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.     

Coordinated interactions between actin and microtubules through crosslinkers in neurite retraction induced by lysophosphatidic acid

Neurite development requires rearrangement of cytoskeletal elements, which are mechanically and functionally integrated with each other. Although the process of how an extracellular signal induces rearrangement of a single element has been closely examined, the mechanisms by which the signal regulates cytoskeletal integration during cell shape changes are poorly understood. We previously reported that lysophosphatidic acid (LPA) induces actin polymerization-dependent microtubule (MT) rearrangement, leading to neurite retraction in cultured neurons. Here we examined whether the crosslinker proteins were involved in LPA-induced neurite retraction using immortalized mouse neuroblast TR cells. When the MT-binding domains of MACF (MT actin-crosslinking factor) were exogenously expressed in TR cells, MTs were found to be stabilized and become resistant to exposure to LPA. On the other hand, expression of MT-associated protein 2c showed no effect on LPA-induced neurite retraction. These findings suggest that MACF is involved in actin-dependent MT rearrangement during LPA-induced neurite retraction.     

Regulation of neuronal cytoskeleton by lysophosphatidic acid: role of GSK-3

Neurite retraction is a crucial process during nervous system development and neurodegeneration. This process implies reorganization of the neuronal cytoskeleton. Some bioactive lipids such as lysophosphatidic acid (LPA) induce neurite retraction. The reorganization of the actin cytoskeleton during neurite retraction is one of the best-characterized effects of LPA. However, less information is available regarding the reorganization of the microtubule (MT) network in response to LPA in neuronal cells. Here, we first give an overview of the roles of cytoskeleton during neurite outgrowth, and subsequently, we review some of the data from different laboratories concerning LPA-induced cytoskeletal rearrangement in neuronal cells. We also summarize our own recent results about modifications of MTs during LPA-induced neurite retraction. We have shown that LPA induces changes in tubulin pools and increases in the phosphorylation levels of microtubule-associated proteins (MAPs), such as Tau. Tau hyperphosphorylation in response to LPA is mediated by the activation of glycogen synthase kinase-3 (GSK-3). The upregulation of GSK-3 activity by LPA seems to be a general process as it occurs in diverse neuronal cells of different species in correlation with the neurite retraction process.     

Lysophosphatidic acid induces neurite retraction in differentiated neuroblastoma cells via GSK-3�� activation

Lysophosphatidic acid (LPA) is a lipid growth factor that exerts diverse biological effects, including rapid neurite retraction and cell migration. Alterations in cell morphology, including neurite retraction, in neurodegenerative disorders such as Alzheimer's disease involve hyperphosphorylation of the cytoskeletal protein tau. Since LPA has been shown to induce neurite retraction in various cultured neural cells and the detailed underlying molecular mechanisms have not yet been elucidated, we investigated whether LPA induced neurite retraction through taumediated signaling pathways in differentiated neuroblastoma cells. When Neuro2a cells differentiated with retinoic acid (RA) were exposed to LPA, cells exhibited neurite retraction in a time-dependent manner. The retraction of neurites was accompanied by the phosphorylation of tau. The LPA-induced neurite retraction and tau phosphorylation in differentiated Neuro2a cells were significantly abolished by the glycogen synthase kinase-3β (GSK-3β) inhibitor lithium chloride. Interestingly, the LPA-stimulated tau phosphorylation and neurite retraction were markedly prevented by the administration of H89, an inhibitor of both cyclic-AMP dependent protein kinase (PKA) and cyclic-AMP response element-binding protein (CREB). Transfection of the dominant-negative CREBs, K-CREB and A-CREB, failed to prevent LPA-induced tau phosphorylation and neurite retraction in differentiated Neuro2a cells. Taken together, these results suggest that GSK-3β and PKA, rather than CREB, play important roles in tau phosphorylation and neurite retraction in LPA-stimulated differentiated Neuro2a cells.     

