Recent development of in vivo cryotechnique to cryobiopsy for living animals

Various microscopic methods have been used to analyze the morphology and molecular distribution of cells and tissues. Using conventional procedures, however, ischemic or anoxic artifacts are inevitably caused by tissue-resection or perfusion-fixation. The in vivo cryotechnique (IVCT) was developed to overcome these problems, and was found to be useful with light microscopy for analyses of the distribution of water-soluble molecules without anoxic effects at high time resolution. But there are limitations to the application of IVCT, such as exposure of target organs of living small animals and immunoreactivity of lipid-soluble molecules owing to freeze-substitution with acetone. Recently, a new cryotechnique called "cryobiopsy" has been developed, which enables one to obtain tissue specimens of large animals including humans without ischemia or anoxia, and has almost the same technical advantages as IVCT. Both IVCT and cryobiopsy complement other live-imaging techniques, and are useful for not only the morphological observation of cells and tissues under normal conditions, but also the preservation of all components in frozen tissue specimens. Therefore, morphofunctional information in vivo would be obtained by freeze-substituion for light or electron microscopy, and also by other analytical methods, such as freeze-fracture replication, X-ray microanalyses, or Raman microscopy. Considering the merits of both IVCT and cryobiopsy, their application should be expanded into other microscopic fields and also from experimental animal studies to clinical medicine.     

Immunohistochemical detection of hypoxia in mouse liver tissues treated with pimonidazole using “in vivo cryotechnique”

To evaluate hypoxic cells in live mouse liver tissues, immunohistochemistry for protein adducts of reductively activated pimonidazole (PARaPi) was performed using the “in vivo cryotechnique (IVCT)” followed by freeze-substitution fixation. This method was used because cryotechniques have some merits for examining biological events in living animal organs with improved time-resolution compared to conventional perfusion and/or immersion chemical fixation. Pimonidazole was intraperitoneally injected into living mice, and then after various times of hypoxia, their livers were quickly frozen by IVCT. The frozen liver tissues were freeze-substituted in acetone containing 2% paraformaldehyde, and routinely embedded in paraffin wax. De-paraffinized sections were immunostained for PARaPi. In liver tissues of mice without hypoxia, almost no immunostained cells were detected. However, in liver tissues with 30 s of hypoxia, some hepatocytes in the pericentral zones were strongly immunostained. After 60 s of hypoxia, many hepatocytes were immunostained with various degrees of staining intensity in all lobular zones, indicating different reactivities of pimonidazole in the hepatocytes to hypoxia. At this time, the general immunoreactivity also appeared to be stronger around the central veins than other portal areas. Although many hepatocytes were immunostained for PARaPi in the liver tissues with perfusion fixation via heart, those with perfusion via portal vein were not immunostained. Thus, IVCT is useful to detect time-dependent hypoxic states with pimonidazole treatment in living animal organs.     

Involvement of protein kinase C in the phosphorylation of rhodopsin

The possibility that protein kinase C is involved in phototransduction by phosphorylating rhodopsin was explored in situ and in vitro. Pretreatment of intact retinas with phorbol myristate acetate markedly increased the light-dependent phosphorylation of rhodopsin, with the greatest effects observed at lower light levels. Phorbol myristate acetate treatment did not affect rhodopsin phosphorylation in retinas not exposed to light, suggesting that protein kinase C modulates the phosphorylation state of rhodopsin in a light-dependent manner. Limited proteolysis of rhodopsin phosphorylated in situ indicates that protein kinase C modifies rhodopsin on a domain distinct from that recognized by rhodopsin kinase. In vitro, protein kinase C purified from bovine retinas phosphorylated unbleached and bleached rhodopsin. Our results are consistent with protein kinase C phosphorylating unbleached rhodopsin in response to low light, suggesting that protein kinase C plays a role in light adaptation.     

