scholarly journals UNC-6 (netrin) stabilizes oscillatory clustering of the UNC-40 (DCC) receptor to orient polarity

2014 ◽  
Vol 206 (5) ◽  
pp. 619-633 ◽  
Author(s):  
Zheng Wang ◽  
Lara M. Linden ◽  
Kaleb M. Naegeli ◽  
Joshua W. Ziel ◽  
Qiuyi Chi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Membrane ◽  
Cell Invasion ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Time Lapse ◽  
Oscillatory Behavior ◽  
Deleted In Colorectal Cancer ◽  
Family Protein ◽  
Anchor Cell

The receptor deleted in colorectal cancer (DCC) directs dynamic polarizing activities in animals toward its extracellular ligand netrin. How DCC polarizes toward netrin is poorly understood. By performing live-cell imaging of the DCC orthologue UNC-40 during anchor cell invasion in Caenorhabditis elegans, we have found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin). Time-lapse analyses revealed that UNC-40 clusters assemble, disassemble, and reform at periodic intervals in different regions of the cell membrane. This oscillatory behavior indicates that UNC-40 clusters through a mechanism involving interlinked positive (formation) and negative (disassembly) feedback. We show that endogenous UNC-6 and ectopically provided UNC-6 orient and stabilize UNC-40 clustering. Furthermore, the UNC-40–binding protein MADD-2 (a TRIM family protein) promotes ligand-independent clustering and robust UNC-40 polarization toward UNC-6. Together, our data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering. We propose that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.

scholarly journals ADF/cofilin promotes invadopodial membrane recycling during cell invasion in vivo

2014 ◽  
Vol 204 (7) ◽  
pp. 1209-1218 ◽  
Author(s):  
Elliott J. Hagedorn ◽  
Laura C. Kelley ◽  
Kaleb M. Naegeli ◽  
Zheng Wang ◽  
Qiuyi Chi ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Cell Invasion ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Actin Dynamics ◽  
Membrane Structures ◽  
Tumor Dissemination ◽  
Anchor Cell ◽  
Dynamic Structures

Invadopodia are protrusive, F-actin–driven membrane structures that are thought to mediate basement membrane transmigration during development and tumor dissemination. An understanding of the mechanisms regulating invadopodia has been hindered by the difficulty of examining these dynamic structures in native environments. Using an RNAi screen and live-cell imaging of anchor cell (AC) invasion in Caenorhabditis elegans, we have identified UNC-60A (ADF/cofilin) as an essential regulator of invadopodia. UNC-60A localizes to AC invadopodia, and its loss resulted in a dramatic slowing of F-actin dynamics and an inability to breach basement membrane. Optical highlighting indicated that UNC-60A disassembles actin filaments at invadopodia. Surprisingly, loss of unc-60a led to the accumulation of invadopodial membrane and associated components within the endolysosomal compartment. Photobleaching experiments revealed that during normal invasion the invadopodial membrane undergoes rapid recycling through the endolysosome. Together, these results identify the invadopodial membrane as a specialized compartment whose recycling to form dynamic, functional invadopodia is dependent on localized F-actin disassembly by ADF/cofilin.

scholarly journals Time-Lapse, Live-Cell Imaging of Potassium Efflux in Cell Exposed to Nanosecond Pulsed Electric Fields

2021 ◽  
Vol 120 (3) ◽  
pp. 223a
Author(s):  
Flavia Mazzarda ◽  
Esin B. Sozer ◽  
Julia L. Pittaluga ◽  
Claudia Muratori ◽  
P. Thomas Vernier
Keyword(s):  
Electric Fields ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Pulsed Electric Fields ◽  
Time Lapse ◽  
Live Cell ◽  
Potassium Efflux ◽  
Nanosecond Pulsed Electric Fields

Dye selection for live cell imaging of intact siRNA

2012 ◽  
Vol 393 (1-2) ◽  
pp. 23-35 ◽  
Author(s):  
Markus Hirsch ◽  
Dennis Strand ◽  
Mark Helm
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Lapse ◽  
Live Cell ◽  
High Yield ◽  
Ph Variation ◽  
Rnai Efficiency

Abstract Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.

scholarly journals Time-lapse live cell imaging to monitor doxorubicin release from DNA origami nanostructures

2018 ◽  
Vol 6 (11) ◽  
pp. 1605-1612 ◽  
Author(s):  
Yun Zeng ◽  
Jiajun Liu ◽  
Shuo Yang ◽  
Wenyan Liu ◽  
Liang Xu ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Time Lapse ◽  
Dna Origami ◽  
Live Cell ◽  
Doxorubicin Release

DNA origami nanostructures can serve as a promising carrier for drug delivery due to the outstanding programmability and biocompatibility.

scholarly journals Intracellular mechanisms of fungal space searching in microenvironments

2019 ◽  
Vol 116 (27) ◽  
pp. 13543-13552 ◽  
Author(s):  
Marie Held ◽  
Ondřej Kašpar ◽  
Clive Edwards ◽  
Dan V. Nicolau
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Turgor Pressure ◽  
Fungal Growth ◽  
Time Lapse ◽  
Growth Direction ◽  
Medical Applications ◽  
Initial Growth ◽  
Space Partitioning ◽  
Constrained Environments

