Graduate Students Mini Symposium II & III 2020

Program:

13:00 Ilijana Vojnovic, AG Endesfelder

Investigation of the kinetochore structure by quantitative multi-color single-molecule localization microscopy imaging

A key element for proper DNA segregation during mitosis is the kinetochore, a multi-protein complex that links the centromeric chromatin to the microtubules attached to the spindle pole body [1]. Our work aims at constructing a detailed map of the human-like regional kinetochore of Schizosaccharomyces pombe by single-molecule localization microscopy (SMLM). For data acquisition, we set each kinetochore protein of interest (POI) into relation to two reference proteins (cnp1 at the centromere and sad1 at the spindle pole). This triple-color arrangement allows to triangulate the position of each POI and in time, to build a kinetochore model; assisted by visual analytic tools and resolved over the cell cycle at a nanometer resolution. We rely on our recently developed mEos3.2-A69T [2,3] fluorescent protein variant, as it can be photoconverted by a novel mechanism using blue and infrared light, termed primed conversion (PC). This enables us to perform aberration-free multi-color imaging by combining mEos3.2-A69T with the UV-photoactivating protein PAmCherry to image two targets in the same spectral detection channel using the orthogonal PC and UV-activation modes [3]. The orientations of the complexes are determined by the conventional FP mScarlet-I, marking the spindle poles. This way we determine protein stoichiometry, elucidate patterns in protein deposition and turnover, measure protein-protein distances and analyze cluster shapes and distributions. With this protein map we can visualize the assembly and regulation of the in situ kinetochore architecture for the very first time.

13:30 Alexander Balinovic, AG Endesfelder

Development and characterization of new fluorophores and methods for advanced SMLM imaging schemes

Single-molecule localization microscopy (SMLM) enables to unravel the molecular architecture of biological structures by localizing single molecules. Many fluorophores and techniques suitable for SMLM have been established in the past years and the topic remains highly relevant to this day. To date, SMLM still has several major limitations, among which I focus on two of them in my PhD work: 1) Phototoxic damage in the biological sample which is currently caused by the high irradiation doses and light illuminations by short wavelengths required for SMLM image acquisition. 2) Methods to control for optimal image quality in SMLM experiments.

14:00 Kyra Geyer, AG Erb

Understanding substrate selectivity of phoslactomycin polyketide synthase using reconstituted in vitro systems

Polyketide synthases (PKS) use simple extender units to synthesize complex natural products. A fundamental question is how different extender units are site-specifically incorporated into the growing polyketide. Here we established phoslactomycin (Pn) PKS that incorporates malonyl- and ethylmalonyl‑CoA as an in vitro model to study substrate specificity. While malonyl-CoA modules of Pn PKS exclusively accept their natural substrate, the ethylmalonyl-CoA module PnC tolerates different α‑substituted derivatives, but discriminates against malonyl‑CoA. We show that the ratio of extender transacylation to hydrolysis controls incorporation in PnC, explaining site-specific selectivity and promiscuity in the natural context of Pn PKS.

14:30-14:45 Coffee break

14:45 Bailey Milne-Davies, AG Diepold

Infection of host cells by T3SS-utilizing bacteria – cell culture and effector translocation

The type III secretion system (T3SS) is utilized by many Gram-negative bacteria to promote survival in a variety of symbiotic relationships. Many pathogenic bacteria use their T3SS, commonly called the injectisome, to translocate bacterial effector proteins to manipulate host cells and promote infection. The injectisome consists of a membrane-spanning nanosyringe that translocates effector proteins from the bacterial cytosol to the host cytoplasm. We are interested in studying how Yersinia enterocolitica regulates effector translocation during infection. Using growth and sensitive enzymatic assays we have gained insight into the life after secretion, demonstrating that Y. enterocolitica can quickly adapt to different environmental conditions. Additionally, we have established cell culture-based infection assays to test the controlled protein translocation by the T3SS.

15:15 Stephan Wimmi, AG Diepold

Impact of T3SS effector proteins on molecular dynamics of Yersinia enterocolitica cytosolic components

Pathogenic bacteria are known to modify their hosts by translocating molecular toxins, called effector proteins. One of the most direct and widespread translocation mechanisms in Gram- bacteria is via the type III secretion system (T3SS). The T3SS resembles a molecular syringe, which establishes a cytosolic connection between bacteria and host cells. While the structure of the T3SS is well-defined, little is known about its molecular function and regulation, and especially the interaction of the effector proteins and the T3SS. We thus studied the dynamics of the cytosolic components which exchange between a free diffusing cytoplasmic and a T3SS bound state. Recent publications showed that dynamics is linked to the functions of the injectisome. We therefore analyzed the diffusion of the essential cytosolic T3SS component SctQ with single particle tracking photoactivated localization microscopy (sptPALM) in different deletion strain backgrounds to investigate the impact that the presence of effector proteins has on the localization and diffusion of SctQ. Our data shows that SctQ diffuses in two populations and that the presence of effector proteins slows down that speed. This suggests that the cytosolic components are not only essential for secretion but participate actively in effector shuttling and recruitment.