journal
MENU ▼
Read by QxMD icon Read
search

Methods in Molecular Biology

journal
https://www.readbyqxmd.com/read/28324492/detection-of-hypoxia-induced-and-iron-depletion-induced-mitophagy-in-mammalian-cells
#1
Shun-Ichi Yamashita, Tomotake Kanki
Mitochondrial quality and quantity are not only regulated by mitochondrial fusion and fission but also by mitochondria degradation. Mitophagy, an autophagy specific for damaged or unnecessary mitochondria, is believed to be an important pathway for mitochondrial homeostasis. To date, several stimuli are known to induce mitophagy. Some of these stimuli, however, including hypoxia, iron depletion, and nitrogen starvation, induce mild mitophagy, which is difficult to detect through decreased mitochondrial mass...
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324491/monitoring-mitophagy-during-aging-in-caenorhabditis-elegans
#2
Nikolaos Charmpilas, Konstantinos Kounakis, Nektarios Tavernarakis
Mitochondria constitute the main energy-producing centers of eukaryotic cells. In addition, they are involved in several crucial cellular processes, such as lipid metabolism, calcium buffering, and apoptosis. As such, their malfunction can be detrimental for proper cellular physiology and homeostasis. Mitophagy is a mechanism that protects and maintains cellular function by sequestering harmful or dysfunctional mitochondria to lysosomes for degradation. In this report, we present experimental procedures for quantitative, in vivo monitoring of mitophagy events in the nematode Caenorhabditis elegans...
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324490/assessment-of-mitophagy-in-ips-cell-derived-neurons
#3
Kei-Ichi Ishikawa, Akihiro Yamaguchi, Hideyuki Okano, Wado Akamatsu
Aberrant mitochondrial function is associated with many neurological diseases. Mitophagy is a key mechanism for the elimination of damaged mitochondria and maintenance of mitochondrial homeostasis. Induced pluripotent stem (iPS) cell technologies developed over the last decade have allowed us to analyze functions of the human neuron. Here we describe an efficient induction method from human iPS cells to neurons, followed by an image-based mitophagy assay.
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324489/monitoring-mitochondrial-changes-by-alteration-of-the-pink1-parkin-signaling-in-drosophila
#4
Tsuyoshi Inoshita, Kahori Shiba-Fukushima, Hongrui Meng, Nobutaka Hattori, Yuzuru Imai
Mitochondrial quality control is a key process in tissues with high energy demands, such as the brain and muscles. Recent studies using Drosophila have revealed that the genes responsible for familial forms of juvenile Parkinson's disease (PD), PINK1 and Parkin regulate mitochondrial function and motility. Cell biological analysis using mammalian cultured cells suggests that the dysregulation of mitophagy by PINK1 and Parkin leads to neurodegeneration in PD. In this chapter, we describe the methods to monitor mitochondrial morphology in the indirect flight muscles of adult Drosophila and Drosophila primary cultured neurons and the methods to analyze the motility of mitochondria in the axonal transport of living larval motor neurons...
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324488/flow-cytometer-monitoring-of-bnip3-and-bnip3l-nix-dependent-mitophagy
#5
Matilda Šprung, Ivan Dikic, Ivana Novak
Mitochondria are organelles with numerous vital roles in cellular metabolism. Impaired or damaged mitochondria are degraded in autophagolysosomes in a process known as mitophagy. Given the fundamental role of mitophagy in maintenance of cellular homeostasis, methods and techniques with which to study this process are constantly evolving and emerging. So far, mitophagy flux was mostly monitored using fluorescently labeled LC3 protein on autophagosomal membrane and any of the labeled outer mitochondrial membrane proteins...
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324487/mitophagy-in-yeast-a-screen-of-mitophagy-deficient-mutants
#6
Kentaro Furukawa, Tomotake Kanki
Mitochondrial autophagy (mitophagy) is a process that selectively degrades mitochondria via autophagy. Recent studies have shown that mitophagy plays an important role in mitochondrial homeostasis by degrading damaged or excess mitochondria. The budding yeast Saccharomyces cerevisiae is a powerful model organism that has been employed to study several biological phenomena. Recently, there has been significant progress in the understanding of mitophagy in yeast following the identification of Atg32, a mitochondrial outer membrane receptor protein for mitophagy...