Filamentous fungi that colonize microenvironments, such as animal or plant tissue or soil, must find optimal paths through their habitat, but the biological basis for negotiating growth in constrained environments is unknown. We used time-lapse live-cell imaging of Neurospora crassa in microfluidic environments to show how constraining geometries determine the intracellular processes responsible for fungal growth. We found that, if a hypha made contact with obstacles at acute angles, the Spitzenkörper (an assembly of vesicles) moved from the center of the apical dome closer to the obstacle, thus functioning as an internal gyroscope, which preserved the information regarding the initial growth direction. Additionally, the off-axis trajectory of the Spitzenkörper was tracked by microtubules exhibiting “cutting corner” patterns. By contrast, if a hypha made contact with an obstacle at near-orthogonal incidence, the directional memory was lost, due to the temporary collapse of the Spitzenkörper–microtubule system, followed by the formation of two “daughter” hyphae growing in opposite directions along the contour of the obstacle. Finally, a hypha passing a lateral opening in constraining channels continued to grow unperturbed, but a daughter hypha gradually branched into the opening and formed its own Spitzenkörper–microtubule system. These observations suggest that the Spitzenkörper–microtubule system is responsible for efficient space partitioning in microenvironments, but, in its absence during constraint-induced apical splitting and lateral branching, the directional memory is lost, and growth is driven solely by the isotropic turgor pressure. These results further our understanding of fungal growth in microenvironments relevant to environmental, industrial, and medical applications.

scholarly journals Live cell imaging of macrophage/bacterium interaction demonstrates cell lysis induced by Corynebacterium diphtheriae and Corynebacterium ulcerans

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Dulanthi Weerasekera ◽  
Jonas Hahn ◽  
Martin Herrmann ◽  
Andreas Burkovski
Keyword(s):  
Fluorescence Microscopy ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Time Lapse ◽  
Corynebacterium Diphtheriae ◽  
Live Cell ◽  
Human Macrophage ◽  
Data Description ◽  
Corynebacterium Ulcerans ◽  
Microscopy Data

Abstract Objectives In frame of a study to characterize the interaction of human macrophage-like cells with pathogenic corynebacteria, Corynebacterium diphtheriae and Corynebacterium ulcerans, live cell imaging experiments were carried out and time lapse fluorescence microscopy videos were generated, which are presented here. Data description The time lapse fluorescence microscopy data revealed new insights in the interaction of corynebacteria with human macrophage-like THP-1 cells. In contrast to uninfected cells and infections with non-pathogenic C. glutamicum used as a control, pathogenic C. diphtheriae and C. ulcerans showed highly detrimental effects towards human cells and induction of cell death of macrophages.

scholarly journals Keeping Life in Focus New Systems Prevent Z-axis Drift in Time Lapse Studies

2006 ◽  
Vol 14 (4) ◽  
pp. 42-46 ◽  
Author(s):  
Edward Lachica
Keyword(s):  
Fluorescent Probes ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Time Lapse ◽  
Living Cells ◽  
Dynamic Processes ◽  
Biological Structure ◽  
Camera Lucida ◽  
Developmental Anatomy ◽  
Organ Level

Just two decades ago, life scientists studied biological structure, developmental anatomy and intracellular processes by describing individual snapshots of kinetic events. Today, with so much bioscience research focusing on dynamic processes that occur on the molecular, cellular and whole organ level, it is important to record events as they happen, over seconds, minutes or hours, in living cells. Photographs and camera lucida drawings of fixed, stained cells have given way to live cell imaging using fluorescent probes, warming trays to promote cell viability and cinemicrography as a method of recording events.

scholarly journals Ionomycin-induced calcium influx induces neurite degeneration in mouse neuroblastoma cells: analysis of a time-lapse live cell imaging system

2016 ◽  
Vol 50 (11) ◽  
pp. 1214-1225 ◽  
Author(s):  
Saki Nakamura ◽  
Ayumi Nakanishi ◽  
Minami Takazawa ◽  
Shunsuke Okihiro ◽  
Shiro Urano ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging System ◽  
Calcium Influx ◽  
Neuroblastoma Cells ◽  
Time Lapse ◽  
Live Cell ◽  
Mouse Neuroblastoma ◽  
Neurite Degeneration

scholarly journals PKCε Phosphorylates and Mediates the Cell Membrane Localization of RhoA

ISRN Oncology ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-9
Author(s):  
Tizhi Su ◽  
Samuel Straight ◽  
Liwei Bao ◽  
Xiujie Xie ◽  
Caryn L. Lehner ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Cell Membrane ◽  
Cell Invasion ◽  
Time Course ◽  
Time Lapse ◽  
Kinase C ◽  
Phosphorylation Event ◽  
Phosphopeptide Mapping ◽  
First Time ◽  
Initial Peak

Protein kinase Cε (PKCε) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKCε and RhoA was defined. Phosphopeptide mapping revealed that PKCε phosphorylates RhoA at T127 and S188. Recombinant PKCε bound to recombinant RhoA in the absence of ATP indicating that the association between PKCε and RhoA does not require an active ATP-docked PKCε conformation. Activation of PKCε resulted in a dramatic coordinated translocation of PKCε and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKCε and RhoA is a biphasic event, an initial peak at the cytoplasm and a gradual prolonged increase at the cell membrane for the entire time-course (12.5 minutes). These results suggest that the PKCε-RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKCε is able to recruit RhoA to the cell membrane indicating that the association between PKCε and RhoA is proximal to the active catalytic site and perhaps independent of a PKCε-RhoA phosphorylation event. This work demonstrates, for the first time, that PKCε phosphorylates and modulates the cell membrane translocation of RhoA.

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