Vivo

Active mechanics of sea star oocytes

The cytoskeleton has the remarkable ability to self-organize into active materials which underlie diverse cellular processes ranging from motility to cell division. Actomyosin is a canonical example of an active material, which generates cellularscale contractility in part through the forces exerted by myosin motors on actin filaments. While the molecular players underlying actomyosin contractility have been well characterized, how cellular-scale deformation in disordered actomyosin networks emerges from filament-scale interactions is not well understood. In this talk, I’ll present work done in collaboration with Sebastian Fürthauer and Nikta Fakhri addressing this question in vivo using the meiotic surface contraction wave seen in oocytes of the bat star Patiria miniata as a model system. By perturbing actin polymerization, we find that the cellular deformation rate is a nonmonotonic function of cortical actin density peaked near the wild type density. To understand this, we develop an active fluid model coarse-grained from filament-scale interactions and find quantitative agreement with the measured data. The model makes further predictions, including the surprising prediction that deformation rate decreases with increasing motor concentration. We test these predictions through protein overexpression and find quantitative agreement. Taken together, this work is an important step for bridging the molecular and cellular length scales for cytoskeletal networks in vivo.

Energy landscapes, order and disorder, and protein sequence coevolution: From proteins to chromosome structure

In vivo, the human genome folds into a characteristic ensemble of 3D structures. The mechanism driving the folding process remains unknown. A theoretical model for chromatin (the minimal chromatin model) explains the folding of interphase chromosomes and generates chromosome conformations consistent with experimental data is presented. The energy landscape of the model was derived by using the maximum entropy principle and relies on two experimentally derived inputs: a classification of loci into chromatin types and a catalog of the positions of chromatin loops. This model was generalized by utilizing a neural network to infer these chromatin types using epigenetic marks present at a locus, as assayed by ChIP-Seq. The ensemble of structures resulting from these simulations completely agree with HI-C data and exhibits unknotted chromosomes, phase separation of chromatin types, and a tendency for open chromatin to lie at the periphery of chromosome territories. Although this theoretical methodology was trained in one cell line, the human GM12878 lymphoblastoid cells, it has successfully predicted the structural ensembles of multiple human cell lines. Finally, going beyond Hi-C, our predicted structures are also consistent with microscopy measurements. Analysis of both structures from simulation and microscopy reveals that short segments of chromatin make two-state transitions between closed conformations and open dumbbell conformations. For gene active segments, the vast majority of genes appear clustered in the linker region of the chromatin segment, allowing us to speculate possible mechanisms by which chromatin structure and dynamics may be involved in controlling gene expression. * Supported by the NSF

Light-degradable hydrogels as dynamic triggers for implantable devices

Triggerable materials capable of being degraded by selective stimuli stand to transform our capacity to precisely control biomedical device activity and performance while reducing the need for invasive interventions. This talk will cover the development of a modular and tunable light-triggerable hydrogel capable of interfacing with implantable devices. We have applied these materials to two applications in the gastrointestinal (GI) tract and demonstrated biocompatibility and on-demand triggering of the material in vitro, ex vivo, and in vivo. Light-triggerable hydrogels have the potential to be applied broadly throughout the GI tract and other anatomic areas. By demonstrating the first use of light-degradable hydrogels in vivo, we provide biomedical engineers and clinicians with a previously unavailable, safe, dynamically deliverable, and precise tool to design dynamically actuated implantable devices.

Anatomical decision-making by cellular collectives: bioelectrical pattern memories, regeneration, and synthetic living organisms

A key question for basic biology and regenerative medicine concerns the way in which evolution exploits physics toward adaptive form and function. While genomes specify the molecular hardware of cells, what algorithms enable cellular collectives to reliably build specific, complex, target morphologies? Our lab studies the way in which all cells, not just neurons, communicate as electrical networks that enable scaling of single-cell properties into collective intelligences that solve problems in anatomical feature space. By learning to read, interpret, and write bioelectrical information in vivo, we have identified some novel controls of growth and form that enable incredible plasticity and robustness in anatomical homeostasis. In this talk, I will describe the fundamental knowledge gaps with respect to anatomical plasticity and pattern control beyond emergence, and discuss our efforts to understand large-scale morphological control circuits. I will show examples in embryogenesis, regeneration, cancer, and synthetic living machines. I will also discuss the implications of this work for not only regenerative medicine, but also for fundamental understanding of the origin of bodyplans and the relationship between genomes and functional anatomy.

