Nuclear Envelope Protein MAN1 Regulates the Drosophila Circadian Clock via Period

Almost all organisms exhibit ~24-h rhythms, or circadian rhythms, in a plentitude of biological processes.These rhythms are driven by endogenous molecular clocks consisting of a series of transcriptional and translational feedback loops. Previously, we have shown that the inner nuclear membrane protein MAN1 regulates this clock and thus the locomotor rhythm in flies, but the mechanism remains unclear. Here, we further confirmed the previous findings and found that knocking down MAN1 in the pacemaker neurons of adult flies is sufficient to lengthen the period of the locomotor rhythm. Molecular analysis revealed that knocking down MAN1 led to reduced m RNA and protein levels of the core clock gene period(per),likely by reducing its transcription. Over-expressing per rescued the long period phenotype caused by MAN1 deficiency whereas per mutation had an epistatic effect on MAN1, indicating that MAN1 sets the pace of the clock by targeting per.

Alp7-Mtol and Alpl4 synergize to promote interphase microtubule regrowth from the nuclear envelope

Microtubules grow not only from the centrosome but also from various noncentrosomal microtubule-organizing centers(MTOCs),including the nuclear envelope(NE)and pre-existing microtubules.The evolutionarily conserved proteins Mtol/CDK5RAP2 and Alpl4/TOG/XMAP215 have been shown to be involved in promoting microtubule nucleation.However,it has remained elusive as to how the microtubule nucleation promoting factors are specified to various noncentrosomal MTOCs,particularly the NE,and how these proteins coordinate to organize microtubule assembly.Here,we demonstrate that in the fission yeast Schizosaccharomyces pombe,efficient interphase microtubule growth from the NE requires Alp7/TACC,Alpl4/TOG/XMAP215,and Mtol/CDK5RAP2.The absence of Alp7,A lp l4 t or Mtol compromises microtubule regrowth on the NE in cells undergoing microtubule repolymerization.We further demonstrate that Alp7 and Mtol interdependently localize to the NE in cells without microtubules and that A lp l4 localizes to the NE in an Alp7 and Mtol-dependent manner.Tethering Mtol to the NE in cells lacking Alp7 partially restores microtubule number and the efficiency of microtubule generation from the NE.Hence,our study delineates that Alp7,A lpl4,and Mtol work in concert to regulate interphase microtubule regrowth on the NE.

LINC complex independent perinuclear actin organization and cell migration

The link of the metazoan nucleus to the actin cytoskeleton is highly important for actin polymerization and migration of multiple cell types as well as for mechanotransduction and even affects the cellular transcriptome.Several mechanisms of organization of actin filaments next to the nuclear envelope have been identified.Among these mechanisms the most studied one is the Linker of nucleoskeleton and cytoskeleton(LINC)complex-dependent perinuclear actin organization.However,recently additional mechanisms have been identified:an Actin-related protein-2/3(Arp2/3)-dependent perinuclear actin polymerization during migration of dendritic cells and a perinuclear actin rim that is formed in response to external force application or migration cues.In parallel,there are also reports on cancer cells that migrate in a LINC complex independent manner and on cancers with reduced expression of the LINC complex components.Thus,suggesting that LINC complex independent migration may be associated with tumour formation.

Mechanism underlying carbon tetrachloride-inhibited protein synthesis in liver

AIM: To study the mechanism underlying carbon tetrachloride (CCl4)-induced alterations of protein synthesis in liver. METHODS: Male Sprague-Dawley rats were given CCl4 (1 mL/100 g body weight) and 3H-leucine incorporation. Malondialdehyde (MDA) level in the liver, in vitro response of hepatocyte nuclei nucleotide triphosphatase (NTPase) to free radicals, and nuclear export of total mRNA with 3'-poly A+ were measured respectively. Survival response of HepG2 cells to CCl4 treatment was assessed by methyl thiazolyl tetrazolium. Km and Vmax values of nuclear envelope NTPase activity in liver of rats treated with CCl4 were assayed by a double-reciprocal plot. RESULTS: The protein synthesis was inhibited while the MDA level was signif icantly increased in liver of rats treated with CCl4. In addition, CCl4 decreased the NTPase binding capacity of nuclear envelope (Km value) in cultured HepG2 cells. Moreover, in vitro ferrous radicals from Fenton's system suppressed the NTPase activity of liver nuclear envelope in a dose-dependent manner. Down-regulation of the nuclear envelope NTPase activity indicated a lower energy provision for nucleocytoplasmic transport of mRNA molecules, an evidence in CCl4-treated HepG2 cells correspondingly supported by the nuclear sequestration of poly (A)+ mRNA molecules in morphological hybridization research. CONCLUSION: Inhibition of mRNA transport, suggestive of decreased NTPase activity of the nuclear envelope, may be involved in carbon tetrachloride-inhibited protein synthesis in liver.

Loop Structures and Barrier Elements from D. melanogaster 87А7 Heat Shock Locus

The three-dimensional organization of the genome is closely related to its functioning. Interactions between parts of the genome located at large distances from each other have been detected within the chromosomes of different organisms, which led to the discovery of topologically associated domains (TADs). Methods that reveal such interactions between chromosomal loci imply detection of both protein-protein and protein-DNA interactions. We investigated the possibility of involvement of the direct DNA-DNA interactions in the structural and functional organization of Drosophila melanogaster chromosomal 87A7 locus, containing genes hsp70Aa and hsp70Ab, with the sequence analysis method. Our results indicate that the functional organization of 87A7 locus may involve different elements: chromosomal DNA fragments that attach chromosomes to the nuclear envelope, short polypurine/polypyrimidine tracts, insulators and their proteins. The combination of interactions of these elements may cause different functional states of 87A7 locus.

Stay in touch with the endoplasmic reticulum

The endoplasmic reticulum(ER),which is composed of a continuous network of tubules and sheets,forms the most widely distributed membrane system in eukaryotic cells.As a result,it engages a variety of organelles by establishing membrane contact sites(MCSs).These contacts regulate organelle positioning and remodeling,including fusion and fission,facilitate precise lipid exchange,and couple vital signaling events.Here,we systematically review recent advances and converging themes on ER-involved organellar contact.The molecular basis,cellular influence,and potential physiological functions for ER/nuclear envelope contacts with mitochondria,Golgi,endosomes,lysosomes,lipid droplets,autophagosomes,and plasma membrane are summarized.

Calcium regulation of nucleocytoplasmic transport

Bidirectional trafficking of macromolecules between the cytoplasm and the nucleus is mediated by the nuclear pore complexes(NPCs)embedded in the nuclear envelope(NE)of eukaryotic cell.The NPC functions as the sole pathway to allow for the passive diffusion of small molecules and the facilitated translocation of larger molecules.Evidence shows that these two transport modes and the conformation of NPC can be regulated by calcium stored in the lumen of nuclear envelope and endoplasmic reticulum.However,the mechanism of calcium regulation remains poorly understood.In this review,we integrate data on the observations of calciumregulated structure and function of the NPC over the past years.Furthermore,we highlight challenges in the measurements of dynamic conformational changes and transient transport kinetics in the NPC.Finally,an innovative imaging approach,single-molecule superresolution fluorescence microscopy,is introduced and expected to provide more insights into the mechanism of calcium-regulated nucleocytoplasmic transport.