Jeffrey Nguyen

Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization

Significance Across all kingdoms of life, maintaining the correct cell shape is critical for behaviors such as sensing, motility, surface attachment, and nutrient acquisition. Maintaining proper shape requires cellular-scale coordination of proteins and feedback systems that enable responses that correct local morphological perturbations. Here, we demonstrate that the MreB cytoskeleton in Escherichia coli preferentially localizes to regions of negative curvature, directing growth away from the poles and actively straightening locally curved regions of the cell. Moreover, our biophysical simulations of curvature-biased growth suggest that cell wall insertion causes surface deformations that could be responsible for the circumferential motion of MreB. Taken together, our work demonstrates that MreB’s local orchestration of persistent, bursty growth enables robust, uniform growth at the cellular scale. Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization.

RodZ links MreB to cell wall synthesis to mediate MreB rotation and robust morphogenesis

Significance The bacterial actin homolog, MreB, is a key determinant of rod-cell shape but the mechanism by which it functions has remained a topic of much debate. Recently it was shown that MreB exists as small polymers that actively rotate around the cell circumference. This rotation is widely conserved, yet its mechanism and function have remained unknown. Here we show that MreB rotates because cytoplasmic MreB filaments are coupled to periplasmic cell wall synthesis through the transmembrane protein RodZ, which acts as a transmembrane linker. Furthermore, by genetically uncoupling MreB rotation from growth we establish MreB rotation acts as a robustness mechanism for rod-like shape determination. This work thus explains the mystery of MreB rotation and suggests a new model for bacterial cell shape maintenance. The rod shape of most bacteria requires the actin homolog, MreB. Whereas MreB was initially thought to statically define rod shape, recent studies found that MreB dynamically rotates around the cell circumference dependent on cell wall synthesis. However, the mechanism by which cytoplasmic MreB is linked to extracytoplasmic cell wall synthesis and the function of this linkage for morphogenesis has remained unclear. Here we demonstrate that the transmembrane protein RodZ mediates MreB rotation by directly or indirectly coupling MreB to cell wall synthesis enzymes. Furthermore, we map the RodZ domains that link MreB to cell wall synthesis and identify mreB mutants that suppress the shape defect of ΔrodZ without restoring rotation, uncoupling rotation from rod-like growth. Surprisingly, MreB rotation is dispensable for rod-like shape determination under standard laboratory conditions but is required for the robustness of rod shape and growth under conditions of cell wall stress.

Automatically tracking neurons in a moving and deforming brain

Advances in optical neuroimaging techniques now allow neural activity to be recorded with cellular resolution in awake and behaving animals. Brain motion in these recordings pose a unique challenge. The location of individual neurons must be tracked in 3D over time to accurately extract single neuron activity traces. Recordings from small invertebrates like C. elegans are especially challenging because they undergo very large brain motion and deformation during animal movement. Here we present an automated computer vision pipeline to reliably track populations of neurons with single neuron resolution in the brain of a freely moving C. elegans undergoing large motion and deformation. 3D volumetric fluorescent images of the animal’s brain are straightened, aligned and registered, and the locations of neurons in the images are found via segmentation. Each neuron is then assigned an identity using a new time-independent machine-learning approach we call Neuron Registration Vector Encoding. In this approach, non-rigid point-set registration is used to match each segmented neuron in each volume with a set of reference volumes taken from throughout the recording. The way each neuron matches with the references defines a feature vector which is clustered to assign an identity to each neuron in each volume. Finally, thin-plate spline interpolation is used to correct errors in segmentation and check consistency of assigned identities. The Neuron Registration Vector Encoding approach proposed here is uniquely well suited for tracking neurons in brains undergoing large deformations. When applied to whole-brain calcium imaging recordings in freely moving C. elegans, this analysis pipeline located 156 neurons for the duration of an 8 minute recording and consistently found more neurons more quickly than manual or semi-automated approaches.

