Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “vesicles”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 343 records · Page 19
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Giantin is required for coordinated production of aggrecan, link protein and type XI collagen during chondrogenesis

Highlights: • Specificity of giantin for intracellular traffic of cartilage ECM is proposed. • Loss of giantin result in decreased of aggrecan, link protein and hyaluronan. • Loss of giantin cause increased type XI collagen. • Giantin is involved in perichondrium differentiation. Extracellular matrix (ECM) constitutes a proper micro-environment for cell proliferation, migration and differentiation, as well as playing pivotal roles in developmental processes including endochondral ossification. Cartilage ECM is mainly composed of fibrous proteins, including collagen, proteoglycan, and hyaluronan. Because almost all ECM components are transported by intracellular vesicular transport systems, molecules that mediate vesicle transport are also important for endochondral ossification. Giantin, encoded by the Golgb1 gene, is a tethering factor for coatomer 1 (COPI) vesicles and functions in the cis-medial Golgi compartments. An insertion mutation in the Golgb1 gene, resulting in a lack of giantin protein expression, has been detected in ocd/ocd rats that exhibit a pleiotropic phenotype including osteochondrodysplasia. To reveal the function of giantin in chondrogenesis, the present study assessed the effects of loss of giantin expression on cartilage ECM and Golgi morphology. Giantin was expressed in normal, but not in ocd/ocd, chondrocytes in the epiphyseal areas of embryonic femurs, whereas GM130 was expressed in both normal and ocd/ocd chondrocytes. The staining intensities of safranin O and azan (aniline blue) were reduced and enhanced, respectively, in epiphyseal cartilage of ocd/ocd femurs. Immunostaining showed that levels of type II collagen and fibronectin were comparable in normal and ocd/ocd cartilage. Levels of type XI collagen were higher, while levels of aggrecan, link protein and hyaluronan were lower, in ocd/ocd than in normal cartilage, although semi-quantitative RT-PCR showed similar levels of type XI collagen, aggrecan and link protein mRNAs in normal and ocd/ocd cartilage. Isolated chondrocytes of ocd/ocd and normal rats showed similar immunostaining patterns for cis-, medial-, and trans-Golgi marker proteins, whereas monolayers of ocd/ocd chondrocytes showed reduced levels of aggrecan and link protein and increased level of type XI collagen in spite of similar transcripts levels. These findings suggest that giantin plays a pivotal role in coordinated production of aggrecan, link protein and type XI collagen in chondrocytes, and that loss of giantin causes osteochondrodysplasia with disturbance of these ECM components.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Bitter tastant quinine modulates glucagon-like peptide-1 exocytosis from clonal GLUTag enteroendocrine L cells via actin reorganization

Highlights: • Various bitter taste receptors were expressed in GLUTag cells. • Quinine increased the intracellular Ca{sup 2+} levels but did not promote GLP-1 secretion. • Quinine caused actin reorganization and suppressed exocytosis of GLP-1 vesicles. • Application of forskolin in quinine-treated GLUTag cells enhanced GLP-1 exocytosis. Enteroendocrine L cells in the gastrointestinal tract secrete glucagon-like peptide-1 (GLP-1), which plays an important role in glucose homeostasis. Here we investigated the effect of bitter tastant quinine on GLP-1 secretion using clonal GLUTag mouse enteroendocrine L cells. We found that GLUTag cells expressed putative quinine receptors at mRNA levels. Although application of quinine resulted in an increase of intracellular Ca{sup 2+} levels, which was mediated by Ca{sup 2+} release from the endoplasmic reticulum and Ca{sup 2+} influx through voltage-sensitive Ca{sup 2+} channels, quinine had little effect on GLP-1 secretion. Total internal reflection fluorescence microscopy and immunocytochemistry revealed that GLP-1-containing vesicles remained unfused with the plasma membrane and facilitated actin polymerization beneath the plasma membrane after application of quinine, respectively. Interestingly, application of forskolin together with quinine induced GLP-1 exocytosis from the cells. These results suggest that quinine does not induce GLP-1 secretion because it facilitates Ca{sup 2+} increase and actin reorganization but not cAMP increase, and both Ca{sup 2+} and cAMP are essential for GLP-1 secretion.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Caspase-1 regulates cellular trafficking via cleavage of the Rab7 adaptor protein RILP

