Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cell basal cortex through LL5α/β

LL5β has been identified as a microtubule-anchoring factor that attaches EB1/CLIP-associating protein (CLASP)–bound microtubule plus ends to the cell cortex. In this study, we show that LL5β and its homologue LL5α (LL5s) colocalize with autocrine laminin-5 and its receptors, integrins α3β1 and α6β4, at the basal side of fully polarized epithelial sheets. Depletion of both laminin receptor integrins abolishes the cortical localization of LL5s, whereas LL5 depletion reduces the amount of integrin α3 at the basal cell cortex. Activation of integrin α3 is sufficient to initiate LL5 accumulation at the cell cortex. LL5s form a complex with the cytoplasmic tails of these integrins, but their interaction might be indirect. Analysis of the three-dimensional distribution of microtubule growth by visualizing EB1-GFP in epithelial sheets in combination with RNA interference reveals that LL5s are required to maintain the density of growing microtubules selectively at the basal cortex. These findings reveal that signaling from laminin–integrin associations attaches microtubule plus ends to the epithelial basal cell cortex.     

Two Golgi-resident 3′-Phosphoadenosine 5′-Phosphosulfate Transporters Play Distinct Roles in Heparan Sulfate Modifications and Embryonic and Larval Development in Caenorhabditis elegans

Synthesis of extracellular sulfated molecules requires active 3′-phosphoadenosine 5′-phosphosulfate (PAPS). For sulfation to occur, PAPS must pass through the Golgi membrane, which is facilitated by Golgi-resident PAPS transporters. Caenorhabditis elegans PAPS transporters are encoded by two genes, pst-1 and pst-2. Using the yeast heterologous expression system, we characterized PST-1 and PST-2 as PAPS transporters. We created deletion mutants to study the importance of PAPS transporter activity. The pst-1 deletion mutant exhibited defects in cuticle formation, post-embryonic seam cell development, vulval morphogenesis, cell migration, and embryogenesis. The pst-2 mutant exhibited a wild-type phenotype. The defects observed in the pst-1 mutant could be rescued by transgenic expression of pst-1 and hPAPST1 but not pst-2 or hPAPST2. Moreover, the phenotype of a pst-1;pst-2 double mutant were similar to those of the pst-1 single mutant, except that larval cuticle formation was more severely defected. Disaccharide analysis revealed that heparan sulfate from these mutants was undersulfated. Gene expression reporter analysis revealed that these PAPS transporters exhibited different tissue distributions and subcellular localizations. These data suggest that pst-1 and pst-2 play different physiological roles in heparan sulfate modification and development.     

Overexpression of glutathione peroxidase attenuates myocardial remodeling and preserves diastolic function in diabetic heart

Oxidative stress plays an important role in the structural and functional abnormalities of diabetic heart. Glutathione peroxidase (GSHPx) is a critical antioxidant enzyme that removes H(2)O(2) in both the cytosol and mitochondia. We hypothesized that the overexpression of GSHPx gene could attenuate left ventricular (LV) remodeling in diabetes mellitus (DM). We induced DM by injection of streptozotocin (160 mg/kg ip) in male GSHPx transgenic mice (TG+DM) and nontransgenic wildtype littermates (WT+DM). GSHPx activity was higher in the hearts of TG mice compared with WT mice, with no significant changes in other antioxidant enzymes. LV thiobarbituric acid-reactive substances measured in TG+DM at 8 wk were significantly lower than those in WT+DM (58 +/- 3 vs. 71 +/- 5 nmol/g, P < 0.05). Heart rate and aortic blood pressure were comparable between groups. Systolic function was preserved normal in WT+DM and TG+DM mice. In contrast, diastolic function was impaired in WT+DM and was improved in TG+DM as assessed by the deceleration time of peak velocity of transmitral diastolic flow and the time needed for relaxation of 50% maximal LV pressure to baseline value (tau; 13.5 +/- 1.2 vs. 8.9 +/- 0.7 ms, P < 0.01). The TG+DM values were comparable with those of WT+Control (tau; 7.8 +/- 0.2 ms). Improvement of LV diastolic function was accompanied by the attenuation of myocyte hypertrophy, interstitial fibrosis, and apoptosis. Overexpression of GSHPx gene ameliorated LV remodeling and diastolic dysfunction in DM. Therapies designed to interfere with oxidative stress might be beneficial to prevent cardiac abnormalities in DM.     

Growth strategy of an emergent macrophyte, Typha orientalis Presl, in comparison with Typha latifolia L. and Typha angustifolia L.

The growth strategy of an emergent plant, Typha orientalis Presl, was examined in experimental ponds in comparison with two other Typha species distributed in Japan, Typha latifolia L. and Typha angustifolia L. T. orientalis showed the greatest ability of vegetative reproduction at the expense of growth in height. T. orientalis started to produce new ramets earlier than T. latifolia and T. angustifolia. These results suggest that T. orientalis should be a rather pioneer-like species and would be restricted to disturbed habitats.     

