Journal of Cell Science

Differentiation of African trypanosomes from replicating slender bloodstream forms to nondividing stumpy forms limits the parasite population size, allowing survival of the mammalian host and establishment of a stable host-parasite relationship. Using a novel in vitro culture system we have shown that slender to stumpy differentiation is induced by parasite density alone and thus is independent of host cues. Here we investigate the density sensing mechanism and show that trypanosomes release a soluble activity of low relative molecular mass, termed stumpy induction factor (SIF), which accumulates in conditioned medium. SIF activity triggers cell cycle arrest in G1/G0 phase and induces differentiation with high efficiency and rapid kinetics. Membrane-permeable derivates of cAMP or the phosphodiesterase inhibitor etazolate perfectly mimic SIF activity. Furthermore, SIF activity elicits an immediate two-to threefold elevation of intracellular cAMP content upon addition to slender forms. We conclude that SIF and hence density sensing operate through the cAMP signalling pathway. Temporal correlation of markers indicates that cell cycle arrest invariably precedes differentiation. Thus, our results indicate that the cell cycle regulation of blood-stream forms is under dominant control of cAMP signalling. Irreversible commitment to the quiescent state is elicited by a cAMP agonist within a period shorter than one complete cell cycle.

Changes in the expression and distribution of nuclear lamins were investigated during C2C12 myoblast differentiation. The expression of most lamins was unchanged during myogenesis. By contrast, lamin-B2 expression increased and LAP2α expression decreased twofold. These changes were correlated with reduced solubility and redistribution of A-type lamins. When C2C12 myoblasts were transfected with a lamin-A mutant that causes autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD), the mutant protein accumulated in the nucleoplasm and exerted dominant influences over endogenous lamins. Myoblasts transfected with wild-type lamins differentiated, albeit more slowly, whereas myoblasts transfected with mutant lamins failed to differentiate. Myoblast differentiation requires dephosphorylation of the retinoblastoma protein Rb. During myogenesis, Rb was rapidly and progressively dephosphorylated. Underphosphorylated Rb formed complexes with LAP2α in proliferating myoblasts and postmitotic myoblasts. In myoblasts transfected with the mutant lamins, this complex was disrupted. These data suggest that remodelling of the nucleoskeleton is necessary for skeletal-muscle differentiation and for correct regulation of Rb pathways.

Characterization of myogenic subpopulations has traditionally been performed independently of their functional performance following transplantation. Using the preplate technique, which separates cells based on their variable adhesion characteristics, we investigated the use of cell surface proteins to potentially identify progenitors with enhanced regeneration capabilities. Based on previous studies, we used cell sorting to investigate stem cell antigen-1 (Sca-1) and CD34 expression on myogenic populations with late adhesion characteristics. We compared the regeneration efficiency of these sorted progenitors, as well as those displaying early adhesion characteristics, by quantifying their ability to regenerate skeletal muscle and restore dystrophin following transplantation into allogenic dystrophic host muscle.

Identification and utilization of late adhering populations based on CD34 expression led to differential regeneration, with CD34-positive populations exhibiting significant improvements in dystrophin restoration compared with both their CD34-negative counterparts and early adhering cell populations. Regenerative capacity was found to correspond to the level of myogenic commitment, defined by myogenic regulatory factor expression, and the rate and degree of induced cell differentiation and fusion. These results demonstrate the ability to separate definable subpopulations of myogenic progenitors based on CD34 expression and reveal the potential implications of defining myogenic cell behavioral and phenotypic characteristics in relation to their regenerative capacity in vivo.

