Regulation of multiple tip formation by caffeine in cellular slime molds

Abstract

BackgroundThe multicellular slug in Dictyostelium has a single tip that acts as an organising centre patterning the rest of the slug. High adenosine levels at the tip are believed to be responsible for this tip dominance and the adenosine antagonist, caffeine overrides this dominance promoting multiple tip formation.ResultsCaffeine induced multiple tip effect is conserved in all the Dictyostelids tested. Two key components of cAMP relay namely, cAMP phosphodiesterase (Pde4) and adenyl cyclase-A (AcaA) levels get reduced during secondary tip formation in Dictyostelium discoideum. Pharmacological inhibition of cAMP phosphodiesterase also resulted in multiple tips. Caffeine reduces cAMP levels by 16.4, 2.34, 4.71 and 6.30 folds, respectively in D. discoideum, D. aureostipes, D. minutum and Polysphondylium pallidum. We propose that altered cAMP levels, perturbed cAMP gradient and impaired signalling may be the critical factors for the origin of multiple tips in other Dictyostelids as well. In the presence of caffeine, slug cell movement gets impaired and restricted. The cell type specific markers, ecmA (prestalk) and pspA (prespore) cells are not equally contributing during additional tip formation. During additional tip emergence, prespore cells transdifferentiate to compensate the loss of prestalk cells.ConclusionCaffeine decreases adenyl cyclase–A (AcaA) levels and as a consequence low cAMP is synthesised altering the gradient. Further if cAMP phosphodiesterase (Pde4) levels go down in the presence of caffeine, the cAMP gradient breaks down. When there is no cAMP gradient, directional movement is inhibited and might favour re-differentiation of prespore to prestalk cells.

An integrated, cross-regulation pathway model involving activating/adaptive and feed-forward/feed-back loops for directed oscillatory cAMP signal-relay/response during the development of Dictyostelium

“…During early development of Dictyostelium , secreted cAMP serves as a chemoattractant to organize cells at centers of aggregation ( Meima and Schaap, 1999 ; Jaiswal et al, 2012a ; Jaiswal et al, 2012b ; Fukujin et al, 2016 ; Scavello et al, 2017 ; Singer et al, 2019 ), The receptors for cAMP (CAR1) are surface chemoattractant GPCRs, coupled to Gα2 and Gβγ subunits, which transduce the extracellular signal for directed cell movement ( Janetopoulos et al, 2001 ; Xu et al, 2010 ; Tariqul Islam et al, 2018 ; Adhikari et al, 2021 ). In addition, CAR1 orchestrates a periodic activation/de-activation/re-activation pathway for cAMP generation and clearing, which relays the cAMP signal ( Louis et al, 1993 ; Insall et al, 1994b ; Hereld et al, 1994 ; Thomason et al, 1999 ; McMains et al, 2008 ; Kriebel and Parent, 2009 ; Xu et al, 2010 ; Brzostowski et al, 2013 ; Cao et al, 2014 ).…”

Serotonin and MAOA enable the organizer and tip dominance in Dictyostelium

“…To now probe the impact of MAOA/serotonin on wave propagation, we treated [RNAi]maoA mounds with caffeine or IBMX (PDE4 inhibitor), both of which are known to induce tips 24 . Adding either one of the compounds to [RNAi]maoA mounds resulted in a cAMP wave propagation pattern that was closer to that of the wildtype, and restored tip formation to a more normal pattern (Fig.…”

“…Serotonin was tested from 100 nM to 1 mM on wild-type cells and 500 µM concentration was found to be the effective dose in causing the mound arrest phenotype. The concentrations of SQ22536 (2 mM), H89 (50 µM), 8-Br-cAMP (5 mM), cAMP (0.2 mM), c-di-GMP (1 mM), DIF (200 nM), adenosine (2 mM), adenosine deaminase (5U), caffeine (2 mM) and IBMX (0.5mM) treatment were based on the literature 24,39,47,48 . All the ne chemicals were from Sigma-Aldrich, USA except as indicated.…”

Glimpses of Dictyostelid research in India

“…Both the compounds, adenosine as well as caffeine, rescue mutants defective in streaming (pde4and pdiA -) implying that adenosine affects both streaming and other stages of aggregation in Dictyostelium. Further, when slugs were transferred to caffeine-containing plates, several tips arise along with the slug axis, each forming a fruiting body of its own (Jaiswal et al, 2012b). Caffeine-induced multiple tip formation is conserved across other slime moulds (Jaiswal et al, 2012b).…”

“…Further, when slugs were transferred to caffeine-containing plates, several tips arise along with the slug axis, each forming a fruiting body of its own (Jaiswal et al, 2012b). Caffeine-induced multiple tip formation is conserved across other slime moulds (Jaiswal et al, 2012b). As caffeine is an antagonist to adenosine, the current view is that excess adenosine in the slug front suppresses another tip organizing center along the slug length and caffeine overrides the action of adenosine, inducing the formation of multiple organizing centers.…”