scholarly journals FIB/SEM-based analysis of Borrelia intracellular processing by human macrophages

2020 ◽  
pp. jcs.252320
Author(s):  
Matthias Klose ◽  
Maximilian Scheungrab ◽  
Manja Luckner ◽  
Gerhard Wanner ◽  
Stefan Linder
Keyword(s):  
Live Cell Imaging ◽  
Focused Ion Beam ◽  
Cell Imaging ◽  
Ion Beam ◽  
Human Macrophages ◽  
Host Cytoplasm ◽  
Intracellular Processing ◽  
Membrane Tubulation ◽  
First Time

Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting primarily skin, joints and nervous system. Successful internalization and intracellular processing of borreliae by immune cells like macrophages is decisive for the outcome of a respective infection. Here, we use for the first time focused ion beam scanning electron microscopy tomography (FIB/SEM tomography) to visualize the interaction of borreliae with primary human macrophages with high resolution. We report that interaction between macrophages and the elongated and highly motile borreliae can lead to formation of membrane tunnels that extend deeper into the host cytoplasm than the actual phagosome, most probably as a result of partial extrication of captured borreliae. We also show that membrane tubulation at borreliae-containing phagosomes, a process suggested earlier as a mechanism leading to phagosome compaction, but hard to visualize in live cell imaging, is apparently a frequent phenomenon. Finally, we demonstrate that the endoplasmic reticulum (ER) forms multiple STIM1-positive contact sites with both membrane tunnels and phagosome tubulations, confirming the important role of the ER during uptake and intracellular processing of borreliae.

An Study of Influence of Imperfections on the Delamination of Diamond-Like Carbon Films

2002 ◽  
Vol 719 ◽  
Author(s):  
Myoung-Woon Moon ◽  
Kyang-Ryel Lee ◽  
Jin-Won Chung ◽  
Kyu Hwan Oh
Keyword(s):  
Cross Sections ◽  
Focused Ion Beam ◽  
Imaging System ◽  
Ion Beam ◽  
Carbon Films ◽  
Diamond Like Carbon ◽  
Glass Substrates ◽  
Atomic Force ◽  
Initiation And Propagation

AbstractThe role of imperfections on the initiation and propagation of interface delaminations in compressed thin films has been analyzed using experiments with diamond-like carbon (DLC) films deposited onto glass substrates. The surface topologies and interface separations have been characterized by using the Atomic Force Microscope (AFM) and the Focused Ion Beam (FIB) imaging system. The lengths and amplitudes of numerous imperfections have been measured by AFM and the interface separations characterized on cross sections made with the FIB. Chemical analysis of several sites, performed using Auger Electron Spectroscopy (AES), has revealed the origin of the imperfections. The incidence of buckles has been correlated with the imperfection length.

scholarly journals Live-Cell Imaging of Caspase Activation for High-Content Screening

2009 ◽  
Vol 14 (8) ◽  
pp. 956-969 ◽  
Author(s):  
Christophe Antczak ◽  
Toshimitsu Takagi ◽  
Christina N. Ramirez ◽  
Constantin Radu ◽  
Hakim Djaballah
Keyword(s):  
Cell Death ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Exposure Period ◽  
Live Cells ◽  
Assay Method ◽  
Caspase Activation ◽  
Fluorogenic Substrate ◽  
Automated Imaging ◽  
First Time

Caspases are central to the execution of programmed cell death, and their activation constitutes the biochemical hallmark of apoptosis. In this article, the authors report the successful adaptation of a high-content assay method using the DEVDNucView488™ fluorogenic substrate, and for the first time, they show caspase activation in live cells induced by either drugs or siRNA. The fluorogenic substrate was found to be nontoxic over an exposure period of several days, during which the authors demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the antiapoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to doxorubicin, etoposide, or cell death siRNA. This method was further validated against 2 well-characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to erlotinib, where the authors show a differential time-dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, the results demonstrate the feasibility of using this newly adapted and validated high-content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells. ( Journal of Biomolecular Screening 2009:956-969)

