Blood vessels are critical to normal mammalian development, tissue repair, and growth and treatment of cancer. Mouse research models enable mechanistic studies of blood vessels. We detail how to perfuse mice with fluorescent tomato lectin or the lipophilic fluorophore DiI. We provide d ...
To understand the molecular events responsible for morphological change requires the ability to examine gene expression in a wide range of organisms in addition to model systems to determine how the differences in gene expression correlate with phenotypic differences. There are app ...
The Drosophila larval neuromuscular junction (NMJ) consists of a presynaptic motor neuron terminal and a postsynaptic muscle cell that offer an accessible and popular model system for the analysis of synaptic growth and function. I describe techniques for visualizing fluorescent ...
Detailed methods are provided for the preparation and confocal imaging of cardiac myocyte development and differentiation. Examples include protocols for the analysis of cultured myocytes as well as vibratome sections of hearts from embryonic and adult tissue. Techniques inclu ...
The nucleus is a complex organelle containing numerous highly dynamic, structurally stable domains and bodies, harboring functions that have only begun to be defined. However, the molecular mechanisms for their formation are still poorly understood. Recently it has been shown that a nu ...
Nucleolus assembly starts in telophase with the benefit of building blocks passing through mitosis and lasts until cytokinesis generating the two independent interphasic cells. Several approaches make it possible to follow the dynamics of fluorescent molecules in live cells. Her ...
The term correlative microscopy denotes the sequential visualization of one and the same cell using various microscopic techniques. Correlative microscopy provides a unique platform to combine the particular strength of each microscopic approach and compensate for its speci ...
Measuring dynamics of nuclear proteins is complicated by the fact that many DNA- and chromatin-binding proteins have separate nucleoplasmic and nuclear membrane pools with distinct mobilities. Moreover, when measuring recoveries in FRAP experiments, it is important to be aware th ...
Investigation of differential gene regulation by protein degradation requires analysis of the spatial and temporal association between proteolysis and transcription. Here, we describe the isochronal visualization of proteasomal proteolysis and transcription in cell c ...
A great number of molecules are constantly being exchanged between the nucleus and the cytoplasm via nuclear pore complexes (NPCs). Importantly, this nucleocytoplasmic trafficking is used to transfer information between the two compartments, thereby permitting the manipulat ...
Gene silencing in eukaryotes is a highly controlled process. It involves the concerted action of histone and DNA-modifying enzymes as well as transcription factors and chromatin-associated proteins. To understand how epigenetic gene silencing is regulated, it is important to iden ...
Recent advances have led to several systems to study transcription from defined loci in living cells. It has now become possible to address long-standing questions regarding the interplay between the processes of DNA damage repair and transcription—two disparate processes that can o ...
The ability of cells to copy their DNA allows them to transmit their genetic information to their progeny. In such, this central biological process preserves the instructions that direct the entire development of a cell. Earlier biochemical analysis in vitro and genetic analysis in yeast la ...
Single-molecule fluorescence microscopy has been used for decades to quantify macromolecular dynamics occurring in specimens that are in direct contact with a coverslip. This has permitted in vitro analysis of single-molecule motion in various biochemically reconstituted s ...
Chromosome tagging using lac or tet operator repeats for in vivo visualization of chromosome dynamics has now become a standard methodology used in a range of organisms. One variation of this approach has been to build transgene arrays creating artificial chromosome blocks to study vario ...
The eukaryotic genome is packaged within the nucleus as poly-nucleosome 10 nm chromatin fibres. The nucleosome core particle, the fundamental chromatin subunit, consists of a DNA molecule wrapped around a histone octamer. Biochemical modifications of both the DNA and histone protei ...
Chromatin-protein interactions are important in determining chromosome structure and function, thereby regulating gene expression patterns. Most chromatin associated proteins bind chromatin in a transient manner, with residence times on the order of a few seconds to minutes. ...
During development, the genome undergoes drastic reorganization within the nuclear space. To determine tridimensional genome folding, genome-wide techniques (damID/Hi-C) can be applied using cell populations, but these have to be calibrated using microscopy and single-cell ...
The appropriate functioning of living cells depends on a variety of dynamic processes that necessitate delicate motion, transportation, association, and disassociation in time and space. Different dynamic patterns such as directed motion, normal diffusion, and restricted dif ...
Dictyostelium cells have great utility for live imaging of single gene transcriptional dynamics. The cells allow efficient molecular genetics, for targeting of RNA reporters and fluorescent proteins to individual, defined loci. Dictyostelium cells share many signalling, chr ...
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