ER stress in retinal degeneration in S334ter Rho rats

The S334ter rhodopsin (Rho) rat (line 4) bears the rhodopsin gene with an early termination codon at residue 334 that is a model for several such mutations found in human patients with autosomal dominant retinitis pigmentosa (ADRP). The Unfolded Protein Response (UPR) is implicated in the pathophysiology of several retinal disorders including ADRP in P23H Rho rats. The aim of this study was to examine the onset of UPR gene expression in S334ter Rho retinas to determine if UPR is activated in ADRP animal models and to investigate how the activation of UPR molecules leads to the final demise of S334ter Rho photoreceptors. RT-PCR was performed to evaluate the gene expression profiles for the P10, P12, P15, and P21 stages of the development and progression of ADRP in S334ter Rho photoreceptors. We determined that during the P12-P15 period, ER stress-related genes are strongly upregulated in transgenic retinas, resulting in the activation of the UPR that was confirmed using western blot analysis and RT-PCR. The activation of UPR was associated with the increased expression of JNK, Bik, Bim, Bid, Noxa, and Puma genes and cleavage of caspase-12 that together with activated calpains presumably compromise the integrity of the mitochondrial MPTP, leading to the release of pro-apoptotic AIF1 into the cytosol of S334ter Rho photoreceptor cells. Therefore, two major cross-talking pathways, the UPR and mitochondrial MPTP occur in S334ter-4 Rho retina concomitantly and eventually promote the death of the photoreceptor cells.     

Immunoreactivity of Glutamate in Mouse Retina Inner Segment of Photoreceptors With In Vivo Cryotechnique

The purpose of this study was to clarify a previously controversial issue concerning glutamate (Glu) immunoreactivity (IR) in the inner segment (IS) of photoreceptors by using in vivo cryotechnique (IVCT) followed by freeze substitution (FS), which enabled us to analyze the cells and tissues reflecting living states. Eyeballs from anesthetized mice were directly frozen using IVCT. The frozen tissues were processed for FS fixation in acetone containing chemical fixatives, and embedded in paraffin. Deparaffinized sections were immunostained with an anti-Glu antibody. The strongest Glu-IR was obtained in the specimens prepared by FS with paraformaldehyde or a low concentration of glutaraldehyde, whereas no Glu-IR was obtained without the chemical fixatives. The Glu was immunolocalized in the IS, outer and inner plexiform and ganglion cell layers. Thus, the immunolocalization of Glu in the IS was clearly demonstrated using IVCT. (J Histochem Cytochem 57:883–888, 2009)     

Phosphorylation of GRK7 by PKA in cone photoreceptor cells is regulated by light

The retina-specific G protein-coupled receptor kinases, GRK1 and GRK7, have been implicated in the shutoff of the photoresponse and adaptation to changing light conditions via rod and cone opsin phosphorylation. Recently, we have defined sites of phosphorylation by cAMP-dependent protein kinase (PKA) in the amino termini of both GRK1 and GRK7 in vitro. To determine the conditions under which GRK7 is phosphorylated in vivo, we have generated an antibody that recognizes GRK7 phosphorylated on Ser36, the PKA phosphorylation site. Using this phospho-specific antibody, we have shown that GRK7 is phosphorylated in vivo and is located in the cone inner and outer segments of mammalian, amphibian and fish retinas. Using Xenopus laevis as a model, GRK7 is phosphorylated under dark-adapted conditions, but becomes dephosphorylated when the animals are exposed to light. The conservation of phosphorylation at Ser36 in GRK7 in these different species (which span a 400 million-year evolutionary period), and its light-dependent regulation, indicates that phosphorylation plays an important role in the function of GRK7. Our work demonstrates for the first time that cAMP can regulate proteins involved in the photoresponse in cones and introduces a novel mode of regulation for the retinal GRKs by PKA.     

Morphological study of erythrocyte shapes in red pulp of mouse spleens revealed by an in vivo cryotechnique

The purpose of this study was to clarify erythrocyte shapes in splenic cords of living mouse spleens, using our in vivo cryotechnique followed by scanning (SEM) or transmission (TEM) electron microscopy. Some spleens of mice were quickly frozen by the in vivo cryotechnique while their hearts were beating under anesthesia. In contrast, other spleens were prepared by an in vitro freezing method after they were taken out from the abdominal cavity. They were routinely freeze-substituted, and prepared for SEM and TEM. A few mouse spleens were also routinely fixed and embedded in Quetol-812 to obtain conventional morphology. Erythrocytes in living mouse spleens showed a variety of shapes with narrow spaces between them, trapped among reticular fiber tissues. Similar various shapes of erythrocytes were kept in the red pulp even after blocking normal blood circulation, as prepared by the in vitro freezing method. In comparison to the above-mentioned findings, some erythrocytes were changed to biconcave discoid shapes by the conventional immersion fixation with chemical fixatives. They also showed wide spaces between each other among reticular fiber tissues. Such conventional morphological studies could hardly reveal the in vivo shapes of erythrocytes in functioning spleens with normal blood circulation. In contrast, the various shapes of erythrocytes in the functioning spleens were demonstrated by our in vivo cryotechnique. It is suggested that most erythrocytes congesting in spleens keep their original configuration in spite of microenviromental alteration in splenic blood circulation.     

Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates myosin-binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro. Rho-kinase is thought to regulate the phosphorylation state of the substrates including myosin light chain (MLC), ERM (ezrin/radixin/moesin) family proteins and adducin by their direct phosphorylation and by the inactivation of myosin phosphatase. Here we identified the sites of phosphorylation of MBS by Rho-kinase as Thr-697, Ser-854 and several residues, and prepared antibody that specifically recognized MBS phosphorylated at Ser-854. We found by use of this antibody that the stimulation of MDCK epithelial cells with tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF) induced the phosphorylation of MBS at Ser-854 under the conditions in which membrane ruffling and cell migration were induced. Pretreatment of the cells with Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with Rho functions, or Rho-kinase inhibitors inhibited the TPA- or HGF-induced MBS phosphorylation. The TPA stimulation enhanced the immunoreactivity of phosphorylated MBS in the cytoplasm and membrane ruffling area of MDCK cells. In migrating MDCK cells, phosphorylated MBS as well as phosphorylated MLC at Ser-19 were localized in the leading edge and posterior region. Phosphorylated MBS was localized on actin stress fibers in REF52 fibroblasts. The microinjection of C3 or dominant negative Rho-kinase disrupted stress fibers and weakened the accumulation of phosphorylated MBS in REF52 cells. During cytokinesis, phosphorylated MBS, MLC and ERM family proteins accumulated at the cleavage furrow, and the phosphorylation level of MBS at Ser-854 was increased. Taken together, these results indicate that MBS is phosphorylated by Rho-kinase downstream of Rho in vivo, and suggest that myosin phosphatase and Rho-kinase spatiotemporally regulate the phosphorylation state of Rho-kinase substrates including MLC and ERM family proteins in vivo in a cooperative manner.     

Immunohistochemical analyses on albumin and immunoglobulin in acute hypertensive mouse kidneys by "in vivo cryotechnique"

The purpose of this study is to visualize topographical changes of serum proteins, albumin and immunoglobulin, passing through mouse glomerular capillary loops and their reabsorption in renal proximal tubules by immunohistochemistry in combination with our "in vivo cryotechnique". The "in vivo cryotechnique" was performed on left mouse kidneys under normotensive, experimentally acute hypertensive and heart-arrest conditions. The cryofixed tissues by the technique were routinely processed for freeze-substitution. Serial deparaffinized sections were stained with hematoxylin-eosine and immunostained with anti-mouse albumin, immunoglobulin G (IgG), kappa or lambda light chain and IgG1 heavy chain antibodies. Under the normotensive and heart-arrest conditions, albumin and IgG were clearly immunolocalized in blood vessels and slightly in apical cytoplasmic parts of some proximal tubules. Under the acute hypertensive condition, the albumin and kappa or lambda light chains, but not IgG1 heavy chain, were strongly immunolocalized in the apical cytoplasm of almost all proximal tubules. This study is the first in vivo visualization for glomerular passage of serum proteins and their transtubular absorption. Thus, the "in vivo cryotechnique" with freeze-substitution can be used for clarifying not only the functional morphology of living animal cells, but also in situ immunohistochemical localization of their components.     

The rate of rhodopsin phosphorylation in isolated rentinas of frog and cattle.

Phosphorylation of rhodopsin has been measured in isolated retinas incubated with 32P-phosphate under physiological conditions. The half-time of the light-induced phosphorylation was found to be approximately 2 min with frog retinas at 21 degrees C, and in the order of 1--2 min with cattle retinas at 36 degrees C. It is suggested by this slow rate that the phosphorylation reaction is not directly involved in the chain of events which lead from absorption of a photon to excitation of the photoreceptor cells but may perhaps have a regulatory function in controlling light/dark adaptation.     

Regulation of myosin-bound protein phosphatase by insulin in vascular smooth muscle cells: evaluation of the role of Rho kinase and phosphatidylinositol-3-kinase-dependent signaling pathways

In this study, we examined the molecular mechanism of myosin-bound protein phosphatase (MBP) regulation by insulin and evaluated the role of MBP in insulin-mediated vasorelaxation. Insulin rapidly stimulated MBP in confluent primary vascular smooth muscle cell (VSMC) cultures. In contrast, VSMCs isolated from diabetic and hypertensive rats exhibited impaired MBP activation by insulin. Insulin-mediated MBP activation was accompanied by a rapid time-dependent reduction in the phosphorylation state of the myosin-bound regulatory subunit (MBS) of MBP. The decrease observed in MBS phosphorylation was due to insulin-induced inhibition of Rho kinase activity. Insulin also prevented a thrombin-mediated increase in Rho kinase activation and abolished the thrombin-induced increase in MBS phosphorylation and MBP inactivation. These data are consistent with the notion that insulin inactivates Rho kinase and decreases MBS phosphorylation to activate MBP in VSMCs. Furthermore, treatment with synthetic inhibitors of phosphatidylinositol-3 kinase (PI3-kinase), nitric oxide synthase (NOS), and cyclic guanosine monophosphate (cGMP) all blocked insulin's effect on MBP activation. We conclude that insulin stimulates MBP via its regulatory subunit, MBS partly by inactivating Rho kinase and stimulating NO/cGMP signaling via PI3-kinase as part of a complex signaling network that controls 20-kDa myosin light chain (MLC20) phosphorylation and VSMC contraction.     

Light-mediated activation of diacylglycerol kinase in rat and bovine rod outer segments

The hydrolysis of phosphatidylinositol 4,5-bisphosphate is regulated by light in retinal rod outer segment (ROS) membranes. We recently reported that the activities of phosphatidylinositol synthetase and phosphatidylinositol 3-kinase are also higher in bleached (light-exposed) ROS (B-ROS). In this study, we investigated the effect of bleaching on diacylglycerol (DAG) kinase (DAG-kinase) activity in bovine and rat ROS membranes prepared from dark-adapted (D-ROS) or bleached (B-ROS) retinas. In bovine ROS, DAG-kinase activity toward endogenous DAG substrate was higher in B-ROS than in D-ROS. Quantification of DAG in both sets of membranes showed that the levels were the same, eliminating the possibility that the greater DAG-kinase activity was due to higher levels of endogenous substrate in B-ROS. DAG-kinase activity was also higher in B-ROS against an exogenous, water-soluable substrate (1, 2-didecanoyl-rac-glycerol), which competed with endogenous DAG substrate and saturated at approximately 2 mM. Immunoblot analysis with an anti-DAG-kinase gamma polyclonal antibody demonstrated that the gamma isoform was present in isolated bovine ROS. Immunocytochemistry of frozen bovine retinal sections confirmed the presence of DAG-kinase gamma immunoreactivity in ROS, as well as other retinal cells. Quantification of the immunoreactive products on western blots showed that more DAG-kinase gamma was present in B-ROS than in D-ROS. In an in vivo experiment, ROS prepared from rats exposed to 30 min of room light had greater DAG-kinase activity than ROS prepared from dark-adapted animals. Taken together, these data suggest that light exposure leads to the translocation of DAG-kinase from the cytosol to ROS membranes and that the greater DAG-kinase activity in B-ROS is due to the presence of more protein associated with ROS membranes.     

Tyrosine phosphorylation of BIT on photic stimulation in the rat retina

BIT is a transmembrane glycoprotein with three immunoglobulin-like domains in its extracellular region and tyrosine phosphorylation sites in its cytosolic region. We have previously shown that BIT was tyrosine phosphorylated in the hypothalamic suprachiasmatic nucleus in response to light exposure during the dark period, and suggested that it was involved in the light entrainment of the circadian clock. To further investigate the function of BIT in the nervous system, we examined the effect of photic stimulation on its tyrosine phosphorylation in the rat retina. It was found that the tyrosine phosphorylation level of BIT in the retina was higher in the light period than in the dark period. In addition, a light stimulation during the dark period resulted in a rapid phosphorylation of BIT and a subsequent association of BIT with SHP-2. The phosphorylation state was quickly reverted when the light was turned off. The light-dependent phosphorylation of BIT was also observed in isolated cultured retinas, and this was blocked by a specific Src-family inhibitor, PP-2. Immunohistochemical study showed that BIT was highly enriched in the inner and outer plexiform layers in the retina, where the immunoreactivity to anti-SHP-2 antibody was also detected. These results suggest that tyrosine phosphorylation of BIT is involved in neuronal transmission in the retina.     