Magic numbers in protein phase transitions

Biologists have recently come to appreciate that eukaryotic cells are home to a multiplicity of non-membrane bound compartments, many of which form and dissolve as needed for the cell to function. These dynamical “condensates” enable many central cellular functions – from ribosome assembly, to RNA regulation and storage, to signaling and metabolism. While it is clear that these compartments represent a type of separated phase, what controls their formation, how specific biological components are included or excluded, and how these structures influence physiological and biochemical processes remain largely mysterious. I will discuss recent experiments on phase separated condensates both in vitro and in vivo, and will present theoretical results that highlight a novel “magic number” effect relevant to the formation and control of two-component phase separated condensates.

Holographic control of neuronal circuits

Genetic targeting of neuronal cells with activity reporters (calcium or voltage indicators) has initiated the paradigmatic transition whereby photons have replaced electrons for reading large-scale brain activities at cellular resolution. This has alleviated the limitations of single cell or extracellular electrophysiological probing, which only give access to the activity of at best a few neurons simultaneously and to population activity of unresolved cellular origin, respectively. In parallel, optogenetics has demonstrated that targeting neuronal cells with photosensitive microbial opsins, enables the transduction of photons into electrical currents of opposite polarities thus writing, through activation or inhibition, neuronal signals in a non-invasive way. These progresses have in turn stimulated the development of sophisticated optical methods to increase spatial and temporal resolution, light penetration depth and imaging volume. Today, nonlinear microscopy, combined with spatio-temporal wave front shaping, endoscopic probes engineering or multi scan heads design, enable in vivo in depth, simultaneous recording of thousands of cells in mm 3 volumes at single-spike precision and single-cell resolution. Joint progress in opsin engineering, wave front shaping and laser development have provided the methodology, that we named circuits optogenetics, to control single or multiple target activity independently in space and time with single- neuron and single-spike precision, at large depths. Here, we will review the most significant breakthroughs of the past years, which enable reading and writing neuronal activity at the relevant spatiotemporal scale for brain circuits manipulation, with particular emphasis on the most recent advances in circuit optogenetics.

Biophysics of Mitochondrial Metabolism in vivo

Dynamics of microbiota communities during physical perturbation

The consortium of microbes living in and on our bodies is intimately connected with human biology and deeply influenced by physical forces. Despite incredible gains in describing this community, and emerging knowledge of the mechanisms linking it to human health, understanding the basic physical properties and responses of this ecosystem has been comparatively neglected. Most diseases have significant physical effects on the gut; diarrhea alters osmolality, fever and cancer increase temperature, and bowel diseases affect pH. Furthermore, the gut itself is comprised of localized niches that differ significantly in their physical environment, and are inhabited by different commensal microbes. Understanding the impact of common physical factors is necessary for engineering robust microbiota members and communities; however, our knowledge of how they affect the gut ecosystem is poor. We are investigating how changes in osmolality affect the host and the microbial community and lead to mechanical shifts in the cellular environment. Osmotic perturbation is extremely prevalent in humans, caused by the use of laxatives, lactose intolerance, or celiac disease. In our studies we monitored osmotic shock to the microbiota using a comprehensive and novel approach, which combined in vivo experiments to imaging, physical measurements, computational analysis and highly controlled microfluidic experiments. By bridging several disciplines, we developed a mechanistic understanding of the processes involved in osmotic diarrhea, linking single-cell biophysical changes to large-scale community dynamics. Our results indicate that physical perturbations can profoundly and permanently change the competitive and ecological landscape of the gut, and affect the cell wall of bacteria differentially, depending on their mechanical characteristics.