Rosmarinic Acid-Rich Extracts of Summer Savory ( Satureja hortensis

Summer savory (Satureja hortensis L., Lamiaceae) is used in several regions of the world as a spice and folk medicine. Anti-inflammatory and cytoprotective effects of S. hortensis and of its rosmarinic acid-rich phenolic fraction have been demonstrated in animal trials. However, previous studies of rosmarinic acid in cell models have yielded controversial results. In this study, we investigated the effects of summer savory extracts on H2O2-challenged human lymphoblastoid Jurkat T cells. LC-MS analysis confirmed the presence of rosmarinic acid and flavonoids such as hesperidin and naringin in the phenolic fraction. Adding 25 or 50 µM of H2O2 to the cell culture caused oxidative stress, manifested as generation of superoxide and peroxyl radicals, reduced cell viability, G0/G1 arrest, and enhanced apoptosis. This stress was significantly alleviated by the ethanolic and aqueous extracts of S. hortensis and by the partially purified rosmarinic acid fraction. The application of an aqueous S. hortensis extract doubled the activity of catalase and superoxide dismutase in the cells. The production of IL-2 and IL-10 interleukins was stimulated by H2O2 and was further enhanced by the addition of the S. hortensis extract or rosmarinic acid fraction. The H2O2-challenged Jurkat cells may serve as a model for investigating cellular mechanisms of cytoprotective phytonutrient effects

Kinetochore alignment within the metaphase plate is regulated by centromere stiffness and microtubule depolymerases

During mitosis in most eukaryotic cells, chromosomes align and form a metaphase plate halfway between the spindle poles, about which they exhibit oscillatory movement. These movements are accompanied by changes in the distance between sister kinetochores, commonly referred to as breathing. We developed a live cell imaging assay combined with computational image analysis to quantify the properties and dynamics of sister kinetochores in three dimensions. We show that baseline oscillation and breathing speeds in late prometaphase and metaphase are set by microtubule depolymerases, whereas oscillation and breathing periods depend on the stiffness of the mechanical linkage between sisters. Metaphase plates become thinner as cells progress toward anaphase as a result of reduced oscillation speed at a relatively constant oscillation period. The progressive slowdown of oscillation speed and its coupling to plate thickness depend nonlinearly on the stiffness of the mechanical linkage between sisters. We propose that metaphase plate formation and thinning require tight control of the state of the mechanical linkage between sisters mediated by centromeric chromatin and cohesion

Meeting report--Getting Into and Out of Mitosis

The Company of Biologists Workshop 'Getting Into and Out of Mitosis' was held 10-13 May 2015 at Wiston House in West Sussex, UK. The workshop brought together researchers from wide-ranging disciplines and provided a forum to discuss their latest work on the control of cell division from mitotic entry to exit. This report highlights the main topics and summarises the discussion around the key themes and questions that emerged from the meeting

Additional file 2: of Two-step interphase microtubule disassembly aids spindle morphogenesis

Binding of Ensconsin/MAP7 to microtubules at mitotic entry is regulated by phosphorylation. A) Changes in non-centrosomal microtubule levels relative to NEP. Variance of α-tubulin-GFP signal measured as described in Additional file 1E for control siRNA (blue, five cells) and Cep192 siRNA cells (brown, five cells) for two experiments as in Fig. 2a–c. B) Western blot showing Ensconsin/MAP7 knockdown induced using three different siRNAs targeting Ensconsin/MAP7. C) Representative confocal images (x-y maximum projection) of fixed HeLa cells treated with control siRNA and three siRNAs targeting Ensconsin/MAP7. Boxed areas show regions zoomed in overlays (>10 cells per condition, one experiment). D) Representative confocal images (x-y maximum projection) of fixed MCF10A cells stained to show that Ensconsin/MAP7 is removed from microtubules in prophase compared to interphase. Ensconsin/MAP7 in red, α-tubulin in green and DAPI in blue. Boxed areas show regions zoomed in overlays, in which intensities were adjusted to remove cytoplasmic background signal (six prophase cells, > 20 interphase cells, one experiment). E) Representative confocal images of fixed HeLa cells overexpressing Rap1* in prophase stained to show that the Wt-EMTB-mCherry as well as a corresponding phospho-mimetic mutant (E-EMTB-mCherry) are largely cytoplasmic in prophase, whereas the non-phosphorylatable form (A-EMTB-mCherry) localises to the microtubules. α-Tubulin in green, Wt-EMTB-mCherry, A-EMTB-mCherry, E-EMTB-mCherry in red. Boxed areas are zoomed, shown in inverted greyscale or in overlays, where signal intensities were adjusted to remove cytoplasmic background signal (three cells per condition, one experiment). F) Representative time-lapse confocal images (x-y maximum projection) of flat (Rap1*) HeLa cells stably expressing GFP-α-tubulin and Wt-EMTB-mCherry (upper panel) or its non-phosphorylatable mutant (A-EMTB-mCherry, lower panel) to show that the non-phosphorylatable mutant stays associated with microtubules during prophase, leading to delay in microtubule disassembly at mitotic entry even if its expression level is lower compared to Wt-EMTB-mCherry. Boxed areas show regions zoomed in inverted greyscale or in overlays, where signal intensities were adjusted to remove cytoplasmic background signal. Black arrows indicate interphase microtubules just before or after NEP. Scale bars represent 10 μm. (PDF 5920 kb