Topographic study of arrestin using differential chemical modifications and hydrogen/deuterium exchange.

Arrestin is involved in the quenching of phototransduction by binding to photoactivated and phosphorylated rhodopsin (P-Rho*). To study its conformational changes and regions interacting with P-Rho*, arrestin was subjected to (1) differential acetylation at lysine residues in the presence and absence of P-Rho*, and (2) amide hydrogen/deuterium exchange. Labeled protein was proteolysed and analyzed by mass spectrometry. Three Lys residues, 28, 176, and 211, were protected from acetylation in native arrestin, although they were not located in regions exhibiting slow amide hydrogen exchange rates. The presence of P-Rho* protected lysine 201 from acetylation and partially protected 14 other lysyl residues, including (2, 5), (163, 166, 167), (232, 235, 236, 238), (267, 276), (298, 300), and 367, where parentheses indicate lysine residues found within the same peptide. In contrast, in the C-terminal region of arrestin, lysyl residues (386, 392, 395) were more exposed upon binding to P-Rho*. These data allowed us to identify functional regions in the arrestin molecule.     

Activation of Retinal Tyrosine Hydroxylase In Vitro by Cyclic AMP-Dependent Protein Kinase: Characterization and Comparison to Activation In Vivo by Photic Stimulation

Abstract: Tyrosine hydroxylase in rat retina is activated in vivo as a consequence of photic stimulation. Tyrosine hydroxylase in crude extracts of dark-adapted retinas is activated in vitro by incubation under conditions that stimulate protein phosphorylation by cyclic AMP-dependent protein kinase. Comparison of the activations of the enzyme by photic stimulation in vivo and protein phosphorylation in vitro demonstrated several similarities. Both treatments decreased the apparent K _m of the enzyme for the synthetic pterin cofactor 6MPH₄. Both treatments also produced the same change in the relationships of tyrosine hydroxylase activity to assay pH. When retinal extracts containing tyrosine hydroxylase activated either in vivo by photic stimulation or in vitro by protein phosphorylation were incubated at 25°C, the enzyme was inactivated in a time-dependent manner. The inactivation of the enzyme following both activation in vivo and activation in vitro was partially inhibited by sodium pyrophosphate, an inhibitor of phosphoprotein phosphatase. In addition to these similarities, the activation of tyrosine hydroxylase in vivo by photic stimulation was not additive to the activation in vitro by protein phosphorylation. These data indicate that the mechanism for the activation of tyrosine hydroxylase that occurs as a consequence of light-induced increases of neuronal activity is similar to the mechanism for activation of the enzyme in vitro by protein phosphorylation. This observation suggests that the activation of retinal tyrosine hydroxylase in vivo may be mediated by phosphorylation of tyrosine hydroxylase or some effector molecule associated with the enzyme.     

Influence of UV-B radiation on photosynthetic light-response curves, absorption spectra and motility of four phytoplankton species

Several unicellular algae were exposed to artificial UV-B (280–320 nm) radiation after adaptation to high (43 W m⁻²) and low (19 W m⁻²) visible light. UV-B radiation had different effects on rates of photosynthesis, motility and absorption spectra for these species. Photosynthesis of Euglena gracilis and the diatom Phaeodactylum tricomution was more sensitive to UV-B inhibition than that of the dinoflagellates Heterocapsa triquetra and Prorocentrum minimum . Not only UV-B radiation but also high visible light had a photoinhibitory effect on photosynthesis in all four organisms. The effect on photosynthesis was observed both on the quantum yield and on the light saturation rate of photosynthesis. The dinoflagellates, in contrast to E. gracilis and P. tricorntum , absorbed strongly in the UV region (334 nm) and their absorption peaks increased after growth under high visible light or with or without UV-B radiation for one week. The swimming speed of H. triquetra decreased more after low visible light and UV-B radiation compared to high visible light and UV-B radiation. The negative effects of UV-B radiation on P. minimum and E. gracilis were most pronounced after high visible light.     

The rate of rhodopsin phosphorylation in isolated retinas of frog and cattle

Phosphorylation of rhodopsin has been measured in isolated retinas incubated with ³²P-phosphate under physiological conditions. The half-time of the light-induced phosphorylation was found to be approximately 2 min with frog retinas at 21°C, and in the order of 1–2 min with cattle retinas at 36°C. It is suggested by this slow rate that the phosphorylation reaction is not directly involved in the chain of events which lead from absorption of a photon to excitation of the photoreceptor cells but may perhaps have regulatory function in controlling light/dark adaptation.     

Immunocytochemical localization of cAMP and cGMP in cells of the rat carotid body following natural and pharmacological stimulation.

Although the chemoreceptive function of the carotid body has been known for many decades, the cellular mechanisms of sensory transduction in this organ remain obscure. Common elements in the transductive processes of many cells are the cyclic nucleotide second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Studies from our laboratory have revealed stimulus-induced changes in cyclic nucleotide levels in the carotid body as measured by RIA, but such changes in second messenger levels have not been localized to specific cellular elements in the organ. The present immunocytochemical study utilized the avidin-biotin-peroxidase method to investigate the distribution of cAMP and cGMP in the rat carotid body and to assess changes in the intensity of immunostaining following in vitro stimulation by hypoxia, forskolin, sodium nitroprusside, high potassium, and atrial natriuretic peptide. Both cAMP and cGMP immunoreactivity were localized to type I cells of organs maintained in vivo and fixed by perfusion. Organs exposed to 100% O2-equilibrated media in vitro produced low but visible levels of cAMP immunoreactivity in a majority of type I cells; hypoxia (5% O2-equilibrated media) for 10 min moderately increased the level of immunoreactivity; forskolin (10(-5) M), or forskolin combined with hypoxia, dramatically increased cAMP levels in virtually all cells. Moderate levels of cGMP immunoreactivity in control carotid bodies in vitro were strikingly reduced by hypoxia; a significant increase in cGMP levels occurred following incubation in high potassium (100 mM), and under these conditions, the decrease in cGMP immunoreactivity with hypoxia was much more pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circadian activation of bullfrog retinal mitogen-activated protein kinase associates with oscillator function

The vertebrate retina retains a circadian oscillator, and its oscillation is self-sustained with a period close to 24 h under constant environmental conditions. Here we show that bullfrog retinal mitogen-activated protein kinase (MAPK) exhibits an in vivo circadian rhythm in phosphorylation with a peak at night in a light/dark cycle. The phosphorylation rhythm of MAPK persists in constant darkness with a peak at subjective night, and this self-sustained rhythm is also observed in cultured retinas, indicating its close interaction with the retinal oscillator. The rhythmically phosphorylated MAPK is detected only in a discrete subset of amacrine cells despite ubiquitous distribution of MAPK throughout the retinal layers. Treatment of the cultured retinas with MAPK kinase (MEK) inhibitor PD98059 suppresses MAPK phosphorylation during the subjective night, and this pulse perturbation of MEK activity induces a significant phase delay (4-8 h) of the retinal circadian rhythm in MAPK and MEK phosphorylation. These observations strongly suggest that the site-specific and time-of-day-specific activation of MAPK contributes to the circadian time-keeping mechanism of the retinal clock system.     

Myosin light chain kinase and acto-myosin contractility modulate activation of the ERK cascade downstream of oncogenic Ras

The actin cytoskeleton is recognized as an important component of both adhesion- and growth factor-dependent signaling, but its role in oncogene-dependent signaling has received much less attention. In this study, we investigated the role played by the acto-myosin cytoskeleton and its main regulators, i.e., myosin light chain kinase and Rho kinase, in oncogenic Ki-Ras-induced signaling. We found that activation of the ERK cascade by Ras is dependent on acto-myosin contractility, under the regulation of myosin light chain kinase but not Rho kinase. Inhibition of myosin II or myosin light chain kinase caused a complete loss of ERK phosphorylation in a time- and dose-dependent manner, but proved dispensable for activation of the PI3K pathway. We also provide evidence that the target of myosin light chain kinase lays at the level of Raf activation. Since myosin light chain kinase is a target of ERK, these results suggest a previously uncharacterized signaling pathway involving Ras-mediated alterations of the actin cytoskeleton, which might play a critical role in ERK activation by the Ras oncogene and contribute to aberrant signaling and enhanced cell motility. In addition, restoration of stress fibers following ectopic expression of tropomyosin 2 resulted in reduced levels of ERK phosphorylation. Finally, these studies suggest that myosin light chain kinase but not Rho kinase plays an essential role in the generation of ERK signaling in transformed cells and indicate distinct cellular roles for Rho-kinase and myosin light chain kinase-dependent functions involving the regulation of acto-myosin contractility.