasm. This may, however, be a non-specific result of an insult to the axon, since such changes have also been observed in severed, regenerating nerve fibres. We observed that neuroblastoma neurites respond to MT-depolymerization stimuli by developing lateral filopodia similar to those observed in chick dorsal root ganglion cells. Ultrastructural observation of detergent-lysed, whole mounted neuroblastoma (Neuro 2A) cells indicated that the cytoskeleton remaining after MT depolymerization splayed out perpendicular to the long axis of the neurite. That is, we were able to observe many more cytoskeletal 'ends' after MT depolymerization. The concomitant production of filopodia and the splaying of the cytoskeleton after MT depolymerization supports the hypothesis put forward by Wessels et al. (Exp cell res 117 (1978) 335) that the presence or absence of cytoskeletal ends regulates which region of the cell surface is involved in motile behaviour.     

Microtubule dynamics in nerve cells: analysis using microinjection of biotinylated tubulin into PC12 cells

To study microtubule (MT) dynamics in nerve cells, we microinjected biotin-labeled tubulin into the cell body of chemically fused and differentiated PC12 cells and performed the immunofluorescence or immunogold procedure using an anti-biotin antibody followed by secondary antibodies coupled to fluorescent dye or colloidal gold. Incorporation of labeled subunits into the cytoskeleton of neurites was observed within minutes after microinjection. Serial electron microscopic reconstruction revealed that existing MTs in PC12 neurites incorporated labeled subunits mainly at their distal ends and the elongation rate of labeled segments was estimated to be less than 0.3 micron/min. Overall organization of MTs in the nerve cells was different from that in undifferentiated cells such as fibroblasts. Namely, we have not identified any MT-organizing centers from which labeled MTs are emanating in the cell bodies of the injected cells. Stereo electron microscopy revealed that some fully labeled segments seemed to start in the close vicinity of electron dense material within the neurites. This suggests new nucleation off some structures in the neurites. We have also studied the overall pattern of the incorporation of labeled subunits which extended progressively from the proximal part of the neurites toward their tips. To characterize the mechanism of tubulin incorporation, we have measured mean density of gold labeling per unit length of labeled segments at different parts of the neurites. The results indicate access of free tubulin subunits into the neurites and local incorporation into the neurite cytoskeleton. Our results lead to the conclusion that MTs are not static polymers but dynamic structures that continue to elongate even within the differentiated nerve cell processes.     

Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells

High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.     

Different Roles for RhoA During Neurite Initiation, Elongation, and Regeneration in PC12 Cells

The goal of the present study was to characterize the effects of RhoA at different stages of nerve growth factor (NGF)-induced neuronal differentiation in the PC12 model. This comparative analysis was prompted by previous studies that reported apparently opposite effects for Rho in different models of neuronal differentiation and regeneration. PC12 cells were transfected with activated V14RhoA or dominant negative N19RhoA under the control of either a constitutive or a steroid-regulated promoter. Upon exposure to NGF, V14RhoA cells continued to proliferate and did not extend neurites; however, they remained responsive to NGF, as indicated by the activation of extracellular signal-regulated kinases. This inability to differentiate was reversed by C3 toxin and activation of cyclic AMP signaling, which inactivate RhoA. N19RhoA expression led to an increase in neurite initiation and branching. In contrast, when the RhoA mutants were expressed after NGF priming, only the rate of neurite extension was altered; V14RhoA clones had neurites approximately twice as long, whereas neurites of N19RhoA cells were approximately 50% shorter than those of appropriate controls. The effects of Rho in neurite regeneration mimicked those observed during the initial stages of morphogenesis; activation inhibited, whereas inactivation promoted, neurite outgrowth. Our results indicate that RhoA function changes at different stages of NGF-induced neuronal differentiation and neurite regeneration.     

Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho

Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA- and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.     

Intracellular control of axial shape in non-uniform neurites: a serial electron microscopic analysis of organelles and microtubules in AI and AII retinal amacrine neurites