Highlights: • Viral infection causes cleavage of the trafficking adapter RILP. • RILP cleavage promotes secretion by re-localizing Rab7 vesicles to the cell periphery. • RILP is cleaved by caspase 1 and this prevents its binding to dynein. • Both RILP cleavage and phosphorylation are required for trafficking effects. • RILP is a mechanism of inflammation-induced trafficking changes. Intracellular trafficking is a tightly regulated cellular process, mediated in part by Rab GTPases and their corresponding effector proteins. Viruses have evolved mechanisms to hijack these processes to promote their lifecycles. Here we describe a mechanism by which cleavage of the Rab7 adaptor protein, RILP (Rab interacting lysosomal protein) is induced by viral infection. We report that RILP is directly cleaved by caspase-1 and we have identified a novel caspase-1 recognition site at aspartic acid 75 within the RILP sequence. Alanine substitution at D75 blocks caspase-1-mediated RILP cleavage. Full-length RILP localizes in a tight vesicular structure near the perinuclear region while the cleaved form of RILP re-distributes throughout the cytoplasm. However, cleavage alone was insufficient to re-localize RILP to the cellular periphery and re-localization required specific phosphorylation events near the caspase-1 recognition site. The combination of cleavage and phosphorylation were both needed for release from the dynein component p150{sup Glued} and redistribution of CD63{sup +ve} intracellular vesicles.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The effect of cyclic tensile force on the actin cytoskeleton organization and morphology of human periodontal ligament cells

Highlights: • Tension force upregulates F-actin, Girdin and Akt expression in the PDL. • The actin filament was changed to be regular lines under tension stimulation. • Girdin are required for PDL cell to response to mechanical stimulation. • Tension force upregulates lysosomes and matrix vesicles in Girdin-depleted PDL. To explore Girdin/Akt pathway protein expression and morphology change by cyclic tension in the periodontal ligament cells. Human periodontal ligament cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h though a four-point bending system. Cyclic tension force upregulated F-actin, Girdin and Akt expression in hPDL. In transmission electron microscope assay showed that there are more and bigger mitochondria, more and longer cynapses, more cellular organisms after tension force stimulation than control. The actin filament was changed to be regular lines and pointed to poles of cells. However, we found that the Girdin-depleted cells are small and there are more micro-organisms including more lysosomes and matrix vesicles than control. These finding suggest that the STAT3/Girdin/Akt pathway in PDL to response to mechanical stimulation as well, and Girdin may play a significant role in triggering cell proliferation and migration during orthodontic treatment. It provided an insight into the molecular basis for development of a vitro cell model in studying orthodontic treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Phosphatidylserine Asymmetry Promotes the Membrane Insertion of a Transmembrane Helix

The plasma membrane (PM) contains an asymmetric distribution of lipids between the inner and outer bilayer leaflets. A lipid of special interest in eukaryotic membranes is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM, but PS redistributes to the outer leaflet when the cell is damaged or at the onset of apoptosis. Yet, the influence of PS asymmetry on membrane protein structure and folding are poorly understood. The pH low insertion peptide (pHLIP) adsorbs to the membrane surface at a neutral pH, but it inserts into the membrane at an acidic pH. We have previously observed that in symmetric vesicles, PS affects the membrane insertion of pHLIP by lowering the pH midpoint of insertion. In this work, we researched the effect of PS asymmetry on the membrane interaction of pHLIP. We developed a modified protocol to create asymmetric vesicles containing PS and employed Annexin V labeled with an Alexa Fluor 568 fluorophore as a new probe to quantify PS asymmetry. We observed that the membrane insertion of pHLIP was promoted by the asymmetric distribution of negatively charged PS, which causes a surface charge difference between bilayer leaflets. Our results indicate that lipid asymmetry can modulate the formation of an α-helix on the membrane. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important aspects of protein-membrane interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Nanostructural and rheological transitions of pH-responsive supramolecular systems involving a zwitterionic amphiphile and a triamine

Supramolecular aqueous complexes of a synthesized zwitterionic surfactant, 2-(dimethyl(octadecyl)ammonio)acetate (stearyl betaine) and diethylenetriamine, have been found to display pH-tunable rheological properties without the need for a co-surfactant. For this supramolecular system, the dynamically changing interplay among the constituents is responsible for the rich phase behavior including hollow nanotubules, bilayers, and vesicles. A further hypothesis is that hollow nanotubular structure is highly desirable for achieving chain entanglement geometries with reduced mesh size. The rheological properties of the suspension were measured using a rotational rheometer. An analysis using zero-shear viscosities at various temperatures as well as differential scanning calorimetry(DSC) helped shed light on thermodynamic and phase transitions taking place. The morphology of the suspension was analyzed by using a combination of small angle x-ray scattering(SAXS) and atomic force microscopy(AFM). Large steady-shear viscosities (up to ~160 Pa.s) and viscoelastic behavior was observed in acidic conditions which diminished upon increasing the pH. A novel analysis consisting of measured thermodynamic quantities such as activation energy(E a ) and enthalpy of transition(ΔH T ) further allude to the rich pH-sensitive nanoarchitecture of the system. Rather than commonly observed wormlike micelles, hollow nanotubules, as long as 1.6 µm were observed at pH 4 which disintegrated to bilayer sheets and vesicles upon increasing pH. Finally, gaining a comprehensive understanding from all the results, a transition mechanism between different suspension architectures was proposed.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2014

Prostate Stereotactic Ablative Radiation Therapy Using Volumetric Modulated Arc Therapy to Dominant Intraprostatic Lesions

Purpose: To investigate boosting dominant intraprostatic lesions (DILs) in the context of stereotactic ablative radiation therapy (SABR) and to examine the impact on tumor control probability (TCP) and normal tissue complication probability (NTCP). Methods and Materials: Ten prostate datasets were selected. DILs were defined using T2-weighted, dynamic contrast-enhanced and diffusion-weighted magnetic resonance imaging. Four plans were produced for each dataset: (1) no boost to DILs; (2) boost to DILs, no seminal vesicles in prescription; (3) boost to DILs, proximal seminal vesicles (proxSV) prescribed intermediate dose; and (4) boost to DILs, proxSV prescribed higher dose. The prostate planning target volume (PTV) prescription was 42.7 Gy in 7 fractions. DILs were initially prescribed 115% of the PTV{sub Prostate} prescription, and PTV{sub DIL} prescriptions were increased in 5% increments until organ-at-risk constraints were reached. TCP and NTCP calculations used the LQ-Poisson Marsden, and Lyman-Kutcher-Burman models respectively. Results: When treating the prostate alone, the median PTV{sub DIL} prescription was 125% (range: 110%-140%) of the PTV{sub Prostate} prescription. Median PTV{sub DIL} D50% was 55.1 Gy (range: 49.6-62.6 Gy). The same PTV{sub DIL} prescriptions and similar PTV{sub DIL} median doses were possible when including the proxSV within the prescription. TCP depended on prostate α/β ratio and was highest with an α/β ratio = 1.5 Gy, where the additional TCP benefit of DIL boosting was least. Rectal NTCP increased with DIL boosting and was considered unacceptably high in 5 cases, which, when replanned with an emphasis on reducing maximum dose to 0.5 cm{sup 3} of rectum (Dmax{sub 0.5cc}), as well as meeting existing constraints, resulted in considerable rectal NTCP reductions. Conclusions: Boosting DILs in the context of SABR is technically feasible but should be approached with caution. If this therapy is adopted, strict rectal constraints are required including Dmax{sub 0.5cc}. If the α/β ratio of prostate cancer is 1.5 Gy or less, then high TCP and low NTCP can be achieved by prescribing SABR to the whole prostate, without the need for DIL boosting.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2022

Soft X-ray tomography to map and quantify organelle interactions at the mesoscale

