Research in the Department of Molecular Genetics and Cell Biology

1. Polarity Establishment in Eucaryotic Cells

Establishment of cellular polarity is a key requisite for eucaryotic life. Misfunction in polarity establishment results in severe consequences for the cell and the organism including cell death or development of diseases. Many of the meachnisms underlying polarity establishment are conserved among eucaryotic cells. Due to it's ease in manipulation, we employ the budding yeast Saccharomyces cerevisiae as a model organism to study establishment and maintenance of cellular polarity.  
The assembly of the bud site is a remarkable and hardly understood process. Kick-started by a local pulse of active Cdc42, hundreds of different proteins, lipids, and other macromolecules are attracted to a single spot of less than one micron. After 10 minutes the bud site starts to expand and to grow into a new daughter cell. The structure of the bud site is too complex and probably too amorphous to be assembled by a single linear, step-wise process. The convergence of parallel pathways that influence and inform each other represents an alternative, and probably more realistic view how the bud site forms and thus polarity establishes. 
After bud site establishment, the yeast cells continue polar growth of the bud. This polar growth is the result of a conserved and complex interplay between RhoGTPase-based signalling, cytoskeletal organization, and vesicular traffic and fusion (figure 1.1). 

We investigate the formation, composition and function of protein interaction networks that drive polarity establishment, polar growth and cytokinesis in vivo. Through temporal interaction analysis we are able to describe these networks as time- and localization-dependent conversions of different interaction states inside the living cell.
Experimental strategies in this topic include time resolved fluorescence microscopy, high throughput interaction screens, genetic manipulation of yeast and biochemical assays. 

Figure 1.1 Boi proteins as hubs of polarized secretion and actin filament nucleation in polar growth of yeast cells (figure from Glomb et al. 2020)

2. Cellular Biochemistry of the Septins

The septins were only discovered in the 1970s and represent a class of GTP binding cytoskeletal proteins sharing structural features of the Ras family of GTPases. They form non-polar filaments that are indispensable for eukaryotic life. Dysfunction of septin subunit expression and filament formation is related to severe cellular phenotypes and to diseases such as cancer, male infertility, neurodevelopmental disorders and others.

The human septin SEPT9 is related to several diseases. Identifying novel interaction partners of SEPT9 should help to gain insights into the intracellular processes leading to SEPT9 related cellular misfunctions and disease onset. Applying affinity purification of SEPT9 complexes combined with quantitative mass spectrometry, we have identified 42 interaction partners of SEPT9 (Hecht et al., 2019). Currently we are investigating the role of four novel interaction partners with respect to their SEPT9 dependent function in cellular adhesions, cytokinesis and intracellular vesicle transport (figure 2.1). 
In this project we apply shRNA mediated knock down of candidate genes, immunofluorescence- and live cell microscopy as well as in vitro assays using purified proteins.

 

Septins were discovered in the baker’s yeast Saccharomyces cerevisiae and still today yeast serves as a prime model organism for research on septins due to its ease of genetic manipulation and the limited number of different septin subunits. We aim at understanding the mechanisms of septin filament formation and nucleotide binding by combining biochemical assays, structural biology and cell biology. Recently our laboratory presented the first crystal structure of a yeast septin (Brausemann et al. 2016) (figure 2.2). Currently we study the mechanisms of nucleotide binding and hydrolysis in yeast septins in vitro and aim at translating our results to human septins. A robust purification pipeline for yeast- and human septin rods was therefore established in our laboratory (Renz et al. 2013 and ongoing, unpublished results).

In yeast, septin structures undergo substantial structural transitions during the cell cycle. We suppose that these transitions are driven by post-translational modifications (PTM). We are currently establishing an experimental pipeline to systematically identify PTM of the septins in distinct cell cycle stages. These will allow us to allocate the so far largely unknown functions of these PTM to the structures the septins adopt at the distinct cell cycle stages.

Fig. 2.1 Proposed functions involving the novel interaction partners of SEPT9 (Figure: Matthias Hecht)
Fig. 2.1 Crytsal structure of the yeast septin subunit Cdc11 (PDB-ID: 5AR1) (Figure: Benjamin Grupp)