Postdoc work
My postdoc was an exciting and unique opportunity in that it was not only shared between two labs with complementary expertise in two different countries. In Cees Dekker’s lab (Department of Bionanoscience, Delft University of Technology, Netherlands) I used nanofabrication and soft lithography to design and construct microfluidic devices for immobilizing bacterial cells vertically, then used advanced microscopy in Séamus Holden’s lab (then at Centre for Bacterial Cell Biology, Newcastle University, UK) to image the dynamics of bacterial division machinery at a single-molecule or near-single-molecule level.
In the video below, I give a summary of one paper from my postdoc (Whitley, Jukes et al, Nature Communications 2021). I am very grateful to the Royal Microscopy Society for awarding me with the Early Career Award on this research and the outreach involved.
Mechanism of bacterial cell division
Single molecules of the cell wall synthase PBP2B in Bacillus subtilis during cell division
The emergence and spread of antibiotic-resistant bacteria is a major global health problem. New antibiotics—and other strategies of prevention and treatment—are urgently needed to combat this growing threat. Cell division is a major target for antibiotics, so understanding how bacterial cells divide is important for future antibiotic development. Understanding the mechanism of how bacteria divide has additional applications such as the attempt to build synthetic cells from the bottom up.
Bacterial cell division is a complex biophysical process in which many (nano-scale) proteins have to work together over large distances to build a (micro-scale) wall in the middle of the cell. We use high-resolution microscopy to see how the bacterial cytoskeleton and cell wall synthesis enzymes work together as natural nano-machines to divide cells, using the soil-dwelling bacterium Bacillus subtilis as a model system. One way we see how they work is by ‘breaking them’ – that is, watching what they do when we suddenly inhibit them with specific antibiotics, such as Penicillin G (which targets the cell wall synthesis enzymes) and PC190723 (which targets the bacterial cytoskeleton). Our work on the role of the bacterial cytoskeletal protein FtsZ in cell division is published (Whitley, Jukes et al. Nature Communications 2021), and our more recent work on dynamics of the division cell wall synthesis complex is now on the bioRxiv pre-print server (Whitley et al. 2023).
High-resolution imaging in bacteria
Artwork: Dr. Lizah van der Aart
It’s well-known that bacteria are small, and that you need powerful microscopes to see them. But, if you want to look at individual biomolecules (e.g. proteins) inside them, the situation is even worse – many bacterial cells are ~1000 nm in width, which is not much bigger than the resolution limit of the most powerful light microscopes (~250 nm)! Even worse, most bacteria—including most model systems (e.g. E. coli) and many human pathogens (e.g. M. tuberculosis)—are non-spherical, so they typically lie horizontally flat on microscope coverslips, limiting some key structures to a low-resolution side-on view.
With VerCINI (Vertical Cell Imaging by Nanostructured Immobilization), cells are trapped in tiny holes so they’re forced to stand vertically, making it far easier to image anything that happens along their shorter axes. A prototype of this method was previously developed between the Dekker and Holden labs to look at cell division in the model bacterium Bacillus subtilis (Bisson-Filho et al. Science 2017). After I joined these two labs we optimized this method, and further expanded on it by developing a separate method that combines VerCINI with microfluidics, called μVerCINI (microfluidic VerCINI) (Whitley, Jukes et al. Nature Communications 2021). With μVerCINI, we can image vertical cells in high resolution while simultaneously perturbing them with antibiotics, changing their nutrient supply, fixing them for super-resolution imaging, or many other things. We have now published a detailed protocol for both methods (Whitley et al. Nature Protocols 2022) so they can more easily be used by interested researchers.
PhD work
My PhD work was in single-molecule biophysics in Yann Chemla’s lab (Department of Physics, University of Illinois Urbana-Champaign, USA). I used an instrument combining fluorescence with high-resolution optical tweezers (a.k.a. “fleezers“) to look at both the elastic behavior of nucleic acids (DNA and RNA) and the detailed mechanisms of ‘motor proteins’ that unwind the DNA double-helix (helicases).
Elastic behavior of DNA and RNA
DNA and RNA technologies are rapidly evolving, with enormous gains seen especially in healthcare. It’s well known that DNA and RNA are the genetic material of life on Earth, and that their sequences (A, T/U, G, and C) encode specific gene products like proteins. Less well appreciated is that they are also polymers with unique elastic properties. These properties are important in biology – DNA and RNA are constantly subjected to stretching, bending, and twisting forces as they are processed and packaged into cells and viruses. Understanding the elastic behavior of these molecules is critical not only to understand basic processes in biology, but also for developing next-generation DNA and RNA nanotechnology.
I investigated DNA and RNA elasticity by watching ultra-short strands (~10 bases) bind to one another (hybridize), one molecule at a time. These experiments yielded two separate and significant results. Firstly, we were able to uncover critical determinants of the hybridization reaction (Whitley et al. Nucleic Acids Research 2017), and secondly, we found that even ultra-short DNA and RNA strands (which can barely even be called ‘polymers’ due to their length) still behave as ideal polymers until too much force is applied, at which point this ideal behavior breaks down, possibly from base pairs fraying due to the shearing force (Whitley et al. Physical Review Letters 2018).
Mechanism of DNA helicases
Helicases are ‘motor proteins’ that use chemical energy in cells to unzip the DNA (or RNA) double-helix. They are involved in most processes in cells that involve nucleic acids, from DNA replication to RNA splicing. They are also finding applications in biotechnology as reagents for isothermal PCR and for DNA sequencing with nanopores. A greater understanding of helicase mechanisms has enabled researchers to engineer ‘super-helicases‘ that can unzip thousands of DNA base-pairs without falling off, even against high forces (~60 pN)!
I specifically investigated the mechanism of one large family of helicases (Superfamily 1) using the E. coli helicases UvrD and Rep as model systems. We characterized a regulatory ‘switch’ in these proteins (the 2B domain) that changes their behavior from unzipping the DNA double-helix to re-zipping it (Comstock et al. Science 2015, Ma et al. eLife 2018, Makurath et al. Nucleic Acids Research 2019). More recently we have looked in detail at the mechano-chemical cycle of UvrD by watching individual steps of the motor as it unzips DNA (Carney, Ma et al. Nature Communications 2021).