AI and AII cat retinal amacrine cells have highly varicose non-uniform, neuritic processes. Processes of both types were reconstructed via a computer system using serial electron micrographs. These reconstructions were analyzed for (a) varicosity volume, surface area, and length, (b) "neck" volume, surface area, and length, (c) number of microtubules within the varicosity, (d) number of microtubules within the "neck," and (e) volume and surface area of mitochondria and smooth endoplasmic reticulum and large smooth vesicular bodies within the processes. Correlation of these parameters revealed a linear relationship between the number of microtubules in the necks and mean neck cross-sectional area (rs = 0.780, P less than 0.001), while microtubule number within the varicosities showed no correlation with varicosity volume (rs = 0.239, P greater than 0.2). Varicosity volume did, however, correlate strongly with the summed volume of mitochondria and smooth vesicular bodies contained within the varicosity for both cell types examined. The ratio between membranous organelle volume and varicosity volume for AI amacrine processes of 1:6.97 (rs = 0.927), differed from the ratio of 1:1.80 for the AII amacrine processes (rs = 0.987). Similar relationships were observed in other nonvaricose neurites such as optic tract axons. Membranous organelles appear to contribute an additional obligatory volume to the cytosol that can be as much as seven times the organelles' direct volume. These observations suggest that both the cytoskeletal components, and the membrane organelles play a direct role in determining neurite shape.     

Modeling transport of a pulse of radiolabeled organelles in a Drosophila unipolar motor neuron

Based on published experimental evidence, this paper develops a model for the transport of a pulse of radiolabeled organelles in a unipolar Drosophila motor neuron. In particular, since published data indicate that no microtubules (MTs) travel from the primary neurite into the dendrite, it is investigated how organelles are transported into the dendrite. Analytical solutions describing concentrations of kinesin- and dynein-driven organelles in the primary neurite, axon, and dendrite are obtained. The effects of increasing the width of the pulse and increasing the rate of organelle transition rate from the kinesin-driven to the dynein-driven state are investigated.     

The neurite-initiating effect of microbial extracellular glycolipids in PC12 cells

The effects of several kinds of microbial extracellular glycolipids on neurite initiation in PC12 cells were examined. Addition of mannosylerythritol lipid-A (MEL-A), MEL-B, and sophorose lipid (SL) to PC12 cells caused significant neurite outgrowth. Other glycolipids, such as polyol lipid (PL), rhamnose lipid (RL), succinoyl trehalose lipid-A (STL-A) and STL-B caused no neurite-initiation. MEL-A increased acetylcholine esterase (AChE) activity to an extent similar to nerve growth factor (NGF). However, MEL-A induced one or two long neurites from the cell body, while NGF induced many neurites. In addition, MEL-A-induced differentiation was transient, and after 48 h, percentage of cells with neurites started to decrease in contrast to neurons induced by NGF, which occurred in a time-dependent manner. MEL-A could induce neurite outgrowth after treatment of PC12 cells with an anti-NGF receptor antibody that obstructed NGF action. These results indicate that MEL-A and NGF induce differentiation of PC12 cells through different mechanisms. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ultrastructural effects of nerve growth factor on PC 12 pheochromocytoma cells in spinner culture

PC12 pheochromocytoma cells treated with nerve growth factor (NGF) for two weeks in spinner cultures quickly begin to form processes after plating on an appropriate substrate, while cells freshly exposed to NGF in monolayer culture initiate neurite outgrowth only after a lag period of several days. The present ultrastructural studies indicate that PC 12 cells treated with NGF in spinner cultures do not form neurites, but do form short extensions comparable to those which have been reported within the first two days of exposure to NGF in monolayer cultures. These extensions contain organelles believed to be required for locomotion and for transport of cytoskeletal and membrane components and neurotransmitters. They also form bulbous distensions in which numerous chromaffin-type granules accumulate. These findings suggest that NGF may affect cells in spinner cultures by promoting development or activation of axonal transport mechanisms, and that the existence of these mechanisms may contribute to the neurite outgrowth which the cells exhibit when plated. NGF-treated PC 12 cells in spinner cultures do not accumulate the agranular synaptic-like vesicles, which are typically found in comparably treated monolayer cultures and which have been hypothesized to be sites of acetylcholine storage. These and other data demonstrate that attachment to a substrate can selectively modulate the responses of PC 12 cells to NGF.     

Differential roles of ERK and JNK in early and late stages of neuritogenesis: a study in a novel PC12 model system

The rat pheochromocytoma PC12 cell line has been an invaluable model system for studying neuritogenesis. Nerve growth factor (NGF) elicits multiple aspects of neurite outgrowth in PC12 cells. It is therefore difficult to dissect and assign an individual signaling pathway to each stage of neuritogenesis. We have recently reported the isolation of a variant PC12 cell line, PC12-N1 (N1), which spontaneously extends neuritic processes and exhibits an increased sensitivity to NGF. Here, we show that, under different culture conditions, the cells display three distinct phases of neuritogenesis consisting of neurite initiation, rapid neurite elongation, and a maturation process characterized by the thickening of neurites and increase in cell soma sizes. We demonstrate that signaling through ERK, but not p38 or JNK, is required for the spontaneous neurite initiation and extension. Treatment with low concentrations of NGF induces rapid neurite elongation without affecting neurite branching and cell soma sizes. Such a rapid neurite outgrowth can be blocked by the inhibition of ERK, but not JNK, activities. In the presence of higher concentrations of NGF, the N1 cells undergo further differentiation with many characteristics of mature neurons in culture, e.g. larger cell soma and numerous branches/connections. This process can be completely blocked by inhibiting ERK or JNK activities using specific inhibitors. These results suggest that ERK and JNK signals play different roles in neuritogenesis, and that JNK activity is essential in the late stages of neuritogenesis. Furthermore, our results demonstrate that signaling dosage is important in the activation of a specific pathway, leading to distinctive biological outcomes.     