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324486/mitopho8%C3%AE-60-assay-as-a-tool-to-quantitatively-measure-mitophagy-activity
#7
Zhiyuan Yao, Xu Liu, Daniel J Klionsky
Mitophagy, a selective type of macroautophagy (hereafter referred to as autophagy), specifically mediates the vacuole/lysosome-dependent degradation of damaged or surplus mitochondria. Because this process regulates the number and quality of mitochondria, it is vital for proper cellular homeostasis. Mitophagy also plays critical roles in the clearance of paternal mitochondria in C. elegans embryos, in erythroid cell maturation, and in the prevention of neurodegenerative disease and cancer. In order to study the molecular mechanism and regulation of mitophagy, sensitive assays are necessary to quantitatively measure mitophagy activity...
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324485/maintenance-of-human-embryonic-stem-cells-by-sphingosine-1-phosphate-and-platelet-derived-growth-factor
#8
Raymond C B Wong, Martin F Pera, Alice Pébay
Human embryonic stem cells (hESCs) have historically been cultivated on feeder layers of primary mouse embryonic fibroblasts (MEF) in a medium supplemented with fetal calf serum (FCS). However, serum contains a wide variety of biologically active compounds that might adversely affect hESC growth and differentiation. Thus, cultivation of stem cells in FCS complicates experimental approaches to define the intracellular mechanisms required for hESC maintenance. This chapter describes the serum-free maintenance of hESCs in culture by addition of sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF)...
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324614/fluorescence-based-high-throughput-and-targeted-image-acquisition-and-analysis-for-phenotypic-screening
#9
Manuel Gunkel, Jan Philipp Eberle, Holger Erfle
Applying the right acquisition method in a fluorescence imaging-based screening context is of great importance to obtain an appropriate readout and to select the right scale of the screen. In order to save imaging time and data, we have developed routines for multiscale targeted imaging, providing both a broad overview of a sample and additional in-depth information for targets of interest identified within the screen. These objects can be identified and acquired on-the-fly by an interconnection of image acquisition and image analysis...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324613/analysis-of-protein-kinetics-using-fluorescence-recovery-after-photobleaching-frap
#10
Nickolaos Nikiforos Giakoumakis, Maria Anna Rapsomaniki, Zoi Lygerou
Fluorescence recovery after photobleaching (FRAP) is a cutting-edge live-cell functional imaging technique that enables the exploration of protein dynamics in individual cells and thus permits the elucidation of protein mobility, function, and interactions at a single-cell level. During a typical FRAP experiment, fluorescent molecules in a defined region of interest within the cell are bleached by a short and powerful laser pulse, while the recovery of the fluorescence in the region is monitored over time by time-lapse microscopy...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324612/quantitative-image-analysis-of-single-molecule-mrna-dynamics-in-living-cells
#11
José Rino, Ana C de Jesus, Maria Carmo-Fonseca
Single mRNA molecules can be imaged in living cells by a method that consists in genetically inserting binding sites for a bacteriophage protein in the gene of interest. The resulting reporter transgene is then integrated in the genome of cells that express the phage protein fused to a fluorescent tag. Upon transcription, binding of the fluorescent protein to its target sequence makes the RNA visible. With this approach it is possible to track, in real time, the life cycle of a precursor mRNA at the site of transcription in the nucleus and transport of mature mRNA to the cytoplasm...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324611/automated-analysis-of-intracellular-dynamic-processes
#12
Yao Yao, Ihor Smal, Ilya Grigoriev, Maud Martin, Anna Akhmanova, Erik Meijering
The study of intracellular dynamic processes is of fundamental importance for understanding a wide variety of diseases and developing effective drugs and therapies. Advanced fluorescence microscopy imaging systems nowadays allow the recording of virtually any type of process in space and time with super-resolved detail and with high sensitivity and specificity. The large volume and high information content of the resulting image data, and the desire to obtain objective, quantitative descriptions and biophysical models of the processes of interest, require a high level of automation in data analysis...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324610/designing-image-analysis-pipelines-in-light-microscopy-a-rational-approach
#13
Ignacio Arganda-Carreras, Philippe Andrey
With the progress of microscopy techniques and the rapidly growing amounts of acquired imaging data, there is an increased need for automated image processing and analysis solutions in biological studies. Each new application requires the design of a specific image analysis pipeline, by assembling a series of image processing operations. Many commercial or free bioimage analysis software are now available and several textbooks and reviews have presented the mathematical and computational fundamentals of image processing and analysis...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324609/optical-coherence-microscopy
