Post-exercise cold water immersion attenuates acute anabolic signalling and long-term adaptations in muscle to strength training

We investigated functional, morphological and molecular adaptations to strength training exercise and cold water immersion (CWI) through two separate studies. In one study, 21 physically active men strength trained for 12 weeks (2 d⋅wk–1), with either 10 min of CWI or active recovery (ACT) after each training session. Strength and muscle mass increased more in the ACT group than in the CWI group (P<0.05). Isokinetic work (19%), type II muscle fibre cross-sectional area (17%) and the number of myonuclei per fibre (26%) increased in the ACT group (all P<0.05) but not the CWI group. In another study, nine active men performed a bout of single-leg strength exercises on separate days, followed by CWI or ACT. Muscle biopsies were collected before and 2, 24 and 48 h after exercise. The number of satellite cells expressing neural cell adhesion molecule (NCAM) (10−30%) and paired box protein (Pax7)(20−50%) increased 24–48 h after exercise with ACT. The number of NCAM+ satellitecells increased 48 h after exercise with CWI. NCAM+- and Pax7+-positivesatellite cell numbers were greater after ACT than after CWI (P<0.05). Phosphorylation of p70S6 kinaseThr421/Ser424 increased after exercise in both conditions but was greater after ACT (P<0.05). These data suggest that CWI attenuates the acute changes in satellite cell numbers and activity of kinases that regulate muscle hypertrophy, which may translate to smaller long-term training gains in muscle strength and hypertrophy. The use of CWI as a regular post-exercise recovery strategy should be reconsidered.

The Effects of Nicotine on the Regulation of Neuronal Alpha7 Nicotinic Acetylcholine Receptors and Intracellular Signalling Pathways

Nicotine, the addictive compound of tobacco products, exerts its effects in the brain by binding to neuronal acetylcholine nicotinic receptors (nAChRs). The aim of the present study was to increase the knowledge of nicotine s complex effects, the focus being on homomeric alpha7-nAChRs that are widely expressed in the brain. Nicotinic regulation of differential signalling molecules including transcriptional regulators was also studied. We found that the number of alpha7-nAChRs is increased in specific brain regions in mice, in a time-dependent manner after chronic oral nicotine administration. Our results suggest that in addition to alpha4beta2-nAChRs, the other major nAChR subtype expressed in the brain, the number of alpha7-nAChRs is affected by chronic presence of nicotine. We suggest that when studying the long-term effects of nicotine, the duration on administration is of great importance. Next, we observed that nicotine exposure induces accumulation of cAMP in cell cultures expressing nAChRs. Furthermore, nicotine-induced alpha7-nAChR upregulation was potentiated by treatments enhancing cAMP-signalling, suggesting a role for cAMP in the upregulation process. Protein kinase C (PKC) was found essential for the basal regulation of alpha7-nAChR number. The nicotine-evoked alpha7-nAChR upregulation could be further increased by PKC overexpression. Thirdly, the effects of nicotine on dopamine and cAMP regulated phosphoprotein (DARPP-32) were characterised in rat brain. The results show that DARPP-32 is regulated by both acute and long-term nicotine treatment in the striatal subdivisions. The effect of acute nicotine is dose-dependent and the three striatal regions display differential sensitivities to nicotine. Chronic nicotine is also able to regulate DARPP-32 signalling with prominent effect seen in the nucleus accumbens (NAc), suggesting a role for DARPP-32 in the mediation of long-term effects of nicotine. Finally, the regulation of transcription factors Elk-1 and FosB/deltaFosB by nicotine was investigated. We found that Elk-1 is activated by acute nicotine selectively in the NAc core and hippocampal area CA1, whereas acute nicotine does not affect FosB/deltaFosB. Long-term intermittent or continuous nicotine increases the level of total Elk-1 in the same brain regions as acute nicotine. FosB/deltaFosB is also affected by chronic nicotine. Thus, similarly to other drugs of abuse, nicotine regulates transcriptional regulators Elk-1 and FosB/deltaFosB. These results bring further support for a common mechanism underlying the development of addiction. Nicotine s positive effects on learning and memory might involve the transcription factor Elk-1 based on the changes seen in the hippocampus, the key area in cognitive functions.

