Molecular cell biology

Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102675 ◽

1988 ◽

Vol 46 ◽

pp. 118-119

Author(s):

K. Jacobson ◽

A. Ishihara ◽

B. Holifield ◽

F. Zhang

Keyword(s):

Fluorescence Microscopy ◽

Cell Biology ◽

Extended Period ◽

Dynamic Structure ◽

Living Cells ◽

Membrane Dynamics ◽

Molecular Cell Biology ◽

Image Averaging ◽

Single Living Cells ◽

Single Living

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.

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Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

Journal of Fungi ◽

10.3390/jof7020149 ◽

2021 ◽

Vol 7 (2) ◽

pp. 149

Author(s):

Sarah-Maria Wege ◽

Katharina Gejer ◽

Fabienne Becker ◽

Michael Bölker ◽

Johannes Freitag ◽

...

Keyword(s):

Genome Editing ◽

Cell Biology ◽

Gene Deletion ◽

Fluorescent Protein ◽

Point Mutations ◽

Model Organism ◽

Ustilago Maydis ◽

Genomic Locus ◽

Targeted Gene Deletion ◽

Molecular Cell Biology

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

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