Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles.

Decisions on the fate of cells and their functions are dictated by the spatiotemporal dynamics of molecular signalling networks. However, techniques to examine the dynamics of these intracellular processes remain limited. Here, we show that magnetic nanoparticles conjugated with key regulatory proteins can artificially control, in time and space, the Ran/RCC1 signalling pathway that regulates the cell cytoskeleton. In the presence of a magnetic field, RanGTP proteins conjugated to superparamagnetic nanoparticles can induce microtubule fibres to assemble into asymmetric arrays of polarized fibres in Xenopus laevis egg extracts. The orientation of the fibres is dictated by the direction of the magnetic force. When we locally concentrated nanoparticles conjugated with the upstream guanine nucleotide exchange factor RCC1, the assembly of microtubule fibres could be induced over a greater range of distances than RanGTP particles. The method shows how bioactive nanoparticles can be used to engineer signalling networks and spatial self-organization inside a cell environment.

AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila.

Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing "glue granules" that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1- and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules.

AP-1 controls the trafficking of Notch and Sanpodo toward E-cadherin junctions in sensory organ precursors.

In Drosophila melanogaster, external sensory organs develop from a single sensory organ precursor (SOP). The SOP divides asymmetrically to generate daughter cells, whose fates are governed by differential Notch activation. Here we show that the clathrin adaptor AP-1 complex, localized at the trans Golgi network and in recycling endosomes, acts as a negative regulator of Notch signaling. Inactivation of AP-1 causes ligand-dependent activation of Notch, leading to a fate transformation within sensory organs. Loss of AP-1 affects neither cell polarity nor the unequal segregation of the cell fate determinants Numb and Neuralized. Instead, it causes apical accumulation of the Notch activator Sanpodo and stabilization of both Sanpodo and Notch at the interface between SOP daughter cells, where DE-cadherin is localized. Endocytosis-recycling assays reveal that AP-1 acts in recycling endosomes to prevent internalized Spdo from recycling toward adherens junctions. Because AP-1 does not prevent endocytosis and recycling of the Notch ligand Delta, our data indicate that the DE-cadherin junctional domain may act as a launching pad through which endocytosed Notch ligand is trafficked for signaling.

Role of Gal11, a component of the RNA polymerase II mediator in stress-induced hyperphosphorylation of Msn2 in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the Msn2 transcription factor is a key element in mediating the environmental stress response (ESR), leading to the induction of 100-200 genes through the cis-acting Stress Response Element (STRE) in response to various physico-chemical stresses and nutritional variations. This activation is accompanied by a stress-induced hyperphosphorylation of Msn2. By a systematic screening we identified two proteins essential in this process: (i) the cyclin-dependent Ssn3/Srb10 protein kinase, part of a module of the RNA polymerase II mediator, which has already been shown to be involved in hyperphosphorylation and degradation of Msn2 upon stress, and (ii) Gal11, a component of the mediator. In a gal11 mutant, stress-induced hyperphosphorylation of Msn2 is abolished, stress-induced transcription of Msn2-dependent genes is decreased and Msn2 degradation is impaired. Rgr1, another component of the mediator, is also critical for this hyperphosphorylation, indicating that the integrity of the mediator is required for this process. Moreover the transactivating region of Msn2 interacts in vitro with the N-terminal domain of Gal11. These results point out the role of the mediator, especially its Gal11 subunit, in the hyperphosphorylation and degradation of Msn2 during stress response.

The transcriptional activation region of Msn2p, in Saccharomyces cerevisiae, is regulated by stress but is insensitive to the cAMP signalling pathway.

Msn2p is a transcription factor that mediates a transient cellular response to multiple stresses and to changes in the nutritional environment. It was previously shown that the C-terminal half of Msn2p contains the DNA binding domain, a nuclear localization signal and nuclear export determinants which are activated by stress. In this report, we demonstrate that the N-terminal half of Msn2p contains the transcriptional activation domain(s). In addition, we present evidence that this region of Msn2p is able to mediate both the activation of transcription and export of the protein from the nucleus in response to stress. Interestingly, while the stress response integrated by the components of the C-terminal half that are involved in nucleocytoplasmic localization is reversed by elevated levels of cAMP, the effects of stress on the transcriptional activation domain and the localization determinants present in the N-terminal half of Msn2p are insensitive to variations in the intracellular cAMP concentrations.

Direct activation of genes involved in intracellular iron use by the yeast iron-responsive transcription factor Aft2 without its paralog Aft1.

The yeast Saccharomyces cerevisiae contains a pair of paralogous iron-responsive transcription activators, Aft1 and Aft2. Aft1 activates the cell surface iron uptake systems in iron depletion, while the role of Aft2 remains poorly understood. This study compares the functions of Aft1 and Aft2 in regulating the transcription of genes involved in iron homeostasis, with reference to the presence/absence of the paralog. Cluster analysis of DNA microarray data identified the classes of genes regulated by Aft1 or Aft2, or both. Aft2 activates the transcription of genes involved in intracellular iron use in the absence of Aft1. Northern blot analyses, combined with chromatin immunoprecipitation experiments on selected genes from each class, demonstrated that Aft2 directly activates the genes SMF3 and MRS4 involved in mitochondrial and vacuolar iron homeostasis, while Aft1 does not. Computer analysis found different cis-regulatory elements for Aft1 and Aft2, and transcription analysis using variants of the FET3 promoter indicated that Aft1 is more specific for the canonical iron-responsive element TGCACCC than is Aft2. Finally, the absence of either Aft1 or Aft2 showed an iron-dependent increase in the amount of the remaining paralog. This may provide additional control of cellular iron homeostasis.

Oscillatory nucleocytoplasmic shuttling of the general stress response transcriptional activators Msn2 and Msn4 in Saccharomyces cerevisiae.

Msn2 and Msn4 are two related transcriptional activators that mediate a general response to stress in yeast Saccharomyces cerevisiae by eliciting the expression of specific sets of genes. In response to stress or nutritional limitation, Msn2 and Msn4 migrate from the cytoplasm to the nucleus. Using GFP-tagged constructs and high-resolution time-lapse video microscopy on single cells, we show that light emitted by the microscope also triggers this migration. Unexpectedly, the population of Msn2 or Msn4 molecules shuttles repetitively into and out of the nucleus with a periodicity of a few minutes. A large heterogeneity in the oscillatory response to stress is observed between individual cells. This periodic behavior, which can be induced by various types of stress, at intermediate stress levels, is not dependent upon protein synthesis and persists when the DNA-binding domain of Msn2 is removed. The cAMP-PKA pathway controls the sensitivity of the oscillatory nucleocytoplasmic shuttling. In the absence of PKA, Msn4 continues to oscillate while Msn2 is maintained in the nucleus. We show that a computational model based on the possibility that Msn2 and Msn4 participate in autoregulatory loops controlling their subcellular localization can account for the oscillatory behavior of the two transcription factors.