Release of RASSF1C from the nucleus by Daxx degradation links DNA damage and SAPK/JNK activation

Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) responds to a variety of stress stimuli and controls cell fates such as cell cycle entrance, apoptosis and senescence. Stimuli such as ultraviolet irradiation and chemical reagents that damage genomic DNA induce the activation of the SAPK/JNK signaling pathway. However, it is unclear how the signal arising in the nucleus owing to DNA damage is transmitted to SAPK/JNK in the cytoplasm. Here, we report that the nuclear components Daxx and Ras-association domain family 1C (RASSF1C) link DNA damage to SAPK/JNK activation in HeLa cells. In response to DNA damage, Daxx localized in promyelocytic leukaemia-nuclear bodies (PML-NBs) undergoes ubiquitination and degradation. RASSF1C, a tumor suppressor and newly identified binding partner of Daxx, is constitutively anchored by Daxx in PML-NBs but is released from the nucleus when Daxx is degraded. This released RASSF1C translocates to cytoplasmic microtubules and participates in the activation of SAPK/JNK. Our data define a novel mechanism by which the Daxx-RASSF1C complex in PML-NBs couples nuclear DNA damage to the cytoplasmic SAPK/JNK signaling pathway.     

Crystal structure of the RUN domain of the RAP2-interacting protein x

Rap2-interacting protein x (RPIPx) is a homolog of RPIP8, a specific effector of Rap2 GTPase. The N-terminal region of RPIP8, which contains the RUN domain, interacts with Rap2. Using cell-free synthesis and NMR, we determined that the region encompassing residues 83-255 of mouse RPIPx, which is 40-residues larger than the predicted RUN domain (residues 113-245), is the minimum fragment that forms a correctly folded protein. This fragment, the RPIPx RUN domain, interacted specifically with Rap2B in vitro in a nucleotide-dependent manner. The crystal structure of the RPIPx RUN domain was determined at 2.0 A of resolution by the multiwavelength anomalous dispersion (MAD) method. The RPIPx RUN domain comprises eight anti-parallel alpha-helices, which form an extensive hydrophobic core, followed by an extended segment. The residues in the core region are highly conserved, suggesting the conservation of the RUN domain-fold among the RUN domain-containing proteins. The residues forming a positively charged surface are conserved between RPIP8 and its homologs, suggesting that this surface is important for Rap2 binding. In the crystal the putative Rap2 binding site of the RPIPx RUN domain interacts with the extended segment in a segment-swapping manner.     

C. elegans pharyngeal morphogenesis requires both de novo synthesis of pyrimidines and synthesis of heparan sulfate proteoglycans

The C. elegans pharynx undergoes elongation and morphogenesis to its characteristic bi-lobed shape between the 2- and 3-fold stages of embryogenesis. During this period, the pharyngeal muscles and marginal cells forming the isthmus between the anterior and posterior pharyngeal bulbs elongate and narrow. We have identified the spontaneous mutant pyr-1(cu8) exhibiting defective pharyngeal isthmus elongation, cytoskeletal organization defects, and maternal effect lethality. pyr-1 encodes CAD, a trifunctional enzyme required for de novo pyrimidine synthesis, and pyr-1(cu8) mutants are rescued by supplying exogenous pyrimidines. Similar pharyngeal defects and maternal effect lethality were found in sqv-1, sqv-8, rib-1 and rib-2 mutants, which affect enzymes involved in heparan sulfate proteoglycan (HSPG) synthesis. rib-1 mutant lethality was enhanced in a pyr-1 mutant background, indicating that HSPG synthesis is very sensitive to decreased pyrimidine pools, and HS disaccharides are moderately decreased in both rib-1 and pyr-1 mutants. We hypothesize that HSPGs are necessary for pharyngeal isthmus elongation, and pyr-1 functions upstream of proteoglycan synthesizing enzymes by providing precursors of UDP-sugars essential for HSPG synthesis.     

Postural muscle activity patterns during standing at rest and on an oscillating floor.

Postural muscle activity pattern was examined in the eyes-closed state after adequate adaptation to floor anteroposterior oscillation. Twenty-three subjects were grouped almost evenly according to dominance of anterior or posterior postural muscles in the trunk and thigh during quiet stance. In the posterior-dominant group, this dominance was maintained at every frequency in most subjects. In the anterior-dominant group, this dominance was maintained in most subjects at 0.1 and 0.5 Hz but changed to posterior dominance at 1.0 and 1.5 Hz in about half the subjects. Periodicity of muscle activity was evaluated by EMG amplitude spectrum at the floor oscillation frequency. Periodicity of posterior-dominant muscles in the trunk and thigh increased with increasing oscillatory frequency. In the trunk, the periodicity did not differ significantly between posterior-dominant and anterior-dominant groups. However, in the thigh, periodicity was significantly lower in the anterior-dominant muscles. This was considered to be caused by nonperiodic alternating action of the anterior and posterior muscles. In the lower leg, posterior dominance was observed in quiet stance and at all oscillation frequencies. Periodicity of soleus and gastrocnemius increased at higher frequencies and was higher in gastrocnemius than in soleus. The periodicity difference between both muscles decreased with increasing oscillation frequency.