The genome of Arabidopsis (Arabidopsis thaliana) contains five sequences with high similarity to FLAVONOL SYNTHASE1 (AtFLS1), a previously characterized flavonol synthase gene that plays a central role in flavonoid metabolism. This apparent redundancy suggests the possibility that Arabidopsis uses multiple isoforms of FLS with different substrate specificities to mediate the production of the flavonols, quercetin and kaempferol, in a tissue-specific and inducible manner. However, biochemical and genetic analysis of the six AtFLS sequences indicates that, although several of the members are expressed, only AtFLS1 encodes a catalytically competent protein. AtFLS1 also appears to be the only member of this group that influences flavonoid levels and the root gravitropic response in seedlings under nonstressed conditions. This study showed that the other expressed AtFLS sequences have tissue- and cell type-specific promoter activities that overlap with those of AtFLS1 and encode proteins that interact with other flavonoid enzymes in yeast two-hybrid assays. Thus, it is possible that these “pseudogenes” have alternative, noncatalytic functions that have not yet been uncovered.

Highlights d Inhibitory synapses are composed of nanoscale subsynaptic domains (SSDs) d Gephyrin and GABA A R SSDs closely associate and are dependent on each other d GABA A R SSDs are closely associated with presynaptic active-zone SSDs d Inhibitory synapses recruit additional SSDs during activitydependent growth

The molecular composition of the postsynaptic membrane is sculpted by synaptic activity. During synaptic plasticity at excitatory synapses, numerous structural, signaling and receptor molecules concentrate at the postsynaptic density (PSD) to regulate synaptic strength. We developed an approach that uses light to tune the abundance of specific molecules in the PSD. We used this approach to investigate the relationship between the number of AMPA-type glutamate receptors in the PSD and synaptic strength. Surprisingly, adding more AMPA receptors to excitatory contacts had little effect on synaptic strength. Instead, we observed increased excitatory input through the apparent addition of new functional sites. Our data support a model where adding AMPA receptors is sufficient to activate synapses that had few receptors to begin with, but that additional remodeling events are required to strengthen established synapses. More broadly, this approach introduces the precise spatiotemporal control of optogenetics to molecular control of synaptic function.

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The quality of super resolution images obtained by stochastic single-molecule microscopy critically depends on image analysis algorithms. We find that the choice of background estimator is often the most important determinant of reconstruction quality. A variety of techniques have found use, but many have a very narrow range of applicability depending upon the characteristics of the raw data. Importantly, we observe that when using otherwise accurate algorithms, unaccounted background components can give rise to biases on scales defeating the purpose of super-resolution microscopy. We find that a temporal median filter in particular provides a simple yet effective solution to the problem of background estimation, which we demonstrate over a range of imaging modalities and different reconstruction methods.