Growth differentiation factor 9 signalling in the ovary

In the ovary, two new members of the large TGF-beta superfamily of growth factors were discovered in the 1990s. The oocyte was shown to express two closely related growth factors that were named growth differentiation factor 9 (GDF-9) and growth differentiation factor 9B (GDF-9B). Both of these proteins are required for normal ovarian follicle development although their individual significance varies between species. GDF-9 and GDF-9B mRNAs are expressed in the human oocytes from the primary follicle stage onwards. This thesis project was aimed to define the signalling mechanisms utilized by the oocyte secreted GDF-9. We used primary cultures of human granulosa luteal cells (hGL) as our cell model, and recombinant adenovirus-mediated gene transfer in manipulating the TGF-b family signalling cascade molecules in these cells. Overexpression of the constitutively active forms of the seven type I receptors, the activin receptor-like kinases 1-7 (ALK1-7), using recombinant adenoviruses caused a specific activation of either the Smad1 or Smad2 pathway proteins depending on the ALK used. Activation of both Smad1 and Smad2 proteins also stimulated the expression of dimeric inhibin B protein in hGL cells. Treatment with recombinant GDF-9 protein induced the specific activation of the Smad2 pathway and stimulated the expression of inhibin betaB subunit mRNA as well as inhibin B protein secretion in our cell model. Recombinant GDF-9 also activated the Smad3-responsive CAGA-luciferase reported construct, and the GDF-9 response in hGL cells was markedly potentiated upon the overexpression of Alk5 by adenoviral gene transduction. Alk5 overexpression also enhanced the GDF-9 induced inhibin B secretion by these cells. Similarly, in a mouse teratocarcinoma cell line P19, GDF-9 could activate the Smad2/3 pathway, and overexpression of ALK5 in COS7 cells rendered them responsive to GDF-9. Furthermore, transfection of rat granulosa cells with small interfering RNA for ALK5 or overexpression of the inhibitory Smad7 resulted in dose-dependent suppression of GDF-9 effects. In conclusion, this thesis shows that both Smad1 and Smad2 pathways are involved in controlling the regulation of inhibin B secretion. Therefore, in addition to endocrine control of inhibin production by the pituitary gonadotropins, also local paracrine factors within in the ovary, like the oocyte-derived growth factors, may contribute to controlling inhibin secretion. This thesis shows as well that like other TGF-beta family ligands, also GDF-9 signalling is mediated by the canonical type I and type II receptors with serine/threonine kinase activity, and the intracellular transcription factors, the Smads. Although GDF-9 binds to the BMP type II receptor, its downstream actions are specifically mediated by the type I receptor, ALK5, and the Smad2 and Smad3 proteins.

Dietary modulation of β-catenin signalling in an experimental model of colon cancer

Colorectal cancer is among the major cancers and one of the leading causes of cancer-related deaths in Western societies. Its occurrence is strongly affected by environmental factors such as diet. Thus, for preventative strategies it is vitally important to understand the mechanisms that stimulate adenoma growth and development towards accelerated malignancy or, in contrast, attenuate them to remain in quiescence for periods as long as decades. The main objective of this study was to investigate whether diet is able to modulate β-catenin signalling related to the promotion or prevention of intestinal tumourigenesis in an animal model of colon cancer, the Min/+ mouse. A series of dietary experiments with Min/+ mice were performed where fructo-oligosaccharide inulin was used for tumour promotion and four berries, bilberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), cloudberry (Rubus chamaemorus) and white currant (Ribes x pallidum), were used for tumour prevention. The adenomas (Apc-/-) and surrounding normal-appearing mucosa (Apc+/-) were investigated separately due to their mutational and functional differences. Tumour promotive and preventive diets had opposite effects on β-catenin signalling in the adenomas that was related to the different adenoma growth effects of dietary inulin and berries. The levels of nuclear β-catenin and cyclin D1 combined with size of the adenomas in the treatment groups suggests that diets induced differences in the cancerous process. Adenomas progressing to malignant carcinomas are most likely found in the sub-groups having the highest levels of β-catenin. On the other hand, adenomas staying quiescent for a long period of time are most probably found in the cloudberry or white currant diet groups. The levels of membranous E-cadherin and β-catenin increased as the adenomas in the inulin diet group grew, which could be a result of the overall increase in the protein levels of the cell. Therefore, the increasing levels of membranous β-catenin in Min/+ mice adenomas would be undesirable, due to the simultaneous increase in oncogenic nuclear β-catenin. We propose that the decreased amount of membranous β-catenin in benign adenomas of berry groups also means a decrease in the nuclear pool of β-catenin. Tumour promotion, but not the tumour prevention, influenced β-catenin signalling already in the normal appearing mucosa. Inulin-induced tumour promotion was related to β-catenin signalling in Min/+ mice, and in WT mice changes were also visible. The preventative effects of berries in the initiation phase were not mediated by β-catenin signalling. Our results suggest that, in addition to the number, size, and growth rate of adenomatous polyps, the signalling pattern of the adenomas should be considered when evaluating preventative dietary strategies.