Inter-organelle interactions are a vital part of normal cellular function; however, these have proven difficult to quantify due to the range of scales encountered in cell biology and the throughput limitations of traditional imaging approaches. Here, we demonstrate that soft X-ray tomography (SXT) can be used to rapidly map ultrastructural reorganization and inter-organelle interactions in intact cells. SXT takes advantage of the naturally occurring, differential X-ray absorption of the carbon-rich compounds in each organelle. Specifically, we use SXT to map the spatiotemporal evolution of insulin vesicles and their co-localization and interaction with mitochondria in pancreatic β cells during insulin secretion and in response to different stimuli. We quantify changes in the morphology, biochemical composition, and relative position of mitochondria and insulin vesicles. These findings highlight the importance of a comprehensive and unbiased mapping at the mesoscale to characterize cell reorganization that would be difficult to detect with other existing methodologies.

3D cell mapping↗
DOE OSTI2016

Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy

Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombrero Vesicles – EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses. - Highlights: • Application of EM to understand the complex secretory pathway in human eosinophils. • EM techniques reveal an active vesicular system associated with secretory granules. • Tubular vesicles are involved in the transport of granule-derived immune mediators.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Cytochrome b561, copper, β-cleaved amyloid precursor protein and niemann-pick C1 protein are involved in ascorbate-induced release and membrane penetration of heparan sulfate from endosomal S-nitrosylated glypican-1

Ascorbate-induced release of heparan sulfate from S-nitrosylated heparan sulfate proteoglycan glypican-1 takes place in endosomes. Heparan sulfate penetrates the membrane and is transported to the nucleus. This process is dependent on copper and on expression and processing of the amyloid precursor protein. It remains unclear how exogenously supplied ascorbate can generate HS-anMan in endosomes and how passage through the membrane is facilitated. Here we have examined wild-type, Alzheimer Tg2576 and amyloid precursor protein (-/-) mouse fibroblasts and human fetal and Niemann-Pick C1 fibroblasts by using deconvolution immunofluorescence microscopy, siRNA technology and [S{sup 35}]sulfate-labeling, vesicle isolation and gel chromatography. We found that ascorbate-induced release of heparan sulfate was dependent on expression of endosomal cytochrome b561. Formation and nuclear transport of heparan sulfate was suppressed by inhibition of β-processing of the amyloid precursor protein and formation was restored by copper (I) ions. Membrane penetration was not dependent on amyloid beta channel formation. Inhibition of endosomal exit resulted in accumulation of heparan sulfate in vesicles that exposed the C-terminal of the amyloid precursor protein externally. Endosome-to-nucleus transport was also dependent on expression of the Niemann-Pick C1 protein. We propose that ascorbate is taken up from the medium and is oxidized by cytochrome b561 which, in turn, reduces copper (II) to copper (I) present in the N-terminal, β-cleaved domain of the amyloid precursor protein. Re-oxidation of copper (I) is coupled to reductive, deaminative release of heparan sulfate from glypican-1. Passage through the membrane may be facilitated by the C-terminal, β-cleaved fragment of the amyloid precursor protein and the Niemann-Pick C1 protein. - Highlights: • Cytochrome b561 is a mediator of ascorbate-induced release of heparan sulfate from glypican-1 in endosomes. • Beta-cleavage of the amyloid precursor protein is required for release and membrane penetration of heparan sulfate. • The Niemann-Pick C1 protein is involved in membrane penetration.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

RNA interference screening identifies clathrin-B and cofilin-1 as mediators of MT1-MMP in endometrial cancer

The matrix metalloproteinases (MMPs) are implicated in tumor invasion and metastasis. Given their multiple tumor promoting roles, MMPs are promising targets for the treatment of metastatic cancer. Using a siRNA library screen of 140 membrane trafficking genes, we identified 41 genes in HEC-1B and 36 in Ishikawa cancer cells that decreased metalloproteinases activity. The 16 genes common in both cancer cell lines that decreased MMPs activity are involved in cargo sorting, vesicle formation and vesicle recycling. The top two genes clathrin-B and cofilin-1 were chosen for post hoc functional studies. Higher expression of both genes was confirmed in cancer cells and knockdown with respective siRNAs inhibited their invasive potential and matrix metalloproteinases activity. Membrane Type 1- Matrix Metalloproteinase (MT1-MMP) is a master switch proteinase and regulator of invasion and metastasis. A marked decrease in MT1-MMP expression and activity was seen in clathrin-B and cofilin-1 knockdown cancer cells which was associated with a marked decreased expression of invadopodia formation proteins. Our results suggest that the decreased expression of clathrin-B and cofilin-1 decreases the expression of MT1-MMP and results in attenuation of MT1-MMP at the cell surface, thus inhibiting tumor cell invasion and metastasis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Nucleoporin35 is a novel microtubule associated protein functioning in oocyte meiotic spindle architecture

