Unique N-Terminal Interactions Connect F-BOX STRESS INDUCED (FBS) Proteins to a WD40 Repeat-like Protein Pathway in Arabidopsis

SCF-type E3 ubiquitin ligases provide specificity to numerous selective protein degradation events in plants, including those that enable survival under environmental stress. SCF complexes use F-box (FBX) proteins as interchangeable substrate adaptors to recruit protein targets for ubiquitylation. FBX proteins almost universally have structure with two domains: A conserved N-terminal F-box domain interacts with a SKP protein and connects the FBX protein to the core SCF complex, while a C-terminal domain interacts with the protein target and facilitates recruitment. The F-BOX STRESS INDUCED (FBS) subfamily of plant FBX proteins has an atypical structure, however, with a centrally located F-box domain and additional conserved regions at both the N- and C-termini. FBS proteins have been linked to environmental stress networks, but no ubiquitylation target(s) or biological function has been established for this subfamily. We have identified two WD40 repeat-like proteins in Arabidopsis that are highly conserved in plants and interact with FBS proteins, which we have named FBS INTERACTING PROTEINs (FBIPs). FBIPs interact exclusively with the N-terminus of FBS proteins, and this interaction occurs in the nucleus. FBS1 destabilizes FBIP1, consistent with FBIPs being ubiquitylation targets SCFFBS1 complexes. This work indicates that FBS proteins may function in stress-responsive nuclear events, and it identifies two WD40 repeat-like proteins as new tools with which to probe how an atypical SCF complex, SCFFBS, functions via FBX protein N-terminal interaction events.

Mechanosensitive control of mitotic entry

As cells prepare to divide, they must ensure that enough space is available to assemble the mitotic machinery without perturbing tissue homeostasis. To do so, cells undergo a series of biochemical reactions regulated by cyclin B1-CDK1 that trigger the reorganization of the actomyosin cytoskeleton and ensure the coordination of cytoplasmic and nuclear events. Along with the biochemical events that control mitotic entry, mechanical forces have recently emerged as important players in the regulation of cell cycle events. However, the exact link between mechanical forces and the biochemical events that control mitotic progression remains to be established. Here, we identify a mechanical signal on the nucleus that sets the time for nuclear envelope permeabilization and mitotic entry. This signal relies on nuclear unfolding during the G2-M transition, which activates the stretch-sensitive cPLA2 on the nuclear envelope. This activation upregulates actomyosin contractility, determining the spatiotemporal translocation of cyclin B1 in the nucleus. Our data demonstrate how the mechanosensitive behaviour of cyclin B1 ensures timely and efficient mitotic spindle assembly and prevents chromosomal instability.

NOP53 Suppresses Autophagy through ZKSCAN3-Dependent and -Independent Pathways

Autophagy is an evolutionally conserved process that recycles aged or damaged intracellular components through a lysosome-dependent pathway. Although this multistep process is propagated in the cytoplasm by the orchestrated activity of the mTOR complex, phosphatidylinositol 3-kinase, and a set of autophagy-related proteins (ATGs), recent investigations have suggested that autophagy is tightly regulated by nuclear events. Thus, it is conceivable that the nucleolus, as a stress-sensing and -responding intranuclear organelle, plays a role in autophagy regulation, but much is unknown concerning the nucleolar controls in autophagy. In this report, we show a novel nucleolar–cytoplasmic axis that regulates the cytoplasmic autophagy process: nucleolar protein NOP53 regulates the autophagic flux through two divergent pathways, the ZKSCAN3-dependent and -independent pathways. In the ZKSCAN3-dependent pathway, NOP53 transcriptionally activates a master autophagy suppressor ZKSCAN3, thereby inhibiting MAP1LC3B/LC3B induction and autophagy propagation. In the ZKSCAN3-independent pathway, NOP53 physically interacts with histone H3 to dephosphorylate S10 of H3, which, in turn, transcriptionally downregulates the ATG7 and ATG12 expressions. Our results identify nucleolar protein NOP53 as an upstream regulator of the autophagy process.