At the initial stage of carcinogenesis, transformation occurs in a single cell within an epithelial sheet. However, it remains unknown what happens at the boundary between normal and transformed cells. Using Madin-Darby canine kidney (MDCK) cells transformed with temperature-sensitive v-Src, we have examined the interface between normal and Src-transformed epithelial cells. We show that Src-transformed cells are apically extruded when surrounded by normal cells, but not when Src cells alone are cultured, suggesting that apical extrusion occurs in a cell-context-dependent manner. We also observe apical extrusion of Src-transformed cells in the enveloping layer of zebrafish gastrula embryos. When Src-transformed MDCK cells are surrounded by normal MDCK cells, myosin-II and focal adhesion kinase (FAK) are activated in Src cells, which further activate downstream mitogen-activated protein kinase (MAPK). Importantly, activation of these signalling pathways depends on the presence of surrounding normal cells and plays a crucial role in apical extrusion of Src cells. Collectively, these results indicate that interaction with surrounding normal epithelial cells influences the signalling pathways and behaviour of Src-transformed cells.

In examining how chloral hydrate affects mitosis, we found that extracellular application of 0·l % chloral hydrate produced an abrupt rise in cytosolic free Ca2+. Digitized fluorescence microscopy of Fura-2-loaded, mitotic and interphase PtK cells revealed that Ca2+ rose 15 s after chloral hydrate application, peaked within 1 min at a concentration two-to sevenfold above the basal level and then slowly dropped. Bathing cells in 0·l% chloral hydrate caused metaphase spindles to shorten, starting in 1–2 min, and inhibited spindle elongation without affecting chromosome-to-pole movement during anaphase, as determined by phase-contrast observation of living cells. Spindle elongation and chromosome movement were unaffected by intracellular injection of 7·5% chloral hydrate. Extensive mitotic microtubule breakdown occurred after cells were bathed for 7 min in 0·l% chloral hydrate, while interphase microtubules were unaffected as determined by immunofluorescence. The chloral hydrate-induced microtubule breakdown and metaphase spindle shortening were prevented by 10mM-CoCl2, which has previously been shown to block Ca2+ influx and to stabilize microtubules in vitro. These results imply that disruption of mitotic spindle function and structure by chloral hydrate is due to a rise in cytosolic free Ca2+, and also indicate that mitotic microtubules are more Ca2+-labile than interphase microtubules.

Tau is a group of neuronal microtubule-associated proteins that are formed by alternative mRNA splicing and accumulate in neurofibrillary tangles in Alzheimer's disease (AD) brain. Tau plays a key role in regulating microtubule dynamics, axonal transport and neurite outgrowth, and all these functions of tau are modulated by site-specific phosphorylation. There is significant evidence that a disruption of normal phosphorylation events results in tau dysfunction in neurodegenerative diseases, such as AD, and is a contributing factor to the pathogenic processes. Indeed, the abnormal tau phosphorylation that occurs in neurodegenerative conditions not only results in a toxic loss of function (e.g. decreased microtubule binding) but probably also a toxic gain of function (e.g. increased tau-tau interactions). Although tau is phosphorylated in vitro by numerous protein kinases, how many of these actually phosphorylate tau in vivo is unclear. Identification of the protein kinases that phosphorylate tau in vivo in both physiological and pathological processes could provide potential therapeutic targets for the treatment of AD and other neurodegenerative diseases in which there is tau pathology.

Epithelial cell-cell junctions are specialised structures connecting individual cells in epithelial tissues. They are dynamically and functionally linked to the actin cytoskeleton. Disassembly of these junctions is a key event during physiological and pathological processes, but how this influences gene expression is largely uncharacterised. Here, we investigate whether junction disassembly regulates transcription by serum response factor (SRF) and its coactivator MAL/MRTF. Ca2+-dependent dissociation of epithelial integrity was found to correlate strictly with SRF-mediated transcription. In cells lacking E-cadherin expression, no SRF activation was observed. Direct evidence is provided that signalling occurs via monomeric actin and MAL. Dissociation of epithelial junctions is accompanied by induction of RhoA and Rac1. However, using clostridial cytotoxins, we demonstrate that Rac, but not RhoA, is required for SRF and target gene induction in epithelial cells, in contrast to serum-stimulated fibroblasts. Actomyosin contractility is a prerequisite for signalling but failed to induce SRF activation, excluding a sufficient role of the Rho-ROCK-actomyosin pathway. We conclude that E-cadherin-dependent cell-cell junctions facilitate transcriptional activation via Rac, G-actin, MAL and SRF upon epithelial disintegration.

