The rate, concentration dependence and extent of histamine‐evoked Weibel–Palade body (WPB) exocytosis were investigated with time‐resolved fluorescence microscopy in cultured human umbilical vein ...endothelial cells expressing WPB‐targeted chimeras of enhanced green fluorescent protein (EGFP). Exocytosis of single WPBs was characterized by an increase in EGFP fluorescence, morphological changes and release of WPB contents. The fluorescence increase was due to a rise of intra‐WPB pH from resting levels, estimated as pH 5.45 ± 0.26 (s.d., n= 144), to pH 7.40. It coincided with uptake of extracellular Alexa‐647, indicating the formation of a fusion pore, prior to loss of fluorescent contents. Delays between the increase in intracellular free calcium ion concentration evoked by histamine and the first fusion event were 10.0 ± 4.42 s (n= 9 cells) at 0.3 μm histamine and 1.57 ± 0.21 s (n= 15 cells) at 100 μm histamine, indicating the existence of a slow process or processes in histamine‐evoked WPB exocytosis. The maximum rates of exocytosis were 1.20 ± 0.16 WPB s−1 (n= 9) at 0.3 μm and 3.66 ± 0.45 WPB s−1 at 100 μm histamine (n= 15). These occurred 2–5 s after histamine addition and declined to lower rates with continued stimulation. The initial delays and maximal rate of exocytosis were unaffected by removal of external Ca2+ indicating that the initial burst of secretion is driven by Ca2+ release from internal stores, but sustained exocytosis required external Ca2+. Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 μm), for which there was a delay between calcium elevation and secretion of 1.67 ± 0.24 s (n= 6), and a peak fusion rate of ∼10 WPB s−1.
Abstract Study question Can centromeres be labelled in live human oocytes using Cas9 system and is the labelling efficiency affected by advancing female age? Summary answer Centromeres in human ...oocytes can be labelled using Cas9 system. Signal intensity decreases in oocytes from older patients, suggesting centromeric DNA unravelling as oocytes age. What is known already Aneuploidy in human oocytes is associated with infertility, pregnancy loss or genetic diseases in offspring. It arises due to abnormal chromosome segregation during meiosis, with the risk increasing with advancing female age. This is attributed to age-dependent loss of pericentromere-localised cohesion which holds sister chromatids together. Our work revealed that shugoshin-2 (Sgo2), a protein involved in protection of sister chromatid cohesion is also lost with age. This can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. This hints at more extensive alterations to centromere organisation with advanced maternal age. Study design, size, duration This study uses human oocytes deselected from fertility treatment donated for research purposes with informed consent (HFEA RO155). The oocytes were collected fresh at germinal vesicle (GV), metaphase I (MI) or metaphase II (MII) stage, injected with Cas9 labelling system and live imaged or fixed. Participants/materials, setting, methods Centromeres were labelled in human oocytes using a dead (nuclease-deficient) (d)Cas9-EGFP protein and alpha satellite-specific small guide RNAs (sgRNA). Human oocytes were injected with dCas9-EGFP:sgAlphaSat and live-imaged using dual colour confocal microscopy with SPY555-DNA dye to also visualise chromosomes. High resolution imaging results were analysed with in-house automated kinetochore tracking software. Additional oocytes were fixed and processed for immunofluorescence to determine the high resolution localisation of dCas9-EGFP:sgAlphaSat and Sgo2 molecules. Main results and the role of chance We found that centromeres in human oocytes are efficiently labelled when using Cas9 system. During both meiosis I and II the dCas9-EGFP:sgAlphaSat probe co-localised with pericentromeric bridge and subcentromeric pools of Sgo2. We observed that the dCas9-EGFP:sgAlphaSat signal fades with advancing female age. Quantification reveals that in meiosis II the loss is abrupt at around 35 years of age, resembling Sgo2 behaviour. Initial live imaging indicates that already compromised signal in older oocytes is completely lost about 4hrs after germinal vesicle breakdown, suggesting that advancing age renders centromeres more vulnerable as meiosis I progresses. We have successfully tracked the centromeres in high resolution imaging using the automated software, which allows for detailed investigation of human meiosis chromosome dynamics. This method has been previously validated and successfully used for other cell systems. Limitations, reasons for caution Availability of human oocytes for research purposes is low, limiting our sample size. Moreover, most oocytes used for this study were deselected from treatment of patients diagnosed with infertility, potentially affecting their quality. Wider implications of the findings Our results reveal a large-scale change in centromeric DNA structure in human oocytes with increasing age. Live centromere labelling is a first successful attempt to bivalent tracking in human meiosis with potential to provide insight into age-related loss of fertility in women. Trial registration number n/a
Extracellular regulated kinases (ERKI/II), members of the mitogen-activated protein kinase family, play a role in long-term memory and long-term potentiation (LTP). ERKI/II is required for the ...induction of the early phase of LTP, and we show that it is also required for the late phase of LTP in area CA1 in vitro, induced by a protocol of brief, repeated 100 Hz trains. We also show that ERKI/II is necessary for the upregulation of the proteins encoded by the immediate early genes Zif268 and Homer after the induction of LTP in the dentate gyrus by tetanic stimulation of the perforant path in vivo or by BDNF stimulation of primary cortical cultures. To test whether the induction of persistent synaptic plasticity by stimuli such as BDNF is associated with nuclear translocation of ERKI/II, we expressed enhanced green fluorescent protein (EGFP)-ERKII in PC12 cell lines and primary cortical cultures. In both preparations, we observed translocation of EGFP-ERKII from the cytoplasm to the nucleus in cells exposed to neurotrophic factors. Our results suggest that the induction of late LTP involves translocation of ERKI/II to the nucleus in which it activates the transcription of immediate early genes. The ability to visualize the cellular redistribution of ERKII after induction of long-term synaptic plasticity may provide a method for visualizing neuronal circuits underlying information storage in the brain in vivo.