Stable isotope labeling by amino acids in cell culture-based liquid chromatography–mass spectrometry assay to measure microtubule dynamics in neuronal cell cultures

Microtubules (MTs) are highly dynamic polymers composed of α- and β-tubulin heterodimers. Dysregulation of MT dynamics in neurons may be a contributing factor in the progression of various neurodegenerative diseases. We developed a stable isotope labeling by amino acids in cell culture (SILAC)-based liquid chromatography–mass spectrometry (LC–MS) method to measure the fraction of [¹³C₆]leucine-labeled α-tubulin-derived surrogate peptides. Using this approach, we measured the time course of incorporation of [¹³C₆]leucine label into the MT and dimer pools isolated from cycling cells and rat primary hippocampal neurons. We found that the MT pool is in rapid equilibrium with the dimer pool in the cycling cells, consistent with rapid MT polymerization/depolymerization during cell proliferation. Conversely, in neurons, we found that labeling of the MT pool was rapid, whereas the dimer pool was delayed. These results suggest that newly synthesized α-tubulin is first incorporated into MTs or complexes that co-sediment with MTs and that appearance of labeled α-tubulin in the dimer pool may be a consequence of MT depolymerization or breakdown. Our results demonstrate that a SILAC-based approach can be used to measure MT dynamics and may have utility for exploring MT dysregulation in various models of neurodegenerative disease.     

Cytoskeletal dynamics underlying collateral membrane protrusions induced by neurotrophins in cultured Xenopus embryonic neurons

The establishment and refinement of neuronal connections depend on dynamic modification of the morphology and physiology of developing axons in response to extrinsic factors. In embryonic cultures of Xenopus spinal neurons, acute application of brain-derived neurotrophic factor (BDNF) induced rapid collateral protrusion of filopodium-like microspikes and lamellipodia along the neurite processes, leading to a morphologic alternation of the neuron. Both types of membrane protrusions contained high concentrations of actin filaments and depended on the polymerization of the actin cytoskeleton. Immunofluorescent staining, however, revealed the presence of microtubules (MTs) in lamellipodia induced by BDNF. These MTs appeared to have arisen from debundling of MTs in the neurite shaft at the protrusion sites, splaying and extending in the rapidly protruding lamellipodia. Inhibition of microtubule polymerization by nocodazole largely abolished the formation of lamellipodia but not of microspikes. Taken together, our results suggest that collateral sprouting of microspikes and lamellipodia involve distinctly different cytoskeletal mechanisms. Although the actin cytoskeleton is solely responsible for microspike formation, cooperative efforts by microtubules and actin filaments are essential for lamellipodial protrusion in response to extrinsic factors.     

Different modes of growth cone collapse in NG 108-15 cells

In the fundamental process of neuronal path-finding, a growth cone at the tip of every neurite detects and follows multiple guidance cues regulating outgrowth and initiating directional changes. While the main focus of research lies on the cytoskeletal dynamics underlying growth cone advancement, we investigated collapse and retraction mechanisms in NG108-15 growth cones transiently transfected with mCherry-LifeAct and pCS2+/EMTB-3XGFP for filamentous actin and microtubules, respectively. Using fluorescence time lapse microscopy we could identify two distinct modes of growth cone collapse leading either to neurite retraction or to a controlled halt of neurite extension. In the latter case, lateral movement and folding of actin bundles (filopodia) confine microtubule extension and limit microtubule-based expansion processes without the necessity of a constantly engaged actin turnover machinery. We term this previously unreported second type fold collapse and suggest that it marks an intermediate-term mode of growth regulation closing the gap between full retraction and small scale fluctuations.     

Neuritogenesis in mouse NB2a/d1 neuroblastoma cells: triggering by calcium influx and involvement of actin and tubulin dynamics

Ionophore (A23187)-mediated calcium influx induced rapid neurite outgrowth in NB2a/d1 cells. This outgrowth was prevented by colchicine but not by cycloheximide, demonstrating a requirement for microtubule assembly but not de novo synthesis. Cytochalasin B induced rapid, colchicine-sensitive outgrowth, indicating that depolymerization of the submembrane actin network may be sufficient to allow neurite outgrowth under conditions which permitted microtubule assembly. Neurites induced by serum-deprivation or calcium influx were rapidly retracted by colchicine unless cytochalasin B was first added, indicating that the actin network may provide the retractile force which mediates neurite retraction following microtubule depolymerization. We conclude that neurite outgrowth can be initiated in NB2a/d1 cells by calcium influx, and may involve alterations in actin and microtubule dynamics.     

Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis

In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.     

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.     

Serum- and glucocorticoid-inducible kinase 1 (SGK1) increases neurite formation through microtubule depolymerization by SGK1 and by SGK1 phosphorylation of tau

Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a member of the Ser/Thr protein kinase family that regulates a variety of cell functions. Recently, SGK1 was shown to increase dendritic growth but the mechanism underlying the increase is unknown. Here we demonstrated that SGK1 increased the neurite formation of cultured hippocampal neurons through microtubule (MT) depolymerization via two distinct mechanisms. First, SGK1 directly depolymerized MTs. In vitro MT depolymerization experiments revealed that SGK1, especially N-truncated SGK1, directly disassembled self-polymerized MTs and taxol-stabilized MTs in a dose-dependent and ATP-independent manner. The transfection of sgk1 to HeLa cells also inhibited MT assembly in vivo. Second, SGK1 indirectly depolymerized MTs through the phosphorylation of tau at Ser214. An in vitro kinase assay revealed that active SGK1 phosphorylated tau Ser214 specifically. In vivo transfection of sgk1 also phosphorylated tau Ser214 in HEK293T cells and hippocampal neurons. Further, sgk1 transfection significantly increased the number of primary neurites and shortened the length of the total process in cultured hippocampal neurons. These effects were antagonized by the cotransfection of the tauS214A mutant plasmid. Dexamethasone, a synthetic glucocorticoid, mimics the effect of sgk1 overexpression. Together, these results suggest that SGK1 enhances neurite formation through MT depolymerization by a direct action of SGK1 and by the SGK1 phosphorylation of tau.     

Respective roles of neurofilaments, microtubules, MAP1B, and tau in neurite outgrowth and stabilization.

The respective roles of neurofilaments (NFs), microtubules (MTs), and the microtubule-associated proteins (MAPs) MAP 1B and tau on neurite outgrowth and stabilization were probed by the intracellular delivery of specific antisera into transiently permeabilized NB2a/d1 cells during treatment with dbcAMP. Intracellular delivery of antisera specific for the low (NF-L), middle (NF-M), or extensively phosphorylated high (NF-H) molecular weight subunits did not prevent initial neurite elaboration, nor did it induce retraction of existing neurites elaborated by cells that had been previously treated for 1 d with dbcAMP. By contrast, intracellular delivery of antisera directed against tubulin reduced the percentage of cells with neurites at both these time points. Intracellular delivery of anti-NF-L and anti-NF-M antisera did not induce retraction in cells treated with dbcAMP for 3 d. However, intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, tau, or tubulin induced similar levels of neurite retraction at this time. Intracellular delivery of monoclonal antibodies (RT97 or SMI-31) directed against phosphorylated NF-H induced neurite retraction in cell treated with dbcAMP for 3 d; a monoclonal antibody (SMI-32) directed against nonphosphorylated NF-H did not induce neurite retraction at this time. By contrast, none of the above antisera induced retraction of neurites in cells treated with dbcAMP for 7 d. Neurites develop resistance to retraction by colchicine, first detectable in some neurites after 3 d and in the majority of neurites after 7 d of dbcAMP treatment. We therefore examined whether or not colchicine resistance was compromised by intracellular delivery of the above antisera. Colchicine treatment resulted in rapid neurite retraction after intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, or tau into cells that had previously been treated with dbcAMP for 7 d. By contrast, colchicine resistance was not compromised by the intracellular delivery of antisera directed against NF-L, NF-M, or tubulin. These findings support previous studies indicating that MT polymerization mediates certain aspects of axonal neurite outgrowth and suggest that NFs do not directly participate in these events. These findings further suggest that NFs function in stabilization of the axonal cytoskeleton, apparently by interactions among NFs and MTs that are mediated by NF-H and MAPs.     

Visualization of the stop of microtubule depolymerization that occurs at the high-density region of microtubule-associated protein 2 (MAP2).