TRANSPORT CHARACTERISTICS IN c-AXIS La2-xSrxCuO4 (LSCO) SINGLE CRYSTALS

2005 ◽  
Vol 19 (01n03) ◽  
pp. 447-450
Author(s):  
SANG-JAE KIM ◽  
TAKESHI HATANO
Keyword(s):  
Temperature Dependence ◽  
Single Crystals ◽  
Critical Current ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Etching Method ◽  
First Time ◽  
Small Voltage

c-axis micro-bridges of La 2-x Sr x CuO 4 ( LSCO ) single crystals were fabricated by the focused-ion-beam (FIB) etching method. Small rectangular LSCO pieces were fabricated by cutting and grinding single crystals of underdoped LSCO of x=0.09. The size of LSCO single crystals between electrodes was cut to 20×40μm2 in ab-plane by using the FIB etching method. Superconductor-insulator-superconductor (SIS) like-branch structures on I-V curves of the LSCO stacks were observed for the first time. The branch structures exhibited voltage jumps of several tens mV in the range of from 1.7 K to 5 K with temperature dependence. When the temperature is changed from 5 K to 1.7 K , the critical current and the next branch split into a few of small voltage jumps with the intervals of several mV in the range of from 0.1 mV and 2.0 mV .

scholarly journals LRRK2 mediates tubulation and vesicle sorting from membrane damaged lysosomes

2020 ◽  
Author(s):  
Luis Bonet-Ponce ◽  
Alexandra Beilina ◽  
Chad D. Williamson ◽  
Eric Lindberg ◽  
Jillian H. Kluss ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Leucine Rich Repeat ◽  
Biological Functions ◽  
New Process ◽  
Sporadic Parkinson’S Disease

ABSTRACTMutations in the leucine rich repeat kinase 2 (LRRK2) gene are a cause of familial and sporadic Parkinson’s disease (PD). Nonetheless, the biological functions of LRRK2 remain incompletely understood. Here, we observed that LRRK2 is recruited to lysosomes that have a ruptured membrane. Using unbiased proteomics, we observed that LRRK2 is able to recruit the motor adaptor protein JIP4 to permeabilized lysosomes in a kinase-dependent manner through the phosphorylation of RAB35 and RAB10. Super-resolution live cell imaging microscopy and FIB-SEM revealed that once at the lysosomal membrane, JIP4 promotes the formation of LAMP1-negative lysosomal tubules that release membranous content from ruptured lysosomes. Released vesicular structures are able to interact with other lysosomes. Thus, we described a new process that uses lysosomal tubulation to release vesicular structures from permeabilized lysosomes. LRRK2 orchestrates this process that we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2) that, given the central role of the lysosome in PD, is likely to be disease relevant.

scholarly journals Rapid Morphological and Cytoskeletal Response to Microgravity in Human Primary Macrophages

2019 ◽  
Vol 20 (10) ◽  
pp. 2402 ◽  
Author(s):  
Cora Sandra Thiel ◽  
Svantje Tauber ◽  
Beatrice Lauber ◽  
Jennifer Polzer ◽  
Christian Seebacher ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Imaging ◽  
Live Cell ◽  
Cellular Structures ◽  
Microgravity Environment ◽  
Confocal Laser ◽  
Human Macrophages ◽  
Imaging Experiment

The FLUMIAS (Fluorescence-Microscopic Analyses System for Life-Cell-Imaging in Space) confocal laser spinning disk fluorescence microscope represents a new imaging capability for live cell imaging experiments on suborbital ballistic rocket missions. During the second pioneer mission of this microscope system on the TEXUS-54 suborbital rocket flight, we developed and performed a live imaging experiment with primary human macrophages. We simultaneously imaged four different cellular structures (nucleus, cytoplasm, lysosomes, actin cytoskeleton) by using four different live cell dyes (Nuclear Violet, Calcein, LysoBrite, SiR-actin) and laser wavelengths (405, 488, 561, and 642 nm), and investigated the cellular morphology in microgravity (10−4 to 10−5 g) over a period of about six minutes compared to 1 g controls. For live imaging of the cytoskeleton during spaceflight, we combined confocal laser microscopy with the SiR-actin probe, a fluorogenic silicon-rhodamine (SiR) conjugated jasplakinolide probe that binds to F-actin and displays minimal toxicity. We determined changes in 3D cell volume and surface, nuclear volume and in the actin cytoskeleton, which responded rapidly to the microgravity environment with a significant reduction of SiR-actin fluorescence after 4–19 s microgravity, and adapted subsequently until 126–151 s microgravity. We conclude that microgravity induces geometric cellular changes and rapid response and adaptation of the potential gravity-transducing cytoskeleton in primary human macrophages.