Highlights: • Nup35 concentrated on the spindle during mouse meiotic maturation progression. • Nup35 existed as a putatively phosphorylated form after resumption of meiosis. • Nup35 knockdown significantly compromised the extrusion of first polar body, but not germinal vesicle breakdown. • Nup35 knockdown dissociated some localization signal of p-ERK1/2 from spindle poles to cytoplasm. • Nup35 knockdown weakened kinetochore - microtubule attachment, then normally activated the spindle assembly checkpoint. Nucleoporins (Nups) are a large and diverse family of proteins that mediate nucleocytoplasmic transport at interphase of vertebrate cells. Nups also function in mitosis progression. However, whether Nups are involved in oocyte meiosis progression is still rarely known. In this study, we delineated the roles and regulatory mechanisms of Nucleoporin35 (Nup35) during oocyte meiotic maturation. The immunofluorescent signal of Nup35 was localized in the nuclear membrane at germinal vesicle (GV) stage, the microtubules and spindle at pro-metaphase I (pro-MI), metaphase I (MI), and metaphase II (MII), but to the spindle poles at anaphase I (AI) and telophase I (TI). The dynamic localization pattern of Nup35 during oocyte meiotic maturation implied its specific roles. We also found that Nup35 existed as a putatively phosphorylated form after resumption of meiosis (GVBD), but not at GV stage, implying its functional switch from nuclear membrane to meiotic progression. Further study uncovered that knockdown of Nup35 by specific siRNA significantly compromised the extrusion of first polar body (PBE), but not GVBD, with defects of spindle assembly and chromosome alignment and dissociated some localization signal of p-ERK1/2 from spindle poles to cytoplasm. A defective kinetochore - microtubule attachment (K-MT) was also identified in oocytes after knockdown of Nup35, which activates spindle assembly checkpoint. In conclusion, our results suggest that Nup35 is putatively phosphorylated and released to the cytoplasm after resumption of meiosis, and regulates spindle assembly and chromosome alignment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Antineoplastic activity of Salmonella Typhimurium outer membrane nanovesicles

Highlights: • ST-OMVs is a promising antitumor monotherapy or as an adjuvant to chemotherapies. • ST-OMVs downregulated Ki-67 and upregulated CD49b immune-expression. • ST-OMVs downregulated angiogenesis by inhibiting VEGF gene expression. • ST-OMVs increased tumor cells apoptosis and autophagy (increased caspase-3and Beclin1). Nano-sized Gram-negative bacterial outer membrane vesicles possess unique structural and immunostimulatory effects that could be exploited to regress tumors by alerting the host immune system and reversing the immunosuppressive tumor microenvironment. The current study was conducted to investigate the antitumor activity of the outer membrane vesicles (ST-OMVs) of Salmonella Typhimurium ATCC 14028, in vitro in human colorectal carcinoma (HTC116), breast cancer (MCF-7), and hepatocellular carcinoma (HepG2) cell lines and in vivo in Ehrlich solid carcinoma-bearing mice model either as a mono-immunotherapy or as an adjuvant to a commonly used conventional chemotherapy. In addition, we investigated the safety of ST-OMVs. Adult Swiss albino female mice with transplanted Ehrlich solid carcinoma were treated with either ST-OMVs, paclitaxel or a combination of both. Tumor volume, growth inhibition rate, quantitative RT-PCR of Bax and VEGF genes expression, histopathology and immune-expression of caspase-3, Beclin-1, CD49b and Ki-67 were all analyzed. Our results showed that ST-OMVs significantly decreased tumor volume, significantly increased tumor growth inhibition rate, up-regulated the immunohistochemical expression of caspase-3, Beclin-1, and CD49b (enhanced recruitment of NK cells). Furthermore, ST-OMVs down-regulated the expression of Ki-67, increased Bax gene expression and decreased VEGF gene expression as detected by qRT-PCR analysis. Histologically, ST-OMVs promoted apoptosis, decreased tumor invasion and mitotic activities. Moreover, ST-OMVs showed a remarkable cytotoxic activity in various investigated in vitro cancer cell lines. Our findings demonstrate potential antitumor activity of ST-OMVs that might be used as a promising safe antitumor immunotherapy or an adjuvant to conventional chemotherapeutic drugs, resolving some of their problems.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Identification of protein/mRNA network involving the PSORS1 locus gene CCHCR1 and the PSORS4 locus gene HAX1