Structure-Function Studies of GDNF Family Ligand-RET signalling

Glial cell line-derived neurotrophic factor (GDNF) and its family members neurturin (NRTN), artemin (ARTN) and persephin (PSPN) are growth factors, which are involved in the development, differentiation and maintenance of many neuron types. In addition, they function outside of the nervous system, e.g. in the development of kidney, testis and liver. GDNF family ligand (GFL) signalling happens through a tetrameric receptor complex, which includes two glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor (GFRα) molecules and two RET (rearranged during transfection) receptor tyrosine kinases. Each of the ligands binds preferentially one of the four GFRα receptors: GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The signal is then delivered by RET, which cannot bind the GFLs on its own, but can bind the GFL-GFRα complex. Under normal cellular conditions, RET is only phosphorylated on the cell surface after ligand binding. At least the GDNF-GFRα1 complex is believed to recruit RET to lipid rafts, where downstream signalling occurs. In general, GFRαs consist of three cysteine-rich domains, but all GFRα4s except for chicken GFRα4 lack domain 1 (D1). We characterised the biochemical and cell biological properties of mouse PSPN receptor GFRα4 and showed that it has a significantly weaker capacity than GFRα1 to recruit RET to the lipid rafts. In spite of that, it can phosphorylate RET in the presence of PSPN and contribute to neuronal differentiation and survival. Therefore, the recruitment of RET to the lipid rafts does not seem to be crucial for the biological activity of all GFRα receptors. Secondly, we demonstrated that GFRα1 D1 stabilises the GDNF-GFRα1 complex and thus affects the phosphorylation of RET and contributes to the biological activity. This may be important in physiological conditions, where the concentration of the ligand or the soluble GFRα1 receptor is low. Our results also suggest a role for D1 in heparin binding and, consequently, in the biodistribution of released GFRα1 or in the formation of the GFL-GFRα-RET complex. We also presented the crystallographic structure of GDNF in the complex with GFRα1 domains 2 and 3. The structure differs from the previously published ARTN-GFRα3 structure in three significant ways. The biochemical data verify the structure and reveal residues participating in the interactions between GFRα1 and GDNF, and preliminarily also between GFRα1 and RET and heparin. Finally, we showed that, the precursor of the oncogenic MEN 2B (multiple endocrine neoplasia type 2) form of RET gets phosphorylated already during its synthesis in the endoplasmic reticulum (ER). We also demonstrated that it associates with Src homology 2 domain-containing protein (SHC) and growth factor receptor-bound protein (GRB2) in the ER, and has the capacity to activate several downstream signalling molecules.

GABAa Receptor Mediated Signalling in the Brain: Inhibition, Shunting and Excitation

