Highlights d Cdk4/6 regulates mTORC1 activity via TSC2 d Cdk4/6 binds TSC2 and phosphorylates it on Ser1452 and Ser1217 d Cdk4/6 inhibitors also inhibit mTORC1 d Cdk4/6 couples cell-cycle progression via RB to cell growth via mTORC1

Drosophila Frequenin (Frq) and its mammalian and worm homologue, NCS-1, are Ca2+-binding proteins involved in neurotransmission. Using site-specific recombination in Drosophila, we created two deletions that removed the entire frq1 gene and part of the frq2 gene, resulting in no detectable Frq protein. Frq-null mutants were viable, but had defects in larval locomotion, deficient synaptic transmission, impaired Ca2+ entry and enhanced nerve-terminal growth. The impaired Ca2+ entry was sufficient to account for reduced neurotransmitter release. We hypothesized that Frq either modulates Ca2+ channels, or that it regulates the PI4Kβ pathway as described in other organisms. To determine whether Frq interacts with PI4Kβ with consequent effects on Ca2+ channels, we first characterized a PI4Kβ-null mutant and found that PI4Kβ was dispensable for synaptic transmission and nerve-terminal growth. Frq gain-of-function phenotypes remained present in a PI4Kβ-null background. We conclude that the effects of Frq are not due to an interaction with PI4Kβ. Using flies that were trans-heterozygous for a null frq allele and a null cacophony (encoding the α1-subunit of voltage-gated Ca2+ channels) allele, we show a synergistic effect between these proteins in neurotransmitter release. Gain-of-function Frq phenotypes were rescued by a hypomorphic cacophony mutation. Overall, Frq modulates Ca2+ entry through a functional interaction with the α1 voltage-gated Ca2+-channel subunit; this interaction regulates neurotransmission and nerve-terminal growth.

The conserved Ca 2+ -binding protein Frequenin (homolog of the mammalian NCS-1, neural calcium sensor) is involved in pathologies that result from abnormal synapse number and probability of neurotransmitter release per synapse. Both synaptic features are likely to be co-regulated but the intervening mechanisms remain poorly understood. We show here that Drosophila Ric8a (a homolog of mammalian synembryn, which is also known as Ric8a), a receptor-independent activator of G protein complexes, binds to Frq2 but not to the virtually identical homolog Frq1. Based on crystallographic data on Frq2 and site-directed mutagenesis on Frq1, the differential amino acids R94 and T138 account for this specificity. Human NCS-1 and Ric8a reproduce the binding and maintain the structural requirements at these key positions. Drosophila Ric8a and Gas regulate synapse number and neurotransmitter release, and both are functionally linked to Frq2. Frq2 negatively regulates Ric8a to control synapse number. However, the regulation of neurotransmitter release by Ric8a is independent of Frq2 binding. Thus, the antagonistic regulation of these two synaptic properties shares a common pathway, Frq2-Ric8a-Gas, which diverges downstream. These mechanisms expose the Frq2-Ric8a interacting surface as a potential pharmacological target for NCS-1-related diseases and provide key data towards the corresponding drug design.

The molecular mechanisms regulating animal tissue size during development are unclear. This question has been extensively studied in the Drosophila wing disc. Although cell growth is regulated by the kinase TORC1, no readout exists to visualize TORC1 activity in situ in Drosophila. Both the cell cycle and the morphogen Dpp are linked to tissue growth, but whether they regulate TORC1 activity is not known. We develop here an anti-phospho-dRpS6 antibody that detects TORC1 activity in situ. We find, unexpectedly, that TORC1 activity in the wing disc is patchy. This is caused by elevated TORC1 activity at the cell cycle G/S transition due to CycD/Cdk4 phosphorylating TSC1/2. We find that TORC1 is also activated independently of CycD/Cdk4 when cells with different levels of Dpp signaling or Brinker protein are juxtaposed. We thereby characterize the spatial distribution of TORC1 activity in a developing organ.

Frequenin (Frq) and its mammalian homologue, neuronal calcium sensor 1 (NCS‐1), are important calcium‐binding proteins which enhance neurotransmitter release and facilitation. Here, we report the discovery of a second Frq‐encoding gene (frq2) in Drosophila. The temporal and spatial expression patterns of the two genes are very similar, and the proteins they encode, Frq1 and Frq2, are 95% identical in amino acid sequence. Frq1 is more abundant than Frq2, and is most highly expressed in larva. Loss‐of‐function phenotypes were studied using dominant negative peptides to prevent Frq target binding, RNAi to reduce gene transcription, or both methods. To discriminate chronic from acute loss‐of‐function effects, we compared the effects of transgenic expression and forward‐filling the dominant‐negative peptide into presynaptic terminals. In both cases, a 70% reduction in quantal content per bouton occurred, demonstrating that this trait does not result from homeostatic adaptations of the synapse during development. The chronic treatment also produced more synaptic boutons from MNSNb/d‐Is motorneurons, but fewer active zones per bouton. By contrast, excess‐of‐function conditions yielded a 1.4‐ to 2‐fold increase in quantal content and fewer boutons in the same motorneuron. These synaptic effects resulted in behavioural changes in the Buridan locomotion assay, showing that walking speed is dependent on Frq activity in the nervous system. All the effects were identical for both Frqs, and consistent with excess‐ and loss‐of‐function genotypes. We conclude that Frqs have two distinct functions: one in neurotransmission, regulating the probability of release per synapse, and another in axonal growth and bouton formation.

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BackgroundDrosophila Frequenin (Frq), the homolog of the mammalian Neuronal Calcium Sensor-1 (NCS-1), is a high affinity calcium-binding protein with ubiquitous expression in the nervous system. This protein has an important role in the regulation of neurotransmitter release per synapse, axonal growth and bouton formation. In D. melanogaster, Frequenin is encoded by two genes (frq1 and frq2), a very unexpected feature in the Frq/NCS-1 subfamily. These genes are located in tandem in the same genomic region, and their products are 95% identical in their amino acid sequence, clearly indicating their recent origin by gene duplication. Here, we have investigated the factors involved in this unusual feature by examining the molecular evolution of the two frq genes in Drosophila and the evolutionary dynamics of NCS family in a large set of bilaterian species.ResultsSurprisingly, we have found no amino acid replacements fixed across the twelve Drosophila species surveyed. In contrast, synonymous substitutions have been prevalent in the evolution of the coding region of frq1 and frq2, indicating the presence of strong functional constraints following gene duplication. Despite that, we have detected that significant evolutionary rate acceleration had occurred in Frq1 in early times from the duplication, in which positive selection (likely promoting functional diversification) had probably an important role. The analysis of sequence conservation and DNA topology at the non-coding regions of both genes has allowed the identification of DNA regions candidates to be cis-regulatory elements. The results reveal a possible mechanism of regulatory diversification between frq1 and frq2.ConclusionsThe presence of two Frequenins in Drosophila and the rapid accumulation of amino acid substitutions after gene duplication are very unusual features in the evolution of the Frq/NCS-1 subfamily. Here we show that the action of positive selection in concordance with some extent of regulatory diversification might explain these findings. Selected amino acid substitutions in Frq1 likely contributed to the functional divergence between the two duplicates, which, in turn, should have diverged in their regulation by Ecdysone-induced early genes.

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