Transport of autophagosomes in neurites of PC12 cells during serum deprivation

Autophagy has been linked to various human diseases, including many neurodegenerative disorders. The induction of autophagy has been detected in degenerating neurites initiated by different experimental paradigms and hence is of major interest. Axonal and dendritic degeneration was significantly delayed either by the application of the autophagy inhibitor 3-methyladenine (3-MA) or by knocking down the key autophagy-related genes Atg7 and Beclin 1. In addition, Tomato-LC3-labelled autophagosomes accumulate in neuritic beadings of PC12 cells during nerve growth factor (NGF) deprivation, which might be due to the failure of neurite transport. However, little is known about routes and dynamics of autophagosomes in the neurites of living cells. Here, we further demonstrate that LC3-labelled small autophagosomes are motile and move along the neurites of PC12 cells in both anterograde and retrograde directions after serum deprivation. The autophagosomes paused, re-started, and sometimes changed directions. These results provide valuable insight into neuritic transport of autophagosomes and imply a close relationship between the autophagic process and neurite degeneration.     

Varicones and Growth Cones: Two Neurite Terminals in PC12 Cells

The rat adrenal pheochromocytoma PC12 cell line is one of the traditional models for the study of neurite outgrowth and growth cone behavior. To clarify to what extent PC12 neurite terminals can be compared to neuronal growth cones, we have analyzed their morphology and protein distribution in fixed PC12 cells by immunocytochemistry. Our results show that that PC12 cells display a special kind of neurite terminal that includes a varicosity in close association with a growth cone. This hybrid terminal, or “varicone”, is characterized by the expression of specific markers not typically present in neuronal growth cones. For example, we show that calpain-2 is a specific marker of varicones and can be detected even before the neurite develops. Our data also shows that a fraction of PC12 neurites end in regular growth cones, which we have compared to hippocampal neurites as a control. We also report the extraordinary incidence of varicones in the literature referred to as “growth cones”. In summary, we provide evidence of two different kinds of neurite terminals in PC12 cells, including a PC12-specific terminal, which implies that care must be taken when using them as a model for neuronal growth cones or neurite outgrowth.     

Neuroserpin regulates neurite outgrowth in nerve growth factor-treated PC12 cells

Neuroserpin is a serine protease inhibitor widely expressed in the developing and adult nervous systems and implicated in the regulation of proteases involved in processes such as synaptic plasticity, neuronal migration and axogenesis. We have analysed the effect of neuroserpin on growth factor-induced neurite outgrowth in PC12 cells. We show that small changes in neuroserpin expression result in changes to the number of cells extending neurites and total neurite length following NGF treatment. Increased expression of neuroserpin resulted in a decrease in the number of cells extending neurites and a reduction in total free neurite length whereas reduced levels of neuroserpin led to a small increase in the number of neurite extending cells and a significant increase in total free neurite length compared to the parent cell line. Neuroserpin also altered the response of PC12 cells to bFGF and EGF treatment. Neuroserpin was localised to dense cored secretory vesicles in PC12 cells but was unable to complex with its likely enzyme target, tissue plasminogen activator at the acidic pH found in these vesicles. These data suggest that modulation of neuroserpin levels at the extending neurite growth cone may play an important role in regulating axonal growth.     

Neurite development in PC12 cells cultured on nanopillars and nanopores with sizes comparable with filopodia

We investigated the effect ofnanoscale topography on neurite development in pheochromocytoma (PC12 cells) by culturing the cells on substrates having nanoscale pillars and pores with sizes comparable with filipodia. We found that cells on nanopillars and nanopores developed fewer and shorter neurites than cells on smooth substrates, and that cells on nanopores developed more and longer neurites than cells on nanopillars. These results suggest that PC12 cells were spatially aware of the difference in the nanoscale structures of the underlying substrates and responded differently in their neurite extension. This finding points to the possibility of using nanoscale topographic features to control neurite development in neurons.