Highlights: • CCHCR1 and HAX1 proteins directly interact. • CCHCR1 is a psoriasis candidate gene located on PSORS1 locus. • HAX1 gene is located on PSORS4 locus and is overexpressed in psoriasis. • CCHCR1 and HAX1 co-localize with and affect specific mRNAs in vesicles. • Novel protein/mRNA networks involving CCHCR1 and HAX1 could play a role in psoriasis. CCHCR1 (Coiled-Coil alpha-Helical Rod 1), maps to chromosomal region 6p21.3, within the major psoriasis susceptibility locus PSORS1. CCHCR1 itself is a plausible psoriasis candidate gene, however its role in psoriasis pathogenesis remains unclear. We previously demonstrated that CCHCR1 protein acts as a cytoplasmic docking site for RNA polymerase II core subunit 3 (RPB3) in cycling cells, suggesting a role for CCHCR1 in vesicular trafficking between cellular compartments. Here, we report a novel interaction between CCHCR1 and the RNA binding protein HAX1. HAX1 maps to chromosomal region 1q21.3 within the PSORS4 locus and is over-expressed in psoriasis. Both CCHCR1 and HAX1 share subcellular co-localization with mitochondria, nuclei and cytoplasmic vesicles as P-bodies. By a series of ribonucleoprotein immunoprecipitation (RIP) assays, we isolated a pool of mRNAs complexed with HAX1 and/or CCHCR1 proteins. Among the mRNAs complexed with both CCHCR1 and HAX1 proteins, there are Vimentin mRNA, previously described to be bound by HAX1, and CAMP/LL37 mRNA, whose gene product is over-expressed in psoriasis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Checkpoint kinases are required for oocyte meiotic progression by the maintenance of normal spindle structure and chromosome condensation

Highlights: • Inhibition of Chk1/2 has no significant effects on germinal vesicle breakdown. • Chk1/2 inhibition results in the first polar body extrusion defects. • Chk1/2 is critically involved in meiotic spindle organization. • Inhibition of Chk1/2 leads to abnormal chromosome condensation. • Chk1/2 is important for the development of MII stage oocytes. Checkpoint kinases (Chk) 1/2 are known for DNA damage checkpoint and cell cycle control in somatic cells. According to recent findings, the involvement of Chk1 in oocyte meiotic resumption and Chk2 is regarded as an essential regulator for progression at the post metaphase I stage (MI). In this study, AZD7762 (Chk1/2 inhibitor) and SB218078 (Chk1 inhibitor) were used to uncover the joint roles of Chk1/2 and differentiate the importance of Chk1 and Chk2 during oocyte meiotic maturation. Inhibition of Chk1/2 or Chk1 alone had no significant effect on germinal vesicle breakdown (GVBD) but significantly inhibited the first polar body (PB1). Interestingly, inhibition of Chk1 alone could not increase or completely block the extrusion of PB1 like Chk1/2 inhibition. Also, Chk1/2 inhibition resulted in defective meiotic spindle organization and chromosome condensation both in MI and metaphase II (MII) stages of oocytes. The location of γ-tubulin and Securin were abnormal or missing, while P38 MAPK was activated by Chk1/2 inhibition. Meanwhile, Chk1/2 inhibition reduced the percentage of the second polar body extrusion and pronuclear formation. In conclusion, our results further understand the functions and regulatory mechanism of Chk1/2 during oocyte meiotic maturation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Differential Membrane Binding Mechanics of Synaptotagmin Isoforms Observed in Atomic Detail