Within central nervous system, the simple division of chemical synaptic transmission to depolarizing excitation mediated by glutamate and hyperpolarizing inhibition mediated by γ-amino butyric acid (GABA), is evidently an oversimplification. The GABAa receptor (GABAaR) mediated responses can be of opposite sign within a single resting cell, due to the compartmentalized distribution of cation chloride cotransporters (CCCs). The K+/Cl- cotransporter 2 (KCC2), member of the CCC family, promotes K+ fuelled Cl- extrusion and sets the reversal potential of GABA evoked anion currents typically slightly below the resting membrane potential. The interesting ionic plasticity property of GABAergic signalling emerges from the short-term and long-term alterations in the intraneuronal concentrations of GABAaR permeable anions (Cl- and HCO3-). The short-term effects arise rapidly (in the time scale of hundreds of milliseconds) and are due to the GABAaR activation dependent shifts in anion gradients, whereas the changes in expression, distribution and kinetic regulation of CCCs are underlying the long-term effects, which may take minutes or even hours to develop. In this Thesis, the differences in the reversal potential of GABAaR mediated responses between dopaminergic and GABAergic cell types, located in the substantia nigra, were shown to be attributable to the differences in the chloride extrusion mechanisms. The stronger inhibitory effect of GABA on GABAergic neurons was due to the cell type specific expression of KCC2 whereas the KCC2 was absent from dopaminergic neurons, leading to a less prominent inhibition brought by GABAaR activation. The levels of KCC2 protein exhibited activity dependent alterations in hippocampal pyramidal neurons. Intense neuronal activity, leading to a massive release of brain derived neurotrophic factor (BDNF) in vivo, or applications of tyrosine receptor kinase B (TrkB) agonists BDNF or neurotrophin-4 in vitro, were shown to down-regulate KCC2 protein levels which led to a reduction in the efficacy of Cl- extrusion. The GABAergic transmission is interestingly involved in an increase of extracellular K+ concentration. A substantial increase in interstitial K+ tends to depolarize the cell membrane. The effects that varying ion gradients had on the generation of biphasic GABAaR mediated responses were addressed, with particular emphasis on the novel idea that the K+/Cl- extrusion via KCC2 is accelerated in response to a rapid accumulation of intracellular Cl-. The KCC2 inhibitor furosemide produced a large reduction in the GABAaR dependent extracellular K+ transients. Thus, paradoxically, both the inefficient KCC2 activity (via increased intracellular Cl-) and efficient KCC2 activity (via increased extracellular K+) may promote excitation.

Cytokinin signalling in the regulation of cambial development

Secondary growth of plants is of pivotal importance in terrestrial ecosystems, providing a significant carbon sink in the form of wood. As plant biomass accumulation results largely from the cambial growth, it is surprising that quite little is known about the hormonal or genetic control of this important process in any plant species. The central aim of my thesis studies was to explore the function of cytokinin in the regulation of cambial development. Since their discovery as regulators of plant cell divisions, cytokinins have been assumed to participate in the control of cambial development. Evidence for this action was deduced from hormone treatment experiments, where exogenously applied cytokinin was shown to enhance cambial cell divisions in diverse plant organs and species. In my thesis work, the conservation of cytokinin signalling and homeostasis genes between a herbaceous plant, Arabidopsis, and a hardwood tree species, Populus trichocarpa. Presumably reflecting the ancient origin of cytokinin signalling system, the Populus genome contains orthologs for all Arabidopsis cytokinin signalling and homeostasis genes. Thus, genes belonging to five main families of isopentenyl transferases (IPTs), cytokinin oxidases (CKXs), two-component receptors, histidine containing phosphotransmitters (HPts) and response regulators (RRs) were identified from the Populus genome. Three subfamilies associated with cytokinin signal transduction, the CKI1-like family of two-component receptors, the AHP4-like HPts, and the ARR22-like atypical RRs, were significantly larger in Populus genome than in Arabidopsis. Potential contribution to the extensive secondary development of Populus by the members of these considerably expanded gene families will be discussed. Representatives of all cytokinin signal transduction elements were expressed in the Populus cambial zone, and most of the expressed genes appeared to be slightly more abundant on the phloem side of the meristem. The abundance of cytokinin related genes in the cambium emphasizes the important role of this hormone in the regulation of the extensive secondary growth characteristic of tree species. The function of the pseudo HPts in primary vascular development was studied in Arabidopsis root vasculature. It was demonstrated that the pseudo HPt AHP6 has a role in locally inhibiting cytokinin signalling in the protoxylem position in the Arabidopsis root, thus enabling differentiation of the protoxylem cell file. The possible role of pseudo HPts in cambial development will be discussed. The expression peak of cytokinin signalling genes in the tree cambial zone strongly indicates that cytokinin has a role in the regulation of this meristem function. To address whether cytokinin signalling is required for cambial activity, transgenic Populus trees with modified cytokinin signalling were produced. These trees were expressing a cytokinin catabolic gene from Arabidopsis, CYTOKININ OXIDASE 2, (AtCKX2) under the promoter of a Betula CYTOKININ RECEPTOR 1 (BpCRE1). The pBpCRE1::CKX2 transgenic Populus trees showed a reduced concentration of a biologically active cytokinin, correlating with their impaired cytokinin response. Furthermore, the radial growth of these trees was compromised, as illustrated by a smaller stem diameter than in wild-type trees of the same height. Moreover, the level of cambial cytokinin signalling was down-regulated in these thin-stemmed trees. The reduced signalling correlated with a decreased number of meristematic cambial cells, implicating cytokinin activity as a direct regulator of cambial cell division activity. Together, the results of my study indicate that cytokinins are major hormonal regulators required for cambial development.

