A land­scape with moun­tains and valleys

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A land­scape with moun­tains and valleys

A team led by Martin Lohse has answered a ques­tion that has puzzled sci­ent­ists for some 40 years. In the journal “Cell”, the team explains how cells are able to switch on dif­fer­ent sig­nal­ing path­ways using only one sig­nal­ing molecule: the nuc­le­otide cAMP. To achieve this, the molecule is vir­tu­ally imprisoned in nano­meter-sized spaces.

There are up to a hun­dred dif­fer­ent recept­ors on the sur­face of each cell in the human body. The cell uses these recept­ors to receive extra­cel­lu­lar sig­nals, which it then trans­mits to its interior. Such sig­nals arrive at the cell in vari­ous forms, includ­ing as sens­ory per­cep­tions, neur­o­trans­mit­ters like dopam­ine, or hor­mones like insulin.

One of the most import­ant sig­nal­ing molecules the cell uses to trans­mit such stim­uli to its interior, which then trig­gers the cor­res­pond­ing sig­nal­ing path­ways, is a small molecule called cAMP. This so-called second mes­sen­ger was dis­covered in the 1950s. Until now, exper­i­mental obser­va­tions have assumed that cAMP dif­fuses freely – i.e., that its con­cen­tra­tion is basic­ally the same through­out the cell – and that one sig­nal should there­fore encom­pass the entire cell.

“But since the early 1980s we have known, for example, that two dif­fer­ent heart cell recept­ors release exactly the same amount of cAMP when they receive an external sig­nal, yet com­pletely dif­fer­ent effects are pro­duced inside the cell,” reports Dr. Andreas Bock. Together with Dr. Paolo Anni­bale, Andreas Bock from the Receptor Sig­nal­ing Lab at the Max Del­brück Cen­ter for Molecu­lar Medi­cine in Ber­lin, is first author of the study. Other authors of the study that had been ini­ti­ated in the Lohse labor­at­ory while still in Würzburg include Martin Fal­cke from the MDC, as well as other sci­ent­ists from Ber­lin, Würzburg and Minneapolis.

Like holes in a swiss Cheese

Together with their co-authors, Bock and Anni­bale have now solved this appar­ent con­tra­dic­tion – which has pre­oc­cu­pied sci­ent­ists for almost forty years. The team reports in Cell that, con­trary to pre­vi­ous assump­tions, the major­ity of cAMP molecules can­not move around freely in the cell, but are actu­ally bound to cer­tain pro­teins – par­tic­u­larly pro­tein kinases.

“Due to this pro­tein bind­ing, the con­cen­tra­tion of free cAMP in the cell is actu­ally very low,” says Pro­fessor Martin Lohse, who is senior author of the study. “This gives the rather slow cAMP-degrad­ing enzymes, the phos­phod­i­esterases (PDEs), enough time to form nano­meter-sized com­part­ments around them­selves that are almost free of cAMP.” The sig­nal­ing molecule is then reg­u­lated sep­ar­ately in each of these tiny com­part­ments. “This enables cells to pro­cess dif­fer­ent receptor sig­nals sim­ul­tan­eously in many such com­part­ments,” explains Lohse. The research­ers were able to demon­strate this using the example of the cAMP-depend­ent pro­tein kinase (PKA), which was activ­ated by dif­fer­ent con­cen­tra­tions of cAMP in dif­fer­ent compartments.

“You can ima­gine these cleared-out com­part­ments rather like the holes in a Swiss cheese – or like tiny pris­ons in which the actu­ally rather slow-work­ing PDE keeps watch over the much faster cAMP to make sure it does not break out and trig­ger unin­ten­ded effects in the cell,” explains Anni­bale. “Once the per­pet­rator is locked up, the police no longer have to chase after it.” 

NANOMETER-SCALE MEASUREMENTS

The team iden­ti­fied the move­ments of the sig­nal­ing molecule in the cell using fluor­es­cent cAMP molecules and spe­cial meth­ods of fluor­es­cence spec­tro­scopy – includ­ing fluc­tu­ation spec­tro­scopy and aniso­tropy – which Anni­bale developed and adap­ted for the study. So-called nanor­ulers helped the group to meas­ure the size of the “holes” in which cAMP trig­gers spe­cific sig­nal­ing path­ways. “These nanor­ulers are elong­ated pro­teins that we were able to use like a tiny ruler,” explains Bock, who inven­ted this par­tic­u­lar meas­ure­ment technique.
The team’s meas­ure­ments showed that most com­part­ments are actu­ally smal­ler than 10 nano­met­ers – i.e., 10 mil­lionths of a mil­li­meter. This way, the cell is able to cre­ate thou­sands of dis­tinct cel­lu­lar domains in which it can reg­u­late cAMP inde­pend­ently and thus pro­tect itself from the sig­nal­ing molecule’s unin­ten­ded effects. “We were able to show that a spe­cific sig­nal­ing path­way was ini­tially inter­rup­ted in a hole that was vir­tu­ally cAMP-free,” said Anni­bale. “But when we inhib­ited the PDEs that cre­ate these holes, the path­way went on unobstructed.”

A CHIP RATHER THAN A SWITCH

“This means the cell does not act like a single on/off switch, but rather like an entire chip con­tain­ing thou­sands of such switches,” explains Lohse, sum­mar­iz­ing the find­ings of the research. “The mis­take made in past exper­i­ments was to use cAMP con­cen­tra­tions that were far too high, thus enabling a large amount of the sig­nal­ing molecule to dif­fuse freely in the cell because all bind­ing sites were occupied.”
As a next step, the research­ers want to fur­ther invest­ig­ate the archi­tec­ture of the cAMP “pris­ons” and find out which PDEs pro­tect which sig­nal­ing pro­teins. In the future, med­ical research could also bene­fit from their find­ings. “Many drugs work by alter­ing sig­nal­ing path­ways within the cell,” explains Lohse. “Thanks to the dis­cov­ery of this cell com­part­ment­al­iz­a­tion, we now know there are a great many more poten­tial tar­gets that can be searched for.”
“A study from San Diego, which was pub­lished at the same time as our art­icle in “Cell”, shows that cells begin to pro­lif­er­ate when their indi­vidual sig­nal­ing path­ways are no longer reg­u­lated by spa­tial sep­ar­a­tion,” says Bock. In addi­tion, he adds, it is already known that the dis­tri­bu­tion of cAMP con­cen­tra­tion levels in heart cells changes in heart fail­ure, for example. Their work could there­fore open up new aven­ues for both can­cer and car­di­ovas­cu­lar research.

Text: Anke Brodmerkel

Bock A, Anni­bale P, Kon­rad C, Han­nawacker A, Anton SE, Mai­el­laro I, Zabel U, Sivara­makrish­nan S, Fal­cke M, Lohse MJ (2020) Optical Map­ping of cAMP Sig­nal­ing at the Nano­meter Scale. Cell. 182: 1519–1530.e17.
doi: 10.1016/j.cell.2020.07.035.