Real-time measurements of cAMP production in live<i>Dictyostelium</i>cells

Bagorda, Anna; Das, Satarupa; Rericha, Erin; Chen, David; Davidson, Jean M.; Parent, Carole A.

doi:10.1242/jcs.051987

Cited by 25 publications

(26 citation statements)

References 38 publications

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“…Electron microscopy reveals that signalling cells accumulate and release small vesicles (Maeda and Gerisch, 1977), suggesting that cAMP is released by a conventional exocytic mechanism. Our work contradicts this idea and is consistent with other work showing that cAMP is completely released from cells by lysis in conditions not expected to break small vesicles (Schoen et al, 1989) and that there is a build-up of free cAMP in the cytoplasm during cAMP relay (Bagorda et al, 2009). It seems likely that cAMP is released by a non-exocytic mechanism, possibly through a membrane transporter.…”

Section: Discussionsupporting

confidence: 90%

The exocytic genesecAis required forDictyosteliumcell motility and osmoregulation

Zanchi

Howard

Bretscher

et al. 2010

Journal of Cell Science

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SummaryWe investigated the link between cell movement and plasma membrane recycling using a fast-acting, temperature-sensitive mutant of the Dictyostelium SecA exocytic protein. Strikingly, most mutant cells become almost paralysed within minutes at the restrictive temperature. However, they can still sense cyclic-AMP (cAMP) gradients and polymerise actin up-gradient, but form only abortive pseudopodia, which cannot expand. They also relay a cAMP signal normally, suggesting that cAMP is released by a non-exocytic mechanism. To investigate why SecA is required for motility, we examined membrane trafficking in the mutant. Plasma membrane circulation is rapidly inhibited at the restrictive temperature and the cells acquire a prominent vesicle. Organelle-specific markers show that this is an undischarged contractile vacuole, and we found the cells are correspondingly osmo-sensitive. Electron microscopy shows that many smaller vesicles, probably originating from the plasma membrane, also accumulate at the restrictive temperature. Consistent with this, the surface area of mutant cells shrinks. We suggest that SecA mutant cells cannot move at the restrictive temperature because their block in exocytosis results in a net uptake of plasma membrane, reducing its area, and so restricting pseudopodial expansion. This demonstrates the importance of proper surface area regulation in cell movement.

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Section: Discussionsupporting

confidence: 90%

The exocytic genesecAis required forDictyosteliumcell motility and osmoregulation

Zanchi

Howard

Bretscher

et al. 2010

Journal of Cell Science

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“…Adenylyl Cyclase Assay-The cAR1-mediated activation of ACA was carried out as previously described (30 Live Cell FRET Imaging and Analysis-FRET images were collected at different time points in development as previously described (33). Briefly, to measure FRET, three images were acquired for each set of measurements: (i) YFP fluorescence (514-nm excitation, 530-nm LP emission filter); (ii) CFP fluorescence (458-nm excitation, 475-505-nm BP emission filter); and (iii) FRET (458-nm excitation, 530-nm LP emission filter).…”

Section: Methodsmentioning

confidence: 99%

“…This prompted us to measure the basal intracellular cAMP levels in WT and regA Ϫ cells developed for 5-7 or 3-5 h, respectively. For this purpose, we used a previously characterized cAMP FRET sensor, which harbors the PKA regulatory subunit cAMP-binding domain (B domain) flanked by eYFP and eCFP (33). At low cAMP levels, the probe remains in a closed state, and the FRET response is maximal.…”

Section: Aca Activity Measured Following Subsaturating Camp Stimulatimentioning

confidence: 99%

“…Upon stimulation, binding to the smaller pool of active receptors slightly increases ACA activity producing a relative smaller cAMP increase. We have previously proposed that cAMP secretion is a PKA-regulated event that occurs through the polarized release of exosomes expressing ACA (33,50). We envision that in later development, high PKA activity leads to aberrant secretion, which leads to the formation of disorganized stream, poor chemotaxis to exogenous signals, and the formation of small aggregates.…”

Section: Table 1 Chemotactic Index and Mean Speeds Of Wt And Mutants mentioning

confidence: 99%

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Direct Biochemical Measurements of Signal Relay during Dictyostelium Development