Modulatory roles of microRNAs in the regulation of different signalling pathways in large bowel cancer stem cells

There are emerging data to suggest that microRNAs (miRNAs) have significant roles in regulating the function of normal cells and cancer stem cells (CSCs). This review aims to analyse the roles of miRNAs in the regulation of colon CSCs through their interaction with various signalling pathways. Studies showed a large number of miRNAs that are reported to be deregulated in colon CSCs. However, few of the studies available were able to outline the function of miRNAs in colon CSCs and uncover their signalling pathways. From those miRNAs, which are better described, miR-21 followed by miR-34, miR-200 and miR-215 are the most reported miRNAs to have roles in colon CSC regulation. In particular, miRNAs have been reported to regulate the stemness features of colon CSCs mainly via Wnt/B-catenin and Notch signalling pathways. Additionally, miRNAs have been reported to act on processes involving CSCs through cell cycle regulation genes and epithelial-mesenchymal transition. The relative paucity of data available on the significance of miRNAs in CSCs means that new studies will be of great importance to determine their roles and to identify the signalling pathways through which they operate. Such studies may in future guide further research to target these genes for more effective cancer treatment. miRNAs were shown to regulate the function of cancer stem cells in large bowel cancer by targeting a few key signalling pathways in cells.

Polydimethylsiloxane (PDMS) modulates CD38 expression, absorbs retinoic acid and may perturb retinoid signalling

Polydimethylsiloxane (PDMS) is the most commonly used material in the manufacture of customized cell culture devices. While there is concern that uncured PDMS oligomers may leach into culture medium and/or hydrophobic molecules may be absorbed into PDMS structures, there is no consensus on how or if PDMS influences cell behaviour. We observed that human umbilical cord blood (CB)-derived CD34+ cells expanded in standard culture medium on PDMS exhibit reduced CD38 surface expression, relative to cells cultured on tissue culture polystyrene (TCP). All-trans retinoic acid (ATRA) induces CD38 expression, and we reasoned that this hydrophobic molecule might be absorbed by PDMS. Through a series of experiments we demonstrated that ATRA-mediated CD38 expression was attenuated when cultures were maintained on PDMS. Medium pre-incubated on PDMS for extended durations resulted in a time-dependant reduction of ATRA in the medium and increasingly attenuated CD38 expression. This indicated a time-dependent absorption of ATRA into the PDMS. To better understand how PDMS might generally influence cell behaviour, Ingenuity Pathway Analysis (IPA) was used to identify potential upstream regulators. This analysis was performed for differentially expressed genes in primary cells including CD34+ haematopoietic progenitor cells, mesenchymal stromal cells (MSC), and keratinocytes, and cell lines including prostate cancer epithelial cells (LNCaP), breast cancer epithelial cells (MCF-7), and myeloid leukaemia cells (KG1a). IPA predicted that the most likely common upstream regulator of perturbed pathways was ATRA. We demonstrate here that ATRA is absorbed by PDMS in a time-dependent manner and results in the concomitant reduced expression of CD38 on the cell surface of CB-derived CD34+ cells.

Extracellular vesicles mediate paracrine signalling in androgen deprived prostate cancer

The androgen receptor (AR) is the main therapeutic target for advanced prostate cancer (PCa). Current treatments have focused on inhibiting the transcriptional activity of the AR, however androgens can also induce non-genomic effects by facilitating the initiation of kinase signaling cascades in PCa. Cells, including PCa, secrete extracellular vesicles (EV), which are able to mediate communication between cells and can also contribute towards these processes.

YB-1, a key regulator of androgen receptor signalling

Reactivation of androgen receptor signalling is one of the hallmarks of prostate cancer progression to the terminal castrate resistant stage. A better understanding of mechanisms driving this adaptive response is essential for the development of innovative intervention strategies that effectively delay or halt prostate cancer progression. The Y-box binding protein 1 (YB-1) has been found to be closely associated with prostate cancer progression. By characterising its role in the adaptive process leading to castrate resistance, we aim to promote YB-1 as a novel therapeutic target in advanced prostate cancer.