The time course for cell surface loss of von Willebrand factor (VWF) and the propolypeptide of VWF (proregion) following exocytosis
of individual Weibel-Palade bodies (WPBs) from single human ...endothelial cells was analyzed. Chimeras of enhanced green fluorescent
protein (EGFP) and full-length pre-pro-VWF (VWF-EGFP) or the VWF propolypeptide (proregion-EGFP) were made and expressed in
human umbilical vein endothelial cells. Expression of VWF-EGFP or proregion-EGFP resulted in fluorescent rod-shaped organelles
that recruited the WPB membrane markers P-selectin and CD63. The WPB secretagogue histamine evoked exocytosis of these fluorescent
WPBs and extracellular release of VWF-EGFP or proregion-EGFP. Secreted VWF-EGFP formed distinctive extracellular patches of
fluorescence that were labeled with an extracellular antibody to VWF. The half-time for dispersal of VWF-EGFP from extracellular
patches was 323.5 ± 146.2 s (±S.D., n = 20 WPBs). In contrast, secreted proregion-EGFP did not form extracellular patches but dispersed rapidly from its site of
release. The half-time for dispersal of proregion-EGFP following WPB exocytosis was 2.98 ± 1.88 s (±S.D., n = 32 WPBs). The slow rate of loss of VWF-EGFP is consistent with the adhesive nature of this protein for the endothelial
membrane. The much faster rate of loss of proregion-EGFP indicates that this protein does not interact strongly with extracellular
VWF or the endothelial membrane and consequently may not play an adhesive role at the endothelial cell surface.
Class I myosins are single-headed motor proteins implicated in various motile processes including organelle translocation, ion channel gating, and cytoskeleton reorganization. Dictyostelium ...discoideum myosin-ID belongs to subclass 1α, whose members are thought to be tuned for rapid sliding. The direct analysis of myosin-ID motor activity is made possible by the production of single polypeptide constructs carrying an artificial lever arm. Using these constructs, we show that the motor activity of myosin-ID is activated 80-fold by phosphorylation at the TEDS site. TEDS site phosphorylation acts by stabilizing the actomyosin complex and increasing the coupling between actin binding and the release of hydrolysis products. A surprising effect of Mg2+ ions on in vitro motility was discovered. Changes in the level of free Mg2+ ions within the physiological range are shown to modulate motor activity by inhibiting ADP release. Our results indicate that higher concentrations of free Mg2+ ions stabilize the tension-bearing actin myosin ADP state and shift the system from the production of rapid movement toward the generation of tension.
Class I myosins are single-headed motor proteins, implicated in various motile processes including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Here we describe the ...cellular localization of myosin-IE and its role in the phagocytic uptake of solid particles and cells. A complete analysis of the kinetic and motor properties of Dictyostelium discoideum myosin-IE was achieved by the use of motor domain constructs with artificial lever arms. Class I myosins belonging to subclass IC like myosin-IE are thought to be tuned for tension maintenance or stress sensing. In contrast to this prediction, our results show myosin-IE to be a fast motor. Myosin-IE motor activity is regulated by myosin heavy chain phosphorylation, which increases the coupling efficiency between the actin and nucleotide binding sites tenfold and the motile activity more than fivefold. Changes in the level of free Mg(2+) ions, which are within the physiological range, are shown to modulate the motor activity of myosin-IE by inhibiting the release of adenosine diphosphate.
Nucleoside (NDP) diphosphate kinases are oligomeric enzymes. Most are hexameric, but some bacterial enzymes are tetrameric. Hexamers and tetramers are constructed by assembling identical dimers. The ...hexameric structure is important for protein stability, as demonstrated by studies with natural mutants (the Killer-of-prune mutant of Drosophila NDP kinase and the S120G mutant of the human NDP kinase A in neuroblastomas) and with mutants obtained by site-directed mutagenesis. It is also essential for enzymic activity. The function of the tetrameric structure is unclear.
Calmodulin redistribution in MDCK and HeLa cells subjected to microtubule perturbations by antimitotic drugs was followed using a calmodulin-EGFP fusion protein that preserves the Ca(2+) affinity, ...target binding and activation properties of native calmodulin. CaM-EGFP targeting to spindle structures in normal cell division and upon spindle microtubule disruption allows evaluation of the dynamic redistribution of calmodulin in cell division. Under progressive treatment of stably transfected mammalian cells with nocodazole or vinblastine, the centrosomal matrix at the mitotic poles subdivides into numerous small 'star-like' structures, with the calmodulin concentrated centrally, and partially distinct from the reduced microtubule mass to which kinetochores and chromosomes are attached. Prolonged vinblastine treatment causes the release of localised calmodulin into a uniform cytoplasmic distribution, and tubulin paracrystal formation. By contrast, paclitaxel treatment of metaphase cells apparently causes limited disassembly of the pericentriolar material into a number of multipolar 'ring-like' structures containing calmodulin, each one having multiple attached microtubules terminating in the partially disordered kinetochore/chromosome complex. Thus drugs with opposite effects in either destabilising or stabilising mitotic microtubules cause subdivision of the centrosomal matrix into two distinctive calmodulin-containing structures, namely small punctate 'stars' or larger polar 'rings' respectively. The 'star-like' structures may represent an integral subcomponent for the attachment of kinetochore microtubules to the metaphase centrosome complex. The results imply that microtubules have a role in stabilising the structure of the pericentriolar matrix, involving interaction, either direct or indirect, with one or more proteins that are targets for binding of calmodulin. Possible candidates include the pericentriolar matrix-associated coiled-coil proteins containing calmodulin-binding motifs, such as myosin V, kendrin (PCNT2) and AKAP450.
A novel human nm23/nucleoside diphosphate (NDP) kinase gene, called nm23-H4, was identified by screening a human stomach cDNA library with a probe generated by amplification by reverse ...transcription-polymerase chain reaction. The primers were designed from publicly available database cDNA sequences selected according to their homology to the human nn23-H1 putative metastasis suppressor gene. The full-length cDNA sequence predicts a 187 amino acid protein possessing the region homologous to NDP kinases with all residues crucial for nucleotide binding and catalysis, strongly suggesting that Nm23-H4 possesses NDP kinase activity. It shares 56, 55 and 60% identity with Nm23-H1, Nm23-H2 and DR-Nm23, respectively, the other human Nm23 proteins isolated so far. Compared with these proteins, Nm23-H4 contains an additional NH2-terminal region that is rich in positively charged residues and could indicate routing to mitochondria. The nm23-H4 gene has been localised to human chromosomal band 16p13.3. The corresponding 1.2 kb mRNA is widely distributed and expressed in a tissue-dependent manner, being found at very high levels in prostate, heart, liver, small intestine and skeletal muscle tissues and in low amounts in the brain and in blood leucocytes. Nm23-H4 naturally possesses the Pro-Ser substitution equivalent to the K-pn mutation (P97S) of Drosophila.
The rate, concentration dependence and extent of histamine-evoked WeibelâPalade body (WPB) exocytosis were investigated with
time-resolved fluorescence microscopy in cultured human umbilical vein ...endothelial cells expressing WPB-targeted chimeras
of enhanced green fluorescent protein (EGFP). Exocytosis of single WPBs was characterized by an increase in EGFP fluorescence,
morphological changes and release of WPB contents. The fluorescence increase was due to a rise of intra-WPB pH from resting
levels, estimated as pH 5.45 ± 0.26 ( s.d. , n
= 144), to pH 7.40. It coincided with uptake of extracellular Alexa-647, indicating the formation of a fusion pore, prior
to loss of fluorescent contents. Delays between the increase in intracellular free calcium ion concentration evoked by histamine
and the first fusion event were 10.0 ± 4.42 s ( n
= 9 cells) at 0.3 μ m histamine and 1.57 ± 0.21 s ( n
= 15 cells) at 100 μ m histamine, indicating the existence of a slow process or processes in histamine-evoked WPB exocytosis. The maximum rates
of exocytosis were 1.20 ± 0.16 WPB s â1 ( n
= 9) at 0.3 μ m and 3.66 ± 0.45 WPB s â1 at 100 μ m histamine ( n
= 15). These occurred 2â5 s after histamine addition and declined to lower rates with continued stimulation. The initial
delays and maximal rate of exocytosis were unaffected by removal of external Ca 2+ indicating that the initial burst of secretion is driven by Ca 2+ release from internal stores, but sustained exocytosis required external Ca 2+ . Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 μ m ), for which there was a delay between calcium elevation and secretion of 1.67 ± 0.24 s ( n
= 6), and a peak fusion rate of â¼10 WPB s â1 .
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