Das

Rericha

Bagorda

et al. 2011

Journal of Biological Chemistry

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Upon starvation, individual Dictyostelium discoideum cells enter a developmental program that leads to collective migration and the formation of a multicellular organism. The process is mediated by extracellular cAMP binding to the G proteincoupled cAMP receptor 1, which initiates a signaling cascade leading to the activation of adenylyl cyclase A (ACA), the synthesis and secretion of additional cAMP, and an autocrine and paracrine activation loop. The release of cAMP allows neighboring cells to polarize and migrate directionally and form characteristic chains of cells called streams. We now report that cAMP relay can be measured biochemically by assessing ACA, ERK2, and TORC2 activities at successive time points in development after stimulating cells with subsaturating concentrations of cAMP. We also find that the activation profiles of ACA, ERK2, and TORC2 change in the course of development, with later developed cells showing a loss of sensitivity to the relayed signal. We examined mutants in PKA activity that have been associated with precocious development and find that this loss in responsiveness occurs earlier in these mutants. Remarkably, we show that this loss in sensitivity correlates with a switch in migration patterns as cells transition from streams to aggregates. We propose that as cells proceed through development, the cAMP-induced desensitization and down-regulation of cAMP receptor 1 impacts the sensitivities of chemotactic signaling cascades leading to changes in migration patterns.The directed migration of individual cells or chains or sheets of cells requires cells to transduce external cues into internal signals that regulate cytoskeletal reorganization and cell polarity. Signal transduction is important during development where the transition from single to collective cell migration is regulated by multiple external cues (1). Cell-cell communication, a process that can be manifested through the relay of external chemical signals (2, 3), is critical for a wide range of developmental processes including the survival of the social amoebae Dictyostelium discoideum.Upon starvation, Dictyostelium cells enter a developmental program leading to the transition from cells operating individually to groups of cells operating as a population. Aggregation of starving cells is controlled by signaling centers that emit periodic pulses of 3Ј-5Ј-cAMP. This process is mediated by the binding of extracellular cAMP to the G protein-coupled receptor cAR1, 3 which initiates a signaling cascade leading to, among other things, the activation of ACA (4). When ACA is first activated, rapid cAMP synthesis leads to a burst in internal cAMP levels. Although part of the synthesized cAMP remains inside cells to activate PKA and regulate gene expression, the majority of it is rapidly secreted (5-7). The secreted cAMP binds to cAR1 and, in an autocrine and paracrine amplification step, results in the production and secretion of more cAMP (2, 3). For a spatially extended cell population, the stimulated release of cAMP result...

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“…3A). This finding is not surprising because, although ACA is responsible for most of the chemoattractant-induced cAMP production in Dictyostelium, some cAMP is produced in acaA null cells and, thus, these cells are expected to have residual PKA activity (Bagorda et al, 2009;Kim et al, 1998;Söderbom et al, 1999). Consistent with the observed elevated PKB and PKBR1 activity levels in pkaC null cells, these cells also display higher basal phosphorylation levels and extended cAMP-induced phosphorylation of cellular substrates of PKB kinases compared to those found in wild-type cells (Fig.…”

Section: Pkac Null Cells Are Unable To Perform Chemotaxismentioning

confidence: 97%

Protein kinase A regulates the Ras, Rap1 and TORC2 pathways in response to the chemoattractant cAMP in Dictyostelium

Scavello

Petlick

Ramesh

et al. 2017

Journal of Cell Science

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Efficient directed migration requires tight regulation of chemoattractant signal transduction pathways in both space and time, but the mechanisms involved in such regulation are not well understood. Here, we investigated the role of protein kinase A (PKA) in controlling signaling of the chemoattractant cAMP in Dictyostelium discoideum. We found that cells lacking PKA display severe chemotaxis defects, including impaired directional sensing. Although PKA is an important regulator of developmental gene expression, including the cAMP receptor cAR1, our studies using exogenously expressed cAR1 in cells lacking PKA, cells lacking adenylyl cyclase A (ACA) and cells treated with the PKA-selective pharmacological inhibitor H89, suggest that PKA controls chemoattractant signal transduction, in part, through the regulation of RasG, Rap1 and TORC2. As these pathways control the ACAmediated production of intracellular cAMP, they lie upstream of PKA in this chemoattractant signaling network. Consequently, we propose that the PKA-mediated regulation of the upstream RasG, Rap1 and TORC2 signaling pathways is part of a negative feedback mechanism controlling chemoattractant signal transduction during Dictyostelium chemotaxis.

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Real-time measurements of cAMP production in liveDictyosteliumcells

Cited by 25 publications

References 38 publications

The exocytic genesecAis required forDictyosteliumcell motility and osmoregulation

The exocytic genesecAis required forDictyosteliumcell motility and osmoregulation

Direct Biochemical Measurements of Signal Relay during Dictyostelium Development

Protein kinase A regulates the Ras, Rap1 and TORC2 pathways in response to the chemoattractant cAMP in Dictyostelium

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