Related Experiment Videos
Actin induction during PMA and cAMP-dependent signal pathway activation in Entamoeba histolytica trophozoites
D Ortiz1, M del Carmen Dominguez-Robles, N Villegas-Sepúlveda
1Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN, México, DF, Mexico.
Abstract:
Activation of PKC or cAMP-dependent signalling pathways in Entamoeba histolytica triggers the phosphorylation of proteins involved in actin rearrangements necessary for adhesion and locomotion. Analogous motifs to SRE and CRE sequences--known to respond to PMA and cAMP--were identified within the 5' regulatory region (5'RR) of one of the parasite actin genes. These sequences could be involved in the actin transcriptional upregulation reported during signalling. To test this hypothesis, a plasmid containing the 5'RR of the actin gene fused to the bacterial neomycin gene (neo) was used for stable transfection. Expression of neo and endogenous actin was measured after stimulation of transfected amoebae by PMA and dcAMP. It was found that both compounds induced neo and actin expression and showed a co-operative effect in the induction of neo. Induction by PMA or dcAMP failed if the directing amoebic 5'RR lacked SRE and CRE motifs. Transfection of amoebae with plasmid constructs, containing either progressive deletions of the actin 5'RR or site-directed mutations of the SRE and CRE-like motifs, corroborated that these sequences and a co-ordinated participation of PKC- and PKA-activated transcription factors are responsible for the increments in neo and actin mRNAs. In vivo, these PMA and cAMP-response elements could play an important role in regulating actin expression and organization in signalling processes activated during tissue invasion.
Insights
Signaling pathways in Entamoeba histolytica activate actin gene transcription via specific DNA sequences. This regulation is crucial for parasite adhesion and invasion during tissue infection.
Area of Science:
- Molecular Biology
- Parasitology
- Cell Signaling
Background:
- Protein kinase C (PKC) and cyclic AMP (cAMP)-dependent signaling pathways regulate actin rearrangements in Entamoeba histolytica.
- Actin dynamics are essential for parasite adhesion and locomotion, critical processes during host tissue invasion.
Purpose of the Study:
- To investigate the role of specific regulatory sequences (SRE and CRE) in the 5' regulatory region (5'RR) of an Entamoeba histolytica actin gene.
- To determine if these sequences mediate transcriptional upregulation of actin in response to PKC and cAMP signaling.
Main Methods:
- Stable transfection of Entamoeba histolytica with a plasmid containing the actin gene's 5'RR fused to a reporter gene (neomycin resistance, neo).
- Stimulation of transfected parasites with phorbol 12-myristate 13-acetate (PMA) and dibutyryl cyclic AMP (dcAMP).
- Measurement of neo and endogenous actin gene expression using constructs with intact or mutated/deleted 5'RR sequences.
Main Results:
- PMA and dcAMP induced significant upregulation of both neo and endogenous actin expression.
- A cooperative effect was observed between PMA and dcAMP in inducing neo expression.
- Induction of gene expression was abolished when the SRE and CRE motifs within the 5'RR were absent or mutated.
- Progressive deletions and site-directed mutations confirmed the critical role of SRE and CRE-like motifs in transcriptional regulation.
Conclusions:
- Specific PMA and cAMP-response elements (SRE and CRE-like motifs) within the actin gene's 5'RR are essential for mediating transcriptional activation.
- Coordinated action of PKC- and PKA-activated transcription factors regulates actin mRNA levels.
- These regulatory elements likely play a significant role in controlling actin expression and organization during Entamoeba histolytica's tissue invasion processes.