People with Parkinson's Disease: What Symptoms Do They Most Want to Improve and How Does This Change with Disease Duration?

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative condition with a diverse and complex pattern of motor and non-motor symptoms which change over time with disease duration. OBJECTIVE: The aims of the present study were to discover what symptoms matter most to people with the condition and to examine how these priorities change with disease duration. METHODS: A simple free-text online survey (using SmartSurvey) was developed by Parkinson's UK, which asked participants to identify up to three aspects of the condition they would most like to see improvement in. RESULTS: 790 people participated reporting 2,295 issues related to PD which were grouped into 24 broad symptom domains. Of these, 1,358 (59.1%) were categorised as motor symptoms, 859 (37.4%) as non-motor issues and 78 (3.4%) as medication problems. This study reveals how certain features of PD become more or less important to patients as the condition progresses. Non-motor symptoms were highly cited from the very earliest stages of PD. Problems with walking, balance and falls, speech problems, freezing and dyskinesia become increasingly important as the condition progresses whereas tremor, stiffness and psychological health become decreasingly important as the condition progresses. CONCLUSIONS: The data suggest that the priorities of people affected by PD for improving life are personal and change with duration of the condition. These findings have implications for developing person-centred management and care, as well as for directing future research to improve quality of life.

Phospholipase D2 in prostate cancer: protein expression changes with Gleason score.

BACKGROUND: Phospholipases D1 and D2 (PLD1/2) are implicated in tumorigenesis through their generation of the signalling lipid phosphatidic acid and its downstream effects. Inhibition of PLD1 blocks prostate cell growth and colony formation. Here a role for PLD2 in prostate cancer (PCa), the major cancer of men in the western world, is examined. METHODS: PLD2 expression was analysed by immunohistochemistry and western blotting. The effects of PLD2 inhibition on PCa cell viability and cell motility were measured using MTS, colony forming and wound-healing assays. RESULTS: PLD2 protein is expressed about equally in luminal and basal prostate epithelial cells. In cells from different Gleason-scored PCa tissue PLD2 protein expression is generally higher than in non-tumorigenic cells and increases in PCa tissue scored Gleason 6-8. PLD2 protein is detected in the cytosol and nucleus and had a punctate appearance. In BPH tissue stromal cells as well as basal and luminal cells express PLD2. PLD2 protein co-expresses with chromogranin A in castrate-resistant PCa tissue. PLD2 inhibition reduces PCa cell viability, colony forming ability and directional cell movement. CONCLUSIONS: PLD2 expression correlates with increasing Gleason score to GS8. PLD2 inhibition has the potential to reduce PCa progression.

The putative tumour suppressor protein Latexin is secreted by prostate luminal cells and is downregulated in malignancy.

Loss of latexin (LXN) expression negatively correlates with the prognosis of several human cancers. Despite association with numerous processes including haematopoietic stem cell (HSC) fate, inflammation and tumour suppression, a clearly defined biological role for LXN is still lacking. Therefore, we sought to understand LXN expression and function in the normal and malignant prostate to assess its potential as a therapeutic target. Our data demonstrate that LXN is highly expressed in normal prostate luminal cells but downregulated in high Gleason grade cancers. LXN protein is both cytosolic and secreted by prostate cells and expression is directly and potently upregulated by all-trans retinoic acid (atRA). Whilst overexpression of LXN in prostate epithelial basal cells did not affect cell fate, LXN overexpression in the luminal cancer line LNCaP reduced plating efficiency. Transcriptome analysis revealed that LXN overexpression had no direct effects on gene expression but had significant indirect effects on important genes involved in both retinoid metabolism and IFN-associated inflammatory responses. These data highlight a potential role for LXN in retinoid signaling and inflammatory pathways. Investigating the effects of LXN on immune cell function in the tumour microenvironment (TME) may reveal how observed intratumoural loss of LXN affects the prognosis of many adenocarcinomas.

Phospholipase D inhibitors reduce human prostate cancer cell proliferation and colony formation.