Individual microtubules (MTs) repeat alternating phases of polymerization and depolymerization, a process known as dynamic instability. Microtubule-associated proteins (MAPs) regulate the dynamic instability by increasing the rescue frequency. To explore the influence of MAP2 on in vitro MT dynamics, we correlated the distribution of MAP2 on individual MTs with the dynamic phase changes of the same MTs. MAP2 was modified selectively on its projection region by X-rhodamine iodoacetamide without altering the MT-binding activity. When the labeled MAP2 was added to MTs, the fluorescence was distributed along almost the entire length of individual MTs. However, the inhomogeneity of the distribution gradually became obvious due to the fluorescence bleaching, and the MTs appeared to consist of rapidly bleached portions (RBPs) and slowly bleached portions (SBPs), which were distributed randomly along the MT. By measuring the duration of fluorescence bleaching, the density of MAP2 in SBP was estimated to be approximately 2.5 times higher than the RBP. The average tubulin:MAP2 ratio in SBP was calculated to be 16. When the MT dynamics were observed by dark-field microscopy after determining the MAP2 distribution, rescues were always found to occur only at the SBPs. MTs also displayed intermittent shortening by repeated depolymerization phases separated by pause phases. In these cases, depolymerization phases stopped only at the SBPs. Not every SBP stopped depolymerization, but depolymerization always stopped at an SBP. Taken together, we suggest that there is a minimum density of MAP2 that is necessary to stop depolymerization.     

Microtubule dynamics and the regulation by microtubule-associated proteins (MAPs).

Individual microtubules (MTs) repeat alternating phases of polymerization and depolymerization, a process known as "dynamic instability." The dynamic instability is regulated by various protein factors according to the requirement of cellular conditions. Heat-stable MAPs regulate the dynamic instability by increasing the rescue frequency. To explore the influence of MAP2, a heat-stable MAPs abundant in neuron, on in vitro MT dynamics, the distribution of MAP2 on individual MTs was correlated with the dynamic phase changes of the same MTs by optical microscopy. MAP2 distributed inhomogeneously along the length of MTs by forming high-density regions, clusters. Stops of depolymerization were always found to occur only at the cluster sites. Every cluster did not stop depolymerization, but depolymerization did always stop at a cluster site. We suggest that mode of distribution along MT is an important factor of the function of heat-stable MAPs.     

Phosphatidylinositol 4-phosphate 5-kinase is essential for ROCK-mediated neurite remodeling

Phosphatidylinositol 4-phosphate 5-kinase (PIP-5kin) regulates actin cytoskeletal reorganization through its product phosphatidylinositol 4,5-bisphosphate. In the present study we demonstrate that PIP-5kin is essential for neurite remodeling, which is regulated by actin cytoskeletal reorganization in neuroblastoma N1E-115 cells. Overexpression of wild-type mouse PIP-5kin-alpha inhibits the neurite formation that is normally stimulated by serum depletion, whereas a lipid kinase-defective mutant of PIP-5kin-alpha, D266A, triggers neurite extension even in the presence of serum and blocks lysophosphatidic acid-induced neurite retraction. These results phenocopy those previously reported for the small GTPase RhoA and its effector p160 Rho-associated coiled coil-forming protein kinase (ROCK). However, the ROCK-specific inhibitor Y-27632 failed to block the inhibition by PIP-5kin-alpha of neurite extension, whereas D266A did block the neurite retraction induced by overexpression of ROCK. These results, taken together, suggest that PIP-5kin-alpha functions as a downstream effector for RhoA/ROCK to couple lysophosphatidic acid signaling to neurite retraction presumably through its product phosphatidylinositol 4,5-bisphosphate.     

Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.     

Process formation of podocytes: morphogenetic activity of microtubules and regulation by protein serine/threonine phosphatase PP2A

