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Special methodical expertise

Analysis of protein localization by confocal laser scanning microscopy

Understanding the subcellular and dynamic localization of proteins and protein complexes in cells provides important information about their biological function. Confocal laser scanning microscopy represents a very powerful technique to visualize intracellular protein localization by fluorescence microscopy. This direct and noninvasive method generates high-resolution microscopic images through “optical sectioning” of cells or subcellular structures which have been treated with fluorescent dyes or fluorescent antibodies directed against the protein(s) of interest. The acquired images from “optical sectioning” are reconstructed by a computer allowing subcellular and three-dimensional visualization of protein localization, e.g. at structurally complex objects like the mitotic spindle apparatus (as shown in Figure 1A).

Analysis of protein function by RNA interference (RNAi)-mediated knock down of gene expression

RNA interference (RNAi) represents in many eukaryotes an important genetic system to control whether and to which extent certain genes are expressed in cells and living organisms, e.g. during development. Due to its selective and robust inhibitory effect on gene expression RNAi has become a valuable and very effective research tool to suppress specific genes of interest in order to study their biological function. Technically, small double stranded RNA reagents (dsRNA) directed against the gene of interest can be designed by computational biology. Following introduction into cells by biochemical/biophysical or viral-based methods, dsRNAs bind to their target messenger RNA (mRNA) and activate the so-called RNAi pathway. This mechanism leads to the destruction of the target mRNA and hence prevention of its translation into protein. RNAi has been used for large-scale screens of genes which are necessary for a particular cellular process such as mitotic cell division (Figure 1B). An important gene product for this process is the centrosomal protein TACC3, which RNAi-mediated knock down of gene expression arrests cells in mitosis and prevents their division into two daughter cells (Figure 1C).

Figure Legend:
A: Detection of the mitotic spindle apparatus and microtubules (alpha-Tubulin stained in green) by confocal laser scanning microscopy. The spindle poles are indicated by arrows, the genetic information (chromosomes) is stained in blue.
B: Live cell imaging of a mitotic cell undergoing normal cell division into two daugther cells.
C: In contrast to B, the cell depicted in C lacks the centrosomal protein TACC3 due to RNAi-mediated knock down of gene expression. As a consequence, this cell fails to divide due to the absence of TACC3 as critical regulator of mitotic spindle function.

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