The Reyes Lab aims to understand the function of molecular machines as they work inside the cell
Our group uses single-molecule approaches in live cells to infer the spatial organization and dynamics of proteins and DNA. Our current focus is on the understanding of the molecular machine that duplicate the genome, called the replisome. Surprisingly, there are still fundamental aspects about this important machine that we do not know about. For example, we are still unsure about the architecture of the active replisome. We are also unclear about aspects on the timing at which this machine assembles and disassembles, and what are the factors that determine its stability during the often-long path to completion of chromosomal replication.
We use the unicellular organisms, Escherichia coli and Saccharomyces cerevisiae , as model organisms for bacteria and eukaryotes, respectively. As most of the proteins in the replisome of bacteria do not share common ancestry to those in eukaryotes, even though they accomplish similar tasks, studying both organisms allow us to probe the function of their unrelated machines. Their replisomes are the best characterised by biochemical and genetic methods in their respective phyla. Finally, these organisms are also two of the most widely used genetic models, giving us access to many genetic tools that we use to change the intracellular conditions in a control manner.
Why does our work matter?
DNA replication is crucial for cell proliferation and genome integrity. Hence, its understanding can lead to breakthroughs in the control of unregulated cell proliferation associated with disease, like in the case of bacterial infections and tumour cells. In addition, despite the very high-fidelity associated with this process, DNA replication is still a common source of mutations and genome instability, which can result in genetic diseases. Therefore, it is important to understand the relevant traits that allow high fidelity, the problems during DNA synthesis that generate lesions on DNA, and the coordination of the replisome with other cellular machineries to respond to DNA damage. Outside of its medical relevance, understanding how cells copy their DNA will likely help in the field of Synthetic Biology, where generating cells carrying novel genetic material with extended functionality is desired.
Our expertise in single-molecule microscopy has the potential of contributing to the understanding of many other processes. Among them, we currently work with our collaborators in the study of antibiotic resistance, the regulation of the cell volume in yeast, the activity of RNA polymerase, and the activity of DNA transposases.
What is single-molecule microscopy and why do we use it?
Life is complicated. At every level of organization, too many things happen at the same time. At the lab, very often experiments are only able to measure an average obtained from many molecules or cells. Groups of these cells (or molecules) may be doing different things, hence giving us a mixed picture of what is happening and preventing the correct interpretation of the results. At the fluorescence microscope, this translates into observing at once hundreds to thousands of copies of an identical protein fused fluorescently, although often only a small fraction of these copies is active. Single-molecule microscopy allow us to study individual copies of proteins, and to differentiate between different states present.
Single-molecule microscopy refers to the ability to detect single fluorophores at the microscope. This has been a huge achievement in the evolution of microscopy in the last couple of decades. Single-molecule microscopy has multiple applications. Among them, it is an essential component of the widespread super-resolution techniques PALM and STORM. In our lab, we apply these techniques to study proteins in live-cells. Studying cells is complicated since we are limited by the type of fluorophores we can use, most often they have to be fluorescent proteins (similar to GFP) which are not as bright as other organic fluorescent dyes, and because the cell is a source of noise that hides the dim signal of the single molecules. Therefore, an important part of our job is to continuously search for ways to improve the fluorophores, the microscope, and the image analysis. Use of single-molecule microscopy allows us to study the architecture and activity of multi-protein molecular machines in live cells. With it, we can count copies of molecules at specific points in the cell, determine the fraction of active molecules, study the diffusion of proteins in cells, and estimate the kinetics of binding of proteins to DNA. We use this information to generate models of how proteins work in the cell.