Podocytes possess major processes containing microtubules (MTs) and intermediate filaments and foot processes containing actin filaments (AFs) as core cytoskeletal elements. Although the importance of these cytoskeletal elements for maintaining podocyte processes was previously shown, so far no data are available concerning the developmental regulation of podocyte process formation. A conditionally immortalized mouse podocyte cell line, which can be induced to develop processes similar to those found in vivo, was treated with various reagents to disrupt cytoskeletal elements or to inhibit protein phosphatases. MTs colocalized with vimentin intermediate filaments but not with AFs. After AF disassembly, major processes were maintained, whereas after depolymerization of MTs, podocytes lost their processes, rounded up, and maintained only actin-based peripheral projections. Suppression of MT elongation by nanomolar vinblastine or inhibition of serine/threonine phosphatase PP2A with okadaic acid abolished process formation. PP2A was expressed in undifferentiated but not in differentiated podocytes. One- and two-dimensional western blot analyses revealed a dose-dependent increase in serine/threonine phosphorylation after okadaic acid treatment. Hence, morphogenetic activity of MTs induces podocyte process formation via serine/threonine protein dephosphorylation by PP2A. These results may open new avenues for understanding the signaling mechanism underlying podocyte cytoskeleton alterations during development and in glomerular diseases.     

Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules

Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down-regulation of PP2A methylation and PP2A enzymes containing the B alpha regulatory subunit occur in Alzheimer's disease. In this study, we show that expressed wild-type and methylation- (L309 Delta) and phosphorylation- (T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B', and B'-type regulatory subunits in NIH 3T3 fibroblasts and neuro-2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild-type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309 Delta, and T304D mutants, which are impaired in their ability to associate with the B alpha subunit, induces their loss. Silencing of B alpha subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole-induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal B alpha-containing PP2A heterotrimers contribute to MT destabilization in Alzheimer's disease.     

Disruption of the cytoskeleton during Semaphorin 3A induced growth cone collapse correlates with differences in actin organization and associated binding proteins

Repulsive guidance cues induce growth cone collapse or collapse and retraction. Collapse results from disruption and loss of the actin cytoskeleton. Actin‐rich regions of growth cones contain binding proteins that influence filament organization, such as Arp2/3, cortactin, and fascin, but little is known about the role that these proteins play in collapse. Here, we show that Semaphorin 3A (Sema 3A), which is repulsive to mouse dorsal root ganglion neurons, has unequal effects on actin binding proteins and their associated filaments. The immunofluorescence staining intensity of Arp‐2 and cortactin decreases relative to total protein; whereas in unextracted growth cones fascin increases. Fascin and myosin IIB staining redistribute and show increased overlap. The degree of actin filament loss during collapse correlates with filament superstructures detected by rotary shadow electron microscopy. Collapse results in the loss of branched f‐actin meshworks, while actin bundles are partially retained to varying degrees. Taken together with the known affects of Sema 3A on actin, this suggests a model for collapse that follows a sequence; depolymerization of actin meshworks followed by partial depolymerization of fascin associated actin bundles and their movement to the neurite to complete collapse. The relocated fascin associated actin bundles may provide the substrate for actomyosin contractions that produce retraction. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Possible interrelationship between changes in F-actin and myosin II,protein phosphorylation,and cell volume regulation in Ehrlich ascites tumor cells

Osmotic shrinkage of Ehrlich ascites tumor cells (EATC) elicited translocation of myosin II from the cytosol to the cortical region, and swelling elicits concentration of myosin II in the Golgi region. Rho kinase and p38 both appeared to be involved in shrinkage-induced myosin II reorganization. In contrast, the previously reported shrinkage-induced actin polymerization [Pedersen et al. (1999) Exp. Cell Res. 252, 63-74] was independent of Rho kinase, p38, myosin light chain kinase (MLCK), and protein kinase C (PKC), which thus do not exert their effects on the shrinkage-activated transporters via effects on F-actin. The subsequent F-actin depolymerization, however, appeared MLCK- and PKC-dependent, and the initial swelling-induced F-actin depolymerization was MLCK-dependent; both effects were apparently secondary to kinase-mediated effects on cell volume changes. NHE1 in EATC is activated both by osmotic shrinkage and by the serine/threonine phosphatase inhibitor Calyculin A (CL-A). Both stimuli caused Rho kinase-dependent myosin II relocation to the cortical cytoplasm, but in contrast to the shrinkage-induced F-actin polymerization, CL-A treatment elicited a slight F-actin depolymerization. Moreover, Rho kinase inhibition did not significantly affect NHE1 activation, neither by shrinkage nor by CL-A. Implications for the possible interrelationship between changes in F-actin and myosin II, protein phosphorylation, and cell volume regulation are discussed.