Additional file 4: of Two-step interphase microtubule disassembly aids spindle morphogenesis

Hypo-osmotic shock can be used to mimic changes in tubulin concentration induced by NEP. A) Representative time-lapse confocal images (x-y maximum projection, lower panel: pseudo-color, spectra LUT) of HeLa cells stably expressing H2B-mRFP (not imaged) and mEGFP-α-tubulin, treated with Nocodazole, to show changes in cell diameter and in mEGFP-α-tubulin intensity before and after hypo- or hyper-osmotic shock treatment relative to control treatment. Quantifications of changes in mEGFP-α-tubulin intensity (B) and in cell diameter (C) induced by osmotic shock relative to control treatment. Mean intensity of mEGFP-α-tubulin signal and cell diameter was measured in cells before and after control (blue, seven cells), hypo- (red, eight cells) or hyper- (green, eight cells) osmotic shock treatments (two independent experiments) as described in Methods. Lower panels show comparison between values at –0.5 min and 2 min relative to osmotic shock treatments. Repeated measures two-way ANOVA, Dunnett's multiple comparisons test, ****P = 0.0001. D) Representative time-lapse confocal images (x-y maximum projection, lower panel: pseudo-color, spectra LUT) of HeLa cells stably expressing H2B-mRFP (not imaged) and mEGFP-α-tubulin showing that hypo-osmotic shock affects mitotic spindle, whereas it does not have impact on interphase microtubules. White arrows indicate mitotic spindle. E) Representative time-lapse confocal images (x-y maximum projection) of HeLa cells stably expressing H2B-mRFP and mEGFP-α-tubulin and transiently overexpressing Rap1 treated with Lamin A siRNA and ESCRT-III siRNA during mitotic entry. Boxed areas are zoomed below. Control cell represents a Lamin A siRNA and ESCRT-III siRNA treated cell entering mitosis. The following cells represent accordingly a cell where nuclear envelope rupture was induced in late prophase (close to NEP) followed by immediate disassembly of microtubules and a cell where nuclear envelope rupture was induced in early prophase without triggering immediate disassembly of microtubules. F) Quantifications of timing of changes in centrosomal and non-centrosomal microtubule levels relative to NEP or to nuclear envelope (NE) ablation in cells represented in E as described in Fig. 2b. Scale bars represent 10 μm. (PDF 6682 kb

Additional file 3: of Two-step interphase microtubule disassembly aids spindle morphogenesis

Failure in removal of Ensconsin/MAP7 from microtubules in prophase delays interphase microtubule disassembly and leads to an abnormal-looking mitotic spindle. Movie shows Flat (Rap1*) HeLa cell stably expressing GFP-α-tubulin (green) and non-phosphorylatable Ensconsin/MAP7 microtubule-binding domain (A-EMTB-mCherry, red) during mitotic progression. Scale bar represents 10 μm. (AVI 1091 kb

Kinetochores accelerate centrosome separation to ensure faithful chromosome segregation

At the onset of mitosis, cells need to break down their nuclear envelope, form a bipolar spindle and attach the chromosomes to microtubules via kinetochores. Previous studies have shown that spindle bipolarization can occur either before or after nuclear envelope breakdown. In the latter case, early kinetochore-microtubule attachments generate pushing forces that accelerate centrosome separation. However, until now, the physiological relevance of this prometaphase kinetochore pushing force was unknown. We investigated the depletion phenotype of the kinetochore protein CENP-L, which we find to be essential for the stability of kinetochore microtubules, for a homogenous poleward microtubule flux rate and for the kinetochore pushing force. Loss of this force in prometaphase not only delays centrosome separation by 5-6 minutes, it also causes massive chromosome alignment and segregation defects due to the formation of syntelic and merotelic kinetochore-microtubule attachments. By contrast, CENP-L depletion has no impact on mitotic progression in cells that have already separated their centrosomes at nuclear envelope breakdown. We propose that the kinetochore pushing force is an essential safety mechanism that favors amphitelic attachments by ensuring that spindle bipolarization occurs before the formation of the majority of kinetochore-microtubule attachments

Nonautonomous movement of chromosomes in mitosis

Kinetochores are the central force-generating machines that move chromosomes during cell division. It is generally assumed that kinetochores move in an autonomous manner. However, we reveal here that movements of neighboring sister-kinetochore pairs in metaphase are correlated in a distance-dependent manner. This correlation increases in the absence of kinetochore oscillations or stable end-on attachments. This suggests that periodic movements of bioriented chromosomes limit the correlated motion of nonsisters. Computer simulations show that these correlated movements can occur when elastic crosslinks are placed between the K-fibers of oscillating kinetochores. Strikingly, inhibition of the microtubule crosslinking motor kinesin-5 Eg5 leads to an increase in nonsister correlation and impairs periodic oscillations. These phenotypes are partially rescued by codepletion of the kinesin-12 Kif15, demonstrating a function for kinesin-5 and kinesin-12 motors in driving chromosome movements, possibly as part of a crosslinking structure that correlates the movements of nonsister kinetochores