Dissection of miRNA Pathways Using Arabidopsis Mesophyll Protoplasts

MicroRNAs (miRNAs) control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this article, we describe a transient expression system in Arabidopsis mesophyll protoplasts, which is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, by either overexpression or the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up, and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.

The interplay between neuronal activity and actin dynamics mimic the setting of an LTD synaptic tag

Persistent forms of plasticity, such as long-term depression (LTD), are dependent on the interplay between activity-dependent synaptic tags and the capture of plasticity-related proteins. We propose that the synaptic tag represents a structural alteration that turns synapses permissive to change. We found that modulation of actin dynamics has different roles in the induction and maintenance of LTD. Inhibition of either actin depolymerisation or polymerization blocks LTD induction whereas only the inhibition of actin depolymerisation blocks LTD maintenance. Interestingly, we found that actin depolymerisation and CaMKII activation are involved in LTD synaptic-tagging and capture. Moreover, inhibition of actin polymerisation mimics the setting of a synaptic tag, in an activity-dependent manner, allowing the expression of LTD in non-stimulated synapses. Suspending synaptic activation also restricts the time window of synaptic capture, which can be restored by inhibiting actin polymerization. Our results support our hypothesis that modulation of the actin cytoskeleton provides an input-specific signal for synaptic protein capture.