CAMP gradient

Incubate the dishes at room temperature for 30 min to establish a cAMP gradient. 

The following protocol is for analyzing Ras protein activation during Dictyostelium chemotaxis up to cAMP gradient (Fig. 1). 

The cARl receptor couples to the heterotrimeric G-proteins Ga /GPy to mediate the signaling networks that are responsible for cAMP gradient sensing and cell migration (9,10). In the past 10 years, live cell imaging techniques have been developed to measure dynamics of cARl-mediated signaling network in live D. discoideum cells. 

EstMishinp a steady cAMP gradient. To establish a cAMP gradient, a Femtotip micropipette is filled with 30 pL of solution containing cAMP and Alexa 594 see Note 2) and then attached to the Femtojet microinjector and micromanipulator. The output pressure of the Femtojet is set at Pc = 70 hPa to release a constant and tiny volume of the cAMP/Alexa 594 mixture into a one-well chamber that is filled with 6 mL of DB. Under these conditions, a gradient can be established within 100 pm from the tip of the micropipette and is usually stable for more than 1 h (Fig. 1). 

Suddenly exposinjj a cell to a cAMP jjradient. Two positions of micropipette are preset with TransferMan NK2 micromanipulator Pos. 1, close to the cell Pos. 2, 1,000 pm away from the cells. The micropipette filled with the cAMP/Alexa 594 mixture is connected to the Femtojet microcapillary pressure supply unit and set at the Pos. 2. To quickly expose a cell to a cAMP gradient, the micropipette is moved to Pos. 1 automatically within 100 pm to the cell in less than a second (Fig.lc, eeNote 3). 

Quickly withdraw a cAMP gradient from a cell. To withdraw a gradient from a cell, a micropipette is quickly moved to Pos. 2 (more than 1,000 pm) from a cell (Pos. 1) in less than 1 s (Fig. Ic, eeNote 3). 

The formation of the cAMP gradient was deduced by measuring the diffusion of the fluorescent dye ludferyellow (Mw = 457 Da) using confocal microscopy. The fluorescence intensity at different distances from the pipette was recorded in pixel elements (0.404 x... 

Fig. 4. Experimental equilibrium data of the cAMP gradient with different flow from the micropipettes. A micropipette filled with cAMP and lucifer yellow was applied just above the glass surface in a droplet of cells. The pressure applied was 25,50,80, and 100 hPa. The equilibrium fluorescence intensity was measured by confocal microscopy at 30 s after application of the pipette. The lines are the fitted data using Eq. 13 with and F as indicated in Table 1 the dashed line is Eq. 17 for 50 hPa. The lower panel b) shows the same data as upper panel a), but only at shorter distance from the pipette.

Fig. 9. Inspecting time courses ot intracellular concentrations, (a) The time courses ot membrane-bound PTEN ( PTENbnd in the text), activated Ras, and ot all molecule complexes containing PIP3 and the reporter PH domain in different regions of the simulated cell in a cAMP gradient, (b) A higher detail rendering of the time courses from (a) in the region of the cell whose membrane was exposed to higher concentration of cAMP. It shows the characteristic multiphasic and opposing dynamics of PIP3 and membrane-bound PTEN and the rapid but transient activation of Ras (see ref. 11, for more details).

Camp, T.R. and Stein, P.C., 1943. Velocity gradients and internal work in fluid motion. Journal of the Boston Society of Civil Engineers, 30, 219. 

The tethering of PKA through AKAPs by itself is not sufficient to compartmentalize and control a cAMP/ PKA-dependent pathway. Cyclic AMP readily diffuses throughout the cell. Therefore, discrete cAMP/PKA signalling compartments are only conceivable if this diffusion is limited. Phosphodiesterases (PDE) establish gradients of cAMP by local hydrolysis of the... 

Redox gradients in Au systems of the Yilgarn Craton, Western Australia have been mapped across gold lodes and at camp to district scales using C and S isotopes combined with alteration studies. These gradients can be related to the interplay of oxidized and reduced... 

Binding leads to one of two consequences. If the receptor is coupled to an ion channel, the channel is opened, ions move down electrochemical gradients, and the membrane potential is changed. If the receptor is linked to a G protein, the binding initiates a sequence of biochemical events that result in the production of a second messenger such as cAMP or IP3. These evoke long-term changes that alter excitability of the postsynaptic cell. The complexation process is usually rapidly reversible with an occupancy half-life of 1-20 ms. 

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