BACKGROUND: Phospholipases D1 and D2 (PLD1/2) hydrolyse cell membrane glycerophospholipids to generate phosphatidic acid, a signalling lipid, which regulates cell growth and cancer progression through effects on mTOR and PKB/Akt. PLD expression and/or activity is raised in breast, colorectal, gastric, kidney and thyroid carcinomas but its role in prostate cancer (PCa), the major cancer of men in the western world, is unclear. METHODS: PLD1 protein expression in cultured PNT2C2, PNT1A, P4E6, LNCaP, PC3, PC3M, VCaP, 22RV1 cell lines and patient-derived PCa cells was analysed by western blotting. PLD1 protein localisation in normal, benign prostatic hyperplasia (BPH), and castrate-resistant prostate cancer (CRPC) tissue sections and in a PCa tissue microarray (TMA) was examined by immunohistochemistry. PLD activity in PCa tissue was assayed using an Amplex Red method. The effect of PLD inhibitors on PCa cell viability was measured using MTS and colony forming assays. RESULTS: PLD1 protein expression was low in the luminal prostate cell lines (LNCaP, VCaP, 22RV1) compared with basal lines (PC3 and PC3M). PLD1 protein expression was elevated in BPH biopsy tissue relative to normal and PCa samples. In normal and BPH tissue, PLD1 was predominantly detected in basal cells as well in some stromal cells, rather than in luminal cells. In PCa tissue, luminal cells expressed PLD1. In a PCa TMA, the mean peroxidase intensity per DAB-stained Gleason 6 and 7 tissue section was significantly higher than in sections graded Gleason 9. In CRPC tissue, PLD1 was expressed prominently in the stromal compartment, in luminal cells in occasional glands and in an expanding population of cells that co-expressed chromogranin A and neurone-specific enolase. Levels of PLD activity in normal and PCa tissue samples were similar. A specific PLD1 inhibitor markedly reduced the survival of both prostate cell lines and patient-derived PCa cells compared with two dual PLD1/PLD2 inhibitors. Short-term exposure of PCa cells to the same specific PLD1 inhibitor significantly reduced colony formation. CONCLUSIONS: A new specific inhibitor of PLD1, which is well tolerated in mice, reduces PCa cell survival and thus has potential as a novel therapeutic agent to reduce prostate cancer progression. Increased PLD1 expression may contribute to the hyperplasia characteristic of BPH and in the progression of castrate-resistant PCa, where an expanding population of neuroendocrine-like cells express PLD1.

Adenosine triggers the nuclear translocation of protein kinase C epsilon in H9c2 cardiomyoblasts with the loss of phosphorylation at Ser729.

Adenosine is a major mediator of ischaemic preconditioning (IPC) and cardioprotection. The translocation and activation of protein kinase C epsilon, triggered by adenosine, are essential for these processes. We report here that H9c2 cardiomyoblasts express five PKC isoforms (alpha, beta(I), delta, epsilon and zeta). PKCepsilon is predominantly associated with F-actin fibres in unstimulated H9c2 cells but translocates to the nucleus on stimulation with adenosine. Cytosolic PKCepsilon associated with F-actin fibres is phosphorylated at Ser729 but nuclear PKCepsilon lacks phosphorylation at this site. Adenosine triggers the nuclear translocation after 5 min stimulation. PKCepsilon Ser729Ala and Ser729Glu mutants showed no translocation on adenosine stimulation suggesting both phosphorylation and serine at 729 are critical for this translocation. Among five PKC isoforms (alpha, beta(I), delta, epsilon and zeta) detected, PKCepsilon is the only isoform translocating to the nucleus upon adenosine stimulation. Disruption of microtubules (MTs), but not F-actin-rich fibres, blocked translocation of both endogenous PKCepsilon and overexpressed GFP-PKCepsilon to the nucleus. Ten proteins interacted with cytosolic PKCepsilon; five of which are components of myofibrils. Matrin 3 and vimentin interacted with nuclear PKCepsilon. These findings suggest that adenosine stimulates PKCepsilon translocation to the nucleus in H9c2 cells in a mechanism involving dephosphorylation at Ser729 and MT, which should advance our understanding of the signalling pathways stimulated by adenosine in IPC and cardioprotection.

Phosphorylation at Ser729 specifies a Golgi localisation for protein kinase C epsilon (PKCepsilon) in 3T3 fibroblasts.