Whole-brain calcium imaging with cellular resolution in freely behaving C. elegans

Significance Large-scale neural recordings in freely moving animals are important for understanding how patterns of activity across a population of neurons generates animal behavior. Previously, recordings have been limited to either small brain regions or to immobilized or anesthetized animals exhibiting limited behavior. This work records from neurons with cellular resolution throughout the entire brain of the nematode Caenorhabditis elegans during free locomotion. Neurons are found whose activity correlates with behaviors including forward and backward locomotion and turning. A growing body of evidence suggests that animal behavior is sometimes generated by the collective activity of many neurons. It is hoped that methods like this will provide quantitative datasets that yield insights into how brain-wide neural dynamics encode animal action and perception. The ability to acquire large-scale recordings of neuronal activity in awake and unrestrained animals is needed to provide new insights into how populations of neurons generate animal behavior. We present an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of a freely behaving Caenorhabditis elegans with cellular resolution while simultaneously recording the animal’s position, posture, and locomotion. This instrument provides whole-brain imaging with cellular resolution in an unrestrained and behaving animal. We use spinning-disk confocal microscopy to capture 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s at 6 head-volumes/s. A suite of three cameras monitor neuronal fluorescence and the animal’s position and orientation. Custom software tracks the 3D position of the animal’s head in real time and two feedback loops adjust a motorized stage and objective to keep the animal’s head within the field of view as the animal roams freely. We observe calcium transients from up to 77 neurons for over 4 min and correlate this activity with the animal’s behavior. We characterize noise in the system due to animal motion and show that, across worms, multiple neurons show significant correlations with modes of behavior corresponding to forward, backward, and turning locomotion.

From the Cover: PNAS Plus: Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans

Significance Large-scale neural recordings in freely moving animals are important for understanding how patterns of activity across a population of neurons generates animal behavior. Previously, recordings have been limited to either small brain regions or to immobilized or anesthetized animals exhibiting limited behavior. This work records from neurons with cellular resolution throughout the entire brain of the nematode Caenorhabditis elegans during free locomotion. Neurons are found whose activity correlates with behaviors including forward and backward locomotion and turning. A growing body of evidence suggests that animal behavior is sometimes generated by the collective activity of many neurons. It is hoped that methods like this will provide quantitative datasets that yield insights into how brain-wide neural dynamics encode animal action and perception. The ability to acquire large-scale recordings of neuronal activity in awake and unrestrained animals is needed to provide new insights into how populations of neurons generate animal behavior. We present an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of a freely behaving Caenorhabditis elegans with cellular resolution while simultaneously recording the animal’s position, posture, and locomotion. This instrument provides whole-brain imaging with cellular resolution in an unrestrained and behaving animal. We use spinning-disk confocal microscopy to capture 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s at 6 head-volumes/s. A suite of three cameras monitor neuronal fluorescence and the animal’s position and orientation. Custom software tracks the 3D position of the animal’s head in real time and two feedback loops adjust a motorized stage and objective to keep the animal’s head within the field of view as the animal roams freely. We observe calcium transients from up to 77 neurons for over 4 min and correlate this activity with the animal’s behavior. We characterize noise in the system due to animal motion and show that, across worms, multiple neurons show significant correlations with modes of behavior corresponding to forward, backward, and turning locomotion.

Biophysical Measurements of Bacterial Cell Shape

A bacterias shape plays a large role in determining its mechanism of motility, energy requirements, and ability to avoid predation. Although it is a major factor in cell fitness, little is known about how cell shape is determined or maintained. These problems are made worse by a lack of accurate methods to measure cell shape in vivo, as current methods do not account for blurring artifacts introduced by the microscope. Here, we introduce a method using 2D active surfaces and forward convolution with a measured point spread function to measure the 3D shape of different strains of E. coli from fluorescent images. Using this technique, we are also able to measure the distribution of fluorescent molecules, such as polymers, on the cell surface. This quantification of the surface geometry and fluorescence distribution allow for a more precise measure of 3D cell shape and is a useful tool for measuring protein localization and the mechanisms of bacterial shape control.