In situ high resolution studies of the FG-nucleoporins in the central channel of the nuclear pore complex

The nuclear pore complex (NPC) is the largest protein complex in the nuclear envelope of eukaryotic cells. It provides a permeability barrier for rapid and selective transport of biomolecules in and out of the nucleus. While the scaffold structure and composition of the NPC are well understood, little is known about the permeability barrier, which is formed of multiple copies of about ten different intrinsically disordered nucleoporins. These proteins characteristically contain multiple phenylglycine repeats in their sequence and are referred to as FG-Nups. Due to technical limitations in the study of these highly flexible and dynamic proteins, the conformational dynamics and spatial arrangement of FG-Nups in the permeability barrier of the NPC remain elusive. In this thesis I developed and established two complementary imaging techniques to visualise FG-Nups in the NPC in mammalian cells. To achieve this, I further developed a combination of genetic code expansion using unnatural amino acids and click-chemistry technologies suitable for high resolution fluorescence imaging. This method allows for efficient site-specific labelling of two sites in FG-Nups with small, photostable organic fluorophores with residue precision in situ. The first imaging approach used super-resolution localisation microscopy to precisely map the labelled site of the FG-Nups to a reference in the NPC. This resulted in images showing two-dimensional projection of FG-Nups distribution in NPCs, resolving distances down to 10nm. The second approach involved a complimentary technique based on confocal scanning microscopy: using fluorescence lifetime imaging microscopy (FLIM) to directly measure end-to-end distances between two sites in an FG-Nup less than 10nm apart using Förster resonance energy transfer (FRET). The ultimate goal of my work was to use the approaches to determine the structural arrangement of FG-Nups in the permeability barrier of the NPC. I further applied polymer physics concepts of scaling laws to my results from FLIM-FRET studies. I was able to show that the two major players in the permeability barrier FG-Nups, Nup62 and Nup98, tend to resemble a state between a collapsed coil and an ideal chain. This finding demonstrates the first experimental evidence of the actual scaling of FG-Nups in vivo, which has implications for existing transport models. Aside from its contribution to understanding the NPC permeability barrier function, the developed approach lays the groundwork for a more detailed understanding of disordered protein dynamics, dimensions and functions inside the cell

Electrochemical Nanoprobes for Single-Cell Analysis

The measurement of key molecules in individual cells with minimal disruption to the biological milieu is the next frontier in single-cell analyses. Nanoscale devices are ideal analytical tools because of their small size and their potential for high spatial and temporal resolution recordings. Here, we report the fabrication of disk-shaped carbon nanoelectrodes whose radius can be precisely tuned within the range 5–200 nm. The functionalization of the nanoelectrode with platinum allowed the monitoring of oxygen consumption outside and inside a brain slice. Furthermore, we show that nanoelectrodes of this type can be used to impale individual cells to perform electrochemical measurements within the cell with minimal disruption to cell function. These nanoelectrodes can be fabricated combined with scanning ion conductance microscopy probes, which should allow high resolution electrochemical mapping of species on or in living cells

Biofilm formation on human immune cells is a multicellular predation strategy of Vibrio cholerae

Biofilm formation is generally recognized as a bacterial defense mechanism against environmental threats, including antibiotics, bacteriophages, and leukocytes of the human immune system. Here, we show that for the human pathogen Vibrio cholerae, biofilm formation is not only a protective trait but also an aggressive trait to collectively predate different immune cells. We find that V. cholerae forms biofilms on the eukaryotic cell surface using an extracellular matrix comprising primarily mannose-sensitive hemagglutinin pili, toxin-coregulated pili, and the secreted colonization factor TcpF, which differs from the matrix composition of biofilms on other surfaces. These biofilms encase immune cells and establish a high local concentration of a secreted hemolysin to kill the immune cells before the biofilms disperse in a c-di-GMP-dependent manner. Together, these results uncover how bacteria employ biofilm formation as a multicellular strategy to invert the typical relationship between human immune cells as the hunters and bacteria as the hunted

Electrochemical Nanoprobes for Single-Cell Analysis

The measurement of key molecules in individual cells with minimal disruption to the biological milieu is the next frontier in single-cell analyses. Nanoscale devices are ideal analytical tools because of their small size and their potential for high spatial and temporal resolution recordings. Here, we report the fabrication of disk-shaped carbon nanoelectrodes whose radius can be precisely tuned within the range 5–200 nm. The functionalization of the nanoelectrode with platinum allowed the monitoring of oxygen consumption outside and inside a brain slice. Furthermore, we show that nanoelectrodes of this type can be used to impale individual cells to perform electrochemical measurements within the cell with minimal disruption to cell function. These nanoelectrodes can be fabricated combined with scanning ion conductance microscopy probes, which should allow high resolution electrochemical mapping of species on or in living cells