Synaptotagmin (Syt) is a membrane-associated protein involved in vesicle fusion through the SNARE complex that is found throughout the human body in 17 different isoforms. These isoforms have two membrane-binding C2 domains, which sense Ca 2+ and thereby promote anionic membrane binding and lead to vesicle fusion. Through molecular dynamics simulations using the HMMM acclerated bilayer model, we have investigated how small protein sequence changes in the Ca 2+ binding loops of the C2 domains may give rise to the experimentally determined difference in binding kinetics between Syt-1 and Syt-7 isoforms. Syt-7 C2 domains are found to form more close contacts with anionic phospholipid head groups, particularly in Loop 1, where an additional positive charge in Syt-7 draws the loop closer to the membrane and causes the anchoring residue F167 to insert deeper into the bilayer than the corresponding methionine in Syt-1 (M173). By carrying out additional replica exchange umbrella sampling calculations, we demonstrate that these additional contacts increase the energetic cost of unbinding the Syt-7 C2 domains from the bilayer, causing them to unbind more slowly than their counterparts in Syt-1.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

A Computational Approach for Modeling Neutron Scattering Data from Lipid Bilayers

Biological cell membranes are responsible for a range of structural and dynamical phenomena crucial to a cell's well-being and its associated functions. Due to the complexity of cell membranes, lipid bilayer systems are often used as biomimetic models. These systems have led to signficant insights into vital membrane phenomena such as domain formation, passive permeation and protein insertion. Experimental observations of membrane structure and dynamics are, however, limited in resolution, both spatially and temporally. Importantly, computer simulations are starting to play a more prominent role in interpreting experimental results, enabling a molecular under- standing of lipid membranes. Particularly, the synergy between scattering experiments and simulations offers opportunities for new discoveries in membrane physics, as the length and time scales probed by molecular dynamics (MD) simulations parallel those of experiments. We also describe a coarse-grained MD simulation approach that mimics neutron scattering data from large unilamellar lipid vesicles over a range of bilayer rigidity. Specfically, we simulate vesicle form factors and membrane thickness fluctuations determined from small angle neutron scattering (SANS) and neutron spin echo (NSE) experiments, respectively. Our simulations accurately reproduce trends from experiments and lay the groundwork for investigations of more complex membrane systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Effect of α-Tocopherol on the Microscopic Dynamics of Dimyristoylphosphatidylcholine Membrane

Vitamin E behaves as an antioxidant and is well known for its protective properties of the lipid membrane. The most biologically active form of vitamin E in the human organism is α-tocopherol (aToc). Recently (Marquardt, D.; et al. J. Am. Chem. Soc. 2014, 136, 203₋210) it has been shown that aToc resides near the center of dimyristoylphosphatidylcholine (DMPC) bilayer, which is in stark contrast with other PC membranes, where aToc is located near the lipid₋water interface. Here we report an unusual effect of this exceptional location of aToc on the dynamical behavior of DMPC membrane probed by incoherent elastic and quasielastic neutron scattering. For pure DMPC vesicles, elastic scan data show two step-like drops in the elastic intensity at 288 and 297 K, which correspond to the pre- and main phase transitions, respectively. However, inclusion of aToc into DMPC membrane inhibits the step-like elastic intensity drops, indicating a significant impact of aToc on the phase behavior of the membrane. This observation is supported by our differential scanning calorimetry data, which shows that inclusion of aToc leads to a significant broadening of the main phase transition peak, whereas the peak corresponding to the pretransition disappears. We have performed quasielastic neutron scattering (QENS) measurements on DMPC vesicles with various concentrations of aToc at 280, 293, and 310 K. We have found that aToc affects both the lateral diffusion and the internal motions of the lipid molecules. Below the main phase transition temperature inclusion of aToc accelerates both the lateral and the internal lipid motions. On the other hand, above the main phase transition temperature the addition of aToc restricts only the internal motion, without a significant influence on the lateral motion. To conclude, our results support the finding that the location of aToc in DMPC membrane is deep within the bilayer.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