In pressure or volume overload, hypertrophic growth of the myocardium is associated with myofibroblast differentiation, a process in which cardiac fibroblasts express smooth muscle α-actin (SMA). The signaling mechanisms that mediate force-induced myofibroblast differentiation and SMA expression are not defined. We examined the role of the Rho–Rho-kinase pathway in force-induced SMA expression in fibroblasts using an in vitro model system that applies static tensile forces (0.65 pN/μm2) to integrins via collagen-coated magnetite beads. Force maximally induced RhoA activation at 10 minutes that was localized to force application sites and required intact actin filaments. Force application induced phosphorylation of LIM kinase (5-10 minutes) and an early dephosphorylation of cofilin (5 minutes) that was followed by prolonged cofilin phosphorylation. These responses were blocked by Y27632, an inhibitor of Rho kinase. Force promoted actin filament assembly at force application sites (10-20 minutes), a process that required Rho kinase and cofilin. Force application induced nuclear translocation of the transcriptional co-activator MRTF-A but not MRTF-B. Nuclear translocation of MRTF-A required Rho kinase and intact actin filaments. Force caused 3.5-fold increases of SMA promoter activity that were completely blocked by transfection of cells with dominant-negative MRTF-A or by inhibition of Rho kinase or by actin filament disassembly. These data indicate that mechanical forces mediate actin assembly through the Rho–Rho-kinase–LIMK cofilin pathway. Force-mediated actin filament assembly promotes nuclear translocation of MRTF and subsequent activation of the SMA promoter to enhance SMA expression.

We describe a reproducible method for growing small intestinal epithelium (derived from the suckling rat intestine) in short-term (primary) cultures. Optimal culture conditions were determined by quantitative assays of proliferation (i.e. changes in cellularity and DNA synthesis). Isolation of the epithelia and, significantly, preservation of its three-dimensional integrity was achieved using a collagenase/dispase digestion technique. Purification of the epithelium was also facilitated by the use of a simple differential sedimentation method.

The results presented below support the idea that proliferation of normal gut epithelium ex vivo is initially dependent upon the maintenance of the structural integrity of this tissue and upon factors produced by heterologous mesenchymal cells. Proliferation in vitro was also critically dependent upon the quality of the medium and constituents used.

Cultures reached confluence within 10–14 days and consisted of epithelial colonies together with varying amounts of smooth-muscle-like cells. Cultures have been maintained for periods up to one month, but the longer-term potential for growth by sub-culturing has not been examined. Strategies for reducing the proliferation of these non-epithelial cells are also described.

Since the discovery of the prototypical Sprouty (Spry) protein in Drosophila, there has been an effort to determine how these novel modulators of the Ras/MAP-kinase pathway function. A clue to their mechanism of action comes from the several highly conserved sequences within all the currently known Spry isoforms: an ∼110-residue cysteine-rich sequence in the C-terminal half that directs Spry proteins to a concentration of signaling proteins at the plasma membrane; a small motif surrounding a tyrosine residue(Y55 in human Spry2) that is responsible for interaction with other proteins. In cultured mammalian cells, hSpry2 inhibits epidermal growth factor receptor(EGFR) endocytosis and subsequently sustains the activation of MAP kinase but negatively regulates the same pathway following stimulation of fibroblast growth factor receptors (FGFRs). Current evidence indicates that Cbl is a key protein that interacts directly with Spry2 following activation of receptor tyrosine kinases (RTKs). It appears to be the ability of Cbl to interact as an E3 ubiquitin ligase on specific target proteins and as a docking protein in other contexts that dictates the differential effects Spry2 has on the Ras/MAP-kinase pathway following EGFR and FGFR activation.