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MRC Laboratory of Molecular Biology

5 ARTICLES PUBLISHED IN JoVE

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Biology

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)
Priya Crosby 1, Nathaniel P. Hoyle 1, John S. O'Neill 1
1MRC Laboratory of Molecular Biology

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

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Behavior

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice
Ruben Vale 1,2, Dominic Evans 1,2, Tiago Branco 1,2
1UCL Sainsbury Wellcome Centre for Neural Circuits and Behaviour, 2MRC Laboratory of Molecular Biology

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

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Biology

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry
Thomas Williams 1, Robert R. Kay 1
1MRC-Laboratory of Molecular Biology, Cambridge, UK

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

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Genetics

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria
Peggy Paschke 1, David A. Knecht 2, Thomas D. Williams 1, Peter A. Thomason 3, Robert H. Insall 3, Jonathan R. Chubb 4,5, Robert R. Kay 1, Douwe M. Veltman 1
1MRC Laboratory of Molecular Biology, 2Department of Molecular and Cell Biology, University of Connecticut, 3Cancer Research UK Beatson Institute Glasgow, 4MRC Laboratory for Molecular Cell Biology, University College London, 5Department of Cell and Developmental Biology, University College London

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

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Biology

Imaging ATG9A, a Multi-Spanning Membrane Protein
Alexander R. van Vliet *1,2, Stefano De Tito *1, Eugenia Almacellas *1, Sharon A. Tooze 1
1Molecular Cell Biology of Autophagy, The Francis Crick Institute, 2MRC Laboratory of Molecular Biology

This protocol describes various methods that can help in the study of ATG9A biology, including immunofluorescence followed by image analysis, transient overexpression considerations, and investigating the ATG9A glycosylation status using western blot.

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