Formation of Sub-100 NM GE Wires on Si by E-Beam Evaporation/Lithography

1995 ◽  
Vol 380 ◽  
Author(s):  
C. Deng ◽  
J. C. Wu ◽  
C. J. Barbero ◽  
T. W. Sigmon ◽  
M. N. Wybourne
Keyword(s):  
Cross Section ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Si Substrates ◽  
Novel Technique ◽  
Atomic Force ◽  
Transmission Electron ◽  
First Time ◽  
Scanning Electron

ABSTRACTA fabrication process for sub-100 nm Ge wires on Si substrates is reported for the first time. Wires with a cross section of 6 × 57 nm2 are demonstrated. The wire structures are analyzed by atomic force (AFM), scanning electron (SEM), and transmission electron microscopy (TEM). Sample preparation for TEM is performed using a novel technique using both pre and in situ deposition of multiple protection layers using a Focused Ion Beam (FIB) micromachining system.

scholarly journals EB1 binding restricts STIM1 translocation to ER–PM junctions and regulates store-operated Ca2+ entry

2018 ◽  
Vol 217 (6) ◽  
pp. 2047-2058 ◽  
Author(s):  
Chi-Lun Chang ◽  
Yu-Ju Chen ◽  
Carlo Giovanni Quintanilla ◽  
Ting-Sung Hsieh ◽  
Jen Liou
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Cellular Functions ◽  
Binding Ability ◽  
Trapping Mechanism ◽  
Spatiotemporal Regulation

The endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER–plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) after ER Ca2+ depletion. STIM1 also interacts with EB1 and dynamically tracks microtubule (MT) plus ends. Nevertheless, the role of STIM1–EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with a synthetic construct approach, we found that EB1 binding constitutes a trapping mechanism restricting STIM1 targeting to ER–PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. By trapping STIM1 molecules at dynamic contacts between the ER and MT plus ends, EB1 binding delayed STIM1 translocation to ER–PM junctions during ER Ca2+ depletion and prevented excess SOCE and ER Ca2+ overload. Our study suggests that STIM1–EB1 interaction shapes the kinetics and amplitude of local SOCE in cellular regions with growing MTs and contributes to spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.

Lipid order and molecular assemblies in the plasma membrane of eukaryotic cells

2009 ◽  
Vol 37 (5) ◽  
pp. 1056-1060 ◽  
Author(s):  
Marek Cebecauer ◽  
Dylan M. Owen ◽  
Anna Markiewicz ◽  
Anthony I. Magee
Keyword(s):  
Plasma Membrane ◽  
Physical Properties ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Eukaryotic Cells ◽  
Dynamic Nature ◽  
Membrane Components ◽  
Technological Improvements

Multimolecular assemblies on the plasma membrane exhibit dynamic nature and are often generated during the activation of eukaryotic cells. The role of lipids and their physical properties in helping to control the existence of these structures is discussed. Technological improvements for live cell imaging of membrane components are also reviewed.

scholarly journals How genes find their way inside the cell nucleus

2007 ◽  
Vol 179 (6) ◽  
pp. 1093-1094 ◽  
Author(s):  
Maria Carmo-Fonseca
Keyword(s):  
Cell Nucleus ◽  
Live Cell Imaging ◽  
Gene Locus ◽  
Recent Progress ◽  
Cell Imaging ◽  
Live Cell ◽  
Nuclear Actin ◽  
First Time

Recent progress in live cell imaging suggests a role for nuclear actin in chromatin movement. In this issue, for the first time, a gene locus moving toward a subnuclear compartment was tracked. Motion of the locus is actin dependent, raising the question of whether chromatin movements are random or directed.

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