#14
Rainer A Leitgeb
The present chapter aims at demonstrating the capabilities of optical coherence microscopy (OCM) for applications in biomedical imaging. We furthermore review the functional imaging capabilities of OCM focusing on lable-free optical angiography. We conclude with a section on digital wavefront control and a short outlook on future developments, in particular for contrast enhancement techniques.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324608/two-color-total-internal-reflection-fluorescence-microscopy-of-exocytosis-in-endocrine-cells
#15
Adam J Trexler, Justin W Taraska
We describe a comprehensive method for imaging and analysis of local protein dynamics at single sites of exocytosis in living cultured endocrine cells. This method is well suited to quantitatively map the complex dynamics of individual molecules at single sites of vesicle fusion in live cells.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324607/sted-imaging-in-drosophila-brain-slices
#16
Sandra Fendl, Jesús Pujol-Martí, Joel Ryan, Alexander Borst, Robert Kasper
Super-resolution microscopy is a very powerful tool to investigate fine cellular structures and molecular arrangements in biological systems. For instance, stimulated emission depletion (STED) microscopy has been successfully used in recent years to investigate the arrangement and colocalization of different protein species in cells in culture and on the surface of specimens. However, because of its extreme sensitivity to light scattering, super-resolution imaging deep inside tissues remains a challenge. Here, we describe the preparation of thin slices from the fruit fly (Drosophila melanogaster) brain, subsequent immunolabeling and imaging with STED microscopy...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324606/sample-preparation-and-choice-of-fluorophores-for-single-and-dual-color-photo-activated-localization-microscopy-palm-with-bacterial-cells
#17
Juri N Bach, Giacomo Giacomelli, Marc Bramkamp
Photo-activated localization microscopy (PALM) is one of the light microscopy techniques providing highest resolution. Single photo-activatable or photo-switchable fluorescent molecules are stochastically excited. The point spread function of this event is recorded and the exact fluorophore position is calculated. This chapter describes how bacterial samples can be prepared for PALM to achieve routinely a resolution of ≤30 nm using fluorophores such as mNeonGreen, Dendra2, and PAmCherry. It is also explained how to perform multicolor PALM and combine it with total internal reflection (TIRF) microscopy to increase resolution...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324605/targeted-ablation-using-laser-nanosurgery
#18
Naga Venkata Gayathri Vegesna, Paolo Ronchi, Sevi Durdu, Stefan Terjung, Rainer Pepperkok
Laser-mediated dissection methods have been used for many years to micro-irradiate biological samples, but recent technological progress has rendered this technique more precise, powerful, and easy to use. Today pulsed lasers can be operated with diffraction limited, sub-micrometer precision to ablate intracellular structures. Here, we discuss laser nanosurgery setups and the instrumentation in our laboratory. We describe how to use this technique to ablate cytoskeletal elements in living cells. We also show how this technique can be used in multicellular organisms, to micropuncture and/or ablate cells of interest and finally how to monitor a successful laser nanosurgery...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324604/imaging-the-dynamics-of-cell-wall-polymer-deposition-in-the-unicellular-model-plant-penium-margaritaceum
#19
David Domozych, Anna Lietz, Molly Patten, Emily Singer, Berke Tinaz, Sandra C Raimundo
The unicellular green alga, Penium margaritaceum, represents a novel and valuable model organism for elucidating cell wall dynamics in plants. This organism's cell wall contains several polymers that are highly similar to those found in the primary cell walls of land plants. Penium is easily grown in laboratory culture and is effectively manipulated in various experimental protocols including microplate assays and correlative microscopy. Most importantly, Penium can be live labeled with cell wall-specific antibodies or other probes and returned to culture where specific cell wall developmental events can be monitored...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324603/fret-microscopy-for-real-time-visualization-of-second-messengers-in-living-cells
#20
Axel E Kraft, Viacheslav O Nikolaev
Förster Resonance Energy Transfer (FRET) microscopy is a useful tool in molecular biology and medical research to monitor and quantify real-time dynamics of protein-protein interactions and biochemical processes. Using this well-established technique, many novel signaling mechanisms can be investigated in intact cells or tissues and even in various subcellular compartments. Here, we describe how to perform FRET measurements in living cells expressing FRET-based biosensors and how to evaluate these data. This general protocol can be applied for FRET measurements with various fluorescent biosensors...
2017: Methods in Molecular Biology
journal
journal
31173
1
2
Fetch more papers »
Fetching more papers... Fetching...
Read by QxMD. Sign in or create an account to discover new knowledge that matter to you.
Remove bar
Read by QxMD icon Read
×

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"