Bimodal distribution of motility and cell fate in Dictyostelium discoideum

Pre-starvation amoebae of Dictyostelium discoideum exhibit random movements. Starved cells aggregate by directed movements (chemotaxis) towards cyclic AMP and differentiate into live spores or dead stalk cells. Many differences between presumptive spore and stalk cells precede differentiation. We have examined whether cell motility-related factors are also among them. Cell speeds and localisation of motility-related signalling molecules were monitored by live cell imaging and immunostaining (a) in nutrient medium during growth, (b) immediately following transfer to starvation medium and (c) in nutrient medium that was re-introduced after a brief period of starvation. Cells moved randomly under all three conditions but mean speeds increased following transfer from nutrient medium to starvation medium; the transition occurred within 15 min. The distribution of speeds in starvation medium was bimodal: about 20% of the cells moved significantly faster than the remaining 80%. The motility-related molecules F-actin, PTEN and PI3 kinase were distributed differently in slow and fast cells. Among starved cells, the calcium content of slower cells was lower than that of the faster cells. All differences reverted within 15 min after restoration of the nutrient medium. The slow/fast distinction was missing in Polysphondylium pallidum, a cellular slime mould that lacks the presumptive stalk and spore cell classes, and in the trishanku (triA(center dot)) mutant of D. discoideum, in which the classes exist but are unstable. The transition from growth to starvation triggers a spontaneous and reversible switch in the distribution of D. discoideum cell speeds. Cells whose calcium content is relatively low (known to be presumptive spore cells) move slower than those whose calcium levels are higher (known to be presumptive stalk cells). Slow and fast cells show different distributions of motility-related proteins. The switch is indicative of a bistable mechanism underlying cell motility.

Quorum sensing and pathogenesis: role of small signalling molecules in bacterial persistence

The pathogenesis of Mycobacterium tuberculosis is associated with its ability to survive inside the human host and the bacteria use a variety of mechanism to evade the host's defence. A clearer understanding of the host pathogen interaction is needed to follow the pathogenicity and virulence. Recent advances in the study of inter and intra-cellular communication in bacteria had prompted us to study the role of quorum sensing in bacterial survival and pathogenicity. The cell cell communication in bacteria (quorum sensing) is mediated through the exchange of small molecules called as autoinducers that allow bacteria to modulate their gene expression in response to change in cell-population density. It is a coordinated response that confers multicellularity to a bacterial population in response to stress from external environment. Quorum sensing molecules are the global regulators and regulate a wide range of physiological processes including biofilm formation, motility, cell differentiation, long-term survival and many others. Many bacterial pathogens require quorum sensing to produce the virulence factors in response to host pathogen interaction. Here, we summarize our current understanding on small molecule signalling and their role in the bacterial persistence. New discoveries in these areas have enriched our knowledge on intracellular signalling and their role in the long-term survival of mycobacteria under nutrient starvation.

Imaging cellular signalling: many `moving tales' in MAP kinase odyssey

Cellular signalling events are at the core of every adaptive response. Signalling events link environmental changes to physiological responses, consequently allowing cellular and organismal sustenance and survival. Classical approaches to study cellular signalling have relied on a variety of cell disruptive techniques which yield limited kinetic information, while the underlying events are much more complex. In this article, we discuss how modern live cell imaging microscopy has found increasing utilization in revealing spatio temporal dynamics of various signalling pathways. Utilizing the well studied mitogen-activated protein kinase (MAPK) signalling cascade as a template, the design, construction and utilization of `mobile' (translocation proficient) biosensors, suitable for studying MAPK signalling in living cells are described in detail. Experimental setup and results obtained from these biosensors, based on different proteins involved in the MAPK signalling cascade, have been described along with the setup of a microscope optimal for live cell imaging applications. Utilizing the ability to activate or deactivate signalling pathways using defined activators and specific pharmacological inhibitors, we also show how these sensors can yield unique spatial and temporal kinetic information of signalling in living cells.

Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum

The plane of division of granule neuron progenitors (GNPs) was analysed with respect to the pial surface in P0 to P14 cerebellum and the results showed that there was a significant bias towards the plane of cell division being parallel to pial surface across this developmental window. In addition, the distribution of beta-Catenin in anaphase cells was analysed, which showed that there was a significant asymmetry in the distribution of beta-Catenin in dividing GNPs. Further, inhibition of Sonic Hedgehog (Shh) signalling had an effect on plane of cell division. Asymmetric distribution of beta-Catenin was shown to occur towards the source of a localized extracellular cue.