Physical interaction between VIVID and white collar complex regulates photoadaptation in Neurospora.

Photoadaptation, the ability to attenuate a light response on prolonged light exposure while remaining sensitive to escalating changes in light intensity, is essential for organisms to decipher time information appropriately, yet the underlying molecular mechanisms are poorly understood. In Neurospora crassa, VIVID (VVD), a small LOV domain containing blue-light photoreceptor protein, affects photoadaptation for most if not all light-responsive genes. We report that there is a physical interaction between VVD and the white collar complex (WCC), the primary blue-light photoreceptor and the transcription factor complex that initiates light-regulated transcriptional responses in Neurospora. Using two previously characterized VVD mutants, we show that the level of interaction is correlated with the level of WCC repression in constant light and that even light-insensitive VVD is sufficient partly to regulate photoadaptation in vivo. We provide evidence that a functional GFP-VVD fusion protein accumulates in the nucleus on light induction but that nuclear localization of VVD does not require light. Constitutively expressed VVD alone is sufficient to change the dynamics of photoadaptation. Thus, our results demonstrate a direct molecular connection between two of the most essential light signaling components in Neurospora, VVD and WCC, illuminating a previously uncharacterized process for light-sensitive eukaryotic cells.

Rapid and quantitative imaging of excitation polarized fluorescence reveals ordered septin dynamics in live yeast.

We report an imaging method for fast, sensitive analysis of the orientation of fluorescent molecules by employing a liquid-crystal based universal polarizer in the optical path of a wide-field light microscope. We developed specific acquisition and processing algorithms for measuring the anisotropy and for correcting artifacts caused by fluorescence bleaching, background light, and differential transmission of optical components. We call this approach the Fluorescence LC-PolScope and we used it to analyze the architectural dynamics of septin-green fluorescent protein (septin-GFP) constructs in the neck region of budding yeast. We describe three different states of highly anisotropic septin arrays in which the prevailing orientation of GFP dipoles was either parallel or perpendicular to the mother-bud axis. The transitions between these ordered states were characterized by transient isotropic states. To analyze the patterns of polarized fluorescence, we modeled the alignment of septin-GFP constructs in different stages of septin ring formation. Based on our model, our experimental data are consistent with the formation of paired rather than single filaments and the axis of the α-helical septin terminus linked to a GFP molecule is likely oriented normal to the cell surface. The Fluorescence LC-PolScope combines the molecular specificity of fluorescence tagging with the structural specificity of polarized light analysis.

Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals.

The septins are conserved, GTP-binding proteins important for cytokinesis, membrane compartmentalization, and exocytosis. However, it is unknown how septins are arranged within higher-order structures in cells. To determine the organization of septins in live cells, we developed a polarized fluorescence microscopy system to monitor the orientation of GFP dipole moments with high spatial and temporal resolution. When GFP was fused to septins, the arrangement of GFP dipoles reflected the underlying septin organization. We demonstrated in a filamentous fungus, a budding yeast, and a mammalian epithelial cell line that septin proteins were organized in an identical highly ordered fashion. Fluorescence anisotropy measurements indicated that septin filaments organized into pairs within live cells, just as has been observed in vitro. Additional support for the formation of pairs came from the observation of paired filaments at the cortex of cells using electron microscopy. Furthermore, we found that highly ordered septin structures exchanged subunits and rapidly rearranged. We conclude that septins assemble into dynamic, paired filaments in vivo and that this organization is conserved from yeast to mammals.

Cellular requirements for the small molecule forchlorfenuron to stabilize the septin cytoskeleton.

The septins are filament-forming, GTP-binding proteins that are conserved from yeast to humans. Septins assemble into higher-order structures such as rings, bars, and gauzes with diverse functions including serving as membrane diffusion barriers and scaffolds for cell signaling. The basis for septin filament polymerization and the rules governing septin polymer dynamics are presently not well understood. Pharmacological agents are essential tools in studying such properties of the actin and microtubule cytoskeletons however there are only limited reports of a drug specific to the septin cytoskeleton. Forchlorfenuron (FCF) is a synthetic plant cytokinin used in agriculture which has been shown to alter septin organization in yeast and mammalian tissue culture cells. Here we assess cellular requirements and properties of septin-based structures induced by FCF. Treatment of the filamentous fungus Ashbya gossypii with FCF leads to assembly of extensive septin fibers throughout hyphae which is rapidly reversed upon removal of the drug. These fibers do not exchange or add septin subunits after assembly, indicating that FCF suppresses normal septin dynamics and stabilizes the polymers. While FCF-induced septin fibers do not co-localize to actin or microtubules, a polarized F-actin cytoskeleton is likely required for the assembly of drug-induced septin fibers. Thus, FCF is a potent inducer of septin polymerization and acts as a reversible stabilizer of extended septin polymers. This drug will be a powerful tool for studying mechanisms of septin polymerization and function, particularly in cell types where molecular analyses are complicated by the presence of multiple isoforms and limited genetics.

Regulation of distinct septin rings in a single cell by Elm1p and Gin4p kinases.

Septins are conserved, GTP-binding proteins that assemble into higher order structures, including filaments and rings with varied cellular functions. Using four-dimensional quantitative fluorescence microscopy of Ashbya gossypii fungal cells, we show that septins can assemble into morphologically distinct classes of rings that vary in dimensions, intensities, and positions within a single cell. Notably, these different classes coexist and persist for extended times, similar in appearance and behavior to septins in mammalian neurons and cultured cells. We demonstrate that new septin proteins can add through time to assembled rings, indicating that septins may continue to polymerize during ring maturation. Different classes of rings do not arise from the presence or absence of specific septin subunits and ring maintenance does not require the actin and microtubule cytoskeletons. Instead, morphological and behavioral differences in the rings require the Elm1p and Gin4p kinases. This work demonstrates that distinct higher order septin structures form within one cell because of the action of specific kinases.

The function of the small insertion in the hinge subdomain in the control of constitutive mammalian nitric-oxide synthases.

Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains. This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail. Smaller sequence elements also appear to correlate with control. Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information. In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a beta hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element. Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions. Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors. Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity.