Erdal Toprak

Polarization effect on position accuracy of fluorophore localization

The technique of determining the position of individual fluorescent molecules with nanometer resolution, called FIONA, has become an important tool for several biophysical applications such as studying motility mechanisms of motor proteins. The position determination is usually done by fitting a 2-D Gaussian (x-y vs. photon number) to the emission intensity distribution of the fluorescent molecule. However, the intensity distribution of an emitting molecule depends not only on its position in space, but also on its three-dimensional orientation. Here, we present an extensive numeri-cal study of the achievable accuracy of position determination as a function of molecule orientation. We compare objectives with different numerical apertures and show that an effective pixel size of 100 nm or less per CCD pixel is required to obtain good positional accuracy. Nonetheless, orienta-tion effects can still cause position errors for large anisotropy, as high as 10 nm for high numerical aperture objectives. However, position accuracy is significantly better (< 2.5 nm) when using objectives with a numerical aper-ture of 1.2. Of course, probes with lower anisotropy decrease the positional uncertainty.

Strength of Selection Pressure Is an Important Parameter Contributing to the Complexity of Antibiotic Resistance Evolution

Revealing the genetic changes responsible for antibiotic resistance can be critical for developing novel antibiotic therapies. However, systematic studies correlating genotype to phenotype in the context of antibiotic resistance have been missing. In order to fill in this gap, we evolved 88 isogenic Escherichia coli populations against 22 antibiotics for 3 weeks. For every drug, two populations were evolved under strong selection and two populations were evolved under mild selection. By quantifying evolved populations’ resistances against all 22 drugs, we constructed two separate cross-resistance networks for strongly and mildly selected populations. Subsequently, we sequenced representative colonies isolated from evolved populations for revealing the genetic basis for novel phenotypes. Bacterial populations that evolved resistance against antibiotics under strong selection acquired high levels of cross-resistance against several antibiotics, whereas other bacterial populations evolved under milder selection acquired relatively weaker cross-resistance. In addition, we found that strongly selected strains against aminoglycosides became more susceptible to five other drug classes compared with their wild-type ancestor as a result of a point mutation on TrkH, an ion transporter protein. Our findings suggest that selection strength is an important parameter contributing to the complexity of antibiotic resistance problem and use of high doses of antibiotics to clear infections has the potential to promote increase of cross-resistance in clinics.

Why kinesin is so processive

Kinesin I can walk on a microtubule for distances as long as several micrometers. However, it is still unclear how this molecular motor can remain attached to the microtubule through the hundreds of mechanochemical cycles necessary to achieve this remarkable degree of processivity. We have addressed this issue by applying ensemble and single-molecule fluorescence methods to study the process of kinesin stepping, and our results lead to 4 conclusions. First, under physiologic conditions, ≈75% of processively moving kinesin molecules are attached to the microtubule via both heads, and in this conformation, they are resistant to dissociation. Second, the remaining 25% of kinesin molecules, which are in an “ATP waiting state” and are strongly attached to the microtubule via only one head, are intermittently in a conformation that cannot bind ATP and therefore are resistant to nucleotide-induced dissociation. Third, the forward step in the kinesin ATPase cycle is very fast, accounting for <5% of the total cycle time, which ensures that the lifetime of this ATP waiting state is relatively short. Finally, by combining nanometer-level single-molecule fluorescence localization with higher ATP concentrations than used previously, we have determined that in this ATP waiting state, the ADP-containing head of kinesin is located 8 nm behind the attached head, in a location where it can interact with the microtubule lattice. These 4 features reduce the likelihood that a kinesin I motor will dissociate and contribute to making this motor so highly processive.

Single-molecule fluorescence to study molecular motors

Molecular motors, which use energy from ATP hydrolysis to take nanometer-scale steps with run-lengths on the order of micrometers, have important roles in areas such as transport and mitosis in living organisms. New techniques have recently been developed to measure these small movements at the single-molecule level. In particular, fluorescence imaging has contributed to the accurate measurement of this tiny movement. We introduce three single-molecule fluorescence imaging techniques which can find the position of a fluorophore with accuracy in the range of a few nanometers. These techniques are named after Hollywood animation characters: Fluorescence Imaging with One Nanometer Accuracy (FIONA), Single-molecule High-REsolution Colocalization (SHREC), and Defocused Orientation and Position Imaging (DOPI). We explain new understanding of molecular motors obtained from measurements using these techniques.

Delayed commitment to evolutionary fate in antibiotic resistance fitness landscapes.

Predicting evolutionary paths to antibiotic resistance is key for understanding and controlling drug resistance. When considering a single final resistant genotype, epistatic contingencies among mutations restrict evolution to a small number of adaptive paths. Less attention has been given to multi-peak landscapes, and while specific peaks can be favoured, it is unknown whether and how early a commitment to final fate is made. Here we characterize a multi-peaked adaptive landscape for trimethoprim resistance by constructing all combinatorial alleles of seven resistance-conferring mutations in dihydrofolate reductase. We observe that epistatic interactions increase rather than decrease the accessibility of each peak; while they restrict the number of direct paths, they generate more indirect paths, where mutations are adaptively gained and later adaptively lost or changed. This enhanced accessibility allows evolution to proceed through many adaptive steps while delaying commitment to genotypic fate, hindering our ability to predict or control evolutionary outcomes.

Myosin VI undergoes a 180° power stroke implying an uncoupling of the front lever arm

We simultaneously measure both the step size, via FIONA, and the 3-D orientation, via DOPI, of the light-chain domain of individual dimeric myosin VIs. This allows for the correlation of the change in orientation of the light chain domain to the stepping of the motor. Three different pairs of positions were tested using a rigid bifunctional rhodamine on the calmodulin of the IQ domain. The data for all three labeling positions support the model that the light chain domain undergoes a significant rotation of approximately 180°. Contrary to an earlier study [Sun, Y. et al. (2007) Mol Cell 28, 954–964], our data does not support a model of multiple angles of the lever arm of the lead head, nor “wiggly” walking on actin. Instead, we propose that for the two heads of myosin VI to coordinate their processive movement, the lever arm of the lead head must be uncoupled from the converter until the rear head detaches. More specifically, intramolecular strain causes the myosin VI lever arm of the lead head to uncouple from the motor domain, allowing the motor domain to go through its product-release (phosphate and ADP) steps at an unstrained rate. The lever arm of the lead head rebinds to the motor and attains a rigor conformation when the rear head detaches. By coupling the orientation and position information with previously described kinetics, this allows us to explain how myosin VI coordinates its heads processively while maintaining the ability to move under load with a (semi-) rigid lever arm.

Quantifying the Determinants of Evolutionary Dynamics Leading to Drug Resistance

The emergence of drug resistant pathogens is a serious public health problem. It is a long-standing goal to predict rates of resistance evolution and design optimal treatment strategies accordingly. To this end, it is crucial to reveal the underlying causes of drug-specific differences in the evolutionary dynamics leading to resistance. However, it remains largely unknown why the rates of resistance evolution via spontaneous mutations and the diversity of mutational paths vary substantially between drugs. Here we comprehensively quantify the distribution of fitness effects (DFE) of mutations, a key determinant of evolutionary dynamics, in the presence of eight antibiotics representing the main modes of action. Using precise high-throughput fitness measurements for genome-wide Escherichia coli gene deletion strains, we find that the width of the DFE varies dramatically between antibiotics and, contrary to conventional wisdom, for some drugs the DFE width is lower than in the absence of stress. We show that this previously underappreciated divergence in DFE width among antibiotics is largely caused by their distinct drug-specific dose-response characteristics. Unlike the DFE, the magnitude of the changes in tolerated drug concentration resulting from genome-wide mutations is similar for most drugs but exceptionally small for the antibiotic nitrofurantoin, i.e., mutations generally have considerably smaller resistance effects for nitrofurantoin than for other drugs. A population genetics model predicts that resistance evolution for drugs with this property is severely limited and confined to reproducible mutational paths. We tested this prediction in laboratory evolution experiments using the “morbidostat”, a device for evolving bacteria in well-controlled drug environments. Nitrofurantoin resistance indeed evolved extremely slowly via reproducible mutations—an almost paradoxical behavior since this drug causes DNA damage and increases the mutation rate. Overall, we identified novel quantitative characteristics of the evolutionary landscape that provide the conceptual foundation for predicting the dynamics of drug resistance evolution.

Super-Accuracy and Super-Resolution. Getting Around the Diffraction Limit.

In many research areas such as biology, biochemistry, and biophysics, measuring distances or identifying and counting objects can be of great importance. To do this, researchers often need complicated and expensive tools in order to have accurate measurements. In addition, these measurements are often done under nonphysiological settings. X-ray diffraction, for example, gets Angstrom-level structures, but it requires crystallizing a biological specimen. Electron microscopy (EM) has about 10A resolution, but often requires frozen (liquid nitrogen) samples. Optical microscopy, while coming closest to physiologically relevant conditions, has been limited by the minimum distances to be measured, typically about the diffraction limit, or approximately 200 nm. However, most biological molecules are <5-10nm in diameter, and getting molecular details requires imaging at this scale. In this chapter, we will describe some of the experimental approaches, from our lab and others, that push the limits of localization accuracy and optical resolution in fluorescence microscopy.

Fluidic and microfluidic tools for quantitative systems biology

Understanding genes and their functions is a daunting task due to the level of complexity in biological organisms. For discovering how genotype and phenotype are linked to each other, it is essential to carry out systematic studies with maximum sensitivity and high-throughput. Recent developments in fluid-handling technologies, both at the macro and micro scale, are now allowing us to apply engineering approaches to achieve this goal. With these newly developed tools, it is now possible to identify genetic factors that are responsible for particular phenotypes, perturb and monitor cells at the single-cell level, evaluate cell-to-cell variability, detect very rare phenotypes, and construct faithful in vitro disease models.

Sequence-Specific Targeting of Bacterial Resistance Genes Increases Antibiotic Efficacy

The lack of effective and well-tolerated therapies against antibiotic-resistant bacteria is a global public health problem leading to prolonged treatment and increased mortality. To improve the efficacy of existing antibiotic compounds, we introduce a new method for strategically inducing antibiotic hypersensitivity in pathogenic bacteria. Following the systematic verification that the AcrAB-TolC efflux system is one of the major determinants of the intrinsic antibiotic resistance levels in Escherichia coli, we have developed a short antisense oligomer designed to inhibit the expression of acrA and increase antibiotic susceptibility in E. coli. By employing this strategy, we can inhibit E. coli growth using 2- to 40-fold lower antibiotic doses, depending on the antibiotic compound utilized. The sensitizing effect of the antisense oligomer is highly specific to the targeted gene’s sequence, which is conserved in several bacterial genera, and the oligomer does not have any detectable toxicity against human cells. Finally, we demonstrate that antisense oligomers improve the efficacy of antibiotic combinations, allowing the combined use of even antagonistic antibiotic pairs that are typically not favored due to their reduced activities.