Ultrastructural analysis in yeast reveals a meiosis-specific actin-containing nuclear bundle

Actin polymerises to form filaments/cables for motility, transport, and the structural framework in a cell. Recent studies show that actin polymers are present not only in the cytoplasm but also in the nuclei of vertebrate cells. Here, we show, by electron microscopic observation with rapid freezing and high-pressure freezing, a unique bundled structure containing actin in the nuclei of budding yeast cells undergoing meiosis. The nuclear bundle during meiosis consists of multiple filaments with a rectangular lattice arrangement, often showing a feather-like appearance. The bundle was immunolabelled with an anti-actin antibody and was sensitive to an actin-depolymerising drug. Similar to cytoplasmic bundles, nuclear bundles are rarely seen in premeiotic cells and spores and are induced during meiotic prophase-I. The formation of the nuclear bundle is independent of DNA double-stranded breaks. We speculate that nuclear bundles containing actin play a role in nuclear events during meiotic prophase I.

Separation of Lanthanide Isotopes from Mixed Fission Product Samples

The measurement of radioactive fission products from nuclear events has important implications for nuclear data production, environmental monitoring, and nuclear forensics. In a previous paper, the authors reported the optimization of an intra-group lanthanide separation using LN extraction resin from Eichrom Technologies®, Inc. and a nitric acid gradient. In this work, the method was demonstrated for the separation and quantification of multiple short-lived fission product lanthanide isotopes from a fission product sample produced from the thermal irradiation of highly enriched uranium. The separations were performed in parallel in quadruplicate with reproducible results and high decontamination factors for 153Sm, 156Eu, and 161Tb. Based on the results obtained here, the fission yields for 144Ce, 153Sm, 156Eu, and 161Tb are consistent with published fission yields. This work demonstrates the effectiveness of the separations for the intended application of short-lived lanthanide fission product analysis requiring high decontamination factors.

Standardized measure for phosphatidylinositol, 4,5-bisphosphate by mass spectrometry

Phosphoinositides (PIs) play a fundamental role in many physiological processes such as cell surface signal transduction, membrane trafficking, cytoskeleton regulation, nuclear events and permeability and transport functions of membranes. Levels of PIs vary under different physiological conditions thus PI profiling may be an important step in elucidation of importance in the progression towards many diseases. Previous methods for PI analysis have several disadvantages in time-constraints, use of radioactive samples and inconsistent results due to lack of sensitivity. Mass spectrometry has previously been utilized to quantify low abundance peptides, metabolites and lipids. Here we propose a novel approach for PI quantitation based on inositol head cleavage coupled to high-performance liquid-mass spectrometry (HPLC-MS) to overcome these issues. First, we highlight the extraction and deacylation of PIs from S. Ceravisiae, followed by purification via reverse phase chromatography with a Sep-Pak. Next, using commercially purchased PI (4,5) P2 standards, we created a tune file which provide the correct conditions sensitive enough to identify prolific ion peaks to be utilized in characterization of biological samples. Using the standard tune file, we successfully identified and quantitated the PI (4,5) P2 in cells lacking INP51 which have 2-4 fold increase in PI (4,5)P(subscript 2) and comparing them to the wild type cells. The methods described may form a basis for further optimization of mass spectral based quantitation of PIs.

Standardized measure for phosphatidylinositol, 4,5-bisphosphate by mass spectrometry

Phosphoinositides (PIs) play a fundamental role in many physiological processes such as cell surface signal transduction, membrane trafficking, cytoskeleton regulation, nuclear events and permeability and transport functions of membranes. Levels of PIs vary under different physiological conditions thus PI profiling may be an important step in elucidation of importance in the progression towards many diseases. Previous methods for PI analysis have several disadvantages in time-constraints, use of radioactive samples and inconsistent results due to lack of sensitivity. Mass spectrometry has previously been utilized to quantify low abundance peptides, metabolites and lipids. Here we propose a novel approach for PI quantitation based on inositol head cleavage coupled to high-performance liquid-mass spectrometry (HPLC-MS) to overcome these issues. First, we highlight the extraction and deacylation of PIs from S. Ceravisiae, followed by purification via reverse phase chromatography with a Sep-Pak. Next, using commercially purchased PI (4,5) P2 standards, we created a tune file which provide the correct conditions sensitive enough to identify prolific ion peaks to be utilized in characterization of biological samples. Using the standard tune file, we successfully identified and quantitated the PI (4,5) P2 in cells lacking INP51 which have 2-4 fold increase in PI (4,5)P(subscript 2) and comparing them to the wild type cells. The methods described may form a basis for further optimization of mass spectral based quantitation of PIs.