We demonstrate that GFP-PKCepsilon concentrates at a perinuclear site in living fibroblasts and that cell passage induces rapid translocation of PKCepsilon to the periphery where it appears to colocalise with F-actin. When newly passaged cells have adhered and are proliferating again, GFP-PKCepsilon returns to its perinuclear site. GFP-PKCepsilon co-localises with wheat germ agglutinin suggesting that it is associated with the Golgi at the perinuclear site. In support, PKCepsilon is detected in a Golgi-enriched fraction in pre-passage cells but is lost from the fraction after passage. PKCepsilon at the perinuclear Golgi site is phosphorylated at Ser729 but cell passage induces the loss of the phosphate at this site as reported previously [England et al. (2001) J. Biol. Chem. 276, 10437-10442]. PKCepsilon S729A, S729E and S729T mutants, which are not recognised by a specific antiphosphoPKCepsilon (Ser729) antibody, do not concentrate at a perinuclear/Golgi site in proliferating fibroblasts. This suggests that both phosphorylation and serine rather than threonine are needed at position 729 to locate PKCepsilon at its perinuclear/Golgi site. Phorbol ester induced translocation of PKCepsilon to the nucleus also requires dephosphorylation at Ser729; after translocation nuclear PKCepsilon lacks a phosphate at Ser729. Sulphation and secretion of glycosaminoglycan (GAG) chains from fibroblasts increases on passage and returns to basal as cells proliferate showing that cell passage influences secretory events at the Golgi. The results indicate that Ser729 phosphorylation plays a role in determining PKCepsilon localisation in fibroblasts.

N-WASP regulates extension of filopodia and processes by oligodendrocyte progenitors, oligodendrocytes, and Schwann cells-implications for axon ensheathment at myelination.

The molecular mechanisms used by oligodendrocyte precursor cells (OPCs), oligodendrocytes (OLs), and Schwann cells (SCs) to advance processes for motility in the developing nervous system and to ensheath axons at myelination are currently not well defined. Here we demonstrate that OPCs, OLs, and SCs express the major proteins involved in actin polymerization-driven protrusion; these key proteins including F-actin, the Arp2/3 complex, neural-Wiskott Aldrich Syndrome protein (N-WASP) and WAVE proteins, and the RhoGTPases Rac and Cdc42 are present at the leading edges of processes being extended by OPCs, OLs, and SCs. We reveal by real-time PCR that OLs and SCs have different dominant WAVE isoforms. Inhibition of the WASP/WAVE protein, N-WASP, with wiskostatin that prevents activation of the Arp2/3 complex, blocks process extension by OPCs and SCs. Inhibition of N-WASP also causes OPC and SC process retraction, which is preceded by retraction of filopodia. This implicates filopodia in OPC and SC process stability and also of N-WASP in OPC and SC process dynamics. We also demonstrate that p34 (a component of the Arp2/3 complex), WASP/WAVE proteins, actin, alpha-tubulin, Rac, Cdc42, vinculin, and focal adhesion kinase are detected in water-shocked myelin purified from brain. Inhibition of N-WASP with wiskostatin decreases the number of axons undergoing initial ensheathment in intact optic nerve samples and reduces the Po content of dorsal root ganglia:SC co-cultures. Our findings indicate that OPCs, OLs, and SCs extend processes using actin polymerization-driven protrusion dependent on N-WASP. We hypothesize that inner mesaxons of OLs and SCs use the same mechanism to ensheath axons at myelination.

Neuroglycan C is a novel midkine receptor involved in process elongation of oligodendroglial precursor-like cells.

Midkine is a heparin-binding growth factor that promotes cell attachment and process extension in undifferentiated bipolar CG-4 cells, an oligodendroglial precursor cell line. We found that CG-4 cells expressed a non-proteoglycan form of neuroglycan C, known as a part-time transmembrane proteoglycan. We demonstrated that neuroglycan C before or after chondroitinase ABC treatment bound to a midkine affinity column. Neuroglycan C lacking chondroitin sulfate chains was eluted with 0.5 m NaCl as a major fraction from the column. We confirmed that CG-4 cells expressed two isoforms of neuroglycan C, I, and III, by isolating cDNA. Among three functional domains of the extracellular part of neuroglycan C, the chondroitin sulfate attachment domain and acidic amino acid cluster box domain showed affinity for midkine, but the epidermal growth factor domain did not. Furthermore, cell surface neuroglycan C could be cross-linked with soluble midkine. Process extension on midkine-coated dishes was inhibited by either a monoclonal anti-neuroglycan C antibody C1 or a glutathione S-transferase-neuroglycan C fusion protein. Finally, stable transfectants of B104 neuroblastoma cells overexpressing neuroglycan C-I or neuroglycan C-III attached to the midkine substrate, spread well, and gave rise to cytoskeletal changes. Based on these results, we conclude that neuroglycan C is a novel component of midkine receptors involved in process elongation.

Relative contribution of cell contact pattern, specific PKC isoforms and gap junctional communication in tight junction assembly in the mouse early embryo.

In mouse early development, cell contact patterns regulate the spatial organization and segregation of inner cell mass (ICM) and trophectoderm epithelium (TE) during blastocyst morphogenesis. Progressive membrane assembly of tight junctional (TJ) proteins in the differentiating TE during cleavage is upregulated by cell contact asymmetry (outside position) and suppressed within the ICM by cell contact symmetry (inside position). This is reversible, and immunosurgical isolation of the ICM induces upregulation of TJ assembly in a sequence that broadly mimics that occurring during blastocyst formation. The mechanism relating cell contact pattern and TJ assembly was investigated in the ICM model with respect to PKC-mediated signaling and gap junctional communication. Our results indicate that complete cell contact asymmetry is required for TJ biogenesis and acts upstream of PKC-mediated signaling. Specific inhibition of two PKC isoforms, PKCdelta and zeta, revealed that both PKC activities are required for membrane assembly of ZO-2 TJ protein, while only PKCzeta activity is involved in regulating ZO-1alpha+ membrane assembly, suggesting different mechanisms for individual TJ proteins. Gap junctional communication had no apparent influence on either TJ formation or PKC signaling but was itself affected by changes of cell contact patterns. Our data suggest that the dynamics of cell contact patterns coordinate the spatial organization of TJ formation via specific PKC signaling pathways during blastocyst biogenesis.

Specific PKC isoforms regulate blastocoel formation during mouse preimplantation development.

During early mammalian development, blastocyst morphogenesis is achieved by epithelial differentiation of trophectoderm (TE) and its segregation from the inner cell mass (ICM). Two major interrelated features of TE differentiation required for blastocoel formation include intercellular junction biogenesis and a directed ion transport system, mediated by Na+/K+ ATPase. We have examined the relative contribution of intercellular signalling mediated by protein kinase C (PKC) and gap junctional communication in TE differentiation and blastocyst cavitation. The distribution pattern of four (delta, theta, iota/lambda, zeta) PKC isoforms and PKCmicro/PKD1 showed partial colocalisation with the tight junction marker ZO-1alpha+ in TE and all four PKCs (delta, theta, iota/lambda, zeta) showed distinct TE/ICM staining patterns (predominantly at the cell membrane within the TE and cytoplasmic within the ICM), indicating their potential contribution to TE differentiation and blastocyst morphogenesis. Specific inhibition of PKCdelta and zeta activity significantly delayed blastocyst formation. Although modulation of these PKC isoforms failed to influence the already established programme of epithelial junctional differentiation within the TE, Na+/K+ ATPase alpha1 subunit was internalised from membrane to cytoplasm. Inhibition of gap junctional communication, in contrast, had no influence on any of these processes. Our results demonstrate for the first time that distinct PKC isotypes contribute to the regulation of cavitation in preimplantation embryos via target proteins including Na+/K+ ATPase.

Phorbol ester-induced translocation of PKC epsilon to the nucleus in fibroblasts: identification of nuclear PKC epsilon-associating proteins.

We show that phorbol ester treatment of NIH 3T3 fibroblasts induces rapid translocation of PKC from a perinuclear site to the nucleus, extending findings in PC12 and NG108-15 cells and in myocytes. We have immunoprecipitated the PKC from nuclei isolated from phorbol ester-treated fibroblasts and identified six proteins which associate with nuclear PKC. These have been characterised as matrin 3, transferrin, Rac GTPase activating protein 1, vimentin, beta-actin and annexin II by MALDI-TOF-MS. We have confirmed that these proteins associate with PKC by gel overlay and/or dot blotting assays. The role of these PKC-associating proteins in the nucleus and their interaction with PKC are considered.

PKC signalling regulates tight junction membrane assembly in the pre-implantation mouse embryo.

Epithelial differentiation including tight junction (TJ) formation occurs exclusively within the trophectoderm (TE) lineage of the mouse blastocyst. Here we examine mechanisms by which TJ protein membrane assembly might be regulated by protein kinase C (PKC) in the embryo. To overcome the inherent staging asynchrony of individual blastomeres within intact embryos, we have used isolated inner cell masses (ICMs) from early blastocysts to induce epithelial differentiation in their outer cells responding to their new cell contact pattern. Two TJ proteins examined retain their order of membrane assembly in isolated ICMs in culture as during normal development (early-assembling ZO-2 and late-assembling ZO-1alpha(+)), but this process is highly accelerated. Using six chemical modulators of PKC activity, we show here that PKC signalling is involved in the regulation of TJ membrane assembly. While indolactam-mediated PKC activation stimulates membrane assembly of both TJ proteins, TPA-mediated PKC activation stimulates only that of ZO-1alpha(+). The PKC inhibitors Ro-31-8220, Ro-31-8425 and Gö 6983 suppress the stimulatory effect of both PKC activators on membrane assembly to varying extents according to inhibitor and TJ protein examined. Gö 6983 similarly inhibits ZO-2 and ZO-1alpha(+) membrane assembly. PKC inhibition by Gö 6976 appeared to stimulate TJ membrane assembly. Despite the broad PKC isotype specificity of the inhibitors used, these data suggest that the two TJ proteins are differently regulated by PKC isotypes or subfamilies. As Gö 6983 uniquely affects aPKC (particularly PKCzeta) and we find that both PKCdelta and zeta relocate upon activator treatment to colocalise partially with the TJ proteins in isolated ICMs, we suggest that at least PKCdelta and zeta may play a central role in regulating TJ membrane assembly.

Lysophosphatidic acid induces process retraction in CG-4 line oligodendrocytes and oligodendrocyte precursor cells but not in differentiated oligodendrocytes.

Lysophosphatidic acid is a growth factor-like signalling phospholipid. We demonstrate here that lysophosphatidic acid induces process retraction in central glia-4 cells and oligodendrocyte precursors. This lysophosphatidic acid effect is rapid and concentration-dependent and results in cell rounding. It is inhibited by pre-treatment of cells with C3 exoenzyme, a specific inhibitor of Rho, or with Y-27632, a specific inhibitor of ROCK, a downstream kinase of Rho. Processes of differentiated central glia-4 oligodendrocytes were insensitive to lysophosphatidic acid treatment but cell bodies became phase dark, indicating cell spreading on the poly-l-lysine substratum. RT-PCR and Western blot analyses indicate that oligodendrocyte precursors and mature oligodendrocytes express mRNA and protein for LPA1, one of several LPA receptors. Thus lysophosphatidic acid may be signalling to Rho and stimulating actomyosin contraction in precursor oligodendrocytes by this family of receptors. The results show that lysophosphatidic acid signalling pathways influence retraction of processes in oligodendrocyte precursors but that this effect changes as oligodendrocytes differentiate.

Intestinal sugar absorption is regulated by phosphorylation and turnover of protein kinase C betaII mediated by phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent pathways.

Stimulation of intestinal fructose absorption by phorbol 12-myristate 13-acetate (PMA) results from rapid insertion of GLUT2 into the brush-border membrane and correlates with protein kinase C (PKC) betaII activation. We have therefore investigated the role of phosphatidylinositol 3 (PI3)-kinase and mammalian target of rapamycin in the regulation of fructose absorption by PKC betaII phosphorylation. In isolated jejunal loops, stimulation of fructose absorption by PMA was inhibited by preperfusion with wortmannin or rapamycin, which blocked GLUT2 activation and insertion into the brush-border membrane. Antibodies to the last 18 and last 10 residues of the C-terminal region of PKC betaII recognized several species differentially in Western blots. Extensive cleavage of native enzyme (80/78 kDa) to a catalytic domain product of 49 kDa occurred. PMA and sugars provoked turnover and degradation of PKC betaII by dephosphorylation to a 42-kDa species, which was converted to polyubiquitylated species detected at 180 and 250+ kDa. PMA increased the level of the PKC betaII 49-kDa species, which correlates with the GLUT2 level; wortmannin and rapamycin blocked these effects of PMA. Rapamycin and wortmannin inhibited PKC betaII turnover. PI3-kinase, PDK-1, and protein kinase B were present in the brush-border membrane, where their levels were increased by PMA and blocked by the inhibitors. We conclude that GLUT2-mediated fructose absorption is regulated through PI3-kinase and mammalian target of rapamycin-dependent pathways, which control phosphorylation of PKC betaII and its substrate-induced turnover and ubiquitin-dependent degradation. These findings suggest possible mechanisms for short term control of intestinal sugar absorption by insulin and amino acids.

Microfilament and microtubule organization and dynamics in process extension by central glia-4 oligodendrocytes: evidence for a microtubule organizing center.

Microfilaments in freshly adhering CG-4 cells and differentiated CG-4 oligodendrocytes are concentrated at the tips and edges of rapidly forming processes while microtubules are concentrated in new processes and extend from a concentrated spot of alpha-tubulin staining in the cell body to the cell periphery. In motile bipolar CG-4 cells, microfilaments are heavily concentrated at the flattened end of one process and along the rim of processes and the cell body: microtubules are concentrated along main processes and splay out into process tips and the cell body. In differentiated CG-4 oligodendrocytes, microfilaments are concentrated at the many process tips, in filopodia and in fine processes, but are not obvious in main processes where separate bundles of microtubules, which diverge at process branch points, are concentrated. gamma-tubulin, involved in microtubule nucleation, is concentrated at a small discrete area in the cell body, indicative of a microtubule organizing center. Polymerization of both actin and tubulin is required for initial process elaboration. Depolymerization of microtubules, but not of microfilaments, causes complete retraction of bipolar CG-4 cell processes. This process retraction does not occur if microfilaments are depolymerized first, indicating that process extension/retraction in motile bipolar CG-4 cells may occur by a balance of motor protein-driven forces as suggested for growth cone motility. Cytoskeleton organization in CG-4 cells is very similar to that reported for oligodendrocytes. CG-4 cells are thus a useful model for investigating the signals and mechanisms regulating oligodendrocyte process dynamics.

The 5' untranslated region of protein kinase Cdelta directs translation by an internal ribosome entry segment that is most active in densely growing cells and during apoptosis.

Protein kinase Cdelta (PKCdelta) is a member of the PKC family of phospholipid-dependent serine/threonine kinases and is involved in cell proliferation, apoptosis, and differentiation. Previous studies have suggested that different PKC isoforms might be translationally regulated. We report here that the 395-nt-long 5' untranslated region (5' UTR) of PKCdelta is predicted to form very stable secondary structures with free energies (deltaG values) of around -170 kcal/mol. The 5' UTR of PKCdelta can significantly repress luciferase translation in rabbit reticulocyte lysate but does not repress luciferase translation in a number of transiently transfected cell lines. By using a bicistronic luciferase reporter, we show that the 5' UTR of PKCdelta contains a functional internal ribosome entry segment (IRES). The activity of the PKCdelta IRES is greatest in densely growing cells and during apoptosis, when total protein synthesis and levels of full-length eukaryotic initiation factor 4G are reduced. However, the IRES activity of the 5' UTR of PKCdelta is not enhanced during serum starvation, another condition shown to inhibit cap-dependent translation, suggesting that its potency is dependent on specific cellular conditions. Accumulating data suggest that PKCdelta has a function as proliferating cells reach high density and in early and later events of apoptosis. Our studies suggest a mechanism whereby PKCdelta synthesis can be maintained under these conditions when cap-dependent translation is inhibited.

PKC epsilon is associated with myosin IIA and actin in fibroblasts.

Proteins coimmunoprecipitating with protein kinase C (PKC) epsilon in fibroblasts were identified through matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF m/s). This method identified myosin IIA in PKC epsilon immunoprecipitates, as well as known PKC epsilon binding proteins, actin, beta'Cop and cytokeratin. Myosin is not a substrate for PKC epsilon. Immunofluorescence analysis showed that PKC epsilon is colocalised with actin and myosin in actomyosin stress fibers in fibroblasts. Inhibitors of PKC and myosin ATPase activity, as well as microfilament-disrupting drugs, all inhibited spreading of fibroblasts after passage, suggesting a role for a PKC epsilon